Lipid-lowering Probiotics Modulate Cholesterol Metabolism To Ameliorate Nonalcoholic Fatty Liver Disease In Mice

Background and ObjectivesNon-alcoholic fatty liver disease（NAFLD）is gradually becoming a major cause of liver disease in developed countries and eastern coastal areas of China.The prevalence of adults is between 20%and 30%,of which 10%to 20%have become non-alcoholic steatohepatitis,and even 2%to 3%have developed nonalcoholic steatohepatitis-related cirrhosis.The current research directions for NAFLD treatment include increasing aerobic exercise,limiting energy intake,medication,probiotics,and prebiotic intervention.Our team has found that the combination of probiotics（DM9054+86066 strain）administered intragastrically to rats with fatty liver can regulate the metabolism of bile acids through the abundance and quantity of intestinal microbiota and reduce the blood lipid level in rats,thus improving the status of liver steatosis,reducing liver inflammation and liver cell damage.However,cholesterol has not been studied as an upstream raw material for the production of bile acids,and its effect on the transport and synthesis of a combination of lipid-lowering probiotics has not been studied.MethodsTwenty-four male SPF LDLR^-/-mice were randomly divided into 3 groups（8/group）after 3 weeks of adaptive feeding.The 3 groups were given normal diet+normal saline gavage,high fat diet+saline gavage,high-fat diet+lipid-lowering probiotics(DM9054 and 86066,each 1×10¹⁰cfu)respectively.Every day at 9:00 am,mice were given by gavage using 12#gavage needles.After continuous feeding intervention for 15 weeks,each group of mice was sacrificed by cervical dislocation.Blood was taken from the eyeballs and specimens of serum,liver,and colon were taken.Microplate reader was used to detect serum lipid levels in mice.The protein expression of CYP7A1,FXR,ABCG-8,SR-BI,HMGCR,ABCB-11,FGFR-4,and SREBP-2 in liver of mice was detected by Western blot.real-time PCR was used to evaluate the mRNA expression levels of FXR,ASBT,FGF-15,and ABCG-5 in colon tissues of mice;Results1.Body weight changes in the mice:Compared with the control group,the body weights of the model group mice were significantly increased（p<0.05）.Compared with the model group,the mice in the intervention group lost weight.The difference was statistically significant（p<0.05）.2.Blood lipid changes in the mice:Compared with the control group,the levels of HDL were decreased,and the levels of TC,TG,and LDL in the model group were increased,the differences were statistically significant（p<0.05）.After intervention of lipid-lowering probiotics,the levels of HDL were increased,and the levels of TC,TG,and LDL in the model group were decreased,the difference was statistically significant（p<0.05）.3.Pathological changes in liver of mice:In the control group,the liver specimens were smooth,with bright red color and sharp edges of the liver.Under HE staining,the tissue structure was normal,cells were slightly edematous,and no obvious abnormalities were observed in Sudan III staining.In the model group,the surface of the liver specimens was greasy,covered with yellow spots,yellow and red in color,and the liver margin became blunt.Under HE staining,the histological structure was abnormal.A large number of liver cell steatosis,punctate necrosis and inflammatory cell infiltration in the portal area.Large amounts of orange lipid droplets were observed in Sudan III staining.The liver specimens of the intervention group were smooth in surface,bright red in color,with a few yellow spots and sharp liver margin.Under HE staining microscope,cell edema were observed,and tissue structure were normal,cytoplasm contains a few lipid droplets.Smaller lipid droplets was visible in Sudan III Staining.4.The protein expression levels in liver of mice:Compared with the control group,the model group mice liver FXR,ABCG-8,SR-BI,ABCB-11,FGFR-4protein expression levels were significantly decreased（p<0.05）,SREBP-2 protein expression was significantly increased.Compared with the model group,the expression of SREBP-2 and HMGCR protein in the intervention group was significantly decreased（p<0.05）,and the expression of FGFR-4 protein was significantly increased（p<0.05）.5.The mRNA expression level in intestinal of mice:Compared with the control group,the intestinal ASBT mRNA expression level in the model group was significantly higher（p<0.05）,and ABCG-5 and FGF-15 mRNA expression levels significantly decreased（p<0.05）;Compared with the model group,the intestinal ASBT and ABCG-5 mRNA expression levels in the intervention group were significantly lower（p<0.05）,and the expression level of FGF-15 mRNA was significantly higher（p<0.05）.Conclusions1.Feeding LDLR^-/-mice with a long-term,high-fat diet could successfully establish the NAFLD mouse model,and lipid-lowering probiotic intervention can improve the mice’s NAFLD status;2.Long-term high-fat diet could promote the absorption of cholesterol,inhibit the secretion of cholesterol.Lipid-lowering probiotics intervention can further promote the absorption of cholesterol,and no significant effects were observed on cholesterol secretion;3.Lipid-lowering probiotics could lower the expression of SREBP2,inhibite HMGCR and reduce the synthesis of cholesterol;4.Lipid-lowering probiotic intervention could improve bile acid metabolism by activating the FXR-FGF15 pathway.