The Study On Clinical Efficacy And Mechanism Of Kangxian Ruangan Granule Against Hepatic Fibrosis Based On Gut Microbiota

Objective:1.To observe the clinical efficacy of Kangxianruangan granule(KXRG)on liver fibrosis patients and its regulation effect on gut microbiota,so as to provide more evidence-based medical evidence for the antifibrosis effect of KXRG.2.To observe the effects of KXRG on gut microbiota,intestinal mucosal barrier and immune-related pathways of liver tissue inflammation in CCl₄-induced liver fibrosis mice.Then the pseudo germ-free mouse model was established,and the fecal bacteria of mice in KXRG intervention group were transplanted into pseudo germ-free mouse model to observe whether KXRG affected the expression of Inflammatory and immune-related cytokines in liver by improving gut microbiota,and to further explore the mechanism of KXRG in the treatment of KXRG,in order to provide new research perspectives and targets for prevention and treatment of liver fibrosis.Methods:1.Clinical retrospective study was conducted to study the effects of KXRG on the clinical efficacy and gut microbiota of patients with chronic hepatitis B(CHB)liver fibrosis.80patients with CHB liver fibrosis were divided into two groups:control group were treated with entecavir(ETV),treatment group were treated with ETV,combined with traditional Chinese medicine KXRG,After the intervention of 6 months,Liver function,liver stiffness measurement(LSM)of two groups before and after treatment were compared,the gut microbiota change of two groups after treatment was detected by 16S r RNA sequencing method.2.The liver fibrosis model were established by subcutaneous injection of 40%CCl₄ olive oil solution for 8 weeks,Then treated with water solution of KXRG(3.9g/kg)by gastric gavage.After8 weeks,body weight,liver index,liver function,intestinal and liver pathology were observed in each group.the gut microbiota of mice in each group were detected by 16S r RNA sequencing method.The expression of LPS,IL-1?,IL-6 and TNF-?were detected by enzyme-linked immunosorbent assay(ELISA).Immunohistochemistry and RT-q PCR were used to detect the protein and gene expression of intestinal tight junction proteins,such as Claudin-1,Occludin and ZO-1 in each group.The expression of TLR4,My D88 and NF-?B in liver tissues were detected by Western blotting and RT-q PCR method.3.Pseudo germ-free mice with liver fibrosis were constructed with mix antibiotics(Ampicillin 1g/L,vancomycin 0.5g/L,zincomycin sulfate 1g/L,metronidazole 1g/L)for 7 days.By the method of FMT,the feces solution of normal group,model group,KXRG group in Part?were transplanted to FMT-control group,FMT-model group,FMT-KXRG group with each mouse 200?L,then liver index,liver function and body weight,intestine and liver pathology change of each group were detected,IL-1?,IL-6 and TNF-?were detected by ELISA method.the expression of intestinal tight junction proteins such as Claudin-1,Occludin,ZO-1 were detected with Immunohistochemistry and RT-q PCR method.The expression of TLR4,My D88 and NF-?B in liver tissues were detected by Western blotting and RT-q PCR method.Results:1.Clinical study results:80 patients of liver fibrosis treated with Kangxianruangan granule were collected,including 36 males and 44 females aged 18-65 years.there are 44 patients in treatment group,while 36 cases in control group.(1)Statistical analysis of gender,age,duration of disease,liver function and LSM before treatment in 2 groups showed no statistical significance(P>0.05).(2)The effect of KXRG on liver function in patients showed that:intra-group comparison was made between the treatment group and the control group before and after treatment,the difference of ALT,AST,TBIL,GGT in two groups before treatment was statistically significant(P<0.05).After treatment,ALT,AST,TBIL,GGT of the two groups were compared in two groups.The indexes of the treatment group decreased more obviously than that of the control group,and the difference was statistically significant(P<0.05).(3)The results of the effect of KXRG on LSM of patients with CHB liver fibrosis showed that intra-group comparison before and after treatment showed no statistically significant difference before and after treatment in the control group(P>0.05),while the indexes in the treatment group after treatment decreased significantly than that before treatment,the difference was statistically significant(P<0.05).(4)The influence of KXRG on gut microbiota of 2 groups showed that compared with control group,OTU numbers of treatment group increased significantly(P<0.05),Alpha diversity and Beta diversity of gut microbiota in two groups were changed.According to the classification of different bacterial levels,on phylum level,compared with the control group,the abundance of Bacteroidetes increased significantly,the abundance of Proteobacteria,Fusobacteria decreased significantly,with statistical significance(P<0.05).on family level,compared with control group,the abundance of Lachnospiraceae,Bacteroidaceae in treatment group were significantly up-regulated,the abundance of Enterobacteriaceae,Fusobacteriaceae?Veillonellaceae?Streptococcaceae were down-regulated significantly,with statistical significance(P<0.05).At the genus level,compared with the control group,the abundance of Bifidobacterium,Bacteroides,Lachnospira in treatment group were significantly up-regulated,and the abundance of Escherichia?Veillonella?Fusobacterium were significantly down-regulated,with statistical significance(P<0.05).Based on KEGG database and COG database,it turned out that there are 301functional projections related to differences of gut microbiota in two groups,concluding intestinal infection,apoptosis,invasion,fat polysaccharide biosynthesis,primary bile acid biosynthesis,m TOR signaling pathways,fatty acid metabolism,closely connected,bile secretion,related factor,VEGF signaling pathways,processing and presenting antigen,endoplasmic reticulum protein processing,and the interaction between bacterial toxins,ECM-receptor interaction,circadian rhythm,p53 signaling pathways,etc.2.The results of the first section of the experimental study show that:(1)Compared with control group,weight,liver index of model group was significantly down-regulated,ALT,AST level of model group was up-regulated(P<0.05);compared with model group,weight,liver index of KXRG group was significantly up-regulated,ALT,AST level of model group was down-regulated(P<0.05).(2)KXRG can improve the gut microbiota disturbance induced by CCl₄ in mice.There were differences in OTU index,Alpha diversity and Beta diversity between model group and KXRG group.LEf Se analysis shows:compared with the model group,the abundance of GCA?900066575,Coriobacteriales,Coriobacteriia,Coriobacteriaceae?UCG?002,Atopobiaceae,Deferribacterales,Deferribacteres,Mucispirillum,Deferribacteraceae,Faecalibaculum in KXRG group was significantly down-regulated.The levels of Bacillales,Staphylococcaceae,Staphylococcus,Eubacterium ventriosum group,Prevotella?1,Rhodoplanes,Paenalcaligenes,Adlercreutzia was significantly up-regulated.(3)Compared with control group,there is no significant change in intestinal mucosa in model group,while IHC results show the expression of intestinal tight junction proteins Claudin-1,Occludin and ZO-1 in model group were significantly down-regulated(P<0.05);compared with model group,the expression of Claudin-1,Occludin,ZO-1 in KXRG group was significantly up-regulated(P<0.05).(4)Compared with control group,the liver damage and collagen fibrosis deposition in model group was obvious,meanwhile,the expression of LPS,IL-1?,IL-6,TNF-?was significantly up-regulated(P<0.05),the expression of TLR4,My D88,and NF-?B in liver tissues was significantly up-regulated(P<0.05);compared with model group,the liver damage and collagen fibrosis deposition in KXRG group was improved,the expression of LPS,IL-1?,IL-6,TNF-?was significantly down-regulated(P<0.05),the expression of TLR4,My D88,and NF-?B in liver tissues was significantly down-regulated(P<0.05).3.The results of the second section of the experimental study show that:(1)Compared with control group,the level of ALT,AST and HA,LN,PC??C-?of FMT-model group was significantly up-regulated(P<0.05);Compared with FMT-model group,the level of ALT,AST,HA,LN,PC??C-?of FMT-control group and FMT-KXRG group were obviously down-regulated(P<0.05).(2)Compared with control group,the level of tight junction proteins Claudin-1,Occludin and ZO-1 in the colon tissue of FMT-model group was significantly down-regulated(P<0.05);Compared with FMT-model group,the level of tight junction proteins Claudin-1,Occludin and ZO-1 in the colon tissue of FMT-control group and FMT-KXRG group were obviously up-regulated(P<0.05).(3)Compared with control group,the liver histomathological damage and collagen fiber deposition was obvious,the expression of LPS,IL-1?,IL-6 and TNF-?of FMT-model group was significantly up-regulated(P<0.05),the expression of TLR4,My D88,NF-?B in liver tissue was up-regulated(P<0.05);Compared with FMT-model group,the liver histomathological damage and collagen fiber deposition in FMT-control group and FMT-KXRG group was improved,the expression of LPS,IL-1?,IL-6 and TNF-?in serum of FMT-model group was significantly down-regulated(P<0.05),the expression of TLR4,My D88,NF-?B in liver tissue was down-regulated(P<0.05).Conclusion:1.KXRG can improve liver function and LSM in patients with liver fibrosis,and regulate the structure and abundance of gut microbiota.2.KXRG can improve the intestinal mucosal barrier,down-regulate the expression of TLR4/My D88/NF-?B pathway in the liver,improve inflammation and collagen deposition in the liver by regulating the structure of gut microbiota in liver fibrosis mice,thus play an anti-liver fibrosis role.