The Effect Of Berberine On Intestinal Epithelium Tight Junction

Berberine (BBR), which is found in the root, rhizome and stem bark of Coptidis rhizoma (Huanglian in Chinese), has been used in traditional Eastern medicine for a long time in the treatment of gastroenteritis and diarrhea. Recent developments in the therapy of diabetes and hyperlipidemia have brought berberine into the focus of interest, but the application of BBR on gastrointestinal diseases is still limited. The latest development showed that BBR is a potential phytochemical with multispectrum therapeutic activities in gastrointestinal diseases, and more pharmacological effect should be exploited.Intestinal mucosal barrier is composed of mechanical barrier, biological barrier, chemical barriers and immune barrier. The structural basis of mechanical barrier is intact junctions between intestinal epithelial cells. These junctions include tight junction, adhesion junction and gap junction, and the major constituent is tight junction. The disruption of tight junction lead to increased intestinal permeability, which contribute the initiation and development of many diseases, such as Crohn's disease and ulcerative colitis. Therefore, restoring the integrity of the intestinal tight junction is an important goal in maintaining intestinal barrier function and prevention of bacterial translocation.According to the antibacterial, anti-inflammatory, inhibits secretion effect of BBR, we make the hypothesis that BBR may ameliorate intestinal epithelial tight junction damage. In this article, we used many distinct models of disrupted tight junction in vitro and in vivo, observed function and structure of tight junction, and the distribution of tight junction proteins. We concluded that BBR can attenuate the breakage of tight junction, and the protection effect is mediated by the inhibition of MLCK and iNOS. These findings strongly suggest that BBR treatment should be viewed as a potential novel therapeutics for intestinal diseases.Objectives: Maintenance of the mucosal barrier is a critical function of intestinal epithelial tight junction. Berberine is one of the main constituents of Coptidis rhizoma, which has been used for patients with gastrointestinal disorders for a long time. This study aimed to determine whether berberine could alleviate pro-in?ammatory cytokine-induced intestinal epithelial tight junction damage.Methods: First, Caco-2 monolayers were treated with various concentration of berberine (25, 50, 100, 200μM); we observed the morphology of cells, the integrity of TJs by measuring TEER. We have also taken LDH assay to detect the cellular toxicity in response to different concentration of BBR. After determinated the effective dose of BBR, cells were divided into four groups: control, BBR, TNF-α+IFN-γ, TNF-α+IFN-γ+BBR. Caco-2 monolayers were treated with TNF-αand IFN-γto induce barrier dysfunction, TNF-αand IFN-γincubation was carried out with 100U/ml IFN-γfor 19 h and then 10ng/ml TNF-αwas added. In the experiment group, the cells were treated with 100umol/L BBR for 72h. Control monolayer cells were incubated with DMSO in cell culture medium, and the final concentration of DMSO did not exceed 1%. The protective effect of BBR on TJs was analyzed by measuring TEER. Transmission electron microscopy was used to observe the morphology of tight junction, and subcellular localization of TJ proteins was investigated by immunofluorescence microscopy and Western blot.Results: BBR could significantly increase the TEER and reduce the permeability of macromolecules, and this effect showed a dose depend manner until 100μM, but the effect was weaken when 200μM. Our results demonstrate that BBR showed low cytotoxicity at 200μM concentration, so we chose 100μM as the optimal concentration. Our results show that BBR can significantly prevent pro-inflammatory cytokines induced decrease in TEER. The distortion of tight junction morphology and redistribution of TJ protein occludin were also prevented by berberine treatment. Western blot analysis demonstrated that berberine inhibit the dislocation of occludin from raft fractions to non-raft fractions in membrane microdomains of tight junctions.Conclusions: These findings showed that BBR could reduce epithelial gut permeability, and might help explain the possible mechanisms of anti-diarrhea activity of berberine. We also conclud that BBR can ameliorate pro-inflammatory cytokines induced intestinal epithelia TJ damage in vitro, and BBR may be one of the targeted therapeutic agents that can restore barrier function in intestinal disease states.Objectives: The aim of this study was to establish a mouse model of endotoxin sepsis, and examine the protective effect of pre-treatment and subsequent treatment of BBR in endotoxin-induced intestinal TJ injury.Methods: Twenty male C57BL/6 mice, 6- to 8- weeks old, were randomly divided into into five groups: control, BBR treated alone, LPS injected alone, pretreatement of BBR and administration of BBR after LPS injection. BBR+LPS group mice received 200mg/kg of BBR administered intragastrically once a day for 7days, each dose was dissolved normal saline and diluted to the final concentration of 10 mg/ml. Control, LPS and LPS+BBR group were administered intragastrically with equal volume of normal saline. LPS (10mg/kg) dissolved in normal saline or equal volum of normal saline was injected intraperitoneally 1h after intragastrical treatment on day 7. LPS+BBR group mice received 200mg/kg of BBR administered intragastrically 30 min after injection of LPS. The mice were killed 12h after intraperitoneal injection of LPS or saline. 12h after LPS treatment, clinical condition was assessed as a symptom score, and intestinal permeability of FITC-Dextran was measured by fluorescence spectrophotometer. Then the samples were collected. Ileum and colon adjoining ileocecal junction were obtainedn for histology, confocal microscopy, transmission electron microscope examination. The mucous of samll intestine was scraped and isolated by sucrose density ultracentrifugation. The protein contents were examined by Western blot. Data were analysed via commercial software package SPSS 18.0. All data are presented as Mean±SD and data were analyzed by One-Way ANOVA (LSD) and Dunnett t test. The significance level was accepted at P < 0.05.Results: Ileal mucosal permeability to ?uorescein isothiocyanate dextran assay declared that BBR reduced the increased permeability of TJs in endotoxinemia. Transmission electron microscope showed that pretreatement of BBR partly prevented ultrastructure disruption of TJs by LPS. Immunofluorescence and Western blot were performed; the result showed that pretreatement of BBR partly reversed the redistribution of TJ proteins in colon epithelium and redistribution of TJ proteins in membrane microdomains. Our data also indicated that pretreatement of BBR could suppress cytoplasmic to nuclear translocation of NF-κB and MLCK activation in intestinal epithelium. But the effect of subsequent treatment of BBR is unobvious.Conclusions: Pretreatement of BBR attenuates intestinal epithelium TJ disruption in a mice model of endotoxinemia, this may possibly mediated through down-regulation the NF-κB and MLCK pathway. dissolved normal saline and diluted to the final concentration of 10 mg/ml. Control, LPS and LPS+BBR group were administered intragastrically with equal volume of normal saline. LPS (10mg/kg) dissolved in normal saline or equal volum of normal saline was injected intraperitoneally 1h after intragastrical treatment on day 7. LPS+BBR group mice received 200mg/kg of BBR administered intragastrically 30 min after injection of LPS. The mice were killed 12h after intraperitoneal injection of LPS or saline. 12h after LPS treatment, clinical condition was assessed as a symptom score, and intestinal permeability of FITC-Dextran was measured by fluorescence spectrophotometer. Then the samples were collected. Ileum and colon adjoining ileocecal junction were obtainedn for histology, confocal microscopy, transmission electron microscope examination. The mucous of samll intestine was scraped and isolated by sucrose density ultracentrifugation. The protein contents were examined by Western blot. Data were analysed via commercial software package SPSS 18.0. All data are presented as Mean±SD and data were analyzed by One-Way ANOVA (LSD) and Dunnett t test. The significance level was accepted at P < 0.05.Results: Ileal mucosal permeability to ?uorescein isothiocyanate dextran assay declared that BBR reduced the increased permeability of TJs in endotoxinemia. Transmission electron microscope showed that pretreatement of BBR partly prevented ultrastructure disruption of TJs by LPS. Immunofluorescence and Western blot were performed; the result showed that pretreatement of BBR partly reversed the redistribution of TJ proteins in colon epithelium and redistribution of TJ proteins in membrane microdomains. Our data also indicated that pretreatement of BBR could suppress cytoplasmic to nuclear translocation of NF-κB and MLCK activation in intestinal epithelium. But the effect of subsequent treatment of BBR is unobvious.Conclusions: Pretreatement of BBR attenuates intestinal epithelium TJ disruption in a mice model of endotoxinemia, this may possibly mediated through down-regulation the NF-κB and MLCK pathway. redistribution of TJ proteins both in surface of intestinal epithelium and in lipid raft fractions.Conclusions: Our results suggest that I/R induced intestinal TJ dysfunction can be improved by BBR, and the protective effect of BBR may associated with the suppression of intestinal mucosa iNOS activity. These findings suggested that BBR may have therapeutic potential in intestinal I/R.