Study On The Pathogenesis Of Syndrome Of Damp-heat Of Nonalcoholic Fatty Liver Disease Based On The Gut-liver Axis

Objective: Systematically sort out the research context,development trend and hot spots of the syndrome of damp-heat,study the pathogensis of syndrome of damp-heat of nonalcoholic fatty liver disease(NAFLD)from the perspective of gut-liver axis based on the rat model of syndrome of damp-heat of NAFLD,to explore the biological connotation of damp-heat syndrome.Methods: 1.According to applying CNKI database,using bibliometrics and mapping knowledge method,this paper explores the research status,hot spots and frontier of the syndrome of dampn-heat from the aspects of basic characteristics of literature,cooperation network between researchers and research institutions,keyword cooccurrence and key emergence.2.Experiment research:(1)Replication the model of NAFLD damp-heat syndrome: The NAFLD rat model was established by intragastric administration of high-fat emulsion to simulate the factors of ‘Taiyin internal injury'.The NAFLD rat model was established by intragastric administration of high-fat emulsion to simulate the internal injury of Taiyin,the damp-heat "external evil" factors were produced through the intervention of damp-heat environment,and the NAFLD dampness-heat syndrome model of "internal and external interaction" was duplicated by the combination of the two.Specific grouping: 32 male SD rat were randomly divided into 4 groups with 8 rats in each group.Blank control group: room temperature 20-25 ?,relative humidity 55-65%,normal diet.damp heat environment group: put the artificial climate box for 12 hours a day(time: 8:00-20:00),without affecting the circadian rhythm of rats,the artificial climate box set temperature is 35 ±2 ?,humidity is 90%,during this period,rats are free to eat and drink.NAFLD group: the high fat emulsion was given intragastrically with a dose of 1.5ml/100 g.NAFLD damp-heat syndrome group: damp-heat environment intervention combined with high-fat emulsion intragastric administration.The modeling time for each group was 8 weeks.(2)Detected the serum biochemistry and liver histopathology: The serum liver function,blood lipid,and total bile acid were detected by the automatic biochemical instrument,and the pathological changes of liver tissue were observed by HE staining and oil red O staining.(3)Detected the intestinal flora in model rats: high throughput 16 S r RNA sequencing and bioinformatics analysis were used to study the structure and composition of intestinal flora in model rats.(4)Detected the intestinal mucosal permeability: The expression levels of tight junction proteins ZO-1,Occludin,CLMP in the colonic mucosa of rats were detected by q RT-PCR and Western blot.(5)Detected the inflammatory factors in liver tissue: Immunohistochemistry was used to detect the express of NF-?B in liver.(6)Dectected the AQP in colon and GAS in serum: The expression level of AQP3?AQP4 in the colonic mucosa,and the level of GAS in serum of rats were detected by q RT-PCR,Western blot and ELISA.Result: 1.The results of Mapping knowledge: since 1980 s,the number of literatures on syndrome of damp-heat research has increased year by year,with more than 3000 papers published in recent 10 years;the number of papers published by core authors is 308,which has not yet formed a core research group;the research position is mainly in Guangzhou University of traditional Chinese medicine,and the form of cooperation is mainly between universities and affiliated hospitals;the types of syndrome of damp-heat syndrome that are studied more are as follows : pi-wei damp heat syndreome,damp-heat stasis syndreome,dampn-heat flowing down syndreome,Damp-heat syndrome of liver and gallbladder,sydrome of damp-heat obstruction,spleen deficiency damp heat syndrome,sydrome of dampn-heat in large intestine;the main diseases involved are ulcerative colitis,chronic gastritis,chronic pelvic inflammatory disease,acne,chronic hepatitis B,gouty arthritis,rheumatoid arthritis,eczema,type 2 diabetes mellitus,chronic prostatitis and Helicobacter pylori Bacteria,chronic cholecystitis,children;the biological mechanism mainly focused on the single target of immune inflammatory reaction,metabolism and intestinal flora;research focus and frontier are researches of animal experiments and Chapter on Damp-heat Disease.2.Experiment research:(1)Model construction: NAFLD model was successfully reproduced by intragastric administration of high-fat emulsion.damp heat syndrome model of NAFLD model was reproduced by dampness heat environment + high fat emulsion,which was consistent with clinical manifestations,and the damp heat syndrome model of NAFLD was more significant.(2)Biochemical indexes: there was no significant difference in CHOL,HDL-C,LDL-C,and TG between the damp heat environment group and the blank control group,but the LDL-C in the NAFLD group was significantly higher than that in the blank group(P<0.05).The CHOL and LDL-C in the NAFLD group were significantly higher than those in the damp heat environment group(P<0.01,P<0.05).Compared with the blank control group,CHOL-C,LDL-C,and TBA in the damp heat syndrome model of NAFLD group increased significantly(P<0.05,P<0.01,P<0.01),while HDL-C and TG decreased significantly(P<0.01).Compared with the damp heat environment group,the CHOL-C,and LDL-C in the damp heat syndrome model of NAFLD group increased significantly(P<0.01),TG decreased significantly(P<0.05).(3)Liver pathology: HE staining: in the blank control group,liver was normal;in the damp heat environment group,the structure of hepatic lobule was normal,there were no fat vacuoles,and a little inflammatory cell infiltration could be seen around the central vein.NAFLD group,fat vacuoles of different sizes could be seen in the cytoplasm of hepatocytes,the boundary between cells was blurred and the hepatic sinusoid narrowed.In the damp heat syndrome model of NAFLD group,the arrangement of the hepatic cord was disordered,hepatic sinusoid narrowed,hepatic lobule diffuse hepatocyte vacuolar degeneration,local hepatocyte necrosis,unclear cell boundary,nuclear fragmentation or dissolution,with a small amount of inflammatory cell infiltration.Oil red O staining: there were a large number of red lipid droplets in hepatocytes in the NAFLD group and damp heat syndrome model of NAFLD group,and the number of lipid droplets in damp heat syndrome model of NAFLD group was more than that in NAFLD group.(4)Intestinal microflora:(1)Analysis of Alpha diversity index: compared with the blank control group,Chao1,and ACE index decreased in NAFLD group and damp heat syndrome model of NAFLD group,and the decrease in damp heat syndrome model of NAFLD group was the most significant.There was no significant difference in the Shannon index and Simpson index between the damp heat environment group,NAFLD group,damp heat syndrome model of NAFLD group and blank control group.(2)The results of NMDS analysis showed that the points of blank control group,damp heat environment group and NAFLD group were slightly coincident,while those of damp heat syndrome model of NAFLD group and blank control group did not coincide,indicating that the difference of intestinal flora between damp heat syndrome model of NAFLD model and blank group model was the greatest.(3)Difference species analysis between groups(LEf Se analysis): compared with the blank control group,only the level of Lactobacillus intestinalis decreased in the NAFLD group.The damp heat environment group increased the abundance levels of Lachnospiraceae,Clostridiales,Enterbacteriaceae,and Escherichia coli,and decreased the levels of Faecalibaculum,Romboutsia,Lactobacillaceae and Bacilli.damp heat syndrome model of NAFLD group increased the abundance levels of Prevotellaceae,Lachnospiraceae,and Blautia,and decreased the abundance levels of Lactobacillaceae,Romboutsia,Peptostretococcaceae,and Bacilli.Compared with the damp heat syndrome model of NAFLD group,the damp heat environment group increased the abundance levels of Lachnospiraceae,Blautia,and Prevotellaceae in NAFLD rats,and decreased the abundance levels of Romboutsia and Peptostreptococcaceae in NAFLD group.(5)The level of ZO-1,Occludin,CLMP in colon compared with the blank control group,the expression of ZO-1 m RNA in damp heat environment group,NAFLD group,and damp heat syndrome model of NAFLD group was significantly downregulated(P<0.01).The level of Occludin m RNA in the damp heat environment group was significantly lower than that in the blank control group and NAFLD group(P<0.05).The expression of Occludin protein in the damp heat environment group and damp heat syndrome model of NAFLD group was significantly lower than that in the blank control group,and the difference was statistically significant(P<0.05).The level of CLMP protein in the damp heat syndrome model of NAFLD group was significantly lower than that in the blank control group(P<0.05).(6)The level of NF-?B in liver in damp heat syndrome model of NAFLD group was significant higher than that of blank control group and damp heat enviroment group.(7)The level of APQ3 and APQ4 in colon and GAS in serum: compared with the blank control group,the levels of AQP3 and AQP4 m RNA in the damp heat environment group were significantly increased(P<0.01),while those in the damp heat syndrome model of NAFLD group were significantly decreased(P<0.05,P<0.01);compared with the damp heat environment group,the level of AQP3 and AQP4 m RNA in the NAFLD group,and damp heat syndrome model of NAFLD group were significantly decreased(P<0.01);the AQP4 m RNA expression in the damp heat syndrome model of NAFLD group was significantly lower than that in the NAFLD group(P<0.05).Compared with the blank control group and the damp heat environment group,the expression of AQP4 protein in damp heat syndrome model of NAFLD group decreased significantly(P<0.05).The level of GAS in NAFLD damp heat syndrome model of NAFLD group was significant lower than in blank control group and the damp heat environment group.Conclusion: 1.NAFLD damp heat syndrome model could be successfully established by intragastric administration of high-fat emulsion combined with damp heat environment,and the degree of liver injury in this model was more severe than that in NAFLD model.2.The pathogenesis and progress of NAFLD is related to intestinal water glycerin metabolism and intestinal mucosal barrier dysfunction.The mechanism may be that intestinal water glycerin metabolism disorder(down-regulation of AQP3 and AQP4 expression)affects lipid metabolism and causes hepatic steatosis;intestinal mucosal barrier dysfunction(disorder of intestinal flora and increase of intestinal mucosal permeability)and microbial related molecular model activate the expression of liver inflammatory factors(The expression of NF – ? B was increased),which caused liver inflammation.3.The results of this study suggest that the biological basis of the syndrome of dampheat may be the disorder of intestinal water glycerin metabolism;the biological mechanism of inflammatory reaction induced by the syndrome of damp-heat may be caused by intestinal mucosal barrier dysfunction,intestinal endotoxin translocation and activation of inflammatory cytokines.The results of this study also provide a biological basis for the spleen and stomach as the center of the syndrome of dampn-heat.