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Alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of microRNA-29b in aneurysm patients after endovascular aortic repair

Alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of... Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2017 Volume: 11 Running head verso: Yang et al Drug Design, Development and Therapy Dovepress Running head recto: Alginate oligosaccharide indirectly affects TLR signaling open access to scientific and medical research DOI: 140206 Open access Full Text article O rig inal r esearch alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of microrna- 29b in aneurysm patients after endovascular aortic repair 1–4, Yong Yang * Abstract: Endovascular aortic repair (EVAR) is often followed by aneurysm recurrence. 1–4, Alginate oligosaccharide (AOS) has potential antitumor properties as a natural product while Zhenhuan Ma * 1–4 the related mechanisms remain unclear. Toll-like receptor (TLR) signaling is associated with guokai Yang 1–4 inflammatory activity of aneurysm and may be affected by miR-29b. Thus, inhibitory function Jia Wan of AOS on aneurysms was explored by measuring the important molecules in TLR4 signaling. 1–4 guojian li After EVAR, a total of 248 aortic aneurysm patients were recruited and randomly assigned 1–4 lingjuan Du into two groups: AOS group (AG, oral administration 10-mg AOS daily) and control group 1–4 Ping lu (CG, placebo daily). The size of residual aneurysms, aneurysm recurrence, and side effects Department of Vascular surgery, were investigated. Aneurysm recurrence was determined by Kaplan–Meier analysis. After The s econd People’s h ospital of 2 years, eight and two patients died in the CG and AG, respectively. The sizes of residual Yunnan Province, Kunming, china; Department of Vascular surgery, aneurysms were significantly larger in the CG than in the AG ( P,0.05). The incidence of The Fourth affiliated hospital of aneurysm recurrence was also significantly higher in the CG than in the AG ( P,0.05). AOS Kunming Medical University, Kunming, treatment reduced the levels of miR-29b, TLR4, mitogen-activated protein kinase (MAPK), china; Department of Vascular s urger y, Vascular s urger y c entre in nuclear factor kappa B (NF-kappa B), interleukin 1 (IL-1) beta, and interleukin 6 (IL-6). Yunnan Province, Kunming, china; Overexpression and silence of miR-29b increased and reduced the level of TLR4, phospho-p65 Department of Vascular surgery, NF-kappa B, phospho-p38 MAPK, IL-1 beta, and IL-6. Spearman’s rank correlation analysis abdominal surgery centre in Yunnan Province, Kunming, china shows that the level of miR-29b is positively related to the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 (P,0.05). Thus, AOS represses aneurysm recurrence by indirectly affecting *These authors contributed equally to this work TLR signaling via miR-29b. Keywords: alginate oligosaccharide, outcome assessment, aortic aneurysms, minimally invasive endovascular repair, toll-like receptor signaling pathway, anti-inflammatory agent, microRNA- 29b, mitogen-activated protein kinase Introduction Aortic aneurysm (AA) is a major and emerging threat to public health. Endovascular aortic repair (EVAR) has been developed as a minimally invasive therapy. Although there are some advantages for EVAR, the technique still has some shortcomings: the residual aneurysm often regrows following EVAR and is an important risk factor correspondence: Zhenhuan Ma for the surgery failure; High-rate aneurysm recurrence has been widely reported in Department of Vascular surgery, The EVAR. Stent-assisted coil embolization for large or giant aneurysms has high recur- second People’s hospital of Yunnan Province, 176 Qingnian lu, Kunming rence rates, which may be associated with metal coverage rates of the stents used in 650021, china these procedures. More than 17% patients had aneurysm recurrence and 13% patients Tel +86 871 515 6650 email zhenhuanyy@126.com had residual aneurysms after 2-year segmental artery coil embolization. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 2565–2579 Dovepress © 2017 Yang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/DDDT.S140206 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Yang et al Dovepress In most cases, medical therapy is often considered to con- were measured at 234 nm in a UV-2100PC UV-VIS spec- trol the disease before and after aneurysm therapy. EVAR trophotometer (Shimadzu, Kyoto, Japan). The degrees of infection is usually followed by the technique and will result polymers (DP) were further determined by electrospray in a high risk of aneurysm recurrence. Persistent endoleak is ionization mass spectrometry (ESI-MS). The hydrolysate also a main side effect after EVAR. All the adverse effects was transferred to a carbograph column to remove salt, and signic fi antly affect life quality of AA patients. Most research then concentrated, dried, and resolved in 1 mL methanol. 9–12 13–16 shows that oxidative stress and inflammation are Two-microliter supernatant was injected into a LTQ XL mass associated with the risks of various cancers. Marine algae spectrometer (ThermoFinnigan, Austin, TX, USA). AOS was have been found to be with various bioactive compounds, detected in a positive-ion mode: ion source, 5 kV; capillary such as alginate oligosaccharide (AOS). AOS has been temperature, 280°C–310°C; tube lens, 260 V; sheath gas, 17,18 reported to have antioxidant and anti-inflammatory prop - 35 arbitrary units. The mass spectrometer was set over the 19 20 erties. AOS has been found to have antitumor activities. range m/z 500–1,500. Furthermore, AOS has few side effects as a kind of natural product. AOS may offer novel anticancer agents for various Participants cancers. All the procedures were approved by the Ethical Committee However, the effects of AOS on aneurysm recurrence of the Second People’s Hospital of Yunnan Province and and regrowth, and related molecular mechanisms remain written informed consent was obtained from each patient unclear. Toll-like receptor (TLR) is an important protein for this study. The study was performed according to the in various innate immunity and inflammatory activities. Declaration of Helsinki. A total of 397 patients, experienc- Previous study demonstrates that the silence of TLR4 sig- ing severe chest and back pain, attended the Second People’s naling pathway inhibits the progression of abdominal aortic Hospital of Yunnan Province during the period from January aneurysm (AAA). miRNA, as a small noncoding RNA, is 2013 to December 2014. involved with various biological activities, including cell The diagnosis of thoracic aortic aneurysm (TAA) and 23,24 25 26 development, homeostasis, immune, and ina fl mmatory dissection was confirmed from clinical diagnostic criteria 27,28 33 responses. miRNAs have been reported to be important issued by a previous report. One of the following includ- biomarkers for vascular diseases, while they have been ing criteria were considered: the diameter of aneurysm was considered as potential targets for exploiting therapeutic more than 5 cm; the diameter of aneurysm was 4–5 cm approaches for the disease. Therefore, miRNA is an impor- and increased in size by 0.5 cm in less than half a year; tant regulator for controlling immune and inflammatory the diameter of aneurysm was twofold the diameter of the responses to tumor progression. Present evidence shows normal infrarenal aorta; sudden onset of severe chest pain therapeutic manipulation of miR-29b, which holds a great that had a tearing or ripping quality (classic symptom); promise for controlling AAA development. Therefore, we anterior chest pain, associated with anterior arch or aortic aim to explore the functional role of AOS in AAs’ regression root dissection; neck or jaw pain, with aortic arch involve- by exploring its effects on TLR4 signaling and miR-29b. To ment and extension into the great vessels; tearing or ripping understand safety and efficacy of AOS, the size of residual intrascapular pain, involving the descending aorta; cerebro- aneurysms, aneurysm recurrence, and the side effects were vascular accident symptoms, hemianesthesia, hemiparesis, also investigated. and hemiplegia. The following excluding criteria were used: a family history of AA and dissection; malignancy; Methods psychological disorders. a Os prepared from alginate sodium A computed tomography (CT) scan was performed and The AOS with α-L-guluronate units and β-D-mannuronate the selected patients were revealed with TAA and/or dis- units was bought from Qingdao Qingya Chemical Co., Ltd section. Finally, 248 patients were selected for the present (Qingdao, China). AOS was prepared from alginate sodium study and received modified EVAR. The Zenith TX2 TAA according to an earlier report using alginate lyase, which Endovascular Graft with Pro-Form is a two-piece cylindrical depolymerizes alginate sodium. Briefly, 1 kg sodium alg- endovascular graft with proximal and distal materials (COOK inate was depolymerized in 100 L tap water with 10 mg Medical Inc., Bloomington, IN, USA). The proximal parts alginate lyase at 39°C for 2 h. The lyase was denatured at can be either nontapered or tapered. The stent grafts are made 100°C for 10 min. The amounts of unsaturated saccharides of full-thickness woven polyester fabric sewn to steel Cook-Z submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling stents with polyester and monofilament polypropylene. diameters were measured in infrarenal aorta, which is The Zenith TX2 TAA endovascular graft with ProForm is perpendicular to aortic axis. fully stented to provide stability and the expansible force to enzyme-linked immunosorbent assay open the lumen of the graft during deployment. All endovas- cular grafts with Pro-Form are two cylindrical endovascular analysis grafts with proximal and distal materials. The proximal parts Five-milliliter blood was obtained by vein puncture. Serum was can be either nontapered or tapered. The diameter and length separated by using the centrifugation at 1,500 g, 4°C for 10 min. of each stent-graft was identified preoperatively with use The concentration of tumor necrosis factor (TNF)-alpha was of multislice CT or diagnostic imaging via a catheter with measured by using a human TNF-alpha enzyme-linked immu- a marker. The graft diameter was .10% than normal size nosorbent assay (ELISA) kit (Thermo Fisher Scientic fi , Inc. associated with the diameter of the nondiseased segments of Corporate, Waltham, MA, USA). The concentration of TLR4 aorta under the aneurysm. The length of graft was equal to was measured by human TLR4 ELISA Kit from RayBiotech the distance from the transection site to the nondiseased size (Norcross, GA, USA). The levels of interleukin 1 (IL-1) beta of the descending aorta below the aneurysm. and IL-6 were measured by the ELISA kits from R&D Sys- After surgery, all the patients were randomly and evenly tems (Minneapolis, MN, USA). All the concentrations were assigned into two groups: AOS group (AG), 124 AA patients calculated according to standard calibrating curves. received 10 mg AOS daily and control group (CG), 124 AA The effects of a Os on the viability of patients received a 10 mg placebo daily. endothelial cells end points Endothelial cells (ECs), isolated from human aneurysm, were Outcome criteria were established according to a previous purchased from Shanghai Cell Bank (Shanghai, China) and report for thoracic EVAR. The end points included early cultured in DMEM at 37°C under a 5% CO environment. The morbidities (stroke and paraplegia), respiratory failure, acute cell concentrations were adjusted to 1×10 /cell in a 96-cell kidney injury, need for dialysis, aneurysm-related death, and plate. Cell viability of ECs was determined using Trypan blue reoperation. The proximal attachment zones were determined (Solarbio, Beijing, China) and [ H]thymidine (Amersham based on the proximal attachment site of the proximal edge biosciences, Piscataway, NJ, USA, specific activity of the covered graft; ,2 cm of the left subclavian artery 5 Ci/mmol) uptake after the cells were cultured with differ- (without covering it) is defined as zone 3; it is defined as ent concentrations of AOS (0, 1, 10, 100, and 1,000 ng/mL) zone 4 if the proximal extent of the endograft is .2 cm to for 3 days. The Trypan blue-positive cells were measured the left subclavian artery and ended within the proximal with a hemocytometer under a microscope. For the [ H] half of the TAA (T6 level is near the midpoint of TAA). thymidine absorption, cells were counted after exposure to Aneurysm-related death would be measured at any time. different concentrations of AOS, and 200 μL cell suspension Respiratory failure was determined if mechanical ventila- of 1.0×10 cells/mL for each condition were added to each tion was more than 1 day and reintubation and tracheostomy well of a 96-well plate. After 24-h culture, cells were labeled would be needed. with one Ci/well of [ H]thymidine for 4 h, then collected with an automated sample harvester and measured with a liquid clinical follow-up scintillation counter (Beckman Coulter, Brea, CA, USA). Detailed data were collected using the index admission when surgery was conducted. After discharge, all the patients were cell co-infection followed up at 1, 3, 6, and 12 months and yearly at outpatient Antagomirs and mimics of miR-29b were synthesized by clinics. In addition, a CT angiogram was performed once a Sangon Biotech (Shanghai) Co. Ltd (Shanghai, China). year for 2 years. The cell lines were co-infected with the antagomirs and mimics via Lipofectamine 3000 (Life Technologies, Inc., Carlsbad, The measurement of aa size CA, USA). After 3-day co-infection, the mutant cells were AA size was measured by using an ultrasound scanning picked by using 200 μg/mL G-418. TLR4, nuclear factor system (Xuzhou Forward Medical Instrument Co., Limited, kappa-B (NF-kappa B), IL-1 beta, and IL-6 were measured Xuzhou, China). The ultrasound was sensitive and specific. by ELISA in the cell lines. Meanwhile, cell viability was also Maximum transverse and anterior-posterior external measured using Trypan blue and [ H]thymidine uptake. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress real-time polymerase chain reaction of miR-29b and the levels of TLR4, TNF-alpha, IL-1 beta, and IL-6. Statistical calculation was conducted via SPSS 20 quantification of miR-29b (SPSS Inc., Chicago, IL, USA). There were significantly Total cellular RNA was isolated using an RNA Isolation statistical differences if P-values were ,0.05. Kit (Tiangen, Beijing, China), RNeasy Mini kit (QIA- GEN, Valencia, CA, USA). cDNA was synthesized Results from total RNA by reverse transcription using TaqMan characterization of a Os MicroRNA Reverse Transcription Kit (Applied Biosys- Four main components (DP3, DP4, DP5, and DP6) were tems, Foster City, CA, USA). The levels of miR-29b isolated from alginate sodium after enzyme digestion. were measured by using real-time polymerase chain The isolated fractions of AOS were further confirmed by reaction (Bio-Rad Lab., Hercules, CA, USA). MiR-29b, ESI-MS under the conditions that produced mass spectra forward primer: 5′-GGCTTCAGGAAGCTGGTTT-3′; with [M + K] . The predicted masses DP3 (C18H23O19Na3), reverse primer: 5′-GTGCAGGGTCCGAGGT-3′. U6, forward DP4 (C24H30O25Na4), DP5 (C30H37O31Na5), and DP6 primer: 5′-TTGGTGCTCGCTTCGGCA-3′; reverse primer: (C36H44O37Na6) were 612, 810, 1,008, and 1,206 Da, 5′-GTGCAGGGTCCGAGGT-3′. U6 snRNA was used as an respectively (Figure 1). internal control. The baseline characters of the patients Western blot with Taa and dissection TLR4 can regulate TLR inflammatory response via phospho- After selection, a total of 248 patients with TAA received p65 NF-kappa B p65. Mitogen-activated protein kinase the treatment with modified EVAR. After 2-year therapies (MAPK) as regulatory signal mediators plays a pivotal role in of AOS and placebo, eight patients died in the CG and two organism immunity. To understand the effects of miR-29b patients died in the AG. Thus, 116 patients and 122 patients on TLR4 signaling, phosphorylated MAPK and NF-kappa B finished the experiments of CG and AG, respectively as second messengers in TLR4 signaling were measured by (Figure 2). The baseline characters of all patients are listed Western blot. The antihuman antibodies of TLR4 (ab17942), in Table 1. The number of men was more than women. Most Phospho-p65 anti-NF-kappa B (ab86299), phospho-p38 patients had concomitant diseases, such as cerebrovascular, mitogen-activated protein kinase (MAPK, ab31828), IL-1 ischemic heart, chronic obstructive pulmonary, and renal beta (ab2105), IL-6 (ab6672), β-actin antibody (ab8227), dysfunction. The ages (65.8±14.9 years in AG group vs and secondary antibody Goat Anti-Rabbit IgG H&L (HRP, ab6721) were purchased from Abcam (Beijing, China). Proteins were separated by sodium dodecyl sulfate polyacryl- amide gel electrophoresis and transferred to a polyvinylidene '3 '3 '3 '3 diu fl oride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in the buffer (10 mM Tris [pH 7.5] and 50 mM NaCl) and incubated with primary antibodies overnight at 4°C. The membrane was fur- ther treated with secondary antibodies (1:3,000). Immunore- active bands were visualized by enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA). 5HODWLYHLQWHQVLW\ statistical analysis All data were presented as mean ± SD or frequency and percentage. The variables were compared by the t-test, χ , or P] Fisher’s exact test. Early outcomes and late outcomes were Figure 1 electrospray ionization mass spectrometry analysis of the DP of digested compared using univariate statistics. Overall survival and + aOs from alginate sodium with produced mass spectra as [M + K] . Notes: Mass spectra were visualized following the separation of DP3 ([M + K] =651 being free from aneurysm-related death and reintervention Da); mass spectra were visualized following the separation of DP4 ([M + K] =849 Da); were measured using the Kaplan–Meier and the log-rank mass spectra were visualized following the separation of DP5 ([M + K] =1,047 Da); and mass spectra were visualized following the separation of DP6 ([M + K] =1,245 test. Spearman’s rank correlation coefficient was calculated Da). The mass spectrometer was set over the range m/z 500–1,500. to confirm the strength of correlation between relative levels Abbreviations: aOs, alginate oligosaccharide; DP, degree of polymerization. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling DRUWLFDQHXU\VPSDWLHQWV H[FOXGHG Primary outcomes of eVar GLGQRWPHHWLQFOXVLRQFULWHULD ZHUHORVW CT of the thoracic aorta showed that it could be observed KDGSURWRFROYLRODWLRQ ZLWKGUHZFRQVHQW easily before operation. Deployment of multilayer stent was RWKHUUHDVRQV successfully established in the thoracic aorta after opera- FDVHVXQGHUZHQWUDQGRPL]DWLRQ tion. Three-dimensional reconstruction based on a series of &* FDVHV $* FDVHV angiography images showed TAA bulging in the T2–T5 seg- GLHG $IWHUPRQWKV GLHG ment. The images showed the surgery was successful and no FDVHV FDVHV endoleak was found. Comparatively, emergency CT showed GLHG $IWHUPRQWKV AAA bulging before surgery. Finally, graft implantation and FDVHV FDVHV GLHG $IWHUPRQWKV GLHG artificial vessel were successfully placed in AAs. FDVHV FDVHV end points $QHXU\VPVL]H 6LGHHI IHFWV / 75 1)NDSSD% ,QIODPPDWRU\F\WRNLQHV The average follow-up duration was 24±5 months (see Table 2). Overall, survival was 93.5% and 98.4% in the Figure 2 Present study flowchart. Notes: cg, the patients received 10 mg placebo daily in a control group. ag, the CG and AG, respectively (P=0.05) (Table 2). More patients patients received 10 mg aOs daily in the aOs group. underwent reintervention in the CG than in the AG (P,0.05). Abbreviations: aa, aortic aneurysm; cg, control group; aOs, alginate oligosac- charide; ag, aOs group; Tlr4, toll-like receptor 4; nF-kappa B, nuclear factor Comparatively, the Kaplan–Meier analysis also revealed that kappa B. being free from reintervention was signic fi antly higher in the CG than in the AG during the same period (log rank test, P=0.04). There were 16 patients in the CG and 6 patients in 62.1±15.3 years in AG, P=0.48) and renal insufficiency the AG with aortic reintervention, including endoleak and (33.1% in CG vs 30.6% in AG; P=0.68) were similar in both retrograde dissection in the 2-year follow-up. groups. The incidences of dissection aneurysm, the propor- The covered stent grafts were delivered successfully, and tion of aortic rupture, the maximal diameters of the aneu- complete thrombosis of the aneurysm or entry closure with rysms, and all other parameters were also similar between stable thrombosis was observed in all patients. According two groups (P.0.05) (Table 1). to Kaplan–Meier analysis, the patient survival rates were 93.5% in the CG and 98.4% in the AG at 2-year postopera- Table 1 Baseline characteristics of aortic aneurysm patients tion. During the 2-year follow-up, 10 patients died. Among those, three patients died of aortic reintervention and seven AG (n=124) CG (n=124) P-value patients died of endoleak and retrograde dissection. age, years 65.8±14.9 62.1±15.3 0.48 Male, n (%) 84 (67.7) 86 (69.4) 0.78 alcohol drinking, n (%) 48 (38.7) 44 (35.4) 0.60 side effects smoking, n (%) 60 (48.4) 62 (50.0) 0.80 EVAR-related adverse effects were pain (back or chest), spouse, n (%) 114 (91.9) 112 (90.3) 0.66 Daily calorie intake, calories 2,518.2±456.3 2,476.6±449.4 0.52 fainting, persistent cough, rapid heartbeat, dizziness, sudden Body mass index, m /kg 23.7±3.2 22.6±2.9 0.67 weakness, ischemia of intestines, pulse-less legs, cold arms Diabetes mellitus, n (%) 9 (7.3) 7 (5.6) 0.61 or legs, and so on (Table 3). Among these adverse effects, hypertension, n (%) 78 (62.9) 74 (59.7) 0.61 coronary artery obstructive 15 (12.1) 17 (13.7) 0.70 disorder, n (%) Table 2 late outcomes (n =124 in each group before repair) Renal insufficiency, n (%) 38 (30.6) 41 (33.1) 0.68 aortic pathology Variables AG CG Chi-square P-value Medial degeneration, n (%) 72 (58.3) 61 (48.9) 0.16 (n=122) (n=116) statistic Dissecting aneurysm, n (%) 62 (50.0) 55 (44.4) 0.37 Overall survival 122 (98.4) 116 (93.5) 3.75 0.05 Traumatic dissection, n (%) 8 (6.3) 5 (4.0) 0.39 recurrent aa 6 (4.8) 16 (13.8) 5.58 0.02 Marfan syndrome, n (%) 5 (4.0) 8 (6.5) 0.39 endoleak 2 (1.6) 10 (8.6) 6.05 0.01 aortic rupture, n (%) 5 (4.0) 5 (4.0) 1.00 retrograde dissection 2 (1.6) 3 (2.4) 0.26 0.61 lesion leakage 1 (0.8) 3 (2.4) 1.12 0.29 Zone 3, n (%) 67 (54.0) 74 (59.7) 0.37 Notes: Data presented as n (%). cg, Zenith TX2 aa graft repair and placebo; ag, Zone 4, n (%) 57 (46.0) 50 (40.3) 0.37 Zenith TX2 aa graft repair and aOs. The period of follow-up was 2 years. There aneurysm maximum size (mm) 50.2±4.8 51.0±4.0 0.76 were significantly statistical differences if P,0.05. Note: Data presented as mean ± standard deviation. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- Abbreviations: aOs, alginate oligosaccharide; ag, aOs group; cg, control group. charide; cg, control group. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress Table 3 The side effects between cg and ag, n (%) statistical difference for AA size between the AG and the CG Side effects AG CG Chi-square P-value (P.0.05). Compared with CG, AOS significantly reduced (n=122) (n=116) statistic AA size after 2-year repair (P,0.01) (Figure 3). The results Pain (back or chest) 5 (4.1) 23 (19.8) 14.2 0.01 suggest that long-term AOS consumption will further control Fainting 7 (5.7) 12 (10.3) 1.72 0.19 the growth of residual aneurysms. Persistent cough 7 (5.7) 26 (22.4) 13.85 0.01 rapid heartbeat 5 (4.1) 12 (10.3) 3.50 0.06 a Os reduces the levels of mir-29b in aa Dizziness 7 (5.7) 10 (8. 6) 0.75 0.39 sudden weakness 12 (10.7) 8 (6.9) 0.67 0.41 patients ischemia of intestines 10 (8.2) 15 (12.9) 1.42 0.23 Before AOS treatment, there was no significantly statistical Pulse-less legs 5 (4.1) 14 (12.1) 5.14 0.02 difference for relative level of miR-29b between the AG and cold arms or legs 7 (5.7) 15 (12.9) 3.67 0.06 surgery infection 7 (5.7) 11 (9.4) 1.19 0.27 the CG (P.0.05). After long-term consumption of AOS, it chronic wound 5 (4.1) 20 (17.2) 10.93 0.01 significantly reduced the relative level of miR-29b ( P,0.05) infection (Figure 4). The results suggest that long-term AOS consump- Notes: The period of follow-up was 2 years. There were significantly statistical differences if P,0.05. cg, Zenith TX2 aa graft repair and placebo; ag, Zenith TX2 tion will reduce the relative level of miR-29b. aa graft repair and aOs. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- a Os decreases the levels of Tlr4, nF- charide; cg, control group. kappa B, il-1 beta, and il-6 Before EVAR and AOS treatment, there was no signic fi antly back and chest pain rate, and the rate of persistent cough statistical difference for serum levels of TLR4 (Figure 5A), and wound infection were higher in the AG than in the CG NF-kappa B (Figure 5B), and IL-1 beta (Figure 5C) (P.0.05). (P,0.05). Endovascular therapy has excellent short-term After 2-year follow-up, there was a greater reduction in the effect and stable mid-to-long-term result. AOS can be used as serum levels of TLR4 (Figure 5A), NF-kappa B (Figure 5B), a therapy option such as in an emergency situation or a rescue and IL-1 beta (Figure 5C) in the AG when compared with method by reducing the side effects caused by EVAR. the CG (P,0.05). Before EVAR and AOS treatment, serum levels of IL-6 were lower in the CG than the AG (Figure 5D) a Os reduces aa size (P,0.05). After 2-year follow-up, there was a greater reduc- EVAR surgery reduced the AA size after 2-year follow-up in tion in the serum levels of IL-6 in the AG (Figure 5D) when comparison with the size before surgery in the CG and the AG compared with the CG (P,0.05). The results suggest that (Figure 3). Before AOS treatment, there was no signic fi antly long-term AOS consumption decreases the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6. 7KHVL]HRIDQHXU\VP PP 5HODWLYHOHYHORIPL5E &*% $*% &*$ $*$ Figure 3 scatter dot plot of the aa size variances in different groups. &*% $*% &*$ $*$ Notes: cg-B, before eVar and placebo treatment. ag-B, before eVar and aOs treatment. cg-a, after eVar and 2-year placebo treatment. ag-a, after eVar Figure 4 relative levels of mir-29b in different groups. and 2-year aOs treatment. ag, aa patients received 10 mg aOs daily and cg, Notes: cg-B, before eVar and placebo treatment. ag-B, before eVar and aOs aa patients received 10 mg placebo daily. aa size was measured by ultrasound treatment. cg-a, after eVar and 2-year placebo treatment. ag-a, after eVar and scanning. Maximum transverse and anterior–posterior external diameters were 2-year aOs treatment. n =124, 124, 116, and 122 cases in cg-B, ag-B, cg-a, and measured in infrarenal aorta, which is perpendicular to aortic axis. There was a ag-a groups, respectively. ag, aa patients received 10 mg aOs daily and cg, aa statistical difference if P,0.05. patients received 10 mg placebo daily. There was a statistical difference if P,0.05. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- charide; cg, control group; eVar, endovascular aortic repair. charide; cg, control group; eVar, endovascular aortic repair. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling $ % 7/5 SJP/ 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &*% $*% &*$ $*$ &*% $*% &*$ $*$ ,/ SJP/ ,/EHWD SJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &*% $*% &*$ $*$ &*% $*% &*$ $*$ Figure 5 The effects of aOs on the levels of Tlr4, nF-kappa B, il-1 beta, and il-6 in aa patients. Notes: (A) The effects of aOs on the levels of Tlr4. ( B) The effects of aOs on the levels of nF-kappa B. ( C) The effects of aOs on the levels of il-1 beta. ( D) The effects of aOs on the levels of il-6. n =124, 124, 116, and 122 cases in cg-B, ag-B, cg-a, and ag-a groups, respectively. ag, aa patients received 10 mg aOs daily and cg, aa patients received 10 mg placebo daily. cg-B, the patients before taking placebo. cg-a, the patients after taking placebo for two years. ag-B, the patients before taking aOs. ag-a, the patients after taking aOs for two years. There was a statistical difference if P,0.05. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosaccharide; cg, control group; il-6, interleukin 6; Tlr, toll-like receptor; nF-kappa B, nuclear factor kappa B. exposure to different concentrations of AOS. As shown in The levels of mir-29b are positively Figure 7B, there is no significant reduction in [ H]thymidine associated with the serum levels of Tlr4, uptake that was measured when the concentration was more nF-kappa B, il-1 beta, and il-6 than 1,000 ng/mL (P,0.05). Based on these results, admin- Figure 6 shows that the increase in miR-29b also increases istration of 10 mg daily will not cause toxicity to ECs. the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6. Spearman’s rank correlation test demonstrates that the levels a Os inhibits the growth of ecs but not of miR-29b are positively associated with the serum levels their viabilities of TLR4 (Figure 6A), NF-kappa B (Figure 6B), IL-1 beta ECs were isolated from human aneurysm. Figure 8A shows (Figure 6C), and IL-6 (Figure 6D) (P,0.05) because the rho that AOS inhibits the growth of ECs when its concentration values are 0.82, 0.75, 0.80, and 0.85, respectively. is more than 100 ng/mL. Based on the results, 10 mg/day was used for all patients (the weight of each patient was ,100 kg). effects of a Os toxicity on ecs Cell viability analysis shows that AOS cannot affect cell To avoid the effects of AOS on ECs because of its toxicity, viability by using Trypan Blue (Figure 8B) and [ H]thymi- the concentration of AOS was measured. Trypan blue analy- dine incorporation (Figure 8C) even when the concentration sis showed that exposure of the ECs to AOS, in the presence is 1,000 ng/mL. of more than 100 ng/mL, resulted in loss of cell viability a Os treatment reduces relative levels (Figure 7A; P,0.05). The viability of cells was still more than 95% after 72-h culture when the concentration of AOS of mir-29b was 1,000 ng/mL. The toxicity of AOS was also evaluated Figure 9A showed that AOS could not affect the relative by measuring [ H]thymidine incorporation into DNA after levels of miR-29b when the concentration was ,10 ng/mL. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress $ % 7/5 SJP/ UKR  UKR 3  3 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI 5HODWLYHOHYHORIPL5E 5HODWLYHOHYHORIPL5E & ' ,/ SJP/ UKR  UKR ,/EHWD SJP/ 3  3 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI 5HODWLYHOHYHORIPL5E 5HODWLYHOHYHORIPL5E Figure 6 The relationship between the level of mir-29b and the levels of Tlr4, nF-kappa B, il-1 beta, and il-6. Notes: (A) The relation between the level of mir-29b and the level of Tlr4. ( B) The relation between the level of mir-29b and the level of nF-kappa B. ( C) The relation between the level of mir-29b and the level of il-1 beta. ( D) The relation between the level of mir-29b and the level of il-6. spearman’s rank correlation test was used to compare the significance of two parameters. There is a positive relationship if the value of rho falls between 0.5 and 1. There was a statistical difference if P,0.05. Abbreviations: il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Tlr, toll-like receptor. AOS reduced the relative levels of miR-29b when the a Os treatment reduces the levels of Tlr4, concentration was more than 10 ng/mL and reached the low- nF-kappa B, il-1 beta, and il-6 in ecs est level when the concentration was 1,000 ng/mL. Figure 9B Figure 10A showed that AOS could not affect the levels of showed that the relative levels of miR-29b reached the low- TLR4 when the concentration was ,10 ng/mL. AOS reduced est level when the microRNA was blocked, and reached the the levels of TLR4 when the concentration was more than highest level when the microRNA was overexpressed. 10 ng/mL and reached the lowest level when the concentration $ % FSPî +@WK\PLGLQHXSWDNH 5HODWLYHYLDELOLW\ $26FRQFHQWUDWLRQ QJP/ $26FRQFHQWUDWLRQ QJP/ Figure 7 effects of aOs on the viability of ecs. Notes: (A) cell viability of ecs was measured using Trypan blue. ( B) cell viability of ecs was measured using [ h]thymidine uptake. The data were presented as the mean ± standard deviation. n =8 in each group. Abbreviations: aOs, alginate oligosaccharide; ecs, endothelial cells. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling QJP/ QJP/ QJP/ QJP/ QJP/ FHOOVP/ V &XOWXUHWLPH KRXU % & FSPî +@WK\PLGLQHXSWDNH 5HODWLYHYLDELOLW\ $26FRQFHQWUDWLRQ QJP/ $26FRQFHQWUDWLRQ QJP/ Figure 8 The effects of aOs on the growth of ecs. Notes: (A) real-time analysis of the effects of aOs on the growth of ecs. ( B) Trypan blue analysis of the effects of aOs on the viability of ecs. ( C) [ h]thymidine incorporation analysis of the effects of aOs on the viability of ecs. n =8. Abbreviations: aOs, alginate oligosaccharide; ecs, endothelial cells. was 1,000 ng/mL. Figure 10B showed that AOS could not the lowest level when the concentration was 1,000 ng/mL. affect the levels of NF-kappa B when the concentration Figure 10C showed that AOS could not affect the levels of was ,10 ng/mL. AOS reduced the levels of NF-kappa B IL-1 beta when the concentration was ,10 ng/mL. AOS when the concentration was more than 10 ng/mL and reached reduced the levels of IL-1 beta when the concentration was $ % 5HODWLYHOHYHORIPL5E 5HODWLYHOHYHORIPL5E QJP/ QJP/ QJP/ QJP/ QJP/ &* 2* 6* $26FRQFHQWUDWLRQ Figure 9 relative levels of mir-29b in different groups. Notes: (A) The effects of aOs on the relative levels of mir-29b. ( B) The effects of different treatments on relative levels of mir-29b. n =8 for each group. *P,0.05 vs the cg. Abbreviations: aOs, alginate oligosaccharide; cg, control group; sg, mir-29b was silenced by antagomirs; Og, mir-29b was overexpressed. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress $ % 7/5 SJP/ 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ $26FRQFHQWUDWLRQ $26FRQFHQWUDWLRQ & ' ,/ SJP/ ,/EHWD SJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ $26FRQFHQWUDWLRQ $26FRQFHQWUDWLRQ Figure 10 The effects of aOs on the levels of Tlr4, nF-kappa B, il-1 beta, and il-6 in ecs. Notes: (A) The effects of aOs on the levels of Tlr4. ( B) The effects of aOs on the levels of nF-kappa B. ( C) The effects of aOs on the levels of il-1 beta. ( D) The effects of aOs on the levels of il-6. * P,0.05 vs the cg without aOs addition. Abbreviations: aOs, alginate oligosaccharide; cg, control group; ecs, endothelial cells; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Tlr, toll-like receptor. more than 10 ng/mL and reached the lowest level when the silence of miR-29b reduced the level of TLR4. Figure 11B concentration was 1,000 ng/mL. Figure 10D showed that showed that the overexpression of miR-29b increased the AOS could not affect the levels of IL-6 when the concentra- level of NF-kappa B and the silence of miR-29b reduced the tion was ,100 ng/mL. AOS reduced the levels of IL-6 when level of NF-kappa B. Figure 11C showed that the overex- the concentration was more than 100 ng/mL and reached the pression of miR-29b increased the level of IL-1 beta and the lowest level when the concentration was 1,000 ng/mL. All silence of miR-29b reduced the level of IL-1 beta. Figure 11D the results suggest that AOS treatment reduces the levels of showed that the overexpression of miR-29b increased the TLR4, NF-kappa B, IL-1 beta, and IL-6 in ECs. The results level of IL-6 and the silence of miR-29b reduced the level of can be compared with the levels obtained from the serum IL-6. Therefore, miR-29b level is positively associated with in patients. the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 in ECs, suggesting that the change of miR-29b will affect the levels Mir-29b level is positively associated with of TLR4, NF-kappa B, IL-1 beta, and IL-6. the levels of Tlr4, nF-kappa B, il-1 beta, and il-6 in ecs Western blot MiR-29b mimics and antagomirs were successfully trans- To measure the effects of miR-29b on the changes of fected with ECs and the estimated transfection rate was phospho-p65 NF-kappa B, and phospho-p38 MAPK, between 60% and 80%. Figure 11A showed that the over- Western blot analysis was performed. Western blot analysis expression of miR-29b increased the level of TLR4 and the shows that miR-29b overexpression increases the levels of submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling $ % 7/5 SJP/ 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &* 2* 6* &* 2* 6* & ' ,/ SJP/ ,/EHWD SJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &* 2* 6* &* 2* 6* ( ) FSPî &HOOYLDELOLW\ +@WK\PLGLQHXSWDNH &* 2* 6* &* 2* 6* Figure 11 The effects of mir-29b on ecs. Notes: (A) The effects of mir-29b on the levels of Tlr4. ( B) The effects of mir-29b on the levels of nF-kappa B. ( C) The effects of mir-29b on the levels of il-1 beta. (D) The effects of mir-29b on the levels of il-6. ( E) Trypan blue analysis of the effects of mir-29b on ec viability. ( F) [ h]thymidine uptake analysis of the effects of mir-29b on ec viability. * P,0.05 vs the cg. Abbreviations: cg, control group; ecs, endothelial cells; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Og, mir-29b was overexpressed; sg, mir-29b was silenced by antagomirs; Tlr, toll-like receptor. phospho-p65 NF-kappa B (Figure 12A), phospho-p38 MAPK 248 patients who underwent EVAR, there was no signic fi ant (Figure 12B), TLR4 (Figure 12C), IL-1 beta (Figure 12D), difference for early mortality and neurologic complications and IL-6 (Figure 12E) when compared with the level of between the CG and AG. According to late outcomes, the CGs. In contrast, miR-29b inhibitor reduces the levels of overall survival rate was higher in the AG than in the CG phospho-p65 NF-kappa B (Figure 12A), phospho-p38 MAPK (P,0.05). The results suggest that AOS contributes to (Figure 12B), TLR4 (Figure 12C), IL-1 beta (Figure 12D), AA repair. The reintervention rate and continued presence and IL-6 (Figure 12E) when compared with the level of CGs. of complications were higher in the CG than in the AG All the results suggest that miR-29b can affect the activity (P,0.05). There were significant differences for being free of TLR4 signaling. from aneurysm-related death after two-year therapy between the groups (P,0.05). Discussion AA therapy is still a challenge although a less invasive This was a retrospective research, and the efficacy and method can be commercially available for treating AA. Since suitability of AOS were assessed in AA therapy. Based on introduction of EVAR, it is a safe and feasible alternative submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress $ S1)NDSSD% S0$3. 7/5 ,/EHWD ,/ &* 2* 6* &* 2* 6* &* 2* 6* &* 2* 6* &* 2* 6* %HWDDFWLQ % & SKRVSKRS0$3. 5HODWLYHSURWHLQOHYHORI 5HODWLYHSURWHLQOHYHORI SKRVSKRS1)NDSSD% &* 2* 6* &* 2* 6* OHYHORI7/5 5HODWLYHSURWHLQ 5HODWLYHSURWHLQ OHYHORI,/EHWD &* 2* 6* &* 2* 6* OHYHORI,/ 5HODWLYHSURWHLQ &* 6* 2* Figure 12 (A–F) Western blot analysis of the effects of mir-29b on the expression of Tlr4 signaling molecules. Notes: (A) Western blot analysis for the effects of mir-29b on the expression of p65 nF-kappa B, p38 MaPK, Tlr4, il-1 beta and il-6. ( B) The effects of mir-29b on relative protein level of nF-kappa B. ( C) The effects of mir-29b on the expression of phospho-p38 MaPK. ( D) The effects of mir-29b on the expression of Tlr4. ( E) The effects of mir-29b on the expression of il-1 beta. ( F) The effects of mir-29b on the expression of il-6. * P,0.05 vs the cg. Abbreviations: cg, control group; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Og, mir-29b was overexpressed; sg, mir-29b was silenced by antagomirs; Tlr, toll-like receptor. to conventional open repair. However, the surgery still has thoracotomy, cardiopulmonary bypass and hypothermia some side effects. Respiratory failure is a major complica- inducing an inflammatory reaction also attribute to postop - tion, which affects postoperative morbidity and mortality erative respiratory failure. Because of their less invasive and results in prolonged hospitalization. In addition to methods, respiratory failure was relatively rare in EVAR. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling In the present study, more patients developed respiratory rates. There were eight deaths in the CG and two deaths in failure in the CG than in the AG (P,0.05). AOS reduced the AG related to aortic disease. All these results provide a these side effects significantly ( P,0.05). potential use of the natural products of marine algae for the EVAR has been quickly developed because of its therapy of AA patients (Figure 13). Retrograde dissection improved perioperative mortality and morbidity. However, and endoleak were signic fi ant complications, resulting in the high reintervention is still linked with EVAR. Here, a sig- death of patients. These signic fi ant complications contributed nificantly lower rate of recurrent AA was associated with to an aneurysm-related death rate after EVAR. However, the AOS treatment (16 patients in CG and 6 patients in AG). late aneurysm-related deaths occurred in patients who under- At cell levels, AOS is demonstrated to reduce the levels went EVAR in the r fi st 2 years at our hospital. AOS treatment of miR-29b (Figure 9A), TLR4, NF-kappa B, IL-1 beta, improved EVAR at our institution because aneurysm-related and IL-6 (Figure 10), while the overexpression and silence death rates were decreased when compared with the CG. As of miR-29b will increase and reduce the levels of TLR4, described above, there are advantages in using the combined phospho-p65 NF-kappa B, phospho-p38 MAPK, IL-1 beta, technique, including 1) the reduction in the incidences of AAs and IL-6, respectively (Figure 12). NF-kappa B contains sites by AOS; 2) the reduction in infection rates; 3) the reduction in for phosphorylation, which is important for activating TLR4 the number of deaths; 4) fewer complications such as bleed- signaling pathways. Phosphorylated MAPK also plays an ing, pneumothorax, and stroke; and 5) the reduction due to important role in regulating TLR4 signaling. The reasons AOS in other side effects caused by repair materials. for reducing recurrent AA cases may be caused by AOS, Certainly, there were some limitations for the present which reduced the level of serum miR-29b (Figure 4) and work: 1) four main AOS fractions (DP3, DP4, DP5, and DP6) resulted in the reduction of TLR4, phospho-p65 NF-kappa B, were isolated. We wanted to use one bioactive component for phospho-p38 MAPK, IL-1 beta, and IL-6 (Figure 12). On the subsequent experiment. However, single component cannot other hand, there is a strong positive relationship between be prepared at a scale suitable for a patient population at the miR-29b and the levels of serum TLR4, NF-kappa B, IL-1 moment. On the other hand, oligosaccharides are complex beta, and IL-6 (Figure 6). These results suggest that AOS carbohydrate molecules and the purity will ree fl ct the product treatment reduced the level of serum TLR4, NF-kappa B, quality, so the components were analyzed here. In any way, IL-1 beta, and IL-6 by reducing the level of miR-29b. to confirm the function of specific component, further work is AOS treatment seemed to be associated with the reduc- highly demanded in the future; 2) lack of a negative CG, such tion of long-term mortality rates. The midterm follow-up of as open surgery repair; 3) the effects of AOS on most inflam - patients revealed a significant benefit in cumulative survival matory cytokines and antioxidant enzymes were not explored 2+ 2+ 2+ ± 2+ 2 22& +22& 2+ .HOS 2 2 ± +2 &22 +2 2+ 0DQQXURQDWH *XOXURQDWH *OXFXURQLFDFLG 1D ±  ± 22& 1D 22& 2+ 2+ 2 +2 2+ +2 2 1D ± 2+ 22& $26 $OJLQDWHVRGLXP 7/5 PL5E $QHXU\VP 1RUPDODRUWD 1)NDSSD% ,/EHWD 3 3 ,/ Figure 13 a cartoon description of aOs from marine algae for the therapy of aa patients. The oligosaccharide has been found to prevent the regrowth of aneurysms by inactivating Tlr4, nF-kappa B, il-1 beta, and il-6 via the inhibition of mir-29b. Abbreviations: aa, aortic aneurysm; aOs, alginate oligosaccharide; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Tlr, toll-like receptor. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress 8. Mursalin R, Sakamoto I, Nagayama H, et al. Imaging-based predic- here; 4) present n fi dings demonstrate that AOS inhibits the tors of persistent type II endoleak after endovascular abdominal aortic growth of ECs, which is still difc fi ult to be associated with the aneurysm repair. AJR Am J Roentgenol. 2016;206(6):1335–1340. clinical use of aneurysm therapy. We aimed to control aneu- 9. Park EJ, Venkatesan T, Choi YW, Kim YK. Pinus densiflora stem bark extract induces breast cancer cell death via oxidative stress mediated rysm growth by inhibiting ECs activation via AOS because lysosomal membrane permeabilization. Planta Med. 2016;81(S 01): the ECs were isolated from aneurysm. To further conr fi m S1–S381. 10. Wang Z, Li Z, Ye Y, Xie L, Li W. Oxidative stress and liver the result, animal test will be needed in the future work. The cancer: etiology and therapeutic targets. Oxid Med Cell Longev. epigenetic mechanism of phosphorylation of TLR4 was not 2016;2016:7891574. studied. In any way, further studies will be required to confirm 11. Mani RS, Amin MA, Li X, et al. Inflammation-induced oxidative stress mediates gene fusion formation in prostate cancer. Cell Reports. the therapeutic effects for using AOS. The prospective multi- 2016;17(10):2620–2631. center data are still needed to confirm the present findings. 12. Yadav DK, Rai R, Kumar N, et al. New arylated benzo[h]quinolines induce anti-cancer activity by oxidative stress-mediated DNA damage. Sci Rep. 2016;6:38128. Conclusion 13. Suzuki Y, Okabayashi K, Hasegawa H, et al. Comparison of preopera- Taken together, the present findings demonstrate that AOS tive inflammation-based prognostic scores in patients with colorectal cancer. Ann Surg. Epub 2016 Dec 16. represses the growth of residual aneurysms and reduces aneu- 14. Wheeler TM, Zhao B, Sonpavde G, et al. Antigen-specic fi immunity and rysm recurrence by indirectly affecting TLR signaling via tumor inflammation after vaccination with BPX-101, a drug-activated miR-29b. AOS treatment can be an adjuvant method for AA dendritic cell vaccine for metastatic castration-resistant prostate cancer (mCRPC). J Clin Oncol. 2011;29(7 Suppl):176. repair and control its subsequent development. AOS offers 15. Reyes-Gibby CC, Shete S, Wu X, Kurzrock R, Spitz M. Inflammation an adjuvant method for minimally invasive endovascular genes and pain severity in lung cancer patients. J Clin Oncol. 2009; 27(15 Suppl):9618. repair of AA and dissection. AOS reduced recurrent AA and 16. Oh B, Butow P, Mullan B, et al. Randomized clinical trial of medical its side effects, such as wound infection rates. 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Anti-inflammatory activ - Yunnan Province (No 2014NS048) and (2017NS137). ity of guluronate oligosaccharides obtained by oxidative degradation from alginate in lipopolysaccharide-activated murine macrophage RAW Disclosure 264.7 cells. J Agric Food Chem. 2015;63(1):160–168. 20. Fujihara M, Nagumo T. The effect of the content of D-mannuronic The authors report no conflicts of interest in this work. acid and L-guluronic acid blocks in alginates on antitumor activity. Carbohydr Res. 1992;224:343–347. References 21. Ji J, Wang LC, Wu H, Luan HM. Bio-function summary of marine 1. Butt HZ, Sylvius N, Salem MK, et al. Microarray-based gene expres- oligosaccharides. Int J Biol. 2011;3(1):74. sion profiling of abdominal aortic aneurysm. Eur J Vasc Endovasc Surg. 22. Lai CH, Wang KC, Lee FT, et al. Toll-like receptor 4 is essential in 2016;52(1):47–55. the development of abdominal aortic aneurysm. PLoS One. 2016; 2. Gray C, Goodman P, O’Malley MK, O’Donohoe MK, McDonnell CO. 11(1):e0146565. 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Anti-inflammatory proper - thoracic aortic dissection by noninvasive imaging procedures. N Engl ties of Irisin, mediator of physical activity, are connected with TLR4/ J Med. 1993;328(1):1–9. MyD88 signaling pathway activation. Int J Mol Sci. 2017;18(4):701. 34. Fillinger MF, Greenberg RK, McKinsey JF, Chaikof EL. Society for 41. Li X, Lian LH, Bai T, et al. Cryptotanshinone inhibits LPS-induced Vascular Surgery Ad Hoc Committee on TRS. Reporting standards proinflammatory mediators via TLR4 and TAK1 signaling pathway. for thoracic endovascular aortic repair (TEVAR). J Vasc Surg. 2010; Int Immunopharmacol. 2011;11(11):1871–1876. 52(4):1022–1033, 1033 e1015. Drug Design, Development and Therapy Dovepress Publish your work in this journal Drug Design, Development and Therapy is an international, peer- has also been accepted for indexing on PubMed Central. 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Alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of microRNA-29b in aneurysm patients after endovascular aortic repair

Drug Design, Development and Therapy , Volume 11 – Sep 1, 2017

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© 2017 Yang et al. This work is published and licensed by Dove Medical Press Limited
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1177-8881
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1177-8881
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10.2147/DDDT.S140206
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Abstract

Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2017 Volume: 11 Running head verso: Yang et al Drug Design, Development and Therapy Dovepress Running head recto: Alginate oligosaccharide indirectly affects TLR signaling open access to scientific and medical research DOI: 140206 Open access Full Text article O rig inal r esearch alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of microrna- 29b in aneurysm patients after endovascular aortic repair 1–4, Yong Yang * Abstract: Endovascular aortic repair (EVAR) is often followed by aneurysm recurrence. 1–4, Alginate oligosaccharide (AOS) has potential antitumor properties as a natural product while Zhenhuan Ma * 1–4 the related mechanisms remain unclear. Toll-like receptor (TLR) signaling is associated with guokai Yang 1–4 inflammatory activity of aneurysm and may be affected by miR-29b. Thus, inhibitory function Jia Wan of AOS on aneurysms was explored by measuring the important molecules in TLR4 signaling. 1–4 guojian li After EVAR, a total of 248 aortic aneurysm patients were recruited and randomly assigned 1–4 lingjuan Du into two groups: AOS group (AG, oral administration 10-mg AOS daily) and control group 1–4 Ping lu (CG, placebo daily). The size of residual aneurysms, aneurysm recurrence, and side effects Department of Vascular surgery, were investigated. Aneurysm recurrence was determined by Kaplan–Meier analysis. After The s econd People’s h ospital of 2 years, eight and two patients died in the CG and AG, respectively. The sizes of residual Yunnan Province, Kunming, china; Department of Vascular surgery, aneurysms were significantly larger in the CG than in the AG ( P,0.05). The incidence of The Fourth affiliated hospital of aneurysm recurrence was also significantly higher in the CG than in the AG ( P,0.05). AOS Kunming Medical University, Kunming, treatment reduced the levels of miR-29b, TLR4, mitogen-activated protein kinase (MAPK), china; Department of Vascular s urger y, Vascular s urger y c entre in nuclear factor kappa B (NF-kappa B), interleukin 1 (IL-1) beta, and interleukin 6 (IL-6). Yunnan Province, Kunming, china; Overexpression and silence of miR-29b increased and reduced the level of TLR4, phospho-p65 Department of Vascular surgery, NF-kappa B, phospho-p38 MAPK, IL-1 beta, and IL-6. Spearman’s rank correlation analysis abdominal surgery centre in Yunnan Province, Kunming, china shows that the level of miR-29b is positively related to the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 (P,0.05). Thus, AOS represses aneurysm recurrence by indirectly affecting *These authors contributed equally to this work TLR signaling via miR-29b. Keywords: alginate oligosaccharide, outcome assessment, aortic aneurysms, minimally invasive endovascular repair, toll-like receptor signaling pathway, anti-inflammatory agent, microRNA- 29b, mitogen-activated protein kinase Introduction Aortic aneurysm (AA) is a major and emerging threat to public health. Endovascular aortic repair (EVAR) has been developed as a minimally invasive therapy. Although there are some advantages for EVAR, the technique still has some shortcomings: the residual aneurysm often regrows following EVAR and is an important risk factor correspondence: Zhenhuan Ma for the surgery failure; High-rate aneurysm recurrence has been widely reported in Department of Vascular surgery, The EVAR. Stent-assisted coil embolization for large or giant aneurysms has high recur- second People’s hospital of Yunnan Province, 176 Qingnian lu, Kunming rence rates, which may be associated with metal coverage rates of the stents used in 650021, china these procedures. More than 17% patients had aneurysm recurrence and 13% patients Tel +86 871 515 6650 email zhenhuanyy@126.com had residual aneurysms after 2-year segmental artery coil embolization. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 2565–2579 Dovepress © 2017 Yang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/DDDT.S140206 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Yang et al Dovepress In most cases, medical therapy is often considered to con- were measured at 234 nm in a UV-2100PC UV-VIS spec- trol the disease before and after aneurysm therapy. EVAR trophotometer (Shimadzu, Kyoto, Japan). The degrees of infection is usually followed by the technique and will result polymers (DP) were further determined by electrospray in a high risk of aneurysm recurrence. Persistent endoleak is ionization mass spectrometry (ESI-MS). The hydrolysate also a main side effect after EVAR. All the adverse effects was transferred to a carbograph column to remove salt, and signic fi antly affect life quality of AA patients. Most research then concentrated, dried, and resolved in 1 mL methanol. 9–12 13–16 shows that oxidative stress and inflammation are Two-microliter supernatant was injected into a LTQ XL mass associated with the risks of various cancers. Marine algae spectrometer (ThermoFinnigan, Austin, TX, USA). AOS was have been found to be with various bioactive compounds, detected in a positive-ion mode: ion source, 5 kV; capillary such as alginate oligosaccharide (AOS). AOS has been temperature, 280°C–310°C; tube lens, 260 V; sheath gas, 17,18 reported to have antioxidant and anti-inflammatory prop - 35 arbitrary units. The mass spectrometer was set over the 19 20 erties. AOS has been found to have antitumor activities. range m/z 500–1,500. Furthermore, AOS has few side effects as a kind of natural product. AOS may offer novel anticancer agents for various Participants cancers. All the procedures were approved by the Ethical Committee However, the effects of AOS on aneurysm recurrence of the Second People’s Hospital of Yunnan Province and and regrowth, and related molecular mechanisms remain written informed consent was obtained from each patient unclear. Toll-like receptor (TLR) is an important protein for this study. The study was performed according to the in various innate immunity and inflammatory activities. Declaration of Helsinki. A total of 397 patients, experienc- Previous study demonstrates that the silence of TLR4 sig- ing severe chest and back pain, attended the Second People’s naling pathway inhibits the progression of abdominal aortic Hospital of Yunnan Province during the period from January aneurysm (AAA). miRNA, as a small noncoding RNA, is 2013 to December 2014. involved with various biological activities, including cell The diagnosis of thoracic aortic aneurysm (TAA) and 23,24 25 26 development, homeostasis, immune, and ina fl mmatory dissection was confirmed from clinical diagnostic criteria 27,28 33 responses. miRNAs have been reported to be important issued by a previous report. One of the following includ- biomarkers for vascular diseases, while they have been ing criteria were considered: the diameter of aneurysm was considered as potential targets for exploiting therapeutic more than 5 cm; the diameter of aneurysm was 4–5 cm approaches for the disease. Therefore, miRNA is an impor- and increased in size by 0.5 cm in less than half a year; tant regulator for controlling immune and inflammatory the diameter of aneurysm was twofold the diameter of the responses to tumor progression. Present evidence shows normal infrarenal aorta; sudden onset of severe chest pain therapeutic manipulation of miR-29b, which holds a great that had a tearing or ripping quality (classic symptom); promise for controlling AAA development. Therefore, we anterior chest pain, associated with anterior arch or aortic aim to explore the functional role of AOS in AAs’ regression root dissection; neck or jaw pain, with aortic arch involve- by exploring its effects on TLR4 signaling and miR-29b. To ment and extension into the great vessels; tearing or ripping understand safety and efficacy of AOS, the size of residual intrascapular pain, involving the descending aorta; cerebro- aneurysms, aneurysm recurrence, and the side effects were vascular accident symptoms, hemianesthesia, hemiparesis, also investigated. and hemiplegia. The following excluding criteria were used: a family history of AA and dissection; malignancy; Methods psychological disorders. a Os prepared from alginate sodium A computed tomography (CT) scan was performed and The AOS with α-L-guluronate units and β-D-mannuronate the selected patients were revealed with TAA and/or dis- units was bought from Qingdao Qingya Chemical Co., Ltd section. Finally, 248 patients were selected for the present (Qingdao, China). AOS was prepared from alginate sodium study and received modified EVAR. The Zenith TX2 TAA according to an earlier report using alginate lyase, which Endovascular Graft with Pro-Form is a two-piece cylindrical depolymerizes alginate sodium. Briefly, 1 kg sodium alg- endovascular graft with proximal and distal materials (COOK inate was depolymerized in 100 L tap water with 10 mg Medical Inc., Bloomington, IN, USA). The proximal parts alginate lyase at 39°C for 2 h. The lyase was denatured at can be either nontapered or tapered. The stent grafts are made 100°C for 10 min. The amounts of unsaturated saccharides of full-thickness woven polyester fabric sewn to steel Cook-Z submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling stents with polyester and monofilament polypropylene. diameters were measured in infrarenal aorta, which is The Zenith TX2 TAA endovascular graft with ProForm is perpendicular to aortic axis. fully stented to provide stability and the expansible force to enzyme-linked immunosorbent assay open the lumen of the graft during deployment. All endovas- cular grafts with Pro-Form are two cylindrical endovascular analysis grafts with proximal and distal materials. The proximal parts Five-milliliter blood was obtained by vein puncture. Serum was can be either nontapered or tapered. The diameter and length separated by using the centrifugation at 1,500 g, 4°C for 10 min. of each stent-graft was identified preoperatively with use The concentration of tumor necrosis factor (TNF)-alpha was of multislice CT or diagnostic imaging via a catheter with measured by using a human TNF-alpha enzyme-linked immu- a marker. The graft diameter was .10% than normal size nosorbent assay (ELISA) kit (Thermo Fisher Scientic fi , Inc. associated with the diameter of the nondiseased segments of Corporate, Waltham, MA, USA). The concentration of TLR4 aorta under the aneurysm. The length of graft was equal to was measured by human TLR4 ELISA Kit from RayBiotech the distance from the transection site to the nondiseased size (Norcross, GA, USA). The levels of interleukin 1 (IL-1) beta of the descending aorta below the aneurysm. and IL-6 were measured by the ELISA kits from R&D Sys- After surgery, all the patients were randomly and evenly tems (Minneapolis, MN, USA). All the concentrations were assigned into two groups: AOS group (AG), 124 AA patients calculated according to standard calibrating curves. received 10 mg AOS daily and control group (CG), 124 AA The effects of a Os on the viability of patients received a 10 mg placebo daily. endothelial cells end points Endothelial cells (ECs), isolated from human aneurysm, were Outcome criteria were established according to a previous purchased from Shanghai Cell Bank (Shanghai, China) and report for thoracic EVAR. The end points included early cultured in DMEM at 37°C under a 5% CO environment. The morbidities (stroke and paraplegia), respiratory failure, acute cell concentrations were adjusted to 1×10 /cell in a 96-cell kidney injury, need for dialysis, aneurysm-related death, and plate. Cell viability of ECs was determined using Trypan blue reoperation. The proximal attachment zones were determined (Solarbio, Beijing, China) and [ H]thymidine (Amersham based on the proximal attachment site of the proximal edge biosciences, Piscataway, NJ, USA, specific activity of the covered graft; ,2 cm of the left subclavian artery 5 Ci/mmol) uptake after the cells were cultured with differ- (without covering it) is defined as zone 3; it is defined as ent concentrations of AOS (0, 1, 10, 100, and 1,000 ng/mL) zone 4 if the proximal extent of the endograft is .2 cm to for 3 days. The Trypan blue-positive cells were measured the left subclavian artery and ended within the proximal with a hemocytometer under a microscope. For the [ H] half of the TAA (T6 level is near the midpoint of TAA). thymidine absorption, cells were counted after exposure to Aneurysm-related death would be measured at any time. different concentrations of AOS, and 200 μL cell suspension Respiratory failure was determined if mechanical ventila- of 1.0×10 cells/mL for each condition were added to each tion was more than 1 day and reintubation and tracheostomy well of a 96-well plate. After 24-h culture, cells were labeled would be needed. with one Ci/well of [ H]thymidine for 4 h, then collected with an automated sample harvester and measured with a liquid clinical follow-up scintillation counter (Beckman Coulter, Brea, CA, USA). Detailed data were collected using the index admission when surgery was conducted. After discharge, all the patients were cell co-infection followed up at 1, 3, 6, and 12 months and yearly at outpatient Antagomirs and mimics of miR-29b were synthesized by clinics. In addition, a CT angiogram was performed once a Sangon Biotech (Shanghai) Co. Ltd (Shanghai, China). year for 2 years. The cell lines were co-infected with the antagomirs and mimics via Lipofectamine 3000 (Life Technologies, Inc., Carlsbad, The measurement of aa size CA, USA). After 3-day co-infection, the mutant cells were AA size was measured by using an ultrasound scanning picked by using 200 μg/mL G-418. TLR4, nuclear factor system (Xuzhou Forward Medical Instrument Co., Limited, kappa-B (NF-kappa B), IL-1 beta, and IL-6 were measured Xuzhou, China). The ultrasound was sensitive and specific. by ELISA in the cell lines. Meanwhile, cell viability was also Maximum transverse and anterior-posterior external measured using Trypan blue and [ H]thymidine uptake. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress real-time polymerase chain reaction of miR-29b and the levels of TLR4, TNF-alpha, IL-1 beta, and IL-6. Statistical calculation was conducted via SPSS 20 quantification of miR-29b (SPSS Inc., Chicago, IL, USA). There were significantly Total cellular RNA was isolated using an RNA Isolation statistical differences if P-values were ,0.05. Kit (Tiangen, Beijing, China), RNeasy Mini kit (QIA- GEN, Valencia, CA, USA). cDNA was synthesized Results from total RNA by reverse transcription using TaqMan characterization of a Os MicroRNA Reverse Transcription Kit (Applied Biosys- Four main components (DP3, DP4, DP5, and DP6) were tems, Foster City, CA, USA). The levels of miR-29b isolated from alginate sodium after enzyme digestion. were measured by using real-time polymerase chain The isolated fractions of AOS were further confirmed by reaction (Bio-Rad Lab., Hercules, CA, USA). MiR-29b, ESI-MS under the conditions that produced mass spectra forward primer: 5′-GGCTTCAGGAAGCTGGTTT-3′; with [M + K] . The predicted masses DP3 (C18H23O19Na3), reverse primer: 5′-GTGCAGGGTCCGAGGT-3′. U6, forward DP4 (C24H30O25Na4), DP5 (C30H37O31Na5), and DP6 primer: 5′-TTGGTGCTCGCTTCGGCA-3′; reverse primer: (C36H44O37Na6) were 612, 810, 1,008, and 1,206 Da, 5′-GTGCAGGGTCCGAGGT-3′. U6 snRNA was used as an respectively (Figure 1). internal control. The baseline characters of the patients Western blot with Taa and dissection TLR4 can regulate TLR inflammatory response via phospho- After selection, a total of 248 patients with TAA received p65 NF-kappa B p65. Mitogen-activated protein kinase the treatment with modified EVAR. After 2-year therapies (MAPK) as regulatory signal mediators plays a pivotal role in of AOS and placebo, eight patients died in the CG and two organism immunity. To understand the effects of miR-29b patients died in the AG. Thus, 116 patients and 122 patients on TLR4 signaling, phosphorylated MAPK and NF-kappa B finished the experiments of CG and AG, respectively as second messengers in TLR4 signaling were measured by (Figure 2). The baseline characters of all patients are listed Western blot. The antihuman antibodies of TLR4 (ab17942), in Table 1. The number of men was more than women. Most Phospho-p65 anti-NF-kappa B (ab86299), phospho-p38 patients had concomitant diseases, such as cerebrovascular, mitogen-activated protein kinase (MAPK, ab31828), IL-1 ischemic heart, chronic obstructive pulmonary, and renal beta (ab2105), IL-6 (ab6672), β-actin antibody (ab8227), dysfunction. The ages (65.8±14.9 years in AG group vs and secondary antibody Goat Anti-Rabbit IgG H&L (HRP, ab6721) were purchased from Abcam (Beijing, China). Proteins were separated by sodium dodecyl sulfate polyacryl- amide gel electrophoresis and transferred to a polyvinylidene '3 '3 '3 '3 diu fl oride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in the buffer (10 mM Tris [pH 7.5] and 50 mM NaCl) and incubated with primary antibodies overnight at 4°C. The membrane was fur- ther treated with secondary antibodies (1:3,000). Immunore- active bands were visualized by enhanced chemiluminescence system (Amersham, Arlington Heights, IL, USA). 5HODWLYHLQWHQVLW\ statistical analysis All data were presented as mean ± SD or frequency and percentage. The variables were compared by the t-test, χ , or P] Fisher’s exact test. Early outcomes and late outcomes were Figure 1 electrospray ionization mass spectrometry analysis of the DP of digested compared using univariate statistics. Overall survival and + aOs from alginate sodium with produced mass spectra as [M + K] . Notes: Mass spectra were visualized following the separation of DP3 ([M + K] =651 being free from aneurysm-related death and reintervention Da); mass spectra were visualized following the separation of DP4 ([M + K] =849 Da); were measured using the Kaplan–Meier and the log-rank mass spectra were visualized following the separation of DP5 ([M + K] =1,047 Da); and mass spectra were visualized following the separation of DP6 ([M + K] =1,245 test. Spearman’s rank correlation coefficient was calculated Da). The mass spectrometer was set over the range m/z 500–1,500. to confirm the strength of correlation between relative levels Abbreviations: aOs, alginate oligosaccharide; DP, degree of polymerization. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling DRUWLFDQHXU\VPSDWLHQWV H[FOXGHG Primary outcomes of eVar GLGQRWPHHWLQFOXVLRQFULWHULD ZHUHORVW CT of the thoracic aorta showed that it could be observed KDGSURWRFROYLRODWLRQ ZLWKGUHZFRQVHQW easily before operation. Deployment of multilayer stent was RWKHUUHDVRQV successfully established in the thoracic aorta after opera- FDVHVXQGHUZHQWUDQGRPL]DWLRQ tion. Three-dimensional reconstruction based on a series of &* FDVHV $* FDVHV angiography images showed TAA bulging in the T2–T5 seg- GLHG $IWHUPRQWKV GLHG ment. The images showed the surgery was successful and no FDVHV FDVHV endoleak was found. Comparatively, emergency CT showed GLHG $IWHUPRQWKV AAA bulging before surgery. Finally, graft implantation and FDVHV FDVHV GLHG $IWHUPRQWKV GLHG artificial vessel were successfully placed in AAs. FDVHV FDVHV end points $QHXU\VPVL]H 6LGHHI IHFWV / 75 1)NDSSD% ,QIODPPDWRU\F\WRNLQHV The average follow-up duration was 24±5 months (see Table 2). Overall, survival was 93.5% and 98.4% in the Figure 2 Present study flowchart. Notes: cg, the patients received 10 mg placebo daily in a control group. ag, the CG and AG, respectively (P=0.05) (Table 2). More patients patients received 10 mg aOs daily in the aOs group. underwent reintervention in the CG than in the AG (P,0.05). Abbreviations: aa, aortic aneurysm; cg, control group; aOs, alginate oligosac- charide; ag, aOs group; Tlr4, toll-like receptor 4; nF-kappa B, nuclear factor Comparatively, the Kaplan–Meier analysis also revealed that kappa B. being free from reintervention was signic fi antly higher in the CG than in the AG during the same period (log rank test, P=0.04). There were 16 patients in the CG and 6 patients in 62.1±15.3 years in AG, P=0.48) and renal insufficiency the AG with aortic reintervention, including endoleak and (33.1% in CG vs 30.6% in AG; P=0.68) were similar in both retrograde dissection in the 2-year follow-up. groups. The incidences of dissection aneurysm, the propor- The covered stent grafts were delivered successfully, and tion of aortic rupture, the maximal diameters of the aneu- complete thrombosis of the aneurysm or entry closure with rysms, and all other parameters were also similar between stable thrombosis was observed in all patients. According two groups (P.0.05) (Table 1). to Kaplan–Meier analysis, the patient survival rates were 93.5% in the CG and 98.4% in the AG at 2-year postopera- Table 1 Baseline characteristics of aortic aneurysm patients tion. During the 2-year follow-up, 10 patients died. Among those, three patients died of aortic reintervention and seven AG (n=124) CG (n=124) P-value patients died of endoleak and retrograde dissection. age, years 65.8±14.9 62.1±15.3 0.48 Male, n (%) 84 (67.7) 86 (69.4) 0.78 alcohol drinking, n (%) 48 (38.7) 44 (35.4) 0.60 side effects smoking, n (%) 60 (48.4) 62 (50.0) 0.80 EVAR-related adverse effects were pain (back or chest), spouse, n (%) 114 (91.9) 112 (90.3) 0.66 Daily calorie intake, calories 2,518.2±456.3 2,476.6±449.4 0.52 fainting, persistent cough, rapid heartbeat, dizziness, sudden Body mass index, m /kg 23.7±3.2 22.6±2.9 0.67 weakness, ischemia of intestines, pulse-less legs, cold arms Diabetes mellitus, n (%) 9 (7.3) 7 (5.6) 0.61 or legs, and so on (Table 3). Among these adverse effects, hypertension, n (%) 78 (62.9) 74 (59.7) 0.61 coronary artery obstructive 15 (12.1) 17 (13.7) 0.70 disorder, n (%) Table 2 late outcomes (n =124 in each group before repair) Renal insufficiency, n (%) 38 (30.6) 41 (33.1) 0.68 aortic pathology Variables AG CG Chi-square P-value Medial degeneration, n (%) 72 (58.3) 61 (48.9) 0.16 (n=122) (n=116) statistic Dissecting aneurysm, n (%) 62 (50.0) 55 (44.4) 0.37 Overall survival 122 (98.4) 116 (93.5) 3.75 0.05 Traumatic dissection, n (%) 8 (6.3) 5 (4.0) 0.39 recurrent aa 6 (4.8) 16 (13.8) 5.58 0.02 Marfan syndrome, n (%) 5 (4.0) 8 (6.5) 0.39 endoleak 2 (1.6) 10 (8.6) 6.05 0.01 aortic rupture, n (%) 5 (4.0) 5 (4.0) 1.00 retrograde dissection 2 (1.6) 3 (2.4) 0.26 0.61 lesion leakage 1 (0.8) 3 (2.4) 1.12 0.29 Zone 3, n (%) 67 (54.0) 74 (59.7) 0.37 Notes: Data presented as n (%). cg, Zenith TX2 aa graft repair and placebo; ag, Zone 4, n (%) 57 (46.0) 50 (40.3) 0.37 Zenith TX2 aa graft repair and aOs. The period of follow-up was 2 years. There aneurysm maximum size (mm) 50.2±4.8 51.0±4.0 0.76 were significantly statistical differences if P,0.05. Note: Data presented as mean ± standard deviation. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- Abbreviations: aOs, alginate oligosaccharide; ag, aOs group; cg, control group. charide; cg, control group. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress Table 3 The side effects between cg and ag, n (%) statistical difference for AA size between the AG and the CG Side effects AG CG Chi-square P-value (P.0.05). Compared with CG, AOS significantly reduced (n=122) (n=116) statistic AA size after 2-year repair (P,0.01) (Figure 3). The results Pain (back or chest) 5 (4.1) 23 (19.8) 14.2 0.01 suggest that long-term AOS consumption will further control Fainting 7 (5.7) 12 (10.3) 1.72 0.19 the growth of residual aneurysms. Persistent cough 7 (5.7) 26 (22.4) 13.85 0.01 rapid heartbeat 5 (4.1) 12 (10.3) 3.50 0.06 a Os reduces the levels of mir-29b in aa Dizziness 7 (5.7) 10 (8. 6) 0.75 0.39 sudden weakness 12 (10.7) 8 (6.9) 0.67 0.41 patients ischemia of intestines 10 (8.2) 15 (12.9) 1.42 0.23 Before AOS treatment, there was no significantly statistical Pulse-less legs 5 (4.1) 14 (12.1) 5.14 0.02 difference for relative level of miR-29b between the AG and cold arms or legs 7 (5.7) 15 (12.9) 3.67 0.06 surgery infection 7 (5.7) 11 (9.4) 1.19 0.27 the CG (P.0.05). After long-term consumption of AOS, it chronic wound 5 (4.1) 20 (17.2) 10.93 0.01 significantly reduced the relative level of miR-29b ( P,0.05) infection (Figure 4). The results suggest that long-term AOS consump- Notes: The period of follow-up was 2 years. There were significantly statistical differences if P,0.05. cg, Zenith TX2 aa graft repair and placebo; ag, Zenith TX2 tion will reduce the relative level of miR-29b. aa graft repair and aOs. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- a Os decreases the levels of Tlr4, nF- charide; cg, control group. kappa B, il-1 beta, and il-6 Before EVAR and AOS treatment, there was no signic fi antly back and chest pain rate, and the rate of persistent cough statistical difference for serum levels of TLR4 (Figure 5A), and wound infection were higher in the AG than in the CG NF-kappa B (Figure 5B), and IL-1 beta (Figure 5C) (P.0.05). (P,0.05). Endovascular therapy has excellent short-term After 2-year follow-up, there was a greater reduction in the effect and stable mid-to-long-term result. AOS can be used as serum levels of TLR4 (Figure 5A), NF-kappa B (Figure 5B), a therapy option such as in an emergency situation or a rescue and IL-1 beta (Figure 5C) in the AG when compared with method by reducing the side effects caused by EVAR. the CG (P,0.05). Before EVAR and AOS treatment, serum levels of IL-6 were lower in the CG than the AG (Figure 5D) a Os reduces aa size (P,0.05). After 2-year follow-up, there was a greater reduc- EVAR surgery reduced the AA size after 2-year follow-up in tion in the serum levels of IL-6 in the AG (Figure 5D) when comparison with the size before surgery in the CG and the AG compared with the CG (P,0.05). The results suggest that (Figure 3). Before AOS treatment, there was no signic fi antly long-term AOS consumption decreases the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6. 7KHVL]HRIDQHXU\VP PP 5HODWLYHOHYHORIPL5E &*% $*% &*$ $*$ Figure 3 scatter dot plot of the aa size variances in different groups. &*% $*% &*$ $*$ Notes: cg-B, before eVar and placebo treatment. ag-B, before eVar and aOs treatment. cg-a, after eVar and 2-year placebo treatment. ag-a, after eVar Figure 4 relative levels of mir-29b in different groups. and 2-year aOs treatment. ag, aa patients received 10 mg aOs daily and cg, Notes: cg-B, before eVar and placebo treatment. ag-B, before eVar and aOs aa patients received 10 mg placebo daily. aa size was measured by ultrasound treatment. cg-a, after eVar and 2-year placebo treatment. ag-a, after eVar and scanning. Maximum transverse and anterior–posterior external diameters were 2-year aOs treatment. n =124, 124, 116, and 122 cases in cg-B, ag-B, cg-a, and measured in infrarenal aorta, which is perpendicular to aortic axis. There was a ag-a groups, respectively. ag, aa patients received 10 mg aOs daily and cg, aa statistical difference if P,0.05. patients received 10 mg placebo daily. There was a statistical difference if P,0.05. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosac- charide; cg, control group; eVar, endovascular aortic repair. charide; cg, control group; eVar, endovascular aortic repair. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling $ % 7/5 SJP/ 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &*% $*% &*$ $*$ &*% $*% &*$ $*$ ,/ SJP/ ,/EHWD SJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &*% $*% &*$ $*$ &*% $*% &*$ $*$ Figure 5 The effects of aOs on the levels of Tlr4, nF-kappa B, il-1 beta, and il-6 in aa patients. Notes: (A) The effects of aOs on the levels of Tlr4. ( B) The effects of aOs on the levels of nF-kappa B. ( C) The effects of aOs on the levels of il-1 beta. ( D) The effects of aOs on the levels of il-6. n =124, 124, 116, and 122 cases in cg-B, ag-B, cg-a, and ag-a groups, respectively. ag, aa patients received 10 mg aOs daily and cg, aa patients received 10 mg placebo daily. cg-B, the patients before taking placebo. cg-a, the patients after taking placebo for two years. ag-B, the patients before taking aOs. ag-a, the patients after taking aOs for two years. There was a statistical difference if P,0.05. Abbreviations: aa, aortic aneurysm; ag, aOs group; aOs, alginate oligosaccharide; cg, control group; il-6, interleukin 6; Tlr, toll-like receptor; nF-kappa B, nuclear factor kappa B. exposure to different concentrations of AOS. As shown in The levels of mir-29b are positively Figure 7B, there is no significant reduction in [ H]thymidine associated with the serum levels of Tlr4, uptake that was measured when the concentration was more nF-kappa B, il-1 beta, and il-6 than 1,000 ng/mL (P,0.05). Based on these results, admin- Figure 6 shows that the increase in miR-29b also increases istration of 10 mg daily will not cause toxicity to ECs. the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6. Spearman’s rank correlation test demonstrates that the levels a Os inhibits the growth of ecs but not of miR-29b are positively associated with the serum levels their viabilities of TLR4 (Figure 6A), NF-kappa B (Figure 6B), IL-1 beta ECs were isolated from human aneurysm. Figure 8A shows (Figure 6C), and IL-6 (Figure 6D) (P,0.05) because the rho that AOS inhibits the growth of ECs when its concentration values are 0.82, 0.75, 0.80, and 0.85, respectively. is more than 100 ng/mL. Based on the results, 10 mg/day was used for all patients (the weight of each patient was ,100 kg). effects of a Os toxicity on ecs Cell viability analysis shows that AOS cannot affect cell To avoid the effects of AOS on ECs because of its toxicity, viability by using Trypan Blue (Figure 8B) and [ H]thymi- the concentration of AOS was measured. Trypan blue analy- dine incorporation (Figure 8C) even when the concentration sis showed that exposure of the ECs to AOS, in the presence is 1,000 ng/mL. of more than 100 ng/mL, resulted in loss of cell viability a Os treatment reduces relative levels (Figure 7A; P,0.05). The viability of cells was still more than 95% after 72-h culture when the concentration of AOS of mir-29b was 1,000 ng/mL. The toxicity of AOS was also evaluated Figure 9A showed that AOS could not affect the relative by measuring [ H]thymidine incorporation into DNA after levels of miR-29b when the concentration was ,10 ng/mL. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress $ % 7/5 SJP/ UKR  UKR 3  3 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI 5HODWLYHOHYHORIPL5E 5HODWLYHOHYHORIPL5E & ' ,/ SJP/ UKR  UKR ,/EHWD SJP/ 3  3 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI 5HODWLYHOHYHORIPL5E 5HODWLYHOHYHORIPL5E Figure 6 The relationship between the level of mir-29b and the levels of Tlr4, nF-kappa B, il-1 beta, and il-6. Notes: (A) The relation between the level of mir-29b and the level of Tlr4. ( B) The relation between the level of mir-29b and the level of nF-kappa B. ( C) The relation between the level of mir-29b and the level of il-1 beta. ( D) The relation between the level of mir-29b and the level of il-6. spearman’s rank correlation test was used to compare the significance of two parameters. There is a positive relationship if the value of rho falls between 0.5 and 1. There was a statistical difference if P,0.05. Abbreviations: il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Tlr, toll-like receptor. AOS reduced the relative levels of miR-29b when the a Os treatment reduces the levels of Tlr4, concentration was more than 10 ng/mL and reached the low- nF-kappa B, il-1 beta, and il-6 in ecs est level when the concentration was 1,000 ng/mL. Figure 9B Figure 10A showed that AOS could not affect the levels of showed that the relative levels of miR-29b reached the low- TLR4 when the concentration was ,10 ng/mL. AOS reduced est level when the microRNA was blocked, and reached the the levels of TLR4 when the concentration was more than highest level when the microRNA was overexpressed. 10 ng/mL and reached the lowest level when the concentration $ % FSPî +@WK\PLGLQHXSWDNH 5HODWLYHYLDELOLW\ $26FRQFHQWUDWLRQ QJP/ $26FRQFHQWUDWLRQ QJP/ Figure 7 effects of aOs on the viability of ecs. Notes: (A) cell viability of ecs was measured using Trypan blue. ( B) cell viability of ecs was measured using [ h]thymidine uptake. The data were presented as the mean ± standard deviation. n =8 in each group. Abbreviations: aOs, alginate oligosaccharide; ecs, endothelial cells. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling QJP/ QJP/ QJP/ QJP/ QJP/ FHOOVP/ V &XOWXUHWLPH KRXU % & FSPî +@WK\PLGLQHXSWDNH 5HODWLYHYLDELOLW\ $26FRQFHQWUDWLRQ QJP/ $26FRQFHQWUDWLRQ QJP/ Figure 8 The effects of aOs on the growth of ecs. Notes: (A) real-time analysis of the effects of aOs on the growth of ecs. ( B) Trypan blue analysis of the effects of aOs on the viability of ecs. ( C) [ h]thymidine incorporation analysis of the effects of aOs on the viability of ecs. n =8. Abbreviations: aOs, alginate oligosaccharide; ecs, endothelial cells. was 1,000 ng/mL. Figure 10B showed that AOS could not the lowest level when the concentration was 1,000 ng/mL. affect the levels of NF-kappa B when the concentration Figure 10C showed that AOS could not affect the levels of was ,10 ng/mL. AOS reduced the levels of NF-kappa B IL-1 beta when the concentration was ,10 ng/mL. AOS when the concentration was more than 10 ng/mL and reached reduced the levels of IL-1 beta when the concentration was $ % 5HODWLYHOHYHORIPL5E 5HODWLYHOHYHORIPL5E QJP/ QJP/ QJP/ QJP/ QJP/ &* 2* 6* $26FRQFHQWUDWLRQ Figure 9 relative levels of mir-29b in different groups. Notes: (A) The effects of aOs on the relative levels of mir-29b. ( B) The effects of different treatments on relative levels of mir-29b. n =8 for each group. *P,0.05 vs the cg. Abbreviations: aOs, alginate oligosaccharide; cg, control group; sg, mir-29b was silenced by antagomirs; Og, mir-29b was overexpressed. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress $ % 7/5 SJP/ 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ $26FRQFHQWUDWLRQ $26FRQFHQWUDWLRQ & ' ,/ SJP/ ,/EHWD SJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ QJP/ $26FRQFHQWUDWLRQ $26FRQFHQWUDWLRQ Figure 10 The effects of aOs on the levels of Tlr4, nF-kappa B, il-1 beta, and il-6 in ecs. Notes: (A) The effects of aOs on the levels of Tlr4. ( B) The effects of aOs on the levels of nF-kappa B. ( C) The effects of aOs on the levels of il-1 beta. ( D) The effects of aOs on the levels of il-6. * P,0.05 vs the cg without aOs addition. Abbreviations: aOs, alginate oligosaccharide; cg, control group; ecs, endothelial cells; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Tlr, toll-like receptor. more than 10 ng/mL and reached the lowest level when the silence of miR-29b reduced the level of TLR4. Figure 11B concentration was 1,000 ng/mL. Figure 10D showed that showed that the overexpression of miR-29b increased the AOS could not affect the levels of IL-6 when the concentra- level of NF-kappa B and the silence of miR-29b reduced the tion was ,100 ng/mL. AOS reduced the levels of IL-6 when level of NF-kappa B. Figure 11C showed that the overex- the concentration was more than 100 ng/mL and reached the pression of miR-29b increased the level of IL-1 beta and the lowest level when the concentration was 1,000 ng/mL. All silence of miR-29b reduced the level of IL-1 beta. Figure 11D the results suggest that AOS treatment reduces the levels of showed that the overexpression of miR-29b increased the TLR4, NF-kappa B, IL-1 beta, and IL-6 in ECs. The results level of IL-6 and the silence of miR-29b reduced the level of can be compared with the levels obtained from the serum IL-6. Therefore, miR-29b level is positively associated with in patients. the levels of TLR4, NF-kappa B, IL-1 beta, and IL-6 in ECs, suggesting that the change of miR-29b will affect the levels Mir-29b level is positively associated with of TLR4, NF-kappa B, IL-1 beta, and IL-6. the levels of Tlr4, nF-kappa B, il-1 beta, and il-6 in ecs Western blot MiR-29b mimics and antagomirs were successfully trans- To measure the effects of miR-29b on the changes of fected with ECs and the estimated transfection rate was phospho-p65 NF-kappa B, and phospho-p38 MAPK, between 60% and 80%. Figure 11A showed that the over- Western blot analysis was performed. Western blot analysis expression of miR-29b increased the level of TLR4 and the shows that miR-29b overexpression increases the levels of submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling $ % 7/5 SJP/ 1)NDSSD% QJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &* 2* 6* &* 2* 6* & ' ,/ SJP/ ,/EHWD SJP/ 7KHFRQFHQWUDWLRQRI 7KHFRQFHQWUDWLRQRI &* 2* 6* &* 2* 6* ( ) FSPî &HOOYLDELOLW\ +@WK\PLGLQHXSWDNH &* 2* 6* &* 2* 6* Figure 11 The effects of mir-29b on ecs. Notes: (A) The effects of mir-29b on the levels of Tlr4. ( B) The effects of mir-29b on the levels of nF-kappa B. ( C) The effects of mir-29b on the levels of il-1 beta. (D) The effects of mir-29b on the levels of il-6. ( E) Trypan blue analysis of the effects of mir-29b on ec viability. ( F) [ h]thymidine uptake analysis of the effects of mir-29b on ec viability. * P,0.05 vs the cg. Abbreviations: cg, control group; ecs, endothelial cells; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Og, mir-29b was overexpressed; sg, mir-29b was silenced by antagomirs; Tlr, toll-like receptor. phospho-p65 NF-kappa B (Figure 12A), phospho-p38 MAPK 248 patients who underwent EVAR, there was no signic fi ant (Figure 12B), TLR4 (Figure 12C), IL-1 beta (Figure 12D), difference for early mortality and neurologic complications and IL-6 (Figure 12E) when compared with the level of between the CG and AG. According to late outcomes, the CGs. In contrast, miR-29b inhibitor reduces the levels of overall survival rate was higher in the AG than in the CG phospho-p65 NF-kappa B (Figure 12A), phospho-p38 MAPK (P,0.05). The results suggest that AOS contributes to (Figure 12B), TLR4 (Figure 12C), IL-1 beta (Figure 12D), AA repair. The reintervention rate and continued presence and IL-6 (Figure 12E) when compared with the level of CGs. of complications were higher in the CG than in the AG All the results suggest that miR-29b can affect the activity (P,0.05). There were significant differences for being free of TLR4 signaling. from aneurysm-related death after two-year therapy between the groups (P,0.05). Discussion AA therapy is still a challenge although a less invasive This was a retrospective research, and the efficacy and method can be commercially available for treating AA. Since suitability of AOS were assessed in AA therapy. Based on introduction of EVAR, it is a safe and feasible alternative submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress $ S1)NDSSD% S0$3. 7/5 ,/EHWD ,/ &* 2* 6* &* 2* 6* &* 2* 6* &* 2* 6* &* 2* 6* %HWDDFWLQ % & SKRVSKRS0$3. 5HODWLYHSURWHLQOHYHORI 5HODWLYHSURWHLQOHYHORI SKRVSKRS1)NDSSD% &* 2* 6* &* 2* 6* OHYHORI7/5 5HODWLYHSURWHLQ 5HODWLYHSURWHLQ OHYHORI,/EHWD &* 2* 6* &* 2* 6* OHYHORI,/ 5HODWLYHSURWHLQ &* 6* 2* Figure 12 (A–F) Western blot analysis of the effects of mir-29b on the expression of Tlr4 signaling molecules. Notes: (A) Western blot analysis for the effects of mir-29b on the expression of p65 nF-kappa B, p38 MaPK, Tlr4, il-1 beta and il-6. ( B) The effects of mir-29b on relative protein level of nF-kappa B. ( C) The effects of mir-29b on the expression of phospho-p38 MaPK. ( D) The effects of mir-29b on the expression of Tlr4. ( E) The effects of mir-29b on the expression of il-1 beta. ( F) The effects of mir-29b on the expression of il-6. * P,0.05 vs the cg. Abbreviations: cg, control group; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Og, mir-29b was overexpressed; sg, mir-29b was silenced by antagomirs; Tlr, toll-like receptor. to conventional open repair. However, the surgery still has thoracotomy, cardiopulmonary bypass and hypothermia some side effects. Respiratory failure is a major complica- inducing an inflammatory reaction also attribute to postop - tion, which affects postoperative morbidity and mortality erative respiratory failure. Because of their less invasive and results in prolonged hospitalization. In addition to methods, respiratory failure was relatively rare in EVAR. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Dovepress alginate oligosaccharide indirectly affects Tlr signaling In the present study, more patients developed respiratory rates. There were eight deaths in the CG and two deaths in failure in the CG than in the AG (P,0.05). AOS reduced the AG related to aortic disease. All these results provide a these side effects significantly ( P,0.05). potential use of the natural products of marine algae for the EVAR has been quickly developed because of its therapy of AA patients (Figure 13). Retrograde dissection improved perioperative mortality and morbidity. However, and endoleak were signic fi ant complications, resulting in the high reintervention is still linked with EVAR. Here, a sig- death of patients. These signic fi ant complications contributed nificantly lower rate of recurrent AA was associated with to an aneurysm-related death rate after EVAR. However, the AOS treatment (16 patients in CG and 6 patients in AG). late aneurysm-related deaths occurred in patients who under- At cell levels, AOS is demonstrated to reduce the levels went EVAR in the r fi st 2 years at our hospital. AOS treatment of miR-29b (Figure 9A), TLR4, NF-kappa B, IL-1 beta, improved EVAR at our institution because aneurysm-related and IL-6 (Figure 10), while the overexpression and silence death rates were decreased when compared with the CG. As of miR-29b will increase and reduce the levels of TLR4, described above, there are advantages in using the combined phospho-p65 NF-kappa B, phospho-p38 MAPK, IL-1 beta, technique, including 1) the reduction in the incidences of AAs and IL-6, respectively (Figure 12). NF-kappa B contains sites by AOS; 2) the reduction in infection rates; 3) the reduction in for phosphorylation, which is important for activating TLR4 the number of deaths; 4) fewer complications such as bleed- signaling pathways. Phosphorylated MAPK also plays an ing, pneumothorax, and stroke; and 5) the reduction due to important role in regulating TLR4 signaling. The reasons AOS in other side effects caused by repair materials. for reducing recurrent AA cases may be caused by AOS, Certainly, there were some limitations for the present which reduced the level of serum miR-29b (Figure 4) and work: 1) four main AOS fractions (DP3, DP4, DP5, and DP6) resulted in the reduction of TLR4, phospho-p65 NF-kappa B, were isolated. We wanted to use one bioactive component for phospho-p38 MAPK, IL-1 beta, and IL-6 (Figure 12). On the subsequent experiment. However, single component cannot other hand, there is a strong positive relationship between be prepared at a scale suitable for a patient population at the miR-29b and the levels of serum TLR4, NF-kappa B, IL-1 moment. On the other hand, oligosaccharides are complex beta, and IL-6 (Figure 6). These results suggest that AOS carbohydrate molecules and the purity will ree fl ct the product treatment reduced the level of serum TLR4, NF-kappa B, quality, so the components were analyzed here. In any way, IL-1 beta, and IL-6 by reducing the level of miR-29b. to confirm the function of specific component, further work is AOS treatment seemed to be associated with the reduc- highly demanded in the future; 2) lack of a negative CG, such tion of long-term mortality rates. The midterm follow-up of as open surgery repair; 3) the effects of AOS on most inflam - patients revealed a significant benefit in cumulative survival matory cytokines and antioxidant enzymes were not explored 2+ 2+ 2+ ± 2+ 2 22& +22& 2+ .HOS 2 2 ± +2 &22 +2 2+ 0DQQXURQDWH *XOXURQDWH *OXFXURQLFDFLG 1D ±  ± 22& 1D 22& 2+ 2+ 2 +2 2+ +2 2 1D ± 2+ 22& $26 $OJLQDWHVRGLXP 7/5 PL5E $QHXU\VP 1RUPDODRUWD 1)NDSSD% ,/EHWD 3 3 ,/ Figure 13 a cartoon description of aOs from marine algae for the therapy of aa patients. The oligosaccharide has been found to prevent the regrowth of aneurysms by inactivating Tlr4, nF-kappa B, il-1 beta, and il-6 via the inhibition of mir-29b. Abbreviations: aa, aortic aneurysm; aOs, alginate oligosaccharide; il-6, interleukin 6; nF-kappa B, nuclear factor kappa B; Tlr, toll-like receptor. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress Yang et al Dovepress 8. Mursalin R, Sakamoto I, Nagayama H, et al. Imaging-based predic- here; 4) present n fi dings demonstrate that AOS inhibits the tors of persistent type II endoleak after endovascular abdominal aortic growth of ECs, which is still difc fi ult to be associated with the aneurysm repair. AJR Am J Roentgenol. 2016;206(6):1335–1340. clinical use of aneurysm therapy. We aimed to control aneu- 9. Park EJ, Venkatesan T, Choi YW, Kim YK. Pinus densiflora stem bark extract induces breast cancer cell death via oxidative stress mediated rysm growth by inhibiting ECs activation via AOS because lysosomal membrane permeabilization. Planta Med. 2016;81(S 01): the ECs were isolated from aneurysm. To further conr fi m S1–S381. 10. Wang Z, Li Z, Ye Y, Xie L, Li W. Oxidative stress and liver the result, animal test will be needed in the future work. The cancer: etiology and therapeutic targets. Oxid Med Cell Longev. epigenetic mechanism of phosphorylation of TLR4 was not 2016;2016:7891574. studied. In any way, further studies will be required to confirm 11. Mani RS, Amin MA, Li X, et al. Inflammation-induced oxidative stress mediates gene fusion formation in prostate cancer. Cell Reports. the therapeutic effects for using AOS. The prospective multi- 2016;17(10):2620–2631. center data are still needed to confirm the present findings. 12. Yadav DK, Rai R, Kumar N, et al. New arylated benzo[h]quinolines induce anti-cancer activity by oxidative stress-mediated DNA damage. Sci Rep. 2016;6:38128. Conclusion 13. Suzuki Y, Okabayashi K, Hasegawa H, et al. Comparison of preopera- Taken together, the present findings demonstrate that AOS tive inflammation-based prognostic scores in patients with colorectal cancer. Ann Surg. Epub 2016 Dec 16. represses the growth of residual aneurysms and reduces aneu- 14. Wheeler TM, Zhao B, Sonpavde G, et al. Antigen-specic fi immunity and rysm recurrence by indirectly affecting TLR signaling via tumor inflammation after vaccination with BPX-101, a drug-activated miR-29b. AOS treatment can be an adjuvant method for AA dendritic cell vaccine for metastatic castration-resistant prostate cancer (mCRPC). J Clin Oncol. 2011;29(7 Suppl):176. repair and control its subsequent development. AOS offers 15. Reyes-Gibby CC, Shete S, Wu X, Kurzrock R, Spitz M. Inflammation an adjuvant method for minimally invasive endovascular genes and pain severity in lung cancer patients. J Clin Oncol. 2009; 27(15 Suppl):9618. repair of AA and dissection. AOS reduced recurrent AA and 16. Oh B, Butow P, Mullan B, et al. Randomized clinical trial of medical its side effects, such as wound infection rates. To use AOS qigong on quality of life, fatigue, side effects, mood, status, and inflam - better, further work is still needed to be done to confirm the mation of cancer patients. J Clin Oncol. 2009;27(15 Suppl):9617. 17. Guo JJ, Ma LL, Shi HT, et al. Alginate oligosaccharide prevents acute present therapeutic results in the future. doxorubicin cardiotoxicity by suppressing oxidative stress and endo- plasmic reticulum-mediated apoptosis. Mar Drugs. 2016;14(12):231. 18. Tusi SK, Khalaj L, Ashabi G, Kiaei M, Khodagholi F. Alginate oligo- Acknowledgment saccharide protects against endoplasmic reticulum- and mitochondrial- The work was supported by Scientic fi Research Projects from mediated apoptotic cell death and oxidative stress. Biomaterials. Internal Research Institutions of Medical and Health Units in 2011;32(23):5438–5458. 19. Zhou R, Shi X, Gao Y, Cai N, Jiang Z, Xu X. 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Anti-inflammatory proper - thoracic aortic dissection by noninvasive imaging procedures. N Engl ties of Irisin, mediator of physical activity, are connected with TLR4/ J Med. 1993;328(1):1–9. MyD88 signaling pathway activation. Int J Mol Sci. 2017;18(4):701. 34. Fillinger MF, Greenberg RK, McKinsey JF, Chaikof EL. Society for 41. Li X, Lian LH, Bai T, et al. Cryptotanshinone inhibits LPS-induced Vascular Surgery Ad Hoc Committee on TRS. Reporting standards proinflammatory mediators via TLR4 and TAK1 signaling pathway. for thoracic endovascular aortic repair (TEVAR). J Vasc Surg. 2010; Int Immunopharmacol. 2011;11(11):1871–1876. 52(4):1022–1033, 1033 e1015. Drug Design, Development and Therapy Dovepress Publish your work in this journal Drug Design, Development and Therapy is an international, peer- has also been accepted for indexing on PubMed Central. The manu- reviewed open-access journal that spans the spectrum of drug design script management system is completely online and includes a very and development through to clinical applications. Clinical outcomes, quick and fair peer-review system, which is all easy to use. Visit patient safety, and programs for the development and effective, safe, http://www.dovepress.com/testimonials.php to read real quotes from and sustained use of medicines are the features of the journal, which published authors. Submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2017:11 Dovepress

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