Effect Of Compound Danshen Dripping Pills On Liver Steatosis And Intestinal Flora In NAFLD Mice

BackgroundNonalcoholic fatty liver disease(NAFLD)is a manifestation of metabolic syndrome in the liver,and its prevalence is rising.It is currently the most common chronic liver disease.In recent years,as the theory of intestinal-hepatic axis is put forward,the the study of the pathogenesis of NAFLD with intestinal flora as the target has gradually become a research hotspot.Changes in intestinal flora can cause inflammation and fat accumulation,which in turn leads to NAFLD.Compound Danshen Dripping Pills are mainly composed of tanshinone ?A,cryptotanshinone,salvianolic acid,saponin and so on.Studies have shown that Salvia miltiorrhiza can alleviate hepatic steatosis and fibrosis,and Danshenol borneol can reduce obesity,hepatic steatosis,and low-grade systemic inflammation in mice with high-fat diet by adjusting intestinal flora.However,the specific mechanism of compound Danshen Dripping Pills to improve non-alcoholic fatty liver disease has not yet been elucidated.We hypothesize that the compound Danshen Dripping Pills may improve non-alcoholic fatty liver disease by regulating intestinal flora.ObjectiveThe Objective was to observe the effect of Compound Danshen Dripping Pills on High-fat diet mice non-alcoholic fatty liver disease and intestinal flora,and preliminary explore the relationship among Compound Danshen Dripping Pills,NAFLD and intestinal flora.Method1.Thirty-two KKAy male mice were given high-fat diet for four weeks to establish a NAFLD mouse model,which were randomly divided into a model group,a low-dose Compound Danshen Dripping pills(LCDDP)group,a medium-dose Compound Danshen Dropping Pills(MCDDP)group,and a high-dose Compound Danshen Dropping Pills(HCDDP)group,and eight male C57BL/6 mice were used as control(Con)group and fed with chow diet.LCDDP,MCDDP and HCDDP groups were given respectively 13.5 mg/kg/d,27 mg/kg/d and 40.5 mg/kg/d compound Danshen Dropping Pills by gavage,and at the same time,Model group and Con group were given intragastric administration of distilled water.After 12 weeks of intervention with various doses of compound Danshen Dripping Pills,the serum lipid levels and liver tissue inflammation indicators levels of mice in each group were measured.Hematoxylin-eosin staining(HE),oil red O staining,Masson staining sections and transmission electron microscopy were used to observe the degree of liver lesions in each group.Meanwhile,qRT-PCR and Western blot were used to detect the mRNA and protein expression of TLR4 signaling pathway factors in liver of each group.2.16SrRNA sequencing technology was used to study the effect of Compound Danshen Dripping pills on the structure of intestinal flora in mice with NAFLD.3.The content of short-chain fatty acids(SCFAs)in the feces of each group of mice after the experiment was determined by GC-MS.4.Serum LPS content in each group was determined by ELISA kit.HE staining was used to observe the pathological changes of intestinal mucosa in each group,and transmission electron microscopy was used to observe the changes of the tightly connected structure of ileum mucosa in each group.Result1.Animal experiments showed that Compound Danshen Dripping Pills could significantly reduce serum TCHO levels and increase HDL-C levels in NAFLD mice(P<0.05),and medium and high-dose Compound Danshen Dripping Pills could reduce hepatic tissue tumor necrosis factor ?(TNF-?),interleukin-6(IL-6)and interleukin-1?(IL-1?)levels(P<0.05).Liver tissue oil red O,HE section staining,and Masson staining results showed that Compound Danshen Dripping Pills could significantly improve liver cell lipid deposition and fibrosis in NAFLD mice.MCDDP group and HCDDP group can significantly inhibit TLR4,myeloid differentiation factor 88(MyD88),nuclear factor ?B p65(NF-?B p65)mRNA and protein expression in the liver Toll-like receptor 4(TLR4)signaling pathway of NAFLD mice(P<0.05).2.16srRNA sequencing showed that:? After intervention of Compound Danshen Dripping Pills,the similarity of intestinal flora in NAFLD mice and Con group mice was increased,and the intestinal flora disorder in NAFLD mice was improved.?Compared with the Model group the MCDDP group was reduced the ratio of Firmicues to Bacteroidetes(F/B)in the intestinal flora of NAFLD mice(P<0.05),and the abundance of butyrate-producing bacteria Trichosporaceae(norank-f-Lachnospiraceae)in the MCDDP group increased significantly.3.Compared with the Model group,the content of acetic acid(Ace)in the intestine of mice in the Con group decreased(P<0.05),the content of valeric acid(Val)and hexanoic acid(Hex)increased(P<0.05),and the total short chain fatty acid(SCFAs)decreased(P<0.05);in the LCDDP group,the content of acetic acid(Ace)in the intestine of mice decreased(P<0.05),the content of hexanoic acid(Hex)and isohexic acid(Isohex)increased(P<0.05),and the content of total short-chain fatty acids(SCFAs)decreased(P<0.05);Butyric acid(But)content increased in the MCDDP group(P<0.05);in the MCDDP group and HCDDP group,hexanoic acid(Hex)and isohexic acid(Isohex)content increased(P<0.05).There was no difference in short-chain fatty acids between the Compound Danshen dripping pill group and the Con group.4.Compared with the Model group,the content of serum endotoxin in NAFLD mice was reduced(P<0.05)in the MCDDP.The ileum tissue HE section staining results showed that Compound Danshen Dripping Pills can inhibit the intestinal epithelial cell necrosis and shedding.Transmission electron microscopy showed that Compound Danshen dripping pills could improve the tight junction integrity of intestinal epithelial cells in NAFLD mice.ConclusionCompound Danshen dripping pills can improve liver lipid deposition and inflammation in mice with nonalcoholic fatty liver disease,The mechanism may be to promote the increase of SCFAs such as butyric acid by regulating the structure of the intestinal flora of NAFLD mice,decrease LPS level,promote the recovery of the intestinal mucosal mechanical barrier,and inhibit the expression of TLR4 signaling pathway in the liver.