Study On The Intervention Of Fufang Zhenzhu Tiaozhi Prescription And Its Effective Constituent Berberine On Intestinal Barrier In Mice With Liver Fibrosis

Objective:Liver fibrosis is a progressive wound-healing response resulting from a wide variety of pathogenesis,which is characterized by an excessive accumulation of extracellular matrix?ECM?.Current studies have shown that liver fibrosis is closely related to the function of intestinal barrier.Damage to the intestinal mucosal barrier leads to intestinal inflammatory substances entering the liver via the portal vein,causing liver inflammation and fibrosis.This paper aims to study the intervention effect of Fufang Zhenzhutiaozhi prescription?FTZ?and its effective constituent berberine on intestinal barrier in mice with liver fibrosis and explore its mechanism.Methods:Male C57BL/6 mice were randomly divided into six groups:control group,model group,low-dose FTZ treatment group?1.2g/kg?,high-dose FTZ treatment group?2.4g/kg?,low-dose berberine treatment group?100 mg/kg?and high-dose berberine treatment group?200 mg/kg?.CCl₄was injected intraperitoneally for 6 weeks.At the same time,the treatment groups were treated with FTZ or berberine by gavage,and the control group and the model group were given distilled water with equal dose.After 6weeks,the mice were weighed and sacrificed,then we collected the blood,liver and ileum tissue.The serum Alanine aminotransferase?ALT?activity was measured and then we performed HE and Sirius red staining to observe the pathological morphology of liver.The ileal tissue was sectioned and HE staining was performed to observe intestinal mucosal damage in mice.Using tachypleus chromogenic substrate method to detected serum endotoxin levels.The mRNA levels of?-SMA,TIMP-1,Col1?1,TNF-?,IL-1?in mouse liver and levels of ZO-1,Occludin,Claudin-1 in ileum were detected using RT-PCR.Caco-2 cell cultured in transwell chamber were used as the model of intestinal mucosa barrier.Cells were induced by LPS?Lipopolysaccharides?within the treatment of FTZ or berberine.Trans-epithelial electrical resistance?TEER?was measured and the mRNA levels of ZO-1,Occludin,Claudin-1,IL-1?,TNF-?,IL-6 were detected using RT-PCR.Result:1.Compared with control group,the serum ALT levels of the CCl₄ group were significantly increased and treantment with FTZ or berberine can decrease the ALT levels.2.The HE or Sirius Red staning of liver slices were observed.In CCl₄ group,hepatocellular necrosis occured and there were numbers of inflammatory infiltration and lots of collagen deposition around the portal area.But cells with inflammation or necrosis were significantly reduced in FTZ and berberine administration group compared with the model group,with reduction of collagen synthesis and deposition.3.Both FTZ treatment group and berberine treatment group can down-regulate mRNA expression of?-SMA,Col1?1,TIMP-1,TNF-?,IL-1?compared with CCl₄ group.4.Through the detection of serum endotoxin and the pathological analysis of ileum tissues,we found that berberine treatment can significantly improve the ileum mucosal injury in mice with liver fibrosis and inhibit the level of serum endotoxin.But there was no significant improvement between FTZ group and CCl₄ group.5.Berberine can inhibit the decrease of mRNA levels of ZO-1,Occludin,Claudin-1 in ileum of mice with liver fibrosis.By contrast,the FTZ cannot reverse the reduction of ileum tight junction molecules.6.At the cellular level,LPS significantly increased intestinal epithelial permeability and decreased levels of tight junction molecules ZO-1,Occludin,Claudin-1 compared to the control group.Compared with the LPS group,berberine intervention group markedly decreased the intestinal epithelial permeability and the expression of these tight junction molecules has a sharp increase.But there is no effect of FTZ on the improvement of intestinal barrier injury.7.The mRNA levels of inflammation-associated cytokines IL-1?,TNF-?,IL-6 were significantly down-regulated by LPS.Compared with the LPS group,both FTZ and berberine have the downward trend to these cytokines,but the inhibitory effect of berberine on inflammatory factors was significantly stronger than that of FTZ.Conclusion:1.Both FTZ and its effective constituent berberine can alleviate liver fibrosis in mice induced by CCl₄ and the anti-fibrotic effect of berberine is stronger than FTZ.2.Berberine can improve the intestinal barrier function during liver fibrosis in mice induced by CCl₄,while there is no improvement on the intestinal barrier function by FTZ.3.The mechanism by which berberine improves the intestinal barrier in mice with liver fibrosis may be related to the inhibition of intestinal inflammation and up-regulation of the level of tight junction molecules.