Targeting Autophagy to Restore Intestinal Integrity in Sepsis: Resveratrol’s NOX1/SIRT1-Mediated Protective Effect

Probiotic Bacteria Induce Maturation of Intestinal Claudin 3 Expression and Barrier Function

Time course study of intestinal epithelial barrier disruption in acute mesenteric venous thrombosis

Potential conflict of interest: Nothing to report. See Article on Page 883 An intact intestinal epithelial barrier plays a major role in preventing intestinal invasion of luminal pathogens and antigenic molecules and their subsequent migration to the liver and, potentially, the systemic immune system. The clinical relevance of intestinal barrier dysfunction has been recognized by its pathogenic association with several disease states, ranging from inflammatory bowel disease to rheumatoid arthritis to Alzheimer's disease, and, of direct relevance to this editorial, to alcoholic liver disease (ALD).1 Integrity of intestinal barrier function is regulated, in part, by the epithelial tight junction (TJ) complex composed of proteins such as zonula occludens, occludin, and claudins.3 The TJ complex establishes intestinal barrier integrity by connecting the interepithelial cell spaces and inhibiting the paracellular passage of microbes and microbial products and other luminal contents.4 However, intestinal epithelial barrier dysfunction is frequently observed in several acute and chronic enteropathic disorders, such as inflammatory bowel disease, irritable bowel syndrome, and infectious diarrhea.1 Intestinal production of proinflammatory cytokines, including interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, interleukin (IL)−1β, IL‐6, IL‐13, and the TNF superfamily member, LIGHT, have been documented to promote intestinal epithelial barrier dysfunction.5 Particularly, cytokine‐initiated inflammatory signaling plays a major role in breaching TJ integrity.9 Although the role of cytokines and inflammatory signaling in intestinal epithelial barrier dysfunction has been well documented, the underlying mechanisms are still being defined, and potential therapeutic strategies for reestablishing intestinal barrier function are still under investigation. Work by Chen et al. examined the role of TNF‐α‐induced receptor 1 (TNFR1) signaling as a critical factor in alcohol‐associated loss of intestinal barrier function and the development of liver disease.10 TNF‐α plays a major pathogenic role in many diseases associated with intestinal epithelial barrier dysfunction.11 An interesting aspect of the ALD model of intestinal dysbiosis and barrier dysfunction, in comparison to other enteropathic diseases, such as inflammatory bowel disease, intestinal ischemia, and graft versus host disease, is that the damage to the epithelium is much less extensive, thereby allowing investigation of the pathogenic interaction between proinflammatory signaling and TJ integrity. The data presented in the context of the ALD model showed that chronic alcohol feeding induces intestinal inflammation in the jejunum, as indicated by an increase in TNF‐α production by monocytes and macrophages and an increase in the intestinal permeability. The clinical relevance of these findings was supported by similar findings in duodenal biopsies from patients with chronic alcohol abuse. This is especially important because there are multiple experimental models of ALD (usually early stages of ALD), and it is valuable to connect animal findings to human disease. Chen et al.10 were able to specifically address and establish the causal role of TNF/TNFR1 interaction and signaling in the development of alcohol‐induced barrier dysfunction and intestinal permeability in this model of ALD. Specifically, the investigators were able to demonstrate that TNFR1 mutant mice are protected from alcohol‐induced intestinal barrier dysfunction and liver disease. Importantly, selective reinstatement of TNFR1 expression on intestinal epithelial cells in a TNFR1 mutant mouse carrying a conditional gain‐of‐function allele for this receptor caused a resumption of intestinal pathology and liver disease similar to wild‐type mice. These data certainly support the notion that intestinal TNF/TNFR1 signaling plays an essential pathogenic role in alcohol‐induced intestinal barrier dysfunction and subsequent development of liver disease (Fig. 1). These data also suggest that hepatic TNFR1 is not required for development of ALD in this experimental model system.Figure 1: TNFR1 signaling in the intestine is necessary for subsequent development of alcohol‐induced liver injury. This demonstrates the critical link between alcohol‐induced intestinal inflammation and the subsequent development of alcohol‐induced liver injury (gut:liver axis).In comparison to the research presented by Chen et al., work done by Wang et al.6 investigating intestinal epithelial barrier dysfunction using an IFN‐γ‐primed human intestinal epithelial cell line, Caco‐2 cells, showed that TNFR2, and not TNFR1, is required for TNF‐dependent barrier dysfunction. Findings from these two studies indicate that the type of TNFR relevant for mediating TNF‐dependent TJ disruption could be contextual and dictated by species and/or disease‐type specificity. Importantly, these studies show the need for care in drawing mechanistic conclusions with regard to the development of therapeutic approaches to target inflammatory signaling for reestablishing intestinal epithelial barrier function. Significantly, TNF‐mediated intestinal epithelial TNFR1 signaling in the mouse model of alcohol feeding as well as TNFR2 signaling in a human intestinal epithelial cell line both lead to myosin light chain (MLC) phosphorylation by activation of MLC kinase (MLCK) that plays a major role in the proinflammatory cytokine‐induced intestinal barrier disruption.9 These data indicate that, regardless of which type of receptor is activated, TNF‐dependent disruption of TJ and consequent loss of intestinal barrier function involve MLCK phosphorylation. Work done by Chen et al.10raises some important questions regarding the mechanisms underlying the pathogenesis of ALD. Particularly, an intriguing aspect of the findings is that in TNFR1 knockout animals, restoration of TNFR1 expression only in the intestinal epithelial cells was sufficient to cause liver pathology, even in the absence of TNFR1‐induced inflammatory and cytotoxic signaling in the liver. These data suggest that hepatic TNFR1 activation is not required for the initiation and perpetuation of ALD in this animal model system. Overall, the findings are significant in that they provide an improved understanding of intestinal proinflammatory signaling and malfunction of TJ integrity in the context of chronic alcohol consumption that leads to loss of intestinal epithelial barrier function and development of ALD. These findings raise other important questions, including those related to the best approach to prevent/treat ALD. A quarter of a century ago, we reported increased peripheral blood monocyte production of TNF in human alcoholic hepatitis.12 Chen et al. showed that intestinal monocyte TNF production is increased in experimental ALD and that TNF is increased in intestinal biopsies from human alcoholics. Is it only the intestinal TNF that is important, or is the TNF produced by the liver and systemic cells, which recycles back to activate intestinal TNFR1, also important? Chen et al. also showed that intestinal TNFR1 (but not hepatic TNFR1) was critical for the development of experimental ALD. Multiple approaches, such as antibiotics, probiotics, prebiotics, and several dietary factors, have been shown to stabilize gut‐barrier function, decrease inflammation, and protect against experimental ALD. Perhaps we should be placing greater emphasis on intestinal inflammation and intestinal barrier disruption as a therapeutic target in human ALD.

Ulcerative colitis (UC) is closely associated with disruption of intestinal epithelial tight junction proteins. A variety of studies have confirmed that resveratrol (RSV), a natural polyphenolic compound, has a potential anti-inflammatory effect and can regulate the expression of tight junction proteins. However, the mechanism by which RSV regulates the expression of tight junction proteins in the intestinal epithelium remains unclear. Therefore, we investigated the potential effect of RSV on tight junction proteins in an HT-29 cell model of inflammation induced by lipopolysaccharide (LPS) and explored its mechanism of action. First, the downregulated expression of the tight junction proteins occludin, ZO-1, and claudin-1 in the HT-29 cell model of inflammation induced by LPS was reversed by incubation with RSV, accompanied by a decrease in the expression of tumor necrosis factor α-converting enzyme (TACE). Additionally, the Notch1 pathway was attenuated and the expression of the inflammatory factors IL-6 and TNF-α was decreased by treatment with RSV. Second, after Jagged-1 was used in combination with RSV to reactivate the Notch1 pathway, the protective effects of RSV against the LPS-induced reductions in the expression of the tight junction proteins occludin, ZO-1, and claudin-1 and the decreases in the levels of the inflammatory factors IL-6 and TNF-α were abolished. These results suggest that RSV might regulate the expression of tight junction proteins by attenuating the Notch1 pathway.

Objective To investigate the effect of resveratrol(Res)on the protein expression of reduced form of nicotinamide-adenine dinucleotide phosphate oxidase 4 (NOX4) and glucose-regulated protein 78 (GRP78) and its protective effects on the oxidative stress injuries in the renal cortex of diabetic rats. Methods Fifteen SD rats were given a single intraperitoneal injection of streptozotocin(STZ) to set up animal models of diabetes. Twelve weeks after STZ injection, 12 rat diabetes models were established successfully and were randomized into diabetes mellitus group (group DM, n=6) and Res intervention group (group DR, n=6). Another 6 untreated healthy SD rats served as normal control (group NC). Rats in DR group were administered intragastricly with Res 10 mg·kg-1·d-1 while those in DM group with equal volume of 0.5% sodium carboxymethylcellulose regularly for 2 weeks. At the end of the experiments, the blood glucose (BG), body weight (BW), serum creatinine (Scr), blood urea nitrogen (BUN), 24 urinary albumin excretion(UAE) of all rats were measured. Renal cortex from the groups were embedded in paraffin and sectioned for morphological studies. The activity of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the renal cortex was assayed by chromatometry. The expression of NOX4 and GRP78 proteins in the renal cortex were detected by using Western blotting. Results Compared with those in group NC, the BW in group DM decreased, while BG, Scr, BUN and 24h UAE increased significantly(t=-52.324, -20.487, -20.724, -55.476, all P 0.0167). Moreover, the MDA activity in DM group increased while the SOD and CAT activity decreased significantly as compared with those in group NC(t=-10.661, 8.124, 8.222, all P 0.0167). The glomerular basement membrane was intact in NC group, more glomerular mesangial matrix and cell proliferation was observed in group DM and which were attenuated in the group DR. The expression of NOX4 and GRP78 in the blood vessels of group DM were up-regulated than those in group NC (t=-14.255, -25.179, P<0.0167). After the administration of resveratrol, the expression of NOX4 was still up-regulated while GRP48 was down-regulated in group DR as compared with those in group NC (t=-5.125, -28.017, P<0.0167). Conclusions Oxidative stress is present in the renal cortex of diabetic rats. It suggests that resveratrol protect the renal cortex of diabetic rats by suppressing the expression of NOX4 and GRP78 to attenuate endoplasmic reticulum stress and subsequent oxidative stress injuries during the early stage of diabetes. Key words: Diabetic nephropathies; Resveratrol; Oxidative stress; Endoplasmic reticulum stress; Rats

Objectives Intestinal epithelial barrier function is an important mechanical barrier to maintain intestinal homeostasis and resist the invasion of intestinal pathogens and microorganisms. However, intestinal epithelial barrier function is vulnerable to damage under intestinal ischemia–reperfusion (I/R) injury. Under a category of pathophysiological conditions, including I/R, autophagy plays a crucial role. This study is aimed at discussing the role of autophagy inhibitors and activators in intestinal epithelial barrier function after intestinal I/R by changing autophagy levels. Methods Mice with intestinal IR underwent 45 minutes of surgery for superior mesenteric artery occlusion. The autophagy inhibitor 3-MA and the autophagy inducer rapamycin (RAP) were used to change the level of autophagy, and then, the expressions of tight junction proteins and intestinal barrier function were detected. Results The results showed that the autophagy inhibitor 3-MA aggravated intestinal epithelial barrier dysfunction, while the autophagy inducer RAP attenuated intestinal epithelial barrier dysfunction. In addition, promoting autophagy may promote occludin expression by inhibiting claudin-2 expression. Conclusion Upregulation of autophagy levels by autophagy inducers can enhance intestinal epithelial barrier function after intestinal I/R.

In recent years, the function of plant polyphenols to improve the intestinal barrier has been fully demonstrated. However, the exact mechanisms linking plant polyphenols with the intestinal barrier function have not yet been established. Apple polyphenols (APs) are safe and healthy nutrients, which are extracted from apples and their byproducts. Using pig and IPEC-J2 cell models, this study investigated the effects of dietary AP supplementation on intestinal antioxidant capacity and barrier function. Then, we further explored the role of the Nrf2/Keap1 signaling pathway in maintaining intestinal antioxidant capacity and barrier function. Our study found that dietary AP supplementation improved the intestinal mechanical barrier by promoting the intestinal morphology and intestinal tight junction protein expression, improved the intestinal immune barrier by increasing intestinal secretory immunoglobulin A production, and improved the intestinal biological barrier by increasing probiotics and decreasing the Escherichia coli population. Further research found that dietary AP supplementation increased the intestinal antioxidant capacity and activated the Nrf2/Keap1 signaling pathway. Finally, after treatment with Nrf2-specific inhibitor ML-385, the upregulation effect of APs on antioxidant capacity and tight junction protein expression was reduced in IPEC-J2 cells. Our results suggested that APs promoted intestinal antioxidant capacity and barrier function via the Nrf2/Keap1 signaling pathway.

Impaired intestinal epithelial barrier function is closely associated with the pathogenesis of ulcerative colitis (UC). Atractylenolide-I (AT-I), a major sesquiterpene derived from the herb Atractylodes macrocephala Koidz., has been reported to alleviate DSS-induced colitis in mice. This study aims to investigated the protective effects of AT-1 on intestinal epithelial barrier function and elucidate it's underlying mechanisms. In vivo, an acute colitis model was established in mice, and transcriptomic analysis to identify differentially expressed genes. In vitro, overexpression plasmids and recombinant protein were used to evaluate their effects on intestinal barrier function, and further analysis of its potential mechanisms.The study found that AT-1 ameliorate DSS-induced acute ulcerative colitis, exhibiting protective effects on the intestinal barrier. Transcriptomic analysis revealed that AT-1 significantly modulated the expression of S100A8 and S100A9. Further investigations indicated that S100A9, rather than S100A8, mediated the expression of tight junction proteins, meanwhile, AT-1 reduces neutrophil activation and subsequent release of S100A9. Mechanistically, recombinant human S100A9 protein was found to induce a decrease in intracellular Ca2+ concentration, while AT-1 regulated the expression of tight junction proteins via modulation of the AMPK/mTOR signaling pathway. AT-1 enhances the recovery of DSS-induced intestinal barrier dysfunction by regulating the recombinant human S100A9 protein-mediated AMPK/mTOR signaling pathway. This study provides new insights into the pathogenesis of ulcerative colitis and suggests potential therapeutic strategies for its treatment.

To observe the effect of SIRT1 on intestinal barrier function of epithelial Caco-2 cells under hypoxia and investigate its mechanism. Caco-2 cells were randomly divided into three groups: normoxia group (Nx), hypoxia group (Hx,1%O2 for 6 h) and hypoxia plus 40 μmol/L Resveratrol (agonist of SIRT1) group (Hx+Res). Transepithelial electrical resistance (TER) was determined. mRNA and protein expressions of SIRT1 and tight junctions (ZO-1, Occludin, Claudin-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Both mRNA and protein expressions of SIRT1 were significantly reduced in Hx group as compared with Nx group (0.40±0.02 vs. 0.70±0.07, P=0.001; 0.37±0.03 vs. 0.76±0.03, P=0.001). The mRNA and protein expressions of SIRT1 were significantly increased in Hx+Res group as compared with Hx group(0.50±0.02 vs. 0.40±0.02, P=0.026; 0.54±0.02 vs. 0.37±0.03, P=0.011). The expression levels of ZO-1, Occludin and Claudin-1 in Hx group were lower than those in Nx group (P<0.05), however, pretreatment with Resveratrol could attenuate the decreased expression of above 3 molecules under hypoxia(P<0.05). TERs of Nx group, Hx group and Hx+Res group were (142±7) Ohm/cm(2), (94±3) Ohm/cm(2) and (119±7) Ohm/cm(2) respectively. Compare with the Nx group, the TER of Hx group was significantly decreased(P<0.05). TER of Hx+Res group was significantly increased compare with Hx group, but it was still significantly lower than that in Nx group(P<0.05). Expression of SIRT1 is significantly reduced under hypoxia. Activation of SIRT1 can maintain the epithelial barrier function through regulating the expression of tight junctions under hypoxia.

Clinical application of triptolide (TP), a main active ingredient of the traditional Chinese herb Tripterygium wilfordii Hook f. (TWHF), is limited by a series of severe toxicities, including cardiotoxicity. In previous studies, we found the activation of sirtuin 3 (SIRT3) attenuated TP-induced toxicity in cardiomyocytes. Resveratrol (RSV), a polyphenol from the skins of grapes and red wine, is an activator of SIRT3. The current study aimed to investigate the protective effect of RSV against TP-induced cardiotoxicity and the underlying mechanisms. Mice were treated with a single dose of TP (2.5 mg/kg) via the intragastric (i.g.) route. After 24 h, TP induced abnormal changes of serum biochemistry, activity decrease of antioxidant enzymes and damage of heart tissue such as myocardial fiber rupture, cell swelling and interstitial congestion. In contrast, administration with RSV (50 mg/kg i.g. 12 h before and 2 h after the administration of TP) attenuated the detrimental effects induced by TP in BALB/c mice. Moreover, the cardiomyocyte protective effects of RSV on TP-induced heart injury were associated with the activation of SIRT3 and its downstream targets. In vitro study also indicated that RSV counteracted TP-induced cardiotoxicity through SIRT3-FOXO3 signaling pathway in H9c2 cells. Collectively, these findings suggest the potential of RSV as a promising agent in protecting heart from TP-induced damage.

Reply to: “Uncovering the molecular events associated with increased intestinal permeability in liver cirrhosis: The pivotal role of enterocyte tight junctions and future perspectives”

Effects of POPs-induced SIRT6 alteration on intestinal mucosal barrier function: A comprehensive review.

Acute lung injury (ALI) is a state of inflammation that breaks down the lung endothelial and epithelial cell barriers. In the current study, we investigated the role of resveratrol (RES) in regulating the expression and functions of tight junction proteins (TJP) in epithelial cell responses following exposure to this superantigen. To this end, C3H mice were given resveratrol orally twice prior to intranasal challenge with lethal SEB doses. 16S rRNA results showed that there were microbes transported in the blood in addition to the lung and colonic tissues. For this purpose, we used a reporter E. coli-GFP labeled bacterium to monitor and examine the viability of this bacterium in case of leak from lung into blood and other body compartments during the peak of ALI. The flow cytometry results showed significant reduction in copy numbers of this reporter in the blood in RES-treated mice in comparison to vehicle-treated mice. However, bacteriological examination for viability by culture of the blood showed all were negative for all experimental groups. Mouse transcriptome array showed increase in TJP gene expression in colonic and alveolar epithelial cells of RES-treated mice. Some of these genes were validated with qPCR. We found that there was an increase in the expression of some TJP genes following RES treatment. Commensals and probiotics are also known to decrease intestinal barrier dysfunction caused by inflammatory cytokines. Therefore, mass spectrometry (MS) analysis showed increase in the metabolome in colons of RES-treated group that help in strengthening of intestinal barrier. In conclusion, RES treatment increased directly or indirectly the rigidity of cell boundaries resulting in amelioration of ALI-dependent complications.

Ulcerative colitis (UC) has been identified as one of the inflammatory diseases. Intestinal mucosal barrier function and microflora play major roles in UC. Modified-chitosan products have been consumed as effective and safe drugs to treat UC. The present work aimed to investigate the effect of chitosan (CS) on intestinal microflora and intestinal barrier function in dextran sulfate sodium (DSS)-induced UC mice and to explore the underlying mechanisms. KM (Kunming) mice received water/CS (250, 150 mg/kg) for 5 days, and then received 3% DSS for 5 days to induce UC. Subsequently, CS (250, 150 mg/kg) was administered daily for 5 days. Clinical signs, body weight, colon length, and histological changes were recorded. Alterations of intestinal microflora were analyzed by PCR-DGGE, expressions of TNF-α and tight junction proteins were detected by Western blotting. CS showed a significant effect against UC by the increased body weight and colon length, decreased DAI (disease activity index) and histological injury scores, and alleviated histopathological changes. CS reduced the expression of TNF-α, promoted the expressions of tight junction proteins such as claudin-1, occludin, and ZO-1 to maintain the intestinal mucosal barrier function for attenuating UC in mice. Furthermore, Parabacteroides, Blautia, Lactobacillus, and Prevotella were dominant organisms in the intestinal tract. Blautia and Lactobacillus decreased with DSS treatment, but increased obviously with CS treatment. This is the first time that the effect of original CS against UC in mice has been reported and it is through promoting dominant intestinal microflora such as Blautia, mitigating intestinal microflora dysbiosis, and regulating the expressions of TNF-α, claudin-1, occludin, and ZO-1. CS can be developed as an effective food and health care product for the prevention and treatment of UC.

This study aimed to investigate the effects of conjugated linoleic acid (CLA) on intestinal epithelial barrier function and explore the underlying mechanisms. IPEC-J2 cells and mice were treated with different CLA isomers. The intestinal epithelial barrier function determined by transepithelial electrical resistance (TEER), the expression of tight junction proteins, and the involvement of G-protein coupled receptor 120 (GPR120), intracellular calcium ([Ca2+]i) and myosin light chain kinase (MLCK) were assessed. In vitro, c9, t11-CLA, but not t10, c12-CLA isomer, impaired epithelial barrier function in IPEC-J2 by downregulating the expression of tight junction proteins. Meanwhile, c9, t11-CLA isomer enhanced GPR120 expression, while knockdown of GPR120 eliminated the impaired epithelial barrier function induced by c9, t11-CLA isomer. In addition, c9, t11-CLA isomer increased [Ca2+]i and activated the MLCK signaling pathway in a GPR120-dependent manner. However, chelation of [Ca2+]i reversed c9, t11-CLA isomer-induced MLCK activation and the epithelial barrier function impairment of IPEC-J2. Furthermore, inhibition of MLCK totally abolished the impairment of epithelial barrier function induced by c9, t11-CLA. In vivo, dietary supplementation of c9, t11-CLA rather than t10, c12-CLA isomer decreased the expression of intestinal tight junction proteins and GPR120, increased intestinal permeability, and activated the MLCK signaling pathway in mice. Taken together, our findings showed that c9, t11-CLA, but not t10, c12-CLA isomer, impaired intestinal epithelial barrier function in IPEC-J2 cells and mice through activation of GPR120-[Ca2+]i and the MLCK signaling pathway. These data provided new insight into the regulation of the intestinal epithelial barrier by different CLA isomers and more references for CLA application in humans and animals.