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Background: Endoplasmic reticulum (ER) stress and autophagy are implicated in the pathophysiology of intestinal inflammation; however, their roles in intrauterine growth retardation (IUGR)-induced colon inflammation are unclear. This study explored the protective effects of natural stilbene pterostilbene on colon inflammation using the IUGR piglets and the tumor necrosis factor alpha ( TNF-α)-treated human colonic epithelial cells (Caco-2) by targeting ER stress and autophagy. Results: Both the IUGR colon and the TNF-α-treated Caco-2 cells exhibited inflammatory responses, ER stress, and impaired autophagic flux (P < 0.05). The ER stress inducer tunicamycin and the autophagy inhibitor 3-methyladenine further augmented inflammatory responses and apoptosis in the TNF-α-treated Caco-2 cells (P < 0.05). Conversely, pterostilbene inhibited ER stress and restored autophagic flux in the IUGR colon and the TNF-α-treated cells (P < 0.05). Pterostilbene also prevented the release of inflammatory cytokines and nuclear translocation of nuclear factor kappa B p65, reduced intestinal permeability and cell apoptosis, and facilitated the expression of intestinal tight junc- tion proteins in the IUGR colon and the TNF-α-treated cells (P < 0.05). Importantly, treatment with tunicamycin or autophagosome-lysosome binding inhibitor chloroquine blocked the positive effects of pterostilbene on inflamma- tory response, cell apoptosis, and intestinal barrier function in the TNF-α-exposed Caco-2 cells (P < 0.05). Conclusion: Pterostilbene mitigates ER stress and promotes autophagic flux, thereby improving colon inflammation and barrier dysfunction in the IUGR piglets and the TNF-α-treated Caco-2 cells. Keywords: Autophagic flux, Colon inflammation, Endoplasmic reticulum stress, Intrauterine growth retardation, Piglets common and challenging issues in animal production Introduction [1, 2]. IUGR alters the intestinal morphology, impairs Intrauterine growth retardation (IUGR), which presents intestinal barrier function, and creates feeding intoler- as a failure of fetuses to achieve their intrinsic growth ance, all of which increase the occurrence of intestinal potential during pregnancy, has become one of the most diseases and cause postnatal retardation of growth and development, resulting in high rates of mortality and morbidity [3–5]. IUGR neonates experience a high inci- *Correspondence: email@example.com; firstname.lastname@example.org dence of colitis since they show deficiencies in mucosal College of Animal Science and Technology, Nanjing Agricultural University, immunity and imbalances in T lymphocyte subpopu- Nanjing 210095, China Full list of author information is available at the end of the article lations and lack an efficient colonic barrier [6 –9]. © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 2 of 16 Therefore, protecting the colon against inflammation dimethylated analogue of resveratrol, due to its excellent may be a pivotal requirement for the health of IUGR metabolic stability, intestinal absorption features, and animals. bioavailability [25, 26]. Our previous work indicated that The colon is one site that is vulnerable to endoplasmic pterostilbene had strong protective effects against intesti - reticulum (ER) stress, a physiological response induced nal barrier defects and small intestinal injuries in animals by protein aggregation or misfolding within the ER lumen under the conditions of oxidative stress or immunologi- . A variety of physiological and pathological stress- cal stress [27–29]. Pterostilbene was also found to miti- ors, including bacteria and inflammation tone from the gate colon inflammation in mice fed high-fat diets or intestinal lumen and host, can induce colonic ER stress treated with dextran sulfate sodium . However, the . In addition, the highly secretory cells that occur in potential for attenuation of the IUGR-induced colon the colonic epithelium manufacture a range of complex inflammation by pterostilbene was not documented. proteins and antimicrobial molecules that are currently Whether pterostilbene-mediated protection on intestine recognized as crucial targets for protein misfolding in health is associated with its regulation of ER stress and/ the ER . To counteract these negative effects, the con - or autophagy is not known. The current study explored served signal transduction pathways unfolded protein the mechanism by which pterostilbene protected against responses (UPR) have evolved. They are initially activated the IUGR-induced colon inflammation using a natu - as adaptive responses that strengthen protein folding rally occurring IUGR piglet model. We also revealed the capacity, re-establish ER function, and restore intestinal crosstalk between ER stress and autophagy under inflam - barrier homeostasis . However, once these adaptive mation conditions and the regulation of pterostilbene mechanisms fail to resolve the folding defects, the UPR in these two events in the tumor necrosis factor-alpha will switch to programmed cell death by triggering pro- (TNF-α)-treated human colonic epithelial cells (Caco-2). apoptotic signaling cascades and even induce inflam - matory responses [13, 14]. Indeed, ER stress has been Materials and methods identified as part of the intrinsic machinery of intestinal Reagents and antibodies inflammation [11, 15]. Pterostilbene was obtained from BOC Science (Shirley, Autophagy, a pro-survival mechanism for cells and NY, USA). TNF-α was purchased from Sino Biologi- organisms suffering from starvation or other diverse cal, Inc. (Beijing, China). Tunicamycin, 4-phenylbutyric pathologies, plays an important physiological role in acid (4PBA), and FITC-dextran were purchased from intestinal health . Autophagy modulates the rela- Sigma-Aldrich (St. Louis, MO, USA). Rapamycin and tionship between gut microflora and host immunity and 3-methyladenine (3MA) were purchased from CSN- thereby maintains intestinal barrier function, whereas pharm (Arlington Heights, IL, USA). Chloroquine was impaired autophagy in intestinal epithelium can induce purchased from MedChemExpress (Shanghai, China). intestinal inflammation [17, 18]. Interestingly, autophagy Adenovirus expressing mCherry-GFP-LC3B fusion pro- is recognized as an emerging effector mechanism that tein recognizing CD46 (AdPlus-mCherry-GFP-LC3B) regulates ER homeostasis . The misfolded pro - was purchased from Beyotime (Haimen, Jiangsu, China). teins and damaged ER fragments can be degraded and Dulbecco’s modified Eagle’s medium (DMEM), fetal recycled by autophagy . The UPR branches and bovine serum (FBS), and 1% penicillin and streptomycin UPR-associated transcription factors, such as activat- were obtained from Thermo Fisher Scientific Inc. (Grand ing transcription factor 4 (ATF4) and CCAAT/enhancer Island, NY, USA). binding protein homologous protein (CHOP), have also Antibodies used in Western blot analysis included: been demonstrated to modulate the autophagic process anti-occludin (1:3000; Proteintech; Chicago, IL, USA), . Based on these findings, exploring the exact interac - anti-zonula occludens 1 (ZO-1; 1:1000; Proteintech), tion between ER stress and autophagy in the context of anti-glyceraldehyde phosphate dehydrogenase (GAPDH; inflammation may help to uncover the mechanism and 1:25,000; Proteintech), anti-nuclear factor kappa B p65 identify prospective strategies for the IUGR-induced (NF-κB p65; 1:3000; Proteintech), anti-Lamin B1 (1:5000; colon inflammation. Proteintech), anti-glucose-regulated protein 78 (GRP78; Stilbenes are a class of polyphenolic compounds that 1:3000; Proteintech), anti-CHOP (1:1000; Proteintech), are widely distributed in blueberries, grapes, and other anti-cleaved caspase 12 (c-Casp12; 1:2000; Bioss Bio- medicinal plants . Their multiple biological activities, technology; Beijing, China), anti-phosphorylated pro- especially antioxidant and anti-inflammatory properties, tein kinase RNA-like ER kinase (PERK) (p-PERK; 1:2000; suggest the potential value of stilbenes in the treatment Affinity Biosciences; Cincinnati, OH, USA), anti-total- or prevention of intestinal disorders [22–24]. Recently, PERK (t-PERK; 1:1000; Affinity Biosciences), anti-phos - considerable attention has focused on pterostilbene, a phorylated inositol-requiring kinase 1 alpha (IRE1a) Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 3 of 16 (p-IRE1a; 1:1000; Affinity Biosciences), anti-total-IRE1a paraformaldehyde fixative solution for histological analy - (t-IRE1a; 1:2000; Affinity Biosciences), anti-activating sis. The colonic mucosa was scraped from the remainder transcription factor 6 (ATF6; 1:5000; Proteintech), anti- with a glass microscope slide, snap-frozen in liquid nitro- phosphorylated eukaryotic translational initiation factor gen, and stored at − 80 °C for further analysis. 2 alpha (eIF2α) (p-eIF2α; 1:1000; Bioss Biotechnology), anti-total-eIF2α (t-eIF2α; 1:1000; Bioss Biotechnology), Histopathological analysis anti-Beclin1 (1:3000; Proteintech), anti-LC3 I/II (1:2000; Colon tissues fixed in 4% paraformaldehyde were dehy - Proteintech), anti-p62 (1:4000; Proteintech), anti-Rab7 drated in an ascending alcohol series, cleared with xylene, (1:2000; Affinity Biosciences), and anti-lysosomal associ - embedded in paraffin blocks, cut into 5 μm sections, and ated membrane protein 2 (LAMP2; 1:1000; Proteintech). mounted on glass slides. After deparaffinizing and rehy - drating, the sections were stained with hematoxylin and Animals, experimental design, and sample collection eosin (H&E) buffer, and the histologic alterations were All animal experiments were reviewed and approved observed using a light microscopy (Olympus Corp; Tokyo, by the Institutional Animal Care and Use Committee Japan). The Chiu scoring system was employed to evaluate of Nanjing Agricultural University (SYXK-2017-0027). the colon mucosal injury. The colon goblet cell numbers Approximately 72 healthy sows (Landrace × Yorkshire) were determined by incubating the slices with Alcian blue/ having same parity (second or third) and expected dates periodic acid-Schiff stains as described previously . of confinement (< 4 d) were preselected during pregnancy. The colon goblet cell density was calculated as the goblet At delivery, only sows with about 11–13 live-born piglets cell count divided by the corresponding villus length. were retained. The birth weights (BWs) of newborn pig - lets (Duroc × (Landrace × Yorkshire)) were recorded. Plasma lipopolysaccharide (LPS) concentration Piglets were identified as normal birth weight (NBW) if Plasma LPS levels were measured with an enzyme-linked their BWs were close to the mean litter BW (within 0.5 immunosorbent assay (ELISA) kit obtained from CUSA- standard deviations), whereas those with BWs at least BIO Biotech (Wuhan, Hubei, China). 2 standard deviations lower than the mean litter BW were defined as IUGR [27, 31]. All piglets were permit- Analysis of mucosal immune status ted to suckle naturally up to 21 days of age. After wean- The colon mucosa was homogenized and centrifuged ing, a total of 36 litters that met the selection criteria for at 5000 × g for 5 min, and the concentrations of TNF-α, NBW and IUGR piglets were reserved, and one IUGR interleukin (IL)-1β, IL-4, IL-10, mucin 2, and trefoil fac- and one NBW male piglet were selected from each lit- tor 3 (TFF3) of the supernatants were determined with ter. Half of the selected NBW and IUGR piglets received porcine-specific ELISA kits (CUSABIO Biotech). The a basal diet (NBW-CON and IUGR-CON groups) for 14 colon myeloperoxidase (MPO) activity was measured d and the other half were fed a basal diet supplemented with a commercial kit (Jiancheng Bioengineering Insti- with 300 mg/kg pterostilbene (NBW-PTS and IUGR-PTS tute; Nanjing, Jiangsu, China). The protein levels of the groups) over the same period. Each treatment group con- supernatants were quantified using a bicinchoninic acid sisted of 6 replicate pens with 3 piglets per pen. The basal protein assay kit (Beyotime) for normalization of these diet (Table S1) was formulated according to the nutri- parameters. ent requirements of swine (National Research Council, 2012) . The level of supplemented pterostilbene was Cell culture, treatment, and cell viability analysis determined based on our previous publication [27, 28]. Caco-2 cell line was kindly gifted by Dr. Xiang Hou Diet and water were offered ad libitum during the entire (Jiangsu Academy of Agricultural Sciences; Nanjing, experiment. Jiangsu, China). Caco-2 cells at passage numbers 28–36 The diarrhea rates of piglets were determined as were grown in DMEM medium supplemented with reported by Liu et al. . At the endpoint of the experi- 10% FBS and 1% penicillin and streptomycin at 37 °C mental period, the piglets were weighed, and one piglet in a humidified atmosphere containing 5% CO . The whose BW was close to the average weight of each rep- optimal treatment concentrations of TNF-α and pter- licate was chosen for sampling. Heparinized blood sam- ostilbene were determined by exposing Caco-2 cells ples were withdrawn by anterior vena cava puncture, to TNF-α (0–50 ng/mL) with or without pterostilbene centrifuged at 2500 × g for 10 min at 4 °C, and stored at (0–250 μmol/L) for 24 h. ER stress in Caco-2 cells was − 80 °C for analysis. The piglets were then euthanized, induced and inhibited by tunicamycin (0.5 μg/mL for and the colon tissues were removed. After flushing with 24 h) and 4PBA (1 mmol/L for 24 h), respectively. Rapa- ice-cold PBS, approximately 1 cm of colon samples taken mycin (1 μmol/L for 24 h) and 3MA (5 μmol/L for 24 h) from the middle of the colon were immersed in 4% were used to induce and inhibit autophagy in Caco-2 Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 4 of 16 cells, respectively. Chloroquine (20 μmol/L for 24 h) was chloroquine for 24 h. The supernatants of Caco-2 cells used to obstruct the fusion of autophagosomes and lys- were collected to determine the contents of IL-1β and osomes in Caco-2 cells. IL-6 with ELISA kits (Multi Sciences Biotech; Hang- The Cell Counting Kit-8 (CCK-8; Dojindo Molecular zhou, Zhejiang, China). All procedures strictly followed Technologies; Shanghai, China) was used to evaluate the the manufacturer’s protocols. viability of Caco-2 cells. Briefly, Caco-2 cells were seeded in 96-well microplates at a density of 1 × 10 cells/well. Apoptosis measurement After 24 h, Caco-2 cells were treated with a range of TNF-α Terminal deoxynucleotidyl transferase dUTP nick end or pterostilbene for 24 h. Then, 10 μL CCK-8 reagent was labeling (TUNEL) staining and flow cytometry assays added to each well for 1.5 h, and the absorbance was meas- were carried out to determine the apoptotic index of the ured by a microplate reader (Thermo Fisher Scientific Inc.) colon tissues and Caco-2 cells, respectively. The methods at 450 nm. can be found in our previous study . Analysis of autophagic flux Immunofluorescent staining The autophagic flux was monitored by transfecting Caco-2 cells were plated in 12-well plates at a density of Caco-2 cells with AdPlus-mCherry-GFP-LC3B. The 2 × 10 cells/well for 24 h and then subjected to the study Caco-2 cells were seeded in confocal dishes at a density treatments. Subsequently, the cells were fixed in 4% par - of 2 × 10 cells/well until confluence reached 50%. After aformaldehyde, permeabilized with 0.1% Triton X-100, infection with AdPlus-mCherry-GFP-LC3B dissolved in a and incubated with 3% bovine serum albumin (BSA). complete DMEM medium (multiplicity of infection = 80) The cells were then incubated overnight with the pri - for 24 h, the cells were co-incubated with TNF-α and tuni- mary body against NF-κB p65 (1:200) at 4 °C, followed by camycin, 4PBA, rapamycin, or pterostilbene for another Alexa Fluor 594-conjugated secondary antibody (1:200) 24 h. The expression of GFP and mCherry was visualized for 1 h and 2-(4-amidinophenyl)-6-indolecarbamidine and captured with a laser scanning confocal microscope dihydrochloride (DAPI) for 1 min at room temperature. (Carl Zeiss; Oberkochen, Germany). Images were recorded with a confocal laser scanning microscope (Carl Zeiss). The permeability of Caco‑2 cell monolayers Caco-2 cells (2 × 10 cells/well) were grown in 12-well Total RNA isolation and quantitative real‑time PCR permeable culture chambers (12 mm diameter inserts (qRT‑PCR) analysis and 0.4 μm pore size) with a complete DMEM medium All reagents were purchased from Vazyme (Nanjing, until they formed a tight monolayer with a transepithe- Jiangsu, China). Total RNA from colon samples and Caco-2 lial electrical resistance (TER) of 500 Ω. Thereafter, the cells was isolated with the FastPure Cell/Tissue Total RNA cells were subjected to various treatments, and the TER Isolation Kit and reverse-transcribed into cDNAs with the and FITC-dextran flux were analyzed to determine the HiScript III 1st Strand cDNA Synthesis Kit. The ChamQ permeability of Caco-2 cell monolayers. The TER of each SYBR qPCR Master Mix and the StepOnePlus PCR Sys- culture chamber was tested at 3 different sites using the tem (Applied Biosystems; Carlsbad, CA, USA) were used Millicell-ERS resistance system (Millipore; Bedford, MA, to detect the expression levels of target genes by qRT-PCR USA) and corrected against the blank control. The net analysis. Thermal cycling was initiated at 95 °C for 30 s, fol- resistance was multiplied by the membrane area to give lowed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The the resistance in Ω cm . For FITC-dextran flux analysis, specific primers for GRP78, glucose-regulated protein 94 a complete DMEM medium containing 500 μg/mL FITC- (GRP94), CHOP, ATF4, spliced X-box binding protein-1 dextran was placed in the upper chamber of the tran- (sXBP-1), TNF-α, IL-1β, IL-6, and GAPDH were obtained swells. The FITC-dextran flux was measured by collecting from Sangon Biotech (Shanghai, China). The primer the basal medium every 30 min for 3 h with replacement sequences were provided in Table S2. The results were −ΔΔCT of the sampled volume with fresh medium without FITC- calculated using the 2 method and normalized to dextran at each sampling time and expressed as the flux GAPDH. into the basal chamber as a percentage of the total FITC- dextran initially added to the apical chamber. Analysis of sXBP‑1 mRNA in Caco‑2 cells Real-time PCR with T100 Thermal Cycler (Bio-Rad; Cytokine concentrations in the supernatants of Caco‑2 cells Hercules, CA, USA) was carried out to determine the Caco-2 cells were seeded in 6-well plates at a density of mRNA levels of sXBP-1 in Caco-2 cells. The PCR prod - 5 × 10 cells/well for 24 h and then exposed to TNF-α ucts were separated on 1.8% agarose gel and visualized and pterostilbene with or without tunicamycin and Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 5 of 16 with GelRed DNA stain. The primer sequences can be (Fig. 1c). The histological scores and apoptotic rates were found in Table S2. also higher in the colon of the IUGR piglets than in those of the NBW piglets (P < 0.05; Fig. 1d-f ). Moreover, IUGR Western blot analysis downregulated colon protein levels of occludin and ZO-1 All reagents for protein extraction and determination of piglets (P < 0.05; Fig. 1g). In contrast, dietary pterostil- of protein concentration were obtained from Beyotime. bene administration improved the colon architecture and Total protein from colon samples and Caco-2 cells was blocked hemorrhage and lymphocyte infiltration to the extracted using the lysis buffer containing a cocktail of lamina propria, thereby reducing the histological scores phosphatase and protease inhibitors. A nuclear protein of the IUGR colon (P < 0.05). The increased number of extraction kit was employed to isolate the nuclear protein TUNEL-positive cells and the reduced protein levels of in colon tissues and Caco-2 cells. After measurement of occludin and ZO-1 in the IUGR colon were also miti- the protein contents, 30 μg of protein from each sample gated by pterostilbene (P < 0.05). These data suggest that was separated by SDS-PAGE and electro-transferred to pterostilbene potentially maintains colonic barrier func- the polyvinylidene fluoride membranes. Subsequently, tion of the IUGR piglets. the membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with specific pri - Pterostilbene alleviates inflammatory responses mary antibodies overnight at 4 °C with gentle rocking. and regulates immune function in the IUGR colon After washing with the Tris-buffered saline containing The possible inhibition of the IUGR-induced colon 1% Tween 20, the corresponding secondary antibodies inflammation by pterostilbene was evaluated by meas - were added and incubated for 1.5 h at room temperature. urement of several markers of inflammatory responses. Immunoreactive bands were captured by the Chemi- Compared with the NBW piglets, the IUGR piglets had Doc imaging system (Bio-Rad) and analyzed using the higher concentrations of TNF-α and IL-1β but lower Gel-Pro Analyzer software. concentrations of IL-10 in the colon (P < 0.05; Fig. 2a-d). The MPO activity and nuclear NF-κB p65 protein expres - Statistical analysis sion in the colon were also increased by IUGR (P < 0.05; All statistical analyses were performed using SPSS soft- Fig. 2e and f). Further assessment of colon immune ware version 26 (Chicago, IL, USA). Data from at least function revealed marked reductions in the contents three independent experiments were shown as means of mucin 2 and TFF3 and the number of goblet cells in with their standard errors (SE). For comparison of multi- the IUGR piglets compared to their NBW counterparts ple datasets, one-way analysis of variance (ANOVA) with (P < 0.05; Fig. 2g-j). Conversely, treatment with pterostil- Tukey’s multiple-range test was employed. Values with bene decreased the concentrations of TNF-α and IL-1β, P < 0.05 were considered statistically significant. the MPO activity, and the translocation of NF-κB p65 from the cytoplasm to the nucleus, while increasing the Results IL-10 levels in the IUGR colon (P < 0.05). Pterostilbene Pterostilbene decreases diarrhea rates and improves also promoted the secretion of mucin 2 and TFF3 and colonic barrier function of the IUGR piglets increased the number of goblet cells in the IUGR colon To evaluate the protective potential of pterostilbene on (P < 0.05). These observations indicate that pterostil - intestinal health of the IUGR piglets, we recorded the bene suppresses inflammatory responses and overcomes diarrhea rates and determined the contents of circulating defective immune function in the IUGR colon. LPS, a bacterially generated endotoxin that penetrates the intestinal epithelial barrier and reaches the blood- Pterostilbene inhibits ER stress and restores autophagic stream when the intestinal permeability is increased flux in the IUGR colon . As shown in Fig. 1a and b, the diarrhea rates and ER stress and autophagy are the crucial mechanisms plasma LPS levels were significantly higher in the IUGR implicated in inflammatory bowel diseases [14, 16]. How- piglets than in their NBW counterparts, whereas these ever, an association between ER stress or autophagy and negative effects caused by IUGR were attenuated after the IUGR-induced colon inflammation remains uncon - pterostilbene treatment (P < 0.05). We also assessed the firmed. Our results suggested that IUGR piglets showed colon morphology, apoptosis, and tight junction protein apparent ER stress in the colon, as indicated by the expression of piglets to determine the effects of pterostil - increases in the mRNA abundance of GRP78, GRP94, bene on colon barrier function. H&E staining indicated CHOP, ATF4, and sXBP-1 and the protein levels of extensive damage to the colon epithelium of IUGR pig- GRP78, CHOP, and c-Casp12 (P < 0.05; Fig. 3a and b). We lets, with injuries including hemorrhage, crypt abscesses, also determined the expression of the three arms of the and lymphocyte infiltration to the lamina propria UPR (i.e., IRE1, PERK, and ATF6) and found that IUGR Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 6 of 16 Fig. 1 Pterostilbene decreases diarrhea rates and improves colonic barrier function of the IUGR piglets. a The diarrhea rates of piglets; b Plasma LPS concentration; c Representative photomicrographs of hematoxylin and eosin-stained colonic sections from piglets; d Histologic scores were determined using the Chiu scoring system; e Representative photomicrographs of terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL)-stained colonic sections from piglets; f Quantitative analysis of TUNEL-positive cells in the colon; g The protein levels of colon tight junction proteins were determined by Western blot analysis. Data from at least three independent experiments were presented as mean ± SE (n = 6). P < 0.05 induced the phosphorylation of PERK and IRE1a and p62 and the ratio of LC3 II/I were higher in the IUGR elevated ATF6 protein expression in the colon (P < 0.05). piglets than in the NBW piglets (P < 0.05; Fig . 3c), indi- In contrast, pterostilbene treatment significantly down - cating that IUGR may disturb the process of autophago- regulated the mRNA expression of GRP78, CHOP, and somal degradation in the colon. Pterostilbene treatment sXBP-1 and the protein expression of GRP78, CHOP, and further upregulated the protein expression of Beclin1 c-Casp12 in the IUGR colon (P < 0.05). The phosphor yla - and the ratio of LC3 II/I but markedly suppressed the tion of IRE1a in the IUGR colon was also significantly protein expression of p62 in the IUGR colon (P < 0.05). inhibited by pterostilbene (P < 0.05). To summarize, these results suggest that pterostil- The measurement of autophagy-associated proteins in bene inhibits colon ER stress and reverses impaired the colon showed that the protein levels of Beclin1 and autophagic flux in the IUGR colon. Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 7 of 16 Fig. 2 Pterostilbene alleviates inflammatory responses and regulates immune function in the IUGR colon. a-d The contents of TNF-α, IL-1β, IL-4, and IL-10 in the colon were measured by ELISA assays; e Colon myeloperoxidase (MPO) activity; f Colon protein levels of nuclear NF-κB p65 were determined by Western blot analysis; g Colonic mucin 2 levels; h Colonic trefoil factor 3 ( TFF3) levels; i Representative micrographs of Alcian blue/ periodic acid-Schiff stained colonic sections from piglets; j Goblet cell density of colon. Data from at least three independent experiments were presented as mean ± SE (n = 6). P < 0.05 The crosstalk between ER stress and autophagy ER stress inducer tunicamycin and the autophagy inhibi- in the TNF‑α‑exposed Caco‑2 cells tor 3MA aggravated the TNF-α-induced increases in the Having determined the involvement of ER stress and mRNA abundance of TNF-α, IL-1β, and IL-6, and cell autophagy in the IUGR-induced colon inflammation, apoptosis in Caco-2 cells, whereas the ER stress inhibitor we investigated the potential crosstalk between these 4PBA and the autophagy inducer rapamycin had reversed in the context of inflammation using an in vitro system, actions (P < 0.05; Fig. 4e-h). the Caco-2 cell line. CCK-8 and qRT-PCR assays were To clarify the regulation of ER stress on autophagy conducted to assess the effects of TNF-α on the cell activity under inflammation conditions, autophagy- viability and inflammatory responses in Caco-2 cells, related proteins and autophagic flux in the TNF-α- respectively. TNF-α stimulation at up to 50 ng/mL devel- treated Caco-2 cells were determined after the combined oped no obvious differences in cell viability (P > 0.05) treatment with tunicamycin or 4PBA. TNF-α treatment but a concentration-dependent upregulation (5–50 ng/mL) notably increased the ratio of LC3 II/I and the protein of the mRNA abundance of TNF-α, IL-1β, and IL-6 expression of Beclin1 and p62 but reduced the protein (P < 0.05) in Caco-2 cells (Fig. 4a-d). Thus, an interme - expression of Rab7 and LAMP2 (P < 0.05; Fig. 4i). All diate dosage of TNF-α (10 ng/mL) was chosen to induce of these changes were further aggravated by the com- inflammation in Caco-2 cells. bined treatment with tunicamycin (P < 0.05). Although To assess the effects of ER stress and autophagy on the ratio of LC3 II/I and Beclin1 protein in the TNF-α- inflammatory responses and cell apoptosis, Caco-2 cells exposed cells were upregulated by 4PBA, the protein lev- were co-incubated with TNF-α and the inducers or els of p62, Rab7, and LAMP2 in the TNF-α-exposed cells inhibitors of ER stress and autophagy. Treatment with the were recovered to the control levels by 4PBA (P < 0.05). Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 8 of 16 Fig. 3 Pterostilbene inhibits ER stress and restores autophagic flux in the IUGR colon. a qRT-PCR analysis was performed to determine the mRNA abundance of GRP78, GRP94, CHOP, ATF4, and sXBP-1 in the colon; b, c The protein levels of ER stress markers, UPR sensors, and autophagy-associated molecules were determined by Western blot analysis. Data from at least three independent experiments were presented as mean ± SE (n = 6). P < 0.05 The analysis of autophagic flux of Caco-2 cells illustrated Caco-2 cells, suggesting a blockage of autophagosome basal autophagy in the control cells, which displayed clearance. Treatment with tunicamycin further increased only weak GFP and mCherry signals (Fig. 4j). TNF-α the number of autophagosomes but did not alter the exposure induced the accumulation of autophagosomes number of autolysosomes in the TNF-α-exposed cells. (yellow puncta merged by mCherry and GFP fluores - In contrast, 4PBA treatment of the TNF-α-treated cells cence) and few autolysosomes (mCherry puncta) in increased the number of autolysosomes but reduced the number of autophagosomes. These results suggest that Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 9 of 16 the induction of ER stress worsens the TNF-α-induced ratio of LC3 II/I and the protein levels of Beclin1, Rab7, obstruction of autophagic flux in Caco-2 cells, whereas and LAMP2 and decreased p62 protein in the TNF-α- the inhibition of ER stress exerts opposite effects. exposed cells (P < 0.05; Fig. 5g). Moreover, pterostilbene The effects of autophagy on ER homeostasis in the con - promoted the formation autolysosomes and consump- text of inflammation were ascertained by measurement of tion of autophagosomes in the TNF-α-exposed cells the ER stress markers and the UPR sensors. The mRNA (P < 0.05; Fig. 5h). Taken together, these findings indicate levels of sXBP-1 and the protein levels of GRP78, CHOP, that pterostilbene mitigates ER stress and accelerates and c-Casp12 in Caco-2 cells were increased by TNF-α autophagic flux in the TNF-α-treated cells. treatment (P < 0.05; Fig. 4k and l). TNF-α exposure also increased the phosphorylation of PERK, IRE1a, and, eIF2α, Pterostilbene alleviates the TNF‑α‑induced colon and the protein expression of ATF6 in Caco-2 cells (P < 0.05; inflammation by inhibiting ER stress and promoting Fig. 5m). Treatment with 3MA aggravated the TNF-α- autophagic flux induced elevations in ER stress markers and UPR sensors; To investigate the roles of ER stress and autophagic flux however, rapamycin treatment mitigated these negative in pterostilbene-mediated protection on colonic inflam - effects observed following TNF-α treatment (P < 0.05). mation, Caco-2 cells co-incubated with TNF-α and pterostilbene were treated with the ER stress inducer tunicamycin or the autophagosome-lysosome bind- Pterostilbene inhibits ER stress and promotes autophagic ing inhibitor chloroquine. Treatment with pterostilbene flux in the TNF‑α‑treated Caco‑2 cells improved the TNF-α-induced intestinal barrier dys- We next assessed the effects of pterostilbene on ER stress function in Caco-2 cells, as indicated by the increases and autophagy activity in the TNF-α-treated Caco-2 cells in TER and the protein levels of occludin and ZO-1 and with 4PBA and rapamycin as positive controls. The cell the decrease in FITC-dextran flux (P < 0.05; Fig. 6a-c). viability assay was first conducted to determine the effect Pterostilbene also significantly inhibited cell apopto - of pterostilbene on cell viability in Caco-2 cells. Com- sis, the release of IL-1β and IL-6, and the translocation pared with the untreated cells, treatment with pterostil- of NF-κB p65 from the cytoplasm to the nucleus in the bene at increasing concentrations (0–50 μmol/L) showed TNF-α-treated cells (P < 0.05; Fig. 6d-h). However, both no significant cytotoxicity in Caco-2 cells (P > 0.05; tunicamycin and chloroquine counteracted the pteros- Fig. 5a). Thus, lower than 50 μmol/L pterostilbene was tilbene-mediated beneficial effects on intestinal barrier selected to determine the anti-inflammatory action of function, cell apoptosis, and inflammatory responses pterostilbene in the TNF-α-treated Caco-2 cells. As in the TNF-α-treated Caco-2 cells (P < 0.05), suggesting shown in Fig. 5b-d, pterostilbene (0–5 μmol/L) dose- that the attenuation of pterostilbene on inflammatory dependently blocked the TNF-α-induced elevation of the response and barrier dysfunction depends on the inhibi- mRNA abundance of TNF-α, IL-1β, and IL-6 in Caco-2 tion of ER stress and the promotion of autophagosome- cells (P < 0.05). Therefore, 2.5 μmol/L of pterostilbene was lysosome fusion. chosen for subsequent in vitro studies. As indicated in Fig. 5e and f, pterostilbene was as effec - Discussion tive as 4PBA at suppressing the splicing of XBP-1 mRNA ER stress and autophagy have been recognized as the and downregulating the protein levels of GRP78, CHOP, crucial mechanisms involved in inflammatory bowel dis - c-Casp12, and phosphorylated IRE1a in the TNF-α- ease [12, 17, 18]; however, a role in IUGR-induced colon exposed cells (P < 0.05). In addition, treatment with pter- inflammation has not been established. The present ostilbene had similar effects on autophagy activity to study revealed that IUGR caused the upregulation of ER those seen with rapamycin, as pterostilbene increased the stress indicators (GRP78, CHOP, and c-Casp12) and the (See figure on next page.) Fig. 4 The crosstalk between ER stress and autophagy in the TNF-α-exposed Caco-2 cells. Caco-2 cells were incubated with TNF-α (0–50 ng/mL) for 24 h. a The cell viability was measured by the CCK-8 assay (n = 6); b-d The mRNA abundance of inflammatory cytokines was measured by qRT-PCR analysis (n = 4). Caco-2 cells were co-incubated with TNF-α and tunicamycin ( TM), 4-phenylbutyric acid (4PBA), rapamycin (RAP), or 3-methyladenine (3MA). e-g The mRNA abundance of inflammatory cytokines was measured by qRT-PCR analysis (n = 4); h Representative scatter plots and quantitative analysis of apoptotic cells in experimental groups as analyzed by the flow cytometry (n = 4); i Western blot analysis was conducted to determine the protein levels of autophagy-associated molecules (n = 4); j Autophagic flux of Caco-2 cells was analyzed through transfection with AdPlus-mCherry-GFP-LC3B. The representative immunofluorescent photographs were indicated. GFP dots are green. mCherry dots are red (n = 4); k RT-PCR assay was conducted to detect the mRNA expression of sXBP-1 in Caco-2 cells (n = 4); l, m Western blot analysis was carried out to detect the protein levels of ER stress markers and UPR sensors in Caco-2 cells (n = 4). Data from at least three independent * # experiments were presented as mean ± SE. ns means no significance; P < 0.05 vs. CON group; P < 0.05 vs. TNF-α group Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 10 of 16 Fig. 4 (See legend on previous page.) Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 11 of 16 Fig. 5 Pterostilbene inhibits ER stress and promotes autophagic flux in the TNF-α-treated Caco-2 cells. a Caco-2 cells were incubated with various concentrations of pterostilbene for 24 h and the cell viability was detected by the CCK8 assay (n = 6); b-d Caco-2 cells were co-incubated with TNF-α (10 ng/mL) and pterostilbene (0–5 μmol/L) for 24 h and the mRNA abundance of inflammatory cytokines was measured by qRT-PCR analysis (n = 4). Caco-2 cells were co-incubated with TNF-α and pterostilbene, 4-phenylbutyric acid (4PBA), or rapamycin (RAP) for 24 h. e RT-PCR assay was conducted to detect the mRNA level of sXBP-1 in Caco-2 cells (n = 4); f, g Western blot analysis was carried out to detect the protein levels of ER stress markers, UPR sensors, and autophagy-associated proteins in Caco-2 cells (n = 4); h Autophagic flux of Caco-2 cells was analyzed through transfection with AdPlus-mCherry-GFP-LC3B. The representative immunofluorescent photographs were indicated. GFP dots are green. mCherry * # dots are red (n = 4). Data from at least three independent experiments were presented as mean ± SE. P < 0.05 vs. CON group; P < 0.05 vs. TNF-α group activation of UPR sensors (IRE1a, PERK, and ATF6), and crosstalk between ER stress and autophagy in the TNF-α- altered the expression of the proteins responsible for the treated Caco-2 cells indicated that the autophagy inhibi- formation and degradation of autophagosomes (LC3 II, tor 3MA intensified the TNF-α-induced ER stress, and Beclin1, and p62) in the colon. These changes implicated the ER stress inducer tunicamycin, in turn, augmented ER stress and impaired autophagy in the IUGR-induced the abnormal accumulation of impaired autophago- colon inflammation. In addition, our investigation of the somes caused by TNF-α. In particular, tunicamycin and Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 12 of 16 Fig. 6 Pterostilbene alleviates the TNF-α-induced inflammation and barrier dysfunction by inhibiting ER stress and promoting autophagic flux. Caco-2 cells co-incubated with TNF-α and pterostilbene were treated with tunicamycin ( TM) or chloroquine (CQ) for 24 h. The transepithelial electrical resistance (a) and FITC-dextran flux (b) of Caco-2 cell monolayers were determined; c Western blot analysis was conducted to determine the protein levels of intestinal tight junction complexes in Caco-2 cells; d Representative scatter plots and quantitative analysis of apoptotic cells in various treatments as analyzed by the flow cytometry; e, f ELISA assays were carried out to determine the contents of IL-1β and IL-6 in the supernatants of Caco-2 cells; g Nuclear NF-κB p65 protein levels in Caco-2 cells were detected by Western blot analysis; h Immunofluorescence staining of NF-κB p65 in Caco-2 cells. The red and blue fluorescence represent the NF-κB p65 protein and nucleus, respectively. Data from at least * # $ three independent experiments were presented as mean ± SE (n = 4). P < 0.05 vs. CON group, P < 0.05 vs. TNF-α group, P < 0.05 vs. TNF-α + PTS group 3MA exacerbated the TNF-α-induced inflammatory previously reported that pterostilbene could act as a feed responses and apoptosis in Caco-2 cells, whereas the ER additive in the broiler diet to prevent the growth per- stress inhibitor 4PBA and the autophagy activator rapa- formance descent caused by immunological stress and mycin had opposite effects. These data imply that the oxidative stress [29, 35]. Pterostilbene (300 mg/kg) also regulation of ER stress and autophagy may be the poten- afforded protection against the diquat injection-induced tial therapeutic approaches for the IUGR-induced colon body weight loss of piglets . In this research, how- inflammation. ever, the compromised growth performance, includ- Pterostilbene is a naturally occurring stilbene with vari- ing body weight, average daily gain, and average daily ous biofunctionalities and its effects on animal growth feed intake, of the IUGR piglets was not significantly performance have been investigated [29, 35, 36]. We improved by pterostilbene (300 mg/kg; data no shown). Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 13 of 16 These conflicting findings are probably associated with et al.  have demonstrated that pterostilbene prevented the physiological conditions of animals. the activation of NLR family, pyrin domain containing Although the growth performance of the IUGR pig- three inflammasome (NLRP3) by inducing autophagy in lets was not significantly altered, pterostilbene induced immortalized rat kidney proximal tubular epithelial cells. marked reductions in diarrhea rates and colon inflamma - In the current study, pterostilbene promoted the protein tion of the IUGR piglets. These benefits may result from expression of LC3 II and Beclin1 and recovered the protein the potent anti-inflammation action of pterostilbene. levels of Rab7 and LAMP2 in the TNF-α-treated Caco-2 The available evidence from in vivo and in vitro experi - cells, which not only reveals the strong regulation of pter- ments has indicated that pterostilbene prevents inflam - ostilbene in autophagy but also provides a possible mecha- matory responses by suppressing NF-κB signals [37–39]. nism by which pterostilbene alleviates the TNF-α-induced Consistent with those findings, we observed that pter - inflammatory responses in Caco-2 cells. ostilbene significantly downregulated the expression of Notably, autophagy is a dynamic biological process pro-inflammatory mediators and prevented the nuclear involving autophagosome formation, autophagosome- accumulation of NF-κB p65 in the IUGR piglet colons lysosome fusion, and final degradation. Disruption of any and the TNF-α-treated Caco-2 cells. one of these steps may invoke dysregulated autophagy In addition, the pterostilbene-mediated protection and cause damage to cells and tissues . LC3 is a pro- against ER stress may provide another explanation for tein located in the autophagosomal inner membrane and the attenuation of the IUGR-induced colon inflamma - it, along with Beclin1 protein, plays a pivotal role in the tion. IRE1, a conserved ER transmembrane protein, can formation of autophagosomes . The elevations in LC3 be activated when the ER homeostasis is perturbed. Nota- II in a stable state may result from autophagy activation bly, the kinase domain of IRE1 complexes with the IkB or downstream blockage of autophagic vacuole process- kinase via interaction with TNF-receptor activating fac- ing. P62 is an autophagy substrate, and its reduction is tor 2, which expedites the degradation of IkBα and trig- associated with the promotion of autolysosome degrada- gers NF-κB signals, resulting in inflammatory responses tion . Therefore, pterostilbene-mediated increases in . A previous study with HK-2 cells has reported that the protein levels of LC3 II and Beclin1 and decrease in resveratrol, the parent compound of pterostilbene, pre- p62 protein in the IUGR colon and the TNF-α-exposed vented the LPS- and tunicamycin-induced overproduction Caco-2 cells confirmed that pterostilbene enhanced of inflammatory factors and NF-κB activation by inacti - autophagic activity and accelerated autophagic flux in vation of IRE1 . Liu et al.  have also verified that this study. the knockdown of IRE1 had parallel therapeutic effects Emerging evidence has indicated that the protective to those seen with pterostilbene on the TNF-α-induced effects of natural phytochemicals on autophagy are related inflammatory responses in endothelial cells. In this study, to their regulation of the fusion of lysosomes with mature pterostilbene treatment decreased the phosphorylation autophagosomes, a crucial step for the degradation of of IRE1a in both the IUGR colon and the TNF-α-treated autophagic cargo [48–50]. Wang et al.  have reported Caco-2 cells, which may further inhibit the nuclear trans- that resveratrol mitigated oxidative stress-induced location of NF-κB p65 and the release of inflammatory autophagic dysfunction by promoting the expression of factors. Intriguingly, the beneficial roles of pterostilbene autophagosome-lysosome fusion-promoting protein Rab7. in inactivation of NF-κB signals and downregulation of Pterostilbene was also demonstrated to restore impaired inflammatory cytokines in the TNF-α-treated Caco-2 cells autophagic flux and prevent acetaminophen-induced were abrogated under a combined treatment with the ER liver injury . However, the protective responses were stress inducer tunicamycin. These data suggest the protec - abolished by treatment with chloroquine, the autophago- tive effects of pterostilbene on colon inflammation depend some-lysosome binding inhibitor. The data presented on its suppression of ER stress. herein substantiated the results from the previous studies Autophagy is an indispensable pro-survival process showing that pterostilbene improved the TNF-α-induced under ER stress conditions that allows the misfolded pro- impairment of autophagic flux by upregulating the protein teins and damaged cellular components to undergo lyso- expression of LAMP2 and Rab7 and the number of autolys- some-dependent self-digestion and recycling . Also, osomes in Caco-2 cells. In addition, chloroquine treatment autophagy is strictly interconnected with inflammatory abrogated the positive roles of pterostilbene in inflamma - responses, as the damaged and non-functional mitochon- tory responses and cell apoptosis in the TNF-α-treated dria and aggregated inflammasome structures mainly cells. These findings suggest that autophagosome-lyso - removed through the autophagic process are the crucial some fusion plays a pivotal role in the pterostilbene-medi- mediators for inflammasome activation [43, 44]. Wang ated protection against colonic inflammation. Chen et al. Journal of Animal Science and Biotechnology (2022) 13:125 Page 14 of 16 As the first physical and immunological protective Supplementary Information barrier, intestinal epithelial cells separate the host from The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40104- 022- 00780-6. the external environment and prevent the invasion of bacteria, viruses, and endotoxins . Defects in the Additional file 1: Table S1. Composition and nutrient levels of the basal intestinal barrier may therefore allow these undesired diet. antigens to cross the intestinal epithelium and increase Additional file 2: Table S2. Primer sequences for quantitative real-time PCR the risk of intestinal inflammation. Numerous stud- and real-time PCR analyses. ies have substantiated the potency of pterostilbene in protecting against intestinal barrier damage [22, 30, Authors’ contributions 53]. Here, pterostilbene treatment greatly alleviated HZ, TW, and YC conceived and designed the study. YC, YL, SJ, and PJ performed most experimental work. YC and HZ performed statistical analyses. YC drafted the deficiencies in goblet cell quantity and the secre- the manuscript. HZ and TW provided critical review. All authors read and tion of mucin 2 and TFF3 in the IUGR colon. It also approved the final manuscript. upregulated the protein levels of occludin and ZO-1 Funding and consequently led to a decrease in intestinal epithe- This work was supported by grants from the National Natural Science Founda- lial permeability in both the IUGR colon and the TNF- tion of China (Nos. 31902197 and 31802094) and the Natural Science Founda- α-exposed Caco-2 cells. It is worthwhile to mention tion of Jiangsu Province (No. BK20180531). that ER stress inducer tunicamycin and the autophagic Availability of data and materials flux inhibitor chloroquine largely suppressed the pter- The data used to support the findings of the present study are available from ostilbene-mediated beneficial effects on the intestinal the corresponding author upon request. epithelium permeability and intestinal tight junction protein. These findings indicate that the inhibition of Declarations ER stress and the promotion of autophagic flux are key Ethics approval and consent to participate mechanism by which pterostilbene prevents colon bar- All animal experiments were reviewed and approved by the Institutional Animal rier dysfunction. Care and Use Committee of Nanjing Agricultural University (SYXK-2017-0027). Consent for publication Conclusion Not applicable. This study highlights the roles of ER stress and impaired Competing interests autophagic flux as part of the mechanism of the IUGR- The authors declare no conflicts of interest. induced colon inflammation. Importantly, pterostilbene effectively overcomes ER stress, restores autophagic flux, Author details College of Animal Science and Technology, Nanjing Agricultural University, and further mitigates inflammatory responses and intes - Nanjing 210095, China. Institute of Animal Science, Jiangsu Academy of Agri- tinal barrier dysfunction in both the IUGR colon and the cultural Sciences, Nanjing 210014, China. TNF-α-treated Caco-2 cells. These findings may broaden Received: 8 May 2022 Accepted: 13 September 2022 the understanding of how pterostilbene protects against colon inflammation and help in development of novel nutrition strategies for IUGR animals to improve intes- tinal health. References 1. Resnik R. Intrauterine growth restriction. Obstet Gynecol. 2002;99:490–6. 2. Wu G, Bazer FW, Wallace JM, Spencer TE. Board-invited review: intrauter- Abbreviations ine growth retardation: implications for the animal sciences. J Anim Sci. AdPlus-mCherry-GFP-LC3B: Adenovirus expressing mCherry-GFP-LC3B fusion 2006;84:2316–37. protein recognizing CD46; ANOVA: Analysis of variance; ATF4: Activating tran- 3. 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Pteros- tilbene reduces colonic inflammation by suppressing dendritic cell activation and promoting regulatory T cell development. FASEB J. 2020;34:14810–9. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your ﬁeld rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Journal of Animal Science and Biotechnology – Springer Journals
Published: Nov 4, 2022
Keywords: Autophagic flux; Colon inflammation; Endoplasmic reticulum stress; Intrauterine growth retardation; Piglets
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