Modulation Of The Gut Microbiota In Rats By Hugan Qingzhi Tablets During The Treatment Of High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease

Background and Objective:Nonalcoholic fatty liver disease?NAFLD?is considered to be the metabolic syndrome in the liver with increasing morbidity rate.So far,NAFLD has surpassed chronic hepatitis B as the first major chronic liver disease all over the world.In recent years,with the theory of“Gut-liver axis”proposed,aim for the gut microbiota to study the pathogenesis of NAFLD has aroused more and more attention.Our previous researches demonstrated that 1.08g/kg Hugan Qingzhi Tablet?HQT?could effectively improve insulin resistance,lipid accumulation and chronic inflammation in NAFLD rats.However,its mechanism of action is unknown.In our isobaric tags for relative and absolute quantitation-based proteomics experiments on livers from NAFLD rats,a kyoto encyclopedia of genes and genomes?KEGG?pathway enriclument analysis showed that the gut microbiota is closely related to the pathogenesis of NAFLD.HQT are mainly composed of Rhizoma Alismatis,Fructus Crataegi,Pollen Typhae,Radix Notoginseng and Folium Nelumbinis.The previous study showed that HQT contains a mount of flavonoids such as Isorhamnetin,Quercetin,Rutin and Galactoside with UHPLC-QqQ-Ms method.It is reported that flavonoids could be metabolized by gut microbiota and its biological activity also was affected by the gut microbiota.In turn,flavonoids such as rutin and quercetin could also regulate the disorder of gut microbiota caused by metabolic syndrome.Since HQT is administered orally,it is unavoidable that HQT will come into contact with the gastrointestinal tract.Therefore,we speculate that HQT is able to improve NAFLD by regulating the structure of gut microbiota and protecting the intestinal mucosal barrier.Method:1.Rats were randomly divided into three groups:normal fat diet?NFD?group,HFD group and HQT?HFD and 1.08 g/kg HQT?group.The corresponding drug and diet was administered for 12 weeks while the NAFLD rats were molded.At the end of 12th week,the relevant lipid indexes of liver,inflammatory factor indicators,hematoxylin-eosin?HE?staining and oil red staining were determined to observe the degree of liver tissue lesions.2.16SrRNA sequencing technology was used to study the regulation effect of HQT on the structure of gut microbiota in NAFLD rats.3.The content of SCFAs in the faces of rats at 12th week was determined by GC-MS method.4.The serum Lipopolysaccharide?LPS?content of each group was determined by ELSA kit.The pathological changes of ileum mucosa in each group were observed by HE staining method.The expression and distribution of tight junction protein Claudin-1 and ZO-1 in ileum mucosa of each group were determined by immunohistochemistry method.Meanwhile,qRT-PCR and Western Blotting method were adopted to observe the expression of Toll-like Receptor?TLR4?'s signaling pathway in liver of each group.Results:1.Animal experiments showed that HQT could reduce the level of triglyceride?TG?.,cholesterol?CHOL?and low-density lipoprotein cholesterol?LDLC?]of liver tissue homogenate in NAFLD rats,and significantly increase high-density lipoprotein cholesterol?HDLC?levels.In addition,HQT also could significantly restrain inflammation indicators such as tumor necrosis factor-??TNF-a?,interleukin-6?IL-6?and IL-1? levels.Liver tissue HE and oil red O staining results showed that HQT could significantly improve liver tissue steatosis in NAFLD rats.2.The results of 16SrRNA sequencing of gut microbiota of rat feces showed that the dietary intervention of HQT not only increased the similarity of the OTU composition of the gut microbiota from HFD-fed rat to that of NFD-fed rat,but also reversed the rise of the ratio of Firmicutes to Bacteroidetes caused by the HFD.Besides,HQT enhanced the abundance of some beneficial bacteria such as norank_f<sub>Bacteroidales_S24_7group,Ruminococcaceae,Bifidobacterium.Alistipes and Anaeroplasma,and also restrained the growth of harmful microbes,like Cronobacter,Streptococcus,Holdemanella and Blautia in the intestinal tract.Spearman correlation analysis found that Blautia and Holdemanella exhibited significant?p<0.001?positive correlations with TG,CHOL,LDL-C,IL-6,IL-1?,TNF-a and body weight?BW?and negative correlations with HDL-C?p<0.001?.The norank_f<sub>Bacteroidales_S24-7_group and Alistipes showed opposite trend.Moreover,the HQT group could promote flavonoid biosynthesis compared with the HFD group?p<0.05?.3.Compared with the HFD group,the level of propionic,isobutyric,butyric,valeric,isovaleric,caproic and total SCFAs were significantly increased in the intestinal tract of rats in the HQT group?p<0.01?.4.The intervention of HQT could significantly reduce the content of serum LPS in NAFLD rats?p<0.05?.The HE staining results of ileum tissue showed that HQT could restrain the necrosis and desquamation of intestinal epithelial cells,alleviate the lamina propria edema of the intestinal mucosa and effectively reduce the ratio of inflammatory cells in the intestinal mucosa.HQT could significantly up-regulate the expression of Claudin-1 and ZO-1 in the ileum mucosa of NAFLD rats?p<0.01?.HQT could significantly restrain the expression of TLR4,myeloid differentiation factor 88?MyD88?,Nuclear factor-?B?NF-?B?in the NAFLD rat's liver.Conclusion:The structure of gut microbiota could be regulated by HQT in NAFLD rats.In this way,HQT could restrain harmful bacteria,improve the growth of beneficial bacteria,promote the production of SCFAs such as propionic acid and butyric acid,reduce the production of LPS,up-regulate the expression of intestinal tight junction proteins Claudin-1 and ZO-1,reduce the permeability of intestinal mucosal mechanical barrier,and down regulate the TLR4 signaling pathway in liver.As a result,less of the harmful bacteria and their metabolites could flow through the portal vein into the liver,and liver lipid deposition and inflammation could be alleviated by HQT.