Study On The Effect Of Intestinal Microflora On Intestinal Mucosal Mechanical Barrier In The Study Of The Mechanism Of NAFLD In The Treatment Of Dahuang Zexie Decoction

Nonalcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by diffuse hepatocyte bullous steatosis,which is caused by alcohol and other definite liver damage factors.It includes simple fatty liver(SFL),fatty hepatitis(NASH)and cirrhosis.The latest statistics show that the prevalence of NAFLD in Chinese adults is increasing,showing a trend of younger age,and is closely related to the occurrence of cirrhosis and liver cancer.At present,the first line of NAFLD is to change the way of life,but the effect is not ideal.But the therapeutic effect of traditional Chinese medicine on NAFLD is remarkable and has been widely used.In the previous work,we adopted the rhubarb and Alisma soup to intervene the NAFLD animal model according to the pathogenesis of NAFLD spleen deficiency and the phlegm dampness and turbid stasis.However,the specific mechanism for the treatment of NAFLD is not clear.We hope to make this experiment clear the mechanism of the treatment of NAFLD by Dahuang Zexie Decoction,and to provide ideas and methods for the study of Chinese medicine for the treatment of NAFLD.Objective:1.Through 16sRNA sequencing,immunohistochemistry,Western blotting,to observe the effect of NAFLD in rat intestinal flora,intestinal mucosal mechanical barrier and intestinal tissue of TLR4 signaling pathway in Dahuang Zexie Decoction on high fat diet induced.2.Combined use of-broad-spectrum antibiotics to establish pseudo sterile mouse model constructed by 16sRNA sequencing evaluation model.3.The use of fecal bacteria transplantation(FMT),transplantation of rats feces suspension to pseudo germ free mice,by means of 16sRNA sequencing,immunohistochemistry,Western blotting,TEM,explore the specific mechanism of rhubarb Zexie Decoction improve the intestinal mucosal mechanical barrier NAFLD.Methods:1.Using 45%high fat diet for 8 week old male SD rats for 16 weeks to establish the rat model of NAFLD,with Dahuang Zexie Decoction(5g/kg)intervention in NAFLD rats,to observe the general conditions of rats,body weight,liver function and pathological changes of liver tissue,using high-throughput sequencing to detect each group of rats fecal bacteria 16sRNA,the expression and distribution of intestinal mucosa by immunohistochemical observation of tight junction protein ZO-1 and Occludin,the expression of TLR4 signaling pathway related protein Western in intestinal tissue by blotting;2.The 8 week old SPF male C57BL/6 mice fed with a mixture of antibiotics(ampicillin,neomycin sulfate 1g/1 1 g/L and 1 g/L of metronidazole 500 mg/L)and vancomycin with sterile water for 4 weeks,a pseudo sterile mouse model,weight changes of mice were observed,using high-throughput sequencing method to detect fecal bacteria in 16sRNA,to evaluation model of effect.3.The use of FMT technology,NAFLD rats and normal rats,traditional Chinese medicine intervention rat fecal suspension by gavage 200ul/transplantation,once a week,a total of 8 weeks,the mice were observed the general condition,body weight,liver function and pathological changes of liver tissue were detected.Mice fecal bacteria 16sRNA,expression and distribution of immunohistochemical observation of intestinal ZO-1 and Occludin,to observe the ultrastructure of intestinal mucosa in mice,to detect the expression of intestinal TLR4 signaling pathway related protein Western by blotting.Results:1.(1)Dahuang Zexie Decoction could effectively reduce the weight gain,improve the abnormal function and blood lipid level of NAFLD rats,alleviate the liver lipid deposition and hepatocyte damage induced by high-fat diet in NAFLD rats.(2)Dahuang Zexie Decoction and high-fat diet can lead to the Shannon index,Chao index lower than that in blank group,in different bacterial taxonomic levels,rhubarb Zexie decoction can significantly lower the Firmicutes increased NAFLD rat intestinal flora in(Firmicutes),(Desulfovibrionaceae),Desulfovibrio,Desulfovibrio(Desulfovibrio)Escherichia coli/Shigella(Escherichia/Shigella)at the same time can increase the proportion of bacteria,Bacteroidetes,rumen bacteria(Bacteroidetesother),(Ruminococcaceae),atrial fibrillation(Oscillibacter)is a gram bacillus,Butyricicoccus bacteria ratio;(3)Dahuang Zexie Decoction can ileum and colon tissues in NAFLD rats,ZO-1 Occludin these two kinds of tight junction protein(TJ)expression;(4)Dahuang Zexie Decoction can Alisma TLR4 signaling pathway are upregulated by inhibition of NAFLD in rat ileum and colon tissues The expression of related proteins TLR4,MyD88,p-JNK and p-ERK had no obvious effect on the expression of total JNK and ERK.2.(1)After 4 weeks of sterile water containing combination of broad-spectrum antibiotics,antibiotics decreased body weight of mice than normal SPF mice.(2)16S rRNA and bacterial DNA sequencing showed that the Shannon index and Chao index is far lower than that of normal SPF mice.(3)There is a significant difference in the level of classification of antibiotics in mice and normal SPF mice intestinal bacteria.3.(1)NAFLD rats and normal rats,traditional Chinese medicine intervention of fecal bacteria in rats after transplantation of mice in pseudo germfree mice,there was no difference in body weight.(2)to pseudo sterile mice medicine intervention rat feces after transplantation,transplantation of NAFLD mice with pseudo sterile rat feces showed liver function and a low level of blood lipid,the level and transplantation of normal rat feces pseudo germ-free mice were similar.(3)the three groups of mice fecal bacteria transplantation had no significant change in liver pathology.(4)transplantation medicine intervention rat feces pseudo sterile mice with transplantation of NAFLD rat feces have lower Ruminococcaceae,Desulfovibrionaceae and Desulfovibrio,Desulfovibrio bacteria,and Bacteroides,Oscillibacter is a gram Bacillus bacteria,Butyricicoccus proportion is higher,Compared with the transplantation of normal rats feces pseudo germfree mice similar.(5)mice blood transplantation medicine intervention rat pseudo sterile fecal transplantation of NAFLD rat feces,blood FITC-dextran content lower intestinal mucosal ultrastructure,more complete,ZO-1,expression of occludin distribution is more approximate normal mice.(6)The expression and intervention of Chinese Medicine rat feces of ileal tissue in pseudo germfree mice TLR4,MyD88,p-JNK,p-ERK,lower than NAFLD rat fecal transplantation.Conclusion:Dahuang Zexie Decoction can improve NAFLD rat intestinal flora,the pathogens decreased the proportion of the increase in the proportion of bacteria,reduce intestinal TLR4 ligand LPS,down-regulation of TLR4 signaling pathway activity,expression and distribution so as to improve the intestinal mucosal mechanical barrier tight junction protein,reduce the permeability of the intestinal mucosal barrier,reduce the number of pathogens and its metabolites by intestinal blood flow through the portal vein into the liver,improve the liver inflammation and lipid deposition.