The Experiment Study Of Sympathetic Neurotransmitter NPY Mediates Non-alcoholic Liver Disease Progression Through Regulating Hepatic Cholesterol Metabolism

Objective:With the current epidemic of obesity,the incidence of nonalcoholic fatty liver disease(NAFLD)is increasing.By now,NAFLD has become the most common chronic liver disease and the second most common cause for liver transplantation in the world.However,there exists no effective methods treating NAFLD.So,it is urgently needed to elucidate the mechanism underlying NAFLD progression and find new target in NAFLD treatment and diagnose.The development of NAFLD is partially attributed to cholesterol metabolic disturbance and sympathetic overactivation.Excess release of sympathetic neurotransmitter NPY positively correlated with both NAFLD and cholesterol metabolic disturbance.In the present study,we want to determine the NPY content in NAFLD patients and elucidate the function of NPY in cholesterol metabolic regulation and NAFLD progression.Methods:1.In the first part of this study,subjects with histological proven normal liver,NAFL,and NASH were enrolled.Biomarkers of sympathetic activity and sympathetic neurotransmitter NPY were measured in the serum and liver of enrolled subjects.Furthermore,the hepatic expression of NPY receptors was assessed(transcription and protein level)and related the mRNA expression of NPY receptors to the pathological phenotype of NAFLD.2.In the second part of this study,exogenous NPY was injected through the hepatic portal vein of SD rats and serum and liver were collected 1 hour after injection.In vitro,BRL-3A hepatocytes were treated with NPY,NPY1 R,NPY2R,NPY5 R,or ERK1/2antagonist.Cholesterol content was measured by a coupled enzyme method.Precursor sterolregulatory element binding protein 2(p SREBP2),mature SREBP2(m SREBP2),HMGCR,ERK1/2,p ERK1/2,PKA,p PKA protein expression was examined by Western blotting.3.In the last part of this study,obesity with type 2 diabetes mellitus model was induced by high-fat diet and STZ in SD rats.Our previous study identified obvious hepatic triglyceride and cholesterol accumulation in this rat model.Obese rats with T2 DM were randomly performed sham operation,sham operation combined with food restriction and SG.Body weight,food intake,blood glucose,body composition and cholesterol level were measured at 2 or 4 weeks after operation.Sympathetic nervous biomarker TH and sympathetic neurotransmitter NPY protein content was measured in the liver 2 or 4 weeks after operation.Subsequently,hepatic cholesterol content and both proteins and transcriptional level of SREBP2,HMGCR,and LDLR were measured at 2 and 4 weeks after the operation.Results:1.We demonstrated that there was no difference in blood glucose and hemoglobin A1 C among enrolled subjects.NAFLD was associated with increased serum NE level and hepatic NE and tyrosine hydroxylase(TH)content.The hepatic release of sympathetic neurotransmitter NPY was higher in subjects with NAFLD.Both hepatic mRNA and protein expression of NPY2 R and NPY5 R were higher in subjects with NAFL or NASH.However,in the NAFLD subjects,the hepatic mRNA expression of NPY1 R decreased,but the protein expression increased.NPY1 R,NPY2R,and NPY5 R mRNA expression were correlated with the histological severity of NAFLD.2.In rats,NPY intraportal vein injection rapidly activates p SREBP2,m SREBP2,HMGCR protein expression,and induces hepatic cholesterol accumulation.In the BRA-3A cell line,we observed that NPY increases cholesterogenic protein expression and cholesterol synthesis through Y1 and Y5 receptors.This effect is mediated by the activation of the ERK1/2 signal pathway.3.SG rapidly reduced blood glucose independent of body weight loss and food restriction.SG reduce hepatic TH protein content at 2 weeks but not 4 weeks after operation.Compared with Sham group and Pair fed groups,SG alleviated sympathetic induced NPY release at 2 weeks and 4 weeks after operation.Rats underwent SG exhibited lower total cholesterol(TC)and free cholesterol(FC)in both serum and liver.However,the cholesterol-lowering effect was independent of body weight loss and food restriction just at 2 weeks postoperatively.SREBP2,HMGCR,and LDLR protein and mRNA expression were inhibited 2 weeks postoperatively and recovered 4 weeks after SG.Conclusion: 1.In the subjects with NAFLD,there existed obvious sympathetic overactivation and NPY over release in the liver.NPY1 R,NPY2R and NPY5 R were closely associated with the severity of NAFLD.Thus,NPY system played a pivotal role in NAFLD progression.2.NPY combined with NPY1 R and NPY5 R,promoted ERK1/2phosphorylation,activated SREBP2-HMGCR pathway,and then induced de novo cholesterol synthesis in hepatocytes and accelerated the progression of NAFLD.3.SG alleviated hepatic sympathetic nerve activity and hepatic NPY release,inhibited liver SREBP2 and downstream targets in cholesterol metabolism,reduced hepatic cholesterol de novo synthesis,and thus relieved hypercholesterolemia and hepatic cholesterol accumulation.