| Objective : Based on modern medical theory of "gut-liver axis" and Chinese classic theory "liver-spleen related theory",combined with the the previous clinical and experimental studies,the study aimed to investigate the effect of prevention and treatment for Xiaozhi Yigan Decoction(XZYGD)in mice nonalcoholic fatty liver disease(NAFLD)model induced with high fat diet,and explored the mechanism of "preventing and treating NAFLD focusing on Spleen" from the view of the effect on metabolic inflammation and intestinal mucosal barrier.Methods:1.Theoretical part: The paper reviewed the research progress between intestinal microecology disorder and NAFLD;the correlation between"spleen"and intestinal microflora was analyzed in depth;combined with the academic experience and ideology of the tutor,we put forward the idea of "preventing and treating NAFLD"focusing on Spleen"from the view of etiology and pathogenesis.2.Experimental section:Forty Sprague-Dawley male mice were randomly divided into four groups,including blank control group,model group,XZYGD group and probiotics group,except for the model control group with 16,the other groups were 8 in each.The blank control group was fed with normal diets,the other 3 groups were fed with high fat diet,2 rats in the model controlgroup were killed to evaluate model at random every 4 weeks,the NAFLD models were successfully replicated for 16 weeks.In the 4th week of modeling,the XZYGD group intragastrically treated with XZYGD at a dose of12.5g · kg-1 · d-1;the probiotics group was intragastrically given with Live combined bifidobacterium and lactobacillus tablets 0.7g·kg-1·d-1,and the blank control group and model group were administrated intragastrically with0.9% sodium chloride solution,body weight was measured once a week,and the dosage of administration were adjusted according to body weight,the administration lasted 12 weeks.The rats were totally killed after 24 hours of the last intragastric administration,the blood,liver and ileum tissues were collected,the fecal samples were also collected from the colon.The biochemical analysis method was used to detect serum ALT(alanine aminotransferase,alanine aminotransferase(AST),and Content of triglyceride(TG),FFA(free fatty acid)in liver homogenate.Liver tissue NAS integral were evaluated by hematoxylin eosin staining,and lipid deposition were measured by oil red O staining,liver fibrosis was observed by Masson staining.The mucosal tissue were observed by light microscope and electron microscopy.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of IL-1?and TNF-?in serum,the LPS of portal vein plasma were detected by limulus chromogenic substrate method.Western blot was used to detect the expression of TLR4 and NF-?Bp65 protein in ileum.Real-Time PCR(SYBR)absolute quantification method was used to detect and analyze the content of fecal flora.The expression of Occludin and ZO-1 protein in rat liver tissues were detected by immunohistochemistry.The content of SIgA in intestinal mucus were detected by ELISA.Results:1.The comparison of body weight and liver index in the rat.At the 16 th weeks of experiment,compared with the blank control group,the body weight and liver index of the model group were significantly higher than those of inthe blank control group,after XZYGD intervention,the body weight and liver index of rats decreased significantly.The results showed that XZYGD could improve the body weight of NAFLD rats with obesity.2.The comparison of biochemical indexes in serum and liver homogenate of rats.Compared with the blank control group,the level of serum AST and ALT increased significantly in model control group,especially AST were more significantly,and the contents of TG and FFA in liver homogenate were also increased;While intervented by the XZYGD,the above 4 indicators were significantly decreased.3.Pathological changes of liver in rats.Compared with the blank control group,NAS score in the model control group was significantly higher,after intervention of XZYGD,the NAS score decreased significantly,oil red O staining indicated that lipid droplets of the liver tissue in model group increased significantly,while the lipid droplet decreased significantly in XZYGD group.Masson staining showed that the collagen fiber staining increased significantly in model group,some specimens had fibrous septum,and collagen fiber coloring is significantly reduced in Xiaozhi Yigan Decoction group.The results show that XZYGD could improve liver pathological damage and progression of NAFLD model rats.4.Effect of XZYGD on metabolic inflammatory indexes in NAFLD rats.Compared with the blank control group,the content of LPS in plasma and serum IL-1 ?,TNF-? levels were significantly increased in the model group,after the intervention of XZYGD,the level of indexes above-mentioned were significantly reduced.5.Effect of XZYGD on intestinal micro-ecological barrier in NAFLD rats.The experiments was based on 3 aspects:intestinal mucosal barrier,mechanical barrier and immune barrier.(1)Compared with the blank control group,the number of rat fecal samples of Escherichia coli and Enterobactercloacae increased in model control group,while the number of Bifidobacterium and Lactobacillus decreased,While the population of Escherichia coli and Enterobacter cloacae were decreased in XZYGD group and the number of Bifidobacterium Lactobacillus increased.(2)Compared with the control group,the expression of Occludin and ZO-1 protein in intestinal mucosal(by immunohistochemistry)decreased in model control group,While the expression Occludin,ZO-1 protein was up-regulated in Xiaozhiyigan group.In addition,it was observed tight junction were shortened,desmosomes were loosed by the transmission electron microscope,while The morphology and integrity of the tight junction were improved to some extent after the treatment of XZYGD.(3)Compared with the control group,the expression of TLR4 and NF-?B protein in ileum of rat model was significantly increased,while the XZYGD group showed a significant downward trend.The content of SIgA in model control group of intestinal mucus in rats was significantly reduced,and increased in XZYGD group.Conclusions:XZYGD,based on therapeutic method in theory of Spleen,had good effects in hepatoprotective and lipid metabolism of liver,it also could significantly improve the pathological changes of liver tissue,reduce the degree of liver injury.The possible mechanism might be that:XZYGD could repair the disorder intestinal mucosal micro ecological barrier in NAFLD rat model,reduce the NAFLD rat model of portal vein plasma LPS level and factor IL-1??TNF-?level,so as to inhibit the LPS/TLR4 pathway to reduce secondary liver injury induced by metabolic inflammation. |
PDF Full Text Request