Exploring The Mechanism Of Ursolic Acid Inhibit Liver Fibrosis Based On Intestinal Bacteria-Liver Bile Acids Axis

Background and ObjectiveLiver fibrosis is a reversible process of repair and reconstruction after liver injury caused by various causes.Its sustainable development will lead to the occurrence of liver cirrhosis and liver cancer,which is a serious threat to human health.Although effective antiviral therapy can partially reverse liver fibrosis,there are still no widely effective anti-fibrosis drugs for liver fibrosis caused by other causes in clinic.Due to the existence of"liver-gut axis",the abnormal metabolic reaction in the liver can cause the destruction of intestinal microecology,and the disordered intestinal function can accelerate the progress of liver fibrosis.In view of the reversibility of liver fibrosis,it is of great significance to study the occurrence and development mechanism of liver fibrosis and explore anti-fibrosis drugs.Ursolic acid（UA）,a traditional Chinese medicine,has the effects of protecting hepatocytes,inhibiting liver inflammation and anti-fibrosis.It is recognized as a potential drug against liver fibrosis,but its effect and specific mechanism of reversing liver fibrosis need to be clarified.The purpose of this study was to explore the therapeutic effect and molecular biological mechanism of UA in the occurrence and process of liver fibrosis.Materials and methods:I.Study the effect of UA on the occurrence and process of liver fibrosis in mice1.Construction of CCl4 induced liver fibrosis mouse model and UA treatment:Wild type C57BL/6 male mice were randomly divided into three groups:Control group,CCl4 group and UA treatment group.The control group was injected intraperitoneally with 1ml/kg olive oil for 8 weeks,twice a week;CCl4 group was given intraperitoneal injection of 1ml/kg CCl4（diluted with olive oil 20%）for 8weeks,twice a week;UA group was given intraperitoneal injection of 1ml/kg CCl4（diluted by olive oil 20%）for 8 weeks,twice a week,and UA（50mg/kg/D）was given by gavage for last 2 weeks,7 times/week.After modeling,fasting for 3 days to obtain blood,liver and other samples.2.Construction of HFD induced liver fibrosis mouse model and UA treatment:wild-type C57BL/6 male mice were randomly divided into three groups:LFD group,HFD group and UA treatment group.After 1 week of transitional diet,LFD group was given LFD feed free diet for 24 weeks;HFD group was given HFD feed and free diet for 24 weeks;UA group were given HFD feed free diet for 24 weeks,and UA（50mg/kg/D）was given by gavage for last 6 weeks,3 times/week.After modeling,fasting for 3 days to obtain blood,liver and other samples.3.Construction of MCD induced liver fibrosis mouse model and UA treatment:wild-type C57BL/6 male mice were randomly divided into three groups:MCS group,MCD group and UA treatment group.After 1 week of transitional diet,MCS group was given MCS feed free diet for 8 weeks;MCD group was given MCD feed and free diet for 8 weeks;UA group were given MCD feed free diet for 8 weeks,and UA（50mg/kg/D）was given by gavage for last 2 weeks,7 times/week.After modeling,fasting for 3 days to obtain blood,liver and other samples.4.Molecular biological detection:ALT,AST,hydroxyproline and Triglyceride in mouse serum were detected by ELISA;The glucose tolerance and insulin tolerance of mice were measured by GTT and ITT tests;Liver tissue structure,collagen deposition and lipid deposition were detected by HE staining,Masson staining,Sirius red staining and oil red O staining;Liver fibrosis related genes were detected by immunohistochemistry,immunohistochemistry fluorescence,fluorescence quantitative PCR（q PCR）and Western blot（WB）（α-SMA,Collagen-I,TIMP-1）,hepatocyte apoptosis.II.Study the effect of UA on intestinal bacterial composition and intestinal barrier function in mice with liver fibrosis1.Microbial diversity:Take the intestinal tissues of mice after CCl4 modeling,MCD modeling and corresponding UA treatment（the grouping treatment is the same as before）,and sequence and analyze the intestinal contents with 16Sr RNA intestinal bacteria.The data were obtained by DNA extraction,PCR amplification,Illumina Miseq sequencing and data analysisα-Diversity,β-Diversity,composition of intestinal bacteria and LEf Se analysis were used to screen key bacteria,and to observe whether UA regulates intestinal bacteria and affects the process of liver fibrosis2.Detection of intestinal barrier function:HE staining was used to observe the pathological changes of ileum;The level of LPS in mouse serum was detected by ELISA;The expression of intestinal tight junction protein（claudin-1,Claudin-1）was detected by immunofluorescence,q PCR and WB.3.Liver fibrosis mouse model of intestinal decontamination:After one week of indoor adaptation and two weeks of drinking mixed antibiotic water（amoxicillin 1g/L,vancomycin 0.5g/L,neomycin 1g/L and metronidazole 1g/L）,they were randomly divided into three groups:Control group,CCl4 group and UA treatment group.The modeling process of CCl4 was the same as before;After one week of indoor adaptation and two weeks of drinking mixed antibiotic water（amoxicillin 1g/L,vancomycin 0.5g/L,neomycin 1g/L and metronidazole 1g/L）,they were randomly divided into three groups:Control group,MCD group and UA treatment group.The modeling process of MCD was the same as before.4.Molecular biological detection:ALT,AST,hydroxyproline and Triglyceride in mouse serum were detected by ELISA;Liver tissue structure,collagen deposition and lipid deposition were detected by HE staining,Masson staining,Sirius red staining and oil red O staining;Liver fibrosis related genes were detected by immunohistochemistry,immunohistochemistry fluorescence,fluorescence quantitative PCR（q PCR）and Western blot（WB）（α-SMA,Collagen-I,TIMP-1）,hepatocyte apoptosis.III.Study the effect of UA on spectrum of liver bile acids1.Quantitative determination of bile acids:the liver tissues of mouse after CCl4modeling,MCD modeling and corresponding UA treatment were taken（the grouping treatment was the same as before）,and the liver was sequenced by UPLS-MS.After standard preparation,UPLS-MS and data analysis,the absolute quantification of bile acids was obtained.Principal component analysis（PCA）and OPLS-DA differential metabolite screening were carried out to observe whether UA regulates liver bile acid spectrum and affects the process of liver fibrosis.2.Conjoint Analysis of intestinal bacteria and bile acids:Pearson correlation was used to calculate the correlation between the abundance of corresponding levels of microorganisms and corresponding metabolites.IV.Study the regulation of UA on NOX4/NLRP3 inflammasomes signaling pathway1.Construct CCl4 induced liver fibrosis model and UA treatment based on NOX4^-/-mice:wild-type C57BL/6 male mice were randomly divided into two groups:CCl4 group and CCl4+AP group.CCl4 group was injected intraperitoneally with1ml/kg CCl4（olive oil dilution 20%）for 8 weeks,twice a week;CCl4+AP group was given intraperitoneal injection of 1ml/kg CCl4（olive oil dilution 20%）for 8 weeks,twice a week,and AP（50mg/kg/D）was given by gavage for last 2 weeks,7times/week;C57BL/6 male mice of NOX4^-/-mice were randomly divided into two groups:NOX4^-/-+CCl4 group and NOX4^-/-+CCl4+UA group.The treatment was the same as before.After modeling,they fasted for 3 days to obtain blood,liver,intestinal contents and other samples.2.Construction of CCl4 induced liver fibrosis model and UA treatment based on NLRP3^-/-mice:wild-type C57BL/6 male mice were injected intraperitoneally with1ml/kg CCl4（diluted by olive oil 20%）for 8 weeks,twice a week;C57BL/6 male mice of NLRP3^-/-mice were randomly divided into two groups:NLRP3^-/-+CCl4group and NLRP3^-/-+CCl4+UA group.The treatment was the same as before.After modeling,fasting for 3 days to obtain blood,liver,intestinal contents and other samples.3.Molecular biological detection:ALT,AST and hydroxyproline in mouse serum were detected by ELISA;Liver tissue structure and collagen deposition were detected by HE staining,Masson staining and Sirius red staining;Liver fibrosis related genes were detected by immunohistochemistry,q PCR and WB（α-SMA,Collagen-I,TIMP-1）,NOX4,NLRP3 inflammasomes.Results:I Occurrence and development of reversible liver fibrosis mice treated with UA1.Successfully construct the mouse model of liver fibrosis induced by CCl4intraperitoneal injection and verify the therapeutic effect of UA:After 2 weeks of treatment with UA,compared with CCl4 group,the liver injury,collagen deposition and hepatocyte apoptosis in UA group were significantly reduced,ALT,AST and hydroxyproline decreased significantly,and the expression ofα-SMA,Collagen-I,TIMP-1 and other liver fibrosis related genes decreased significantly.2.Successfully construct the mouse model of liver fibrosis induced by HFD diet and verify the therapeutic effect of UA:After 6 weeks of treatment with UA,compared with HFD group,the weight of mice in UA group was significantly reduced,glucose tolerance and insulin tolerance were improved,liver injury,collagen deposition and lipid deposition were significantly reduced,ALT,AST and hydroxyproline were significantly decreased,and the expression ofα-SMA,Collagen-I,TIMP-1 and other liver fibrosis related genes decreased significantly.3.Successfully construct the mouse model of liver fibrosis induced by MCD diet and verify the therapeutic effect of UA:After two weeks of treatment with UA,compared with the MCD group,the weight of mice in UA group was significantly increased,liver injury,collagen deposition and lipid deposition were significantly reduced,ALT,AST and hydroxyproline were significantly decreased,and the expression ofα-SMA,Collagen-I,TIMP-1 and other liver fibrosis related genes decreased significantly.II Effects of UA on intestinal bacterial composition and intestinal barrier function in mice with liver fibrosis1.Effects of UA on intestinal bacterial composition and intestinal barrier function of mice with liver fibrosis induced by CCl4 intraperitoneal injection:the sequencing results of intestinal content diversity showed that compared with CCl4group,α-diversity of UA group were significantly increase,β-diversity showed significantly differences between groups,and the species composition of microbiota changed significantly.The phylum level showed the increase of Firmicutes and the decrease of Proteobacteria,the genus level showed the decrease of Akkermansia,Helicobacter,Bacteroide and the increase of Lactobacillus and Parabacteroides;Results of ileal barrier function:compared with CCl4 group,the ileal tissue structure of UA group was significantly improved,the level of serum LPS decreased,and the expressions of Occludin and claudin-1 increased significantly;After intestinal decontamination pretreatment,compared with CCl4+ABx group,there were no significant changes in ALT,AST and hydroxyproline,liver injury and collagen deposit in CCl4+UA+ABx group.2.Effects of UA on intestinal bacterial composition and intestinal barrier function of mice with liver fibrosis induced by MCD diet:the sequencing results of intestinal content diversity showed that compared with MCD group,α-diversity of UA group were significantly increase,β-diversity showed significantly differences between groups,and the species composition of microbiota changed significantly.The phylum level showed the increase of Firmicutes and the decrease of Proteobacteria,the genus level showed the decrease of Akkermansia,Staphylococcus,Bacteroide and the increase of Lactobacillus and Parabacteroides;Results of ileal barrier function:compared with MCD group,the ileal tissue structure of UA group was significantly improved,the level of serum LPS decreased,and the expressions of Occludin and claudin-1 increased significantly;After intestinal decontamination pretreatment,compared with MCD+Abx group,there were no significant changes in ALT,AST and hydroxyproline,liver injury and collagen deposit in MCD+UA+ABx group.III.Study the effect of UA on spectrum of liver bile acids1.Effect and Conjoint Analysis of UA on liver bile acids in mice with liver fibrosis induced by CCl4 intraperitoneal injection:the sequencing results of liver bile acids spectrum showed that compared with CCl4 group,the composition of liver bile acids in UA group changed significantly,deoxycholic acid（DCA）,cholic acid（CA）,Taurodeoxycholic acid（TDCA）decreased significantly,and DCA was positively correlated with Bacteroide,Akkermansia,Helicobacter;TDCA was positively correlated with Bacteroide and Akkermansia,and DCA was negatively correlated with Lactobacillus and Parabacteroides.2.Effect and Conjoint Analysis of UA on liver bile acids in mice with liver fibrosis induced by MCD diet:the sequencing results of liver bile acids spectrum showed that compared with MCD group,the composition of liver bile acids in UA group changed significantly,DCA,CA,TCA,TDCA decreased significantly,UDCA increased significantly,and DCA was positively correlated with Bacteroide,Akkermansia,Helicobacter;TDCA was positively correlated with Akkermansia,Helicobacter,UDCA was positively correlated with Lactobacillus,Parabacteroides,and DCA was negatively correlated with Lactobacillus and Parabacteroides.IV.UA regulates NOX4/NLRP3 inflammasome signaling pathway1.UA inhibited the expression of NOX4 and NLRP3 inflammasome in mice with liver fibrosis induced by CCl4 intraperitoneal injection:Immunohistochemical,q PCR and WB results showed that the expression of NOX4 and NLRP3inflammasome in CCl4 group was up-regulated compared with control group;Compared with CCl4 group,the expression of NOX4 and NLRP3 inflammasome in UA group was down-regulated.2.Inhibition of NLRP3 inflammasome can effectively alleviate liver fibrosis:NLRP3^-/-mice were constructed and treated with CCl4 intraperitoneal injection and UA.Compared with WT+CCl4 group,NLRP3^-/-+CCl4 group decreased inflammatory cell infiltration,collagen deposition,ALT,AST and hydroxyproline,and the expression ofα-SMA,Collagen-I,TIMP-1 and other liver fibrosis related genes;Compared with NLRP3^-/-+CCl4 group,there was no significant difference in the above changes in NLRP3^-/-+CCl4+UA group.3.Inhibition of NOX4 can effectively alleviate liver fibrosis:NOX4^-/-mice were constructed and treated with CCl4 intraperitoneal injection and UA or NOX4inhibitor（AP）.Compared with WT+CCl4 group,NOX4^-/-+CCl4 group and WT+AP group decreased inflammatory cell infiltration,collagen deposition,ALT,AST and hydroxyproline,and the expression ofα-SMA,Collagen-I,TIMP-1 and other liver fibrosis related genes;Compared with NOX4^-/-+CCl4 group,there was no significant difference in the above changes in NOX4^-/-+CCl4+UA group.4.NOX4 can effectively stimulate the expression of NLRP3 inflammasome:q PCR and WB results showed that there was no significant change in the expression of NOX4 in NLRP3^-/-+CCl4 group compared with WT+CCl4 group;Compared with WT+CCl4 group,the expression of NLRP3 inflammasome decreased significantly in NOX4^-/-+CCl4 group,NOX4^-/-+CCl4+UA group and WT+AP group.5.Effect of NOX4/NLRP3 inflammasome signaling pathway on intestinal bacteria in mice with liver fibrosis induced by CCl4 intraperitoneal injection:the sequencing results of intestinal content diversity showed that compared with WT+CCl4 group,NLRP3^-/-+CCl4 group and NOX4^-/-+CCl4,α-diversity of UA group were significantly increase,β-diversity showed significantly differences between groups,and the species composition of microbiota changed significantly.The phylum level showed the increase of Firmicutes and the decrease of Proteobacteria,the genus level showed the decrease of Akkermansia and the increase of Lactobacillus.Conclusion:1.UA has obvious therapeutic significance on the progress of liver fibrosis induced by various ways in mice.The injury of liver function,the deposition of collagen and the expression of liver fibrosis related genes decreased.2.The change of intestinal bacteria plays an important role in the process of liver fibrosis in mice,which is often accompanied by the change of intestinal barrier function,and the reversal of liver fibrosis by UA depends on the regulation of intestinal bacteria.3.The changes of bile acids spectrum are involved in the process of liver fibrosis and the regulation of UA reversing liver fibrosis in mice,and are closely related to the changes of intestinal bacteria.4.NOX4/NLRP3 inflammasome signaling pathway is an important mechanism for the progression of liver fibrosis and the reversal of liver fibrosis by UA,and is closely related to the regulation of intestinal bacteria.