The Mechanism Of The Damage Of Intestinal Mucosal Barrier In Rats With Acute Intrahepatic Cholestasis And The Effects Of Probiotics On It

PartⅠInvestigation of the mechanism of the damage of intestinal mucosal barrier in rats with acute intrahepatic cholestasisObjective:To investigate the changes of intestinal mucosal barrier in the rat models with acute intrahepatic cholestasis induced by alpha-haphtylisocyocyanate(ANIT), and explore its mechanisms.Methods:A single dose (50 mg/kg) of a-naphthylisothiocyanate (ANIT) was administered by gavage to each experimental rat to induce intrahepatic cholestasis. At 24h,48h and 72h after gavage, the levels of the serum total bilirubin (TB) and alanine aminotransferase(ALT) were detected and immunohistochemistry and western blot techniques were used to examine the distribution and expression of tight junction proteins-Zonula occludens-1 (ZO-1),Occludin and two cell factors-nuclear transcription factor-kappa B (NF-κB),tumor necrosis factor (TNF-a). And the results of Western blot were quantitative analysised by image analytical system.Results:1. After the administration of ANIT, the levels of serum ALT and TB in the model group increased gradually, reached the pinnache at 48h.The levels of serum ALT and TB in the model group at each time point were obviously higher than those in control group (P<0.01)2. Under the light microscope, the structure of the intestinal mucosa is obviously broken in the model group compared with the control group; By electron microscopy, the bowel mucosal microvilli structure and cell-cell junction is significantly broken in the model group than those in control group.3. ZO-1 and Occludin were localized along the apical region of the lateral plasma membrane representing the region of tight junctions in surface and crypt epithelial cells. The positive stainings of ZO-1 and Occludin of model group at 24h after gavage, were decreased than those of control group, the decrease was most obviosly at 48h, and partly recovered at 72h. Western blot demonstrated consistent and significant reduction with immunohistochemistry. The protein expressions in model group were significantly lower than those in control group (P<0.01)4. The expressions of NF-κB and TNF-a in model group were inverse correlation with the expressions of ZO-1 and Occludin. The expressions of NF-κB and TNF-a in model group were markedly higher than those in control group.Conclusion:When Acute intrahepatic cholestasis induced by ANIT happened, NF-κB was activated and made the levels of TNF-a increased. TNF-a could release other mediators of inflammation and reactivated NF-κB. These could induce inflammatory reaction and the abnormal distribution of tight junction proteins and the alteration of their quantity, which affects the intestinal barrier integrity, resulting in impaired barrier function.PartⅡEffects of probiotics on the intestinal mucosal barrier in rats with experimental acute intrahepatic cholestasis Objective:To study the effects of live combination bifidobacterium, lactobacillus and enterococcus(BIFICO) on the expressions of intestinal mucosal epithelium tight junction protein-ZO-1,Occludin and cytokine-NF-κB,TNF-a in rats with cholestasis and further discuss the protection of the probiotics to intestinal mucosal barrier, so as to provide the academic basis for its clinical uses.Methods:Male SD rats of 3 weeks old were randomly divided into control group, model group and BIFICO group. Before making the animal model, BIFICO(4.2×108 viable bacterium counts/kg/d) were adminsterated to the rats in BIFICO group for 4 days. Model and control groups were fed by normal saline. All groups did not stop being administrated treating agent daily until executed. At the 5th day after administration, after fasting for 12h, model group and BIFICO group were intragastrically administrated ANIT(50mg/kg) for modeling. At 24h,48h and 72h after modeling, every 10 rats in each group were executed for taking abdominal aortal blood and terminal ileum tissue. The levels of serum TB and ALT were detected and immunohistochemistry and western blot techniques were used to examine the distribution and expression of tight junction proteins-ZO-1,Occludin and cell factors-NF-κB,TNF-a. And the results of Western blot were quantitative analysised by image analytical system.Results:1. The levels of serum ALT and TB in the BIFICO group at each time point were obviously lower than those in model group (P<0.05)2. Under the light microscope, the structure of the intestinal mucosa is obviously broken in the model group compared with the control group, but the damage in BIFICO group is obviously reduced; By electron microscopy, the bowel mucosal microvilli structure and cell-cell junction is significantly broken in the model group than those in control group, but the damage in BIFICO group is obviously reduced.3. The positive stainings of ZO-1 and Occludin of model group at 24h after modeling, were decreased than those of control group, the decrease was most obviosly at 48h, and partly recovered at 72h. Western blot demonstrated consistent and significant reduction with immunohistochemistry. The protein expressions in BIFICO group have significant recovery compared to those in model group at each time point (P<0.05)4. The protein expressions of NF-κB and TNF-a in BIFICO group were notably lower than those in model group at each time point (P<0.05)Conclusion:The results suggested that probiotics supplement can improve the expressions of ZO-1 and Occludin and at the same time decrease the expressions of NF-κB and TNF-a in rats with acute intrahepatic cholestasis, thereby it can cut down the probability of intestinal inflammatory reaction and conduce to improve the intestinal barrier function.