The Effect Of Cholesterol-lowering Probiotics On Bile Acid Metabolism Of Non-alcoholic Fatty Liver Disease And Mechanism

Background and ObjectivesNon-alcoholic fatty liver disease(NAFLD) encompasses a spectrum of hepatic histopathological changes ranging from non-inflammatory intracellular fat deposition to non-alcoholic steatohepatitis(NASH), which may progress into hepatic fibrosis, cirrhosis, or hepatocellular carcinoma. The hallmark of NAFLD is the excessive hepatic accumulation of neutral lipids that result from an imbalance between lipid availability and lipid removal. Currently, NAFLD is recognized as the most common form of liver disease worldwide affecting between 25 and 30% of the general population. Notably, NAFLD is highly prevalent among overweight and obese patients and in subjects with type 2 diabetes(T2DM). Moreover, NAFLD is strongly associated with the features of metabolic syndrome, and the hepatic manifestation of this cluster of features linked to an increased cardiometabolic risk.Although the precise pathophysiology of NAFLD is complex and not fully understood, chronic high fat diet, dysfunction of the gut- liver axis, gut microbiota are thought to play a role in NAFLD development. Besides, series of studies have suggested a bidirectional interaction between gut microbiota and the gut- liver axis, namely microbiome-gut-liver axis. It has been demonstrated that probiotics intervention could improve the fat liver and protect liver injury. However, in non-alcoholic fatty liver disease, the pathway through which gut microbime affected the gut- liver axis isn’t clearly revealed.Calory restriction, exercise and drugs are the main therapies for non-alcoholic fatty liver disease, but they don’t work very well. Our previous study has demonstrated that cholesterol-loweing probiotics applied to NAFLD rat models manifest effects of blood lipid reduction and liver protection as well as upregulating the expression of FXR of intestine at mRNA level. It indicates that the cholesterol-lowering probiotics we screened may ameliorate hyperlipoidemia and fatty liver in vivo by regulating bile acids metabolism, but the concrete impact and mechanism remains unclear.Murine NAFLD models were established with chronic high fat diet to investigate the effects of NAFLD on bile acids metabolism and how cholesterol-loweing probiotics improve fatty liver by regulating bile acids metabolism. This would provide theoretical basis for developing new therapies for NAFLD. MethodsTwenty one SD male rats were divided randomly into three groups including control group, model group and probiotics intervention group. The control group received normal diet. The NAFLD model were established by chronic high fat diet(45% of calories derived from fat diet)for 20 weeks. The probiotics intervention group received HFD with cholesterol-lowering probiotics by oral gavage. While mice in control and model groups were treated with equal volume of saline. Blood, ileum and liver were collected for further determinations. At the end of the study, all mice were killed. General index includes body weight, the levels of triglyceride, cholesterol and CK18-M30 in serums samples. The expression of CYP7A1, FXR, FGF15 and ASBT at mRNA level were detected by real time polymerase chain reaction(Real Time-PCR). Western-blot analysis was used to detect protein expression of CYP7A1, FXR in liver and ASBT in ileum. The expression of FXR in liver and ileum were analyzed by immunohistochemistry.Results1. General indexes: Compared with the control group, mice in model group showed a significant increase in physiological parameters and and serological markers(body weight, triglyceride, cholesterol and CK18-M30 in serums samples)(P <0.05). While cholesterol-loweing probiotics intervention can lower these indexes significantly(P <0.05).2. Pathological changes: In contrast with the control group, liver fat accumulation, soakage of inflammation cell in liver and fluff injury of small intestine can be observed in biopsy of the model group. Cholesterol-loweing probiotics intervention can improve these injuries significantly.3. The expression of CYP7A1 in liver at mRNA and protein levels: Compared to the control group, the expression of CYP7A1 at mRNA level decreased in liver significantly in model group(P <0.05); and cholesterol-loweing probiotics intervention can increase the expression of CYP7A1 significantly(P <0.05). The transcription corresponds to the expression at protein level.4. Change of FXR pathway in liver: In comparison with the control group, the expression of FXR at mRNA level increased in liver significantly in model group(P<0.05); and cholesterol-loweing probiotics intervention can decrease the expression of FXR significantly(P <0.05). The transcription corresponds to the expression at protein levels( by western-bloting and immunohistochemistry).5. Change of FXR-FGF15 pathway in ileum: Chronic high fat diet can suppress the FXR-FGF15 pathway in ileum significantly(P <0.05); while cholesterol-loweing probiotics intervention can active the FXR-FGF15 pathway significantly(P <0.05)( by western-bloting and Real-Time PCR).6. The expression of ASBT in ileum at mRNA and protein levels: The expression of ASBT at mRNA level increased in ileum significantly in model group in contrast to the control group(P <0.05); and cholesterol-loweing probiotics intervention can decrease the expression of ASBT significantly(P <0.05). The transcription corresponds to the expression at protein level. Conclusions1. Chronic high fat diet can induce non-alcoholic fatty liver disease in the murine model. Probiotics intervention can improve the fat liver and protect liver injury in rats.2. Chronic high fat diet downregulates the expression of CYP7A1 by aggravating rat hepatocyte injury, activating hepatic FXR pathway and upregulating ASBT in ileum, while probiotics intervention can upregulate the expression of CYP7A1 by improving rat hepatocyte injury, suppressing hepatic FXR pathway and downregulating ASBT in ileum.3. Chronic high fat diet can damage the fluff of small intestine and suppress the FXR-FGF15 pathway in ileum; while probiotics intervention can improve the fluff of small intestine and active the FXR-FGF15 pathway in ileum, which in turn can improve the fat liver.