Effects Of Cholesterol-lowering Probiotics On Bile Acid Pathway In Mice With Nonalcoholic Fatty Liver Disease

Background and ObjectivesThe global prevalence of nonalcoholic fatty liver disease(NAFLD)is estimated to be 25%,and it continues to rise worldwide[1,2]NAFLD is usually a complex progressive liver disease,but the symptoms are not limited to the liver,it is a liver manifestation of the metabolic syndrome[3].It is worrying that NAFLD may be increasingly associated with major complications such as cirrhosis,hepatocellular carcinoma,and increased liver-related overall mortality[4]Due to its serious complications and comorbidities,the medical expenses of NAFLD will continue to increase,which will be a heavy burden on patients and the medical insurance system[5].The pathogenesis of NAFLD is very complicated.From the initial "two strikes"hypothesis to the "multiple strikes" hypothesis,its pathogenic mechanism has been further elaborated[6,7],but it is still not fully understood.At present,there are no effective and long-term oral medications for NAFLD in the clinic.Therefore,actively exploring new therapeutic drugs and approaches has important clinical significance.In recent years,more and more studies on the relationship between NAFLD and bile acids have been reported.Several studies have shown that metabolic disorders of bile acids have promoted the occurrence and development of NAFLD[8,9]In patients with NAFLD or NAFLD animals,serum bile acid levels have been increased,and the ratio of bile acids(primary and secondary bile acids)has changed significantly[10-12].Our previous research results showed that cholesterol-lowering probiotics(Lactobacillus rhamnosus GG and Lactobacillus plantarum WCFS1)can regulate the expression of cholesterol 7?-hydroxylase(CYP7A1)which is a rate-limiting enzyme in bile acid production in NAFLD rats[13].However,the effect of cholesterol-lowering probiotics on the bile acid pathway(FXR pathway)and its metabolism and transport mechanisms is not clear.In this experiment,we preliminary explored the effects of cholesterol-lowering probiotics on the bile acid pathway and its metabolism and transport in high fat diet NAFLD mice,and then provided a basis for elucidating cholesterol-lowering probiotics regulating FXR pathways to improve NAFLD in mice.1.Reproduction and genotype identification of FXR-/-miceMethodThe FXR+/-male and FXR+/-female mice were cohabited in a cage at a ratio of 1:1.The mice genotype were identified by agarose gel electrophoresis after PCR amplification of mice tail DNA.Then Western Blot was used to detect the expression of FXR protein in the liver of different genotype mice.ResultsThere were 18 mice in F1 generation,including 6 FXR+/+mice(4 males and 2 females),9 FXR+/-mice(3 males and 6 females)and 3 FXR-/-mice(1 male and 2 females).There were 8 FXR-/-mice(3 males and 5 females)in F2 generation;Western Blot results suggested that the expression of liver FXR in FX-/-mice was extremely low,indicating that FXR gene was completely knocked out.ConclusionsAgarose gel electrophoresis after PCR amplification of mice tail DNA to identify mice genotypes can smoothly and reliably distinguish FXR+/+,FXR+/-and FXR-/-mice.2.Effects of high fat diet on glycolipid metabolism and liver steatosis in FXR-/-miceMethodND(WT)group and ND(FXR-/-)group(n=6)were fed normal diet for 12 weeks.HFD(WT)group and HFD(FXR-/-)group(n=6)were fed high fat diet for 12 weeks.After the-mice sacrificed,the serum lipids,total bile acid levels and liver function indicators were detected by fully automatic biochemical analyzer;The relative expression levels of liver inflammatory factors and FXR downstream genes at mRNA levels were detected by RT-PCR;HE staining was used to observe liver steatosis.Results(1)There was no difference in body weight between the HFD(WT)group and the HFD(FXR-/-)group,but the HFD(FXR-/-)group showed severe impaired glucose tolerance(P<0.01),lipid Metabolic disorders(P<0.01),impaired liver function(P<0.01)and increased serum bile acid levels(P<0.01).(2)Compared with the HFD(WT)group,the relative expression levels of TNF-?,TLR4 and SR-B1 in the HFD(FXR-/-)group were significantly increased(P<0.01).(3)Compared with WT mice,the relative expression levels of liver SHP in ND(FXR-/-)group and HFD(FXR-/-)group were significantly decreased(P<0.01),while the relative expression of CYP7A1 was significant increase(P<0.01).(4)The liver structure of the ND(WT)group and ND(FXR-/-)group were approximately normal;In the HFD(WT)group,there were disorder of hepatic cord structure,fat vacuoles scattered and no infiltration of inflammatory cells;In the HFD(FXR-/-)group,there were disorder of hepatic cord structure,turbid swelling of hepatocytes,a lot of fat vacuoles and infiltration of very few inflammatory cells.ConclusionsDeletion of FXR causes disorders of glucose and lipid metabolism,abnormal bile acid metabolism,liver inflammation,and fatty degeneration of the liver,but this change requires the induction of high fat diet.Compared with C57BL/6 mice,the high fat diet induced NAFLD animal model established by FXR-/-mice has obvious advantages and can better reflect its disease characteristics.3.Cholesterol-lowering probiotics improve NAFLD in high fat diet FXR-/-miceMethodFXR-/-mice were randomly divided into 3 groups(n=6):The mice were given normal diet and administered with normal saline in the ND group.The mice were given high fat diet and administered with normal saline in the HFD group.The mice were given high fat diet and administered with cholesterol-lowering probiotics in the HFD group.All the mice were intervened for 12 weeks.The serum lipids,total bile acid levels and liver function indicators were detected by fully automatic biochemical analyzer;The relative expression levels of liver and intestine inflammatory factors were detected by RT-PCR;The expression levels of intestinal mucosal barrier protein,FXR axis and bile acid transport related proteins in liver and intestine were detected by Western Blot;The liver and intestinal tissue pathological changes were evaluated by HE staining.Results(1)Compared with the ND group,the HFD group showed significant weight gain(P<0.01),impaired insulin resistance and impaired glucose tolerance(P<0.01),the intervention of cholesterol-lowering probiotics could improve the above symptoms.(2)Compared with the ND group,the serum levels of TC,LDL-C,HDL-C,ALT,AST and bile acid in the HFD group were significantly increased(P<0.01).After the intervention of cholesterol-lowering probiotics,the contents of TC,TG,LDL-C,and bile acid decreased(P<0.05).(3)Compared with the ND group,the relative expression levels of liver TLR4,IL-1 and TNF-? in the HFD group increased(P<0.01),the relative expression levels of SR-B1 decreased(P<0.01).After the intervention of cholesterol-lowering probiotics,the relative expression levels of TLR4,IL-I and TNF-? decreased(P<0.05),the relative expression levels of SR-B1 increased(P<0.01).(4)The liver structure of the ND group was approximately normal;In the HFD group,there were disorder of hepatic cord structure,turbid swelling of hepatocytes and a lot of fat vacuoles;The H+P group alleviated the liver tissue of disorder.(5)Compared with ND group,the expression of liver SHP,FGFR4 and BSEP protein in the HFD group decreased(P<0.01),the expression of CYP7A1 protein increased(P<0.01).After the intervention of cholesterol-lowering probiotics,the expression of SHP did not change,the expression of FGFR4 and BSEP proteins increased(P<0.05),the expression of CYP7A1 protein decreased(P<0.01).(6)Compared with the ND group,the relative expression levels of ileum TLR4,IL-1,IL-6 and TNF-? in the HFD group increased(P<0.01).After intervention with cholesterol-lowering probiotics,the relative expression levels of TLR4,IL-1,IL-6 and TNF-? decreased(P<0.01).(7)The intestine structure of the ND group was approximately normal;HE showed the small intestine villi broken,missing and gap widened,epithelial cell necrosis and shedding in the HFD group;Destruction of the intestinal structure could be improved to some extent by cholesterol-lowering probiotics.(8)Compared with ND group,the expression levels of intestine ZO-1,Claudin-1 and Claudin-3 in the HFD group were decreased(P<0.01).Compared with HFD group,the expression of Claudin-1,Claudin-3 and FGF15 proteins in the H+P group increased(P<0.05).ConclusionsCholesterol-lowering probiotics have similar effects to selective intestinal FXR agonists,which may improve bile acid metabolism and transport by regulating the FXR-FGF15 pathway,thereby alleviating NAFLD in high fat diet FXR-/-mice.