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A frog antioxidant peptide protects against myocardial ischemia reperfusion injury in rats A frog antioxidant peptide protects against myocardial ischemia reperfusion injury in rats
Lin, Zhi; Jiang, Yongliang; Yang, Ping; Sun, Lin; Lu, Di
2020-01-01 00:00:00
ALL LIFE 2020, VOL. 13, NO. 1, 45–53 https://doi.org/10.1080/21553769.2019.1699171 A frog antioxidant peptide protects against myocardial ischemia reperfusion injury in rats a∗ a∗ b a b† Zhi Lin , Yongliang Jiang , Ping Yang , Lin Sun and Di Lu a b Department of Cardiology, the 2nd Affiliated Hospital of Kunming Medical University, Kunming, People’s Republic of China; Technology Transfer Center, Kunming Medical University, Kunming, People’s Republic of China ABSTRACT ARTICLE HISTORY Received 7 August 2019 Excessive reactive oxygen species (ROS) induced by myocardial ischemia reperfusion (IR) injury exert Accepted 20 November 2019 detrimental effects on cardiomyocytes. Antioxidant peptides can scavenge free radicals such as 2,2 - azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS ), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and KEYWORDS nitic oxide (NO) in vitro, but little is known about their functions in vivo. Here, we evaluated the effects Antioxidant peptide; reactive of an antioxidant peptide (antioxidant-RL) used at the beginning of reperfusion on myocardial IR oxygen species; myocardial injury. We performed 2, 3, 5-triphenyltetrazolium chloride staining (TTC) to determine myocardial ischemia reperfusion; infarct size. Creatine kinase isoenzyme-MB (CK-MB), cardiac troponin-T (CTnT), tumor necrosis factor- apoptosis alpha (TNF-α), and interleukin-6 (IL-6) levels were determined with enzyme-linked immunosorbent assays (ELISAs). The levels of ROS were measured with a fluorometric Intracellular ROS kit. The expression levels of apoptosis-related proteins in cardiomyocytes were detected by western blot- ting. Antioxidant-RL (10 mg/kg) reduced the infarct size and levels of CK-MB and CTnT in rats. The levels of TNF-α and IL-6 also decreased. Furthermore, ROS levels were ameliorated and the expres- sion of apoptosis-related proteins in myocytes was down-regulated by antioxidant-RL (100 µg/mL). Antioxidant-RL exerted protective effects on myocardial IR injury by scavenging excessive ROS, sup- pressing inflammatory factors, and inhibiting cardiomyocytes apoptosis. Thus, antioxidant-RL may serve as a potent candidate for the treatment of IR injury. CONTACT Lin Sun sunlinkm@sina.com Department of Cardiology, the 2nd Affiliated Hospital of Kunming Medical University, 374 Dianmian Road, Wuhua District, Kunming, Yunnan 650101, People’s Republic of China; Di Lu ludi20040609@126.com Technology Transfer Center, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, Yunnan 650500, People’s Republic of China * These authors made equal contributions to this work. Zhi Lin and Yongliang Jiang performed the experiments. Lin Sun and Di Lu designed the study and analyzed the data. Zhi Lin and Ping Yang wrote and revised the paper. All authors read and approved the final version of the manuscript. Supplemental data for this article can be accessed here. https://doi.org/10.1080/21553769.2019.1699171 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 46 Z. LIN ET AL. Introduction Here, we investigate the potential of antioxidant-RL to reduce myocardial IR injury by scavenging exces- Acute myocardial infarction is a major cause of sive ROS. We measured the infarct size and the level of disabling morbidity and death worldwide (Hausen- creatine kinase isoenzyme MB (CK-MB) and cardiac loy and Yellon 2013). Effective therapy for acute troponin-T (CTnT) (the most sensitive markers for myocardial infarction involves timely reperfusion minor myocardial damage) in a rat model of myocar- (Reimer et al. 1977), which may also induce car- dial IR injury after treatment with antioxidant-RL. diac ischemia reperfusion (IR) injury (Braunwald and We examined the ability of antioxidant-RL to elimi- Kloner 1985). Reperfusion causes arrhythmias and nate excessive ROS induced by IR injury. In addition, reversible contractile dysfunction (stunning), initiates we determine the profiles of inflammatory markers in a microvascular no-reo fl w, and contributes to myocar- plasma samples and apoptosis rate of cardiomyocytes. dial infarction, thereby acting as a major determi- Our results demonstrate that antioxidant-RL protects nant of prognosis (Heusch 2013). Several pathological rats against IR injury by preventing the IR-induced mechanisms are known to contribute to reperfusion excessive production of ROS. injury (Kloner 1988;Richardetal. 1990;Imahashietal. 2005;Matejikovaetal. 2009;Kaljustoetal. 2010;Gao Materials and methods et al. 2017; Scofield et al. 2019), including excessive for- mation of reactive oxygen species (ROS), intracellular Peptide synthesis calcium overload, mitochondrial dysfunction, activa- Antioxidant-RL (AMRLTYNRPCIYAT) was synthe- tion of intracellular proteolysis, and uncoordinated sized by the peptide synthesizer GL Biochem Ltd excessive contractile activity. ROS exhibit signaling (Shanghai, China). The synthetic peptide was puri- functionsinsmallamountsbutcouldbedetrimental fied and analyzed with high-performance liquid chro- when in excess. Excessive ROS could attack cellular matography (HPLC) and matrix-assisted laser desorp- components and induce apoptosis and necrosis (Mur- tion/ionization (MALDI)-time of ig fl ht (TOF) mass phy and Steenbergen 2008). Therefore, inhibition of spectrometry (MS) to confirm that the purity was excessiveROSmaybeanimportant interventionto higher than 98%. prevent IR injury. However, no clinical antioxidant drug is yet available for cardioprotection. Amphibian antioxidant peptides are a group of fast- Animal and ethics acting antioxidants in the skin. These peptides possess Healthy male Sprague-Dawley (SD) rats weighting the ability to rapidly and constantly eliminate free rad- 220–280 g were obtained from the Kunming Med- icalssuchas2,2 -azinobis(3-ethylbenzothiazoline-6- ical University Animal Experiment Center (license sulfonic acid (ABTS+), 2,2-diphenyl-1-picrylhydrazyl No.53004100000079). These specific pathogen-free (DPPH), and nitic oxide (NO) within a few seconds (SPF)ratswereprovidedwithwater andfood accord- and exert anti-inflammatory effects. In amphibians, ing to experimental animal standards. The study was these peptides protect the animal skin from infection performed in accordance with the recommendations or ROS injury (Yang et al. 2009). Antioxidant-RL, a short 14 residue antioxidant peptide (AMRLTYNR- intheGuidancefor theCareand UseofLaboratory Animals issued by the Laboratory Animal Ethics Com- PCIYAT) extracted from the skin secretions of Rana mittee of Kunming Medical University (Figure 1). pleuraden, has the ability to eliminate ABTS in a few seconds. Investigation of the underlying antioxi- dant mechanism has indicated the crucial role of Tyr6, IR model in rats Cys10, and Tyr12 residues in radical elimination (Liu Rats were anesthetized with sodium pentobarbital et al. 2010). Both Tyr6 and Tyr12 covalently interact (50 mg/kg, intraperitoneal) and intubated before being with ABTS via their tyrosyl groups and form a purple artificially ventilated with room air at a frequency of 80 product. The free thiol group in Cys10 is responsible inflations/min on a tidal volume of 1 mL/100 g. Lead for the rapid scavenging of ABTS through the dona- II of electrocardiogram (ECG) was recorded via cuta- tion of electrons to radicals (Liu et al. 2010). Although neous needle electrodes. Heart was accessed through antioxidant-RL canscavengefreeradicalsinvitro,no left thoracotomy in the fourth intercostal space. The study has evaluated its function in vivo. ALL LIFE 47 Figure 1. Schematic representation of the experimental protocol in rats. Myocardial ischemia was induced by the occlusion of LAD branch. Reperfusion of ischemic myocardium for 120 min after 60 min of ischemia was completed by loosening the silk thread. After reperfusion inception, antioxidant-RL (10 mg/kg) and NAC (150 mg/kg) were administered via the tail vein. pericardium was incised, and the heart was exposed. PBS, Sigma) solution at pH 8.5 and 37°C for 30 min. In the IR group, occlusion of the left anterior descend- The infarcted area showed no staining, while the nor- ing (LAD) branch was maintained for 60 min by lift- mal tissue showed reddish brown appearance. Image ing a 5/0 silk thread passing below the LAD through pro plus 6.0 software was used to calculate the infarct a piece of polyethylene tube. Criteria for confirma- size. tion of successful coronary occlusion included appear- IR model with H9c2 cells ance of pale color in the distal myocardium and ST elevation on ECG (Figure 2(A)). In all IR groups, The rat H9c2 cardiomyocytes were generously pro- reperfusion of ischemic myocardium was allowed for vided by Professor Qiu-Xiong LING (Research Center 120 min after 60 min of ischemia via loosening of of Medical Sciences, Guangdong Academy of Med- the silk thread. Antioxidant-RL (10 mg/kg) and N- ical Sciences, Guangzhou, China). H9c2 cardiomy- acetylcysteine (NAC; 150 mg/kg) were administered ocytes were cultured in Dulbecco’s modified Eagle’s via the tail vein at the beginning of reperfusion. medium (DMEM)/F12 (Gibco/BRL, Gaithersburg, Blood samples were collected to obtain plasma after MD, USA) supplemented with 10% fetal bovine serum 2 h reperfusion. The expression of cardiac markers (FBS; HyClone, Logan, UT, USA) and antibiotics CK-MB and CTnT and serum levels of inflamma- (100 IU/mL penicillin and 100 mg/mL streptomycin) tory cytokines tumor necrosis factor alpha (TNF-α) at a density below 5 × 10 cells/mL and maintained and interleukin-6 (IL-6) were evaluated with enzyme- at 37°C in a humidified incubator with 5% CO .To linked immunosorbent assays (ELISAs). harvest H9c2 cardiomyocytes, cells were trypsinized (0.25% trypsin/ethylenediaminetetraacetic acid [EDTA] in PBS), centrifuged (400 × g for 10 min), and Staining with 2,3,5-triphenyltetrazolium chloride resuspended in serum-free DMEM/F12. Cells were (TTC) to determine infarct area counted with a hemocytometer; trypan blue staining TTC staining accurately reflects the extent of irre- (0.4% trypan blue in PBS) showed more than 98% versible myocardial ischemic damage. After washing viable cells. Cells (approximately 1 × 10 cells/mL) with phosphate-buffered saline (PBS), the left ventri- were seeded in six-well plates for 24 h before treatment cle was frozen at −20°Candcutintoslices(2mm inception. thickness). The sections were incubated with a 1% To simulate IR injury, we performed IR with H9c2 TTC (Solarbio Science & Technology Co., Ltd, Bei- cells. The cultured H9c2 cells were incubated in jing, China) and urethane (0.1 g TTC dissolved in 5 mL DMEM/F12 high glucose medium (4.5 g/L) with 10% 48 Z. LIN ET AL. Figure 2. Antioxidant-RL reduces myocardial IR injury in rats. (A) ST-segment elevation was the most important indicator of LAD branch of coronary artery occlusion. ST-segment was elevated after the occlusion of LAD but reduced after LAD recanalization. No signifi- cant difference was observed between the groups. (B) Antioxidant-RL (5, 10, and 15 mg/kg) and NAC (150 mg/kg) reduced the infarct size, as evident from TTC staining, in rats subjected to ischemia reperfusion (IR) injury. The white regions represent the infracted area. Data are presented as mean ± SD (n = 5). (C and D) Antioxidant-RL lowered the plasma concentrations of CK-MB and CTnT in different groups. *P < 0.05 represents significant differences between IR and Sham group; P < 0.05 represents significant difference between antioxidant-RL + IR versus IR group. FBSfor 24handthen subjectedtooxygen-glucose mimicking the stage of reperfusion). Antioxidant-RL deprivation/reperfusion (OGD/R) to mimic myocar- at100μg/mLand NACat0.1Mwereadded1hbefore dial IR. Experiments in H9c2 cardiomyocytes were mimicking OGD. This time point was chosen to mini- carried outatacelldensityof5 × 10 cells/mL. H9c2 mize the possibility of any direct interactions between cardiomyocytes were cultured in DMEM/F12 supple- antioxidant-RL and serum deprivation condition. mented without glucose and serum for 40 min in a humidified incubator with 0.5% O (for mimicking the Measurement of intracellular ROS production stage of OGD). The cells were cultured in DMEM/F12 supplemented with high glucose and 10% FBS for Intracellular ROS level was measured using a u fl o- 2 h in a humidified incubator with 5% CO (for rometric intracellular ROS assay kit (Sigma-Aldrich, 2 ALL LIFE 49 St. Louis, USA), which provides a sensitive, one- difference method. A value of P < 0.05 was con- step fluorometric assay to detect intracellular ROS sidered statistically significant. All statistical analyses (especially superoxide and hydroxyl radical) in live were performed using SPSS version 17.0 (International cells within 1 h incubation. ROS react with a cell- Business Machines Corp., Armonk, NY, USA). permeable sensor and produce a uo fl rometric product (λex = 540 nm/λem = 570 nm). The intensity of the Results colored product is proportional to the amount of ROS. The assay can be performed in a convenient 96-well or Antioxidant-RL protects the heart against 384-well microtiter-plate format. myocardial IR injury Weobserved an elevationinthe ST-segmentafterthe occlusion of the LAD branch, and a drop in the ST- Western blot analysis segment was reported after the loosening of the LAD After reperfusion, great vessels and atria of the heart branch. No significant difference in the change in the were trimmed away and the ventricles were cut open, ST-segment was observed in different myocardial IR weighed, snap-frozen, and stored at −80°C until use. groups (Figure 2(A)). At the end of reperfusion, proteins from cardiomy- We investigated the eeff ct of antioxidant-RL treat- ocytes and the heart were extracted with ice-cold lysis ment on myocardial IR injury through the anal- buer ff and centrifuged at 12,000 × g for 10 min. The ysis of infarct size and CK-MB and CTnT lev- resultant supernatant was assayed using bicinchoninic els. The infarcted myocardium appeared white after acid (BCA) protein assay kit and bovine serum albu- TTC staining. The infarct size was significantly min(BSA)wasusedasastandard.Equal amountsof larger in IR group (65.98% ± 2.00%) than in Sham total protein (40 μg) were loaded, separated by sodium group (9.58% ± 0.39%) (Figure 2(B and C), P < 0.05). dodecyl sulfate polyacrylamide gel electrophoresis However, aremarkabledecreaseinthe infarctsize (SDS-PAGE), and transferred onto polyvinylidene was observed in antioxidant-RL + IR and NAC + IR u fl oride (PVDF) membranes. The membranes were groups (45.34% ± 2.38% and 41.92% ± 1.51%, respec- blocked with 5% tris-buffered saline and Tween-20 tively)ascomparedtothatinIRgroup(Figure 2(B and (TBST) fat-free milk (blocking buffer) for 2 h, briefly C), P < 0.05). washed, and incubated overnight at 4°C with primary The levels of CK-MB and CTnT were higher antibodies against caspase-3 (1:2000, no. ab2758369, in IR group (CK-MB: 18.08 ± 0.56 ng/mL; CTnT: Abcam, Cambridge, MA, USA), caspase-8 (1:2000, no. 765.40 ± 15.19 pg/mL) than in Sham group (CK- ab25901, Abcam, Cambridge, MA, USA), and caspase- MB: 2.32 ± 0.14 ng/mL; CTnT: 172.40 ± 12.05 pg/mL) 9 (1:2000, ab32539, Abcam, Cambridge, MA, USA). (Figure 2(D and E), P < 0.05), but the antioxidant- Blotswerethenprobedwithananti-rabbit secondary RL treatment ameliorated the elevated levels of CK- antibody (1:1000) for 2 h. Proteins were detected using MB and CTnT (CK-MB: 8.44 ± 0.20 ng/mL; CTnT: a chemiluminescence system, according to the man- 347.20 ± 10.68 pg/mL). The levels of CK-MB (79.4 ± ufacturer’s instructions. Band intensities were quan- 1.81 ng/mL) and CTnT (348.4 ± 6.46 pg/mL) in NAC tified using Imaging System Analysis software (Ver- + IR group also decreased as compared to those saDocMp5000; Bio-Rad). Relevant band intensities reported in IR group (Figure 2(D and E), P < 0.05). were quantified after normalization to the amount of β-actin proteinasapositivecontrol. Antioxidant-RL suppresses the levels of inflammatory factors Statistical analysis We observed an increase in the level of TNF- Allvaluesareexpressedasthe mean ± standard α in IR group (156.80 ± 8.19 pg/mL) as compared deviation (S.D). Comparison between multiple-group with that in Sham group (29.60 ± 1.77 pg/mL). How- means was performed using one-way analysis of ever, antioxidant-RL could reduce the elevated lev- variance (ANOVA). Multiple comparison between els of TNF-α caused by IR (108.80 ± 7.37 pg/mL) groups was performed using the least significant (Figure 3(A), P < 0.05). 50 Z. LIN ET AL. Figure 3. Antioxidant-RL reduces inflammatory factors in rats. Antioxidant-RL (10 mg/kg) reduced the plasma concentrations of TNF-α and IL-6 in different groups. *P < 0.05 represents significantly difference between IR and Sham group; P < 0.05 represents significant difference between antioxidant-RL + IR and IR group. The level of IL-6 in IR group (604.56 ± 15.74 pg/mL) Sham group. Antioxidant-RL decreased the expression was higher than that in Sham group (138.2 ± of caspase-3, caspase-8, and csapase-9 in IR H9C2 car- 6.09 pg/mL), but the treatment with antioxidant-RL diomyocytes (Figure 5(A–D), P < 0.05). Similarly, it reduced the elevated level of IL-6 (354.64 ± 9.93 pg/mL) also decreased the expression of caspase-3, caspase-8, (Figure 3(B), P < 0.05). and csapase-9 in myocardial IR rats (Figure 5(E–H), P < 0.05). Antioxidant-RL alleviates oxidative stress in IR H9C2 cardiomyocytes Discussion ROS overproduction and oxidative stress have detri- Myocardial reperfusion injury is the process that can mental eects ff on the heart in myocardial IR injury. induce cardiomyocyte death, and no effective ther- Thus, we performed studies to understand the rela- apy is yet approved against this pathophysiology. tionship between ROS level and myocardial IR injury. In the present study, the function of an amphib- We found that ROS level greatly increased after ian antioxidant peptide (antioxidant-RL) was evalu- myocardial IR (21.94% ± 1.10%) as compared with ated in a rat model of myocardial IR injury. Dieff r- Sham group (3.06% ± 0.49%). However, these elevated ent doses of antioxidant-RL (5, 10, and 15 mg/kg) ROS levels decreased after antioxidant-RL treatment were used to investigate the cardioprotective effects (9.00% ± 0.40%) (Figure 4(A and B), P < 0.05). in rats after myocardial IR injury. The results indi- catedthattheeeff ctofantioxidant-RLonreduc- ing myocardial infarct size was more obvious at Antioxidant-RL suppresses myocardial cell 10 mg/kg than at 5 mg/kg; however, the highest dose apoptosis in IR injury of antioxidant-RL (15 mg/kg) had no extra pro- The primary mechanism underlying cell death in tective eeff cts. These observations demonstrate that myocardial IR is apoptosis. Thus, we explored the antioxidant-RL (10 mg/kg) could reduce myocardial eects ff of antioxidant-RL treatment on myocardial IR infarct size and decrease the levels of two IR injury rats and H9C2 cardiomyocytes through the analy- markers (CK-MB and CTnT), indicative of its cardio- sis of the expression levels of apoptosis-related pro- protective role in vivo. teins, cleaved caspase-3, caspase-8, and caspase-9. As Oxidative stress is one of key mechanisms in a result, we found that the expression levels of cleaved myocardial IR injury (Bagheri et al. 2016; Sinning et al. caspase-3, caspase-8, and casapase-9 were higher for 2017). Excessive ROS can increase myocardial infarct H9c2 cardiomyocytes from IR group than those from size and deteriorate ventricular function (Chen et al. ALL LIFE 51 Figure 4. Antioxidant-RL reduces the level of ROS induced by IR injury in H9C2 cardiomyocytes. Antioxidant-RL (100 μg/mL) decreased IR-induced ROS levels in H9C2 cardiomyocytes. *P < 0.05 represents significant difference between IR and control group; P < 0.05 represents significant difference between antioxidant-RL + IR and IR group. Figure 5. Antioxidant-RL reduces the apoptosis of cardiomyocytes after IR injury. (A–D) The expression levels of apoptosis-related pro- teins, caspase-8, caspase-9, and cleaved caspase-3, in different H9C2 cardiomyocyte groups. Antioxidant-RL (100 μg/mL) treatment reduced the expression of apoptosis-related proteins caspase-8, caspase-9, and cleaved caspase-3 in H9c2 cardiomyocytes. (E–H) The results of the expression of caspase-8, caspase-9, and cleaved caspase-3 in myocardial IR rats were similar to those reported in H9C2 cardiomyocytes. Antioxidant-RL (10 mg/kg) decreased the levels of caspase-8, caspase-9, and cleaved caspase-3 in SD rats. *P < 0.05 represents significant difference between IR and Sham group; P < 0.05 represents significant difference between antioxidant-RL + IR and IR group. 2018). Overproduction of ROS after myocardial IR level of ROS and subsequently alleviate myocar- can damage cells by lipid peroxidation, protein denat- dial IR injury. Our data show that antioxidant-RL uration, and nucleic acid deterioration and severely decreased the level of ROS induced by IR injury in car- aeff ct the overall metabolism (Cadenas 2018). Thus, diomyocytes (Figure 4), and exerted protective effects antioxidant peptides with rapid radical-scavenging on rat hearts through the scavenging of excessive ability may serve as attractive agents to reduce the ROS. 52 Z. LIN ET AL. Inflammationisconsideredasthe most important the treatment of IR injury. Further investigations are causeoftissueinjuryinorganssubjectedtoischemia warranted to determine the mechanisms underlying (Eefting 2004; Ballard-Croft et al. 2008). Antioxidant- the eeff cts of antioxidant-RL on myocardial apoptosis RL reduced the levels of TNF-α and IL-6 in myocar- upon IR injury. dial IR injury (Figure 3). The suppression of inflam- In conclusion, our data demonstrate that antioxi- matory factors could lead to alleviation of myocar- dant-RL could reduce myocardial infarct size, decrease dial IR injury. Apoptosis is a crucial event that can levels of markers of IR injury (CK-MB and CTnT), initiate reperfusion-induced inflammation and subse- and suppress inflammatory factors (TNF- α and IL-6) quent tissue injury (Hamacher-Brady et al. 2006;Peng and apoptosis-associated proteins (cleaved caspase-3, et al. 2011). Therefore, inhibition of myocardial apop- caspase-8, and csapase-9) in myocardial IR injury. The tosis is a key protective mechanism against myocar- protective effects of antioxidant-RL on IR injury were dial IR injury. The process of apoptosis mainly relies mediated through the amelioration of oxidative stress, on the activation of caspases (Kaufmann and Hen- inflammatory factors, and myocardial apoptosis. The gartner 2001). The downstream caspases (caspase-3, combination of these properties deems antioxidant-RL caspase-6, and caspase-7) are activated to stimulate the as an attractive candidate for the treatment of myocar- dial IR injury. caspase cascade and induce cell death upon the acti- vation of the upstream caspases (caspase-8, caspase-9, and caspase-10) (Kavurma et al. 2005). Caspase-8 Disclosure statement and caspase-9 are activated in myocardial IR injury No potential conflict of interest was reported by the authors. and subsequently trigger the activation of downstream effectors caspases such as caspase-3. Caspase-3 is mainly responsible for mediating the typical patho- Ethical approval physiological and morphological changes in the cells All animal procedures were approved by and performed as undergoing apoptosis (Brar et al. 2001). Antioxidant- per the Guidelines for the Care and Use of Animals as stipu- RL (AMRLTYNRPCIYAT) is a cationic host defense lated by Kunming Medical University. The experiments were peptide (HDP) with a net charge of +2. HDPs per- also performed according to the Chinese National Institutes of Health Guidelines for the Use of Laboratory Animals. Animal form multifaceted functions during inflammation and Certificate Number: 5300410000079. immune modulation (Hancock et al. 2016). Recent studies have reported that endogenous cathelicidins (CRAMP and LL-37), a major family of HDPs, are Funding involved in cardiovascular diseases through the reg- This work was supported by the National Natural Science ulation of inflammatory response, microvascular dys- Foundation of China [grant number 81373380, 31760292, function (Zheng et al. 2019), and myocardial apoptosis 81560050]. upon IR injury (Bei et al. 2019). The CRAMP peptide was shown to inhibit cardiomyocyte apoptosis through References the activation of protein kinase B (Akt) and extra- cellular signal-regulated kinase (ERK1/2) and phos- BagheriF,KhoriV,Alizadeh AM,Khalighfard S,Khodayari S, Khodayari H. 2016.Reactiveoxygenspecies-mediated phorylation and nuclear export of FoxO3a (Bei et al. cardiac-reperfusion injury: mechanisms and therapies. Life 2019). 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