Targeting GGPPS For Screening Natural Compounds In The Treatment Of Nonalcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD),characterized by ectopic accumulation of triglycerides(TG)in hepatocytes.Its development process usually includes simple steatosis of the liver,fatty hepatitis,liver fibrosis and other serious diseases,which can lead to cirrhosis,liver failure and even hepatocellular carcinoma.In western countries,NAFLD affects about 30%general population.In recent 20 years,the incidence of the disease in Asian countries has been increasing rapidly,and it has a trend of being younger.In Shanghai,Guangzhou,Hongkong and other developed areas,the prevalence of adult NAFLD is about 15%.The prevalence of the disease continues to increase globally,paralleled with the increase in obesity,has become a serious public health problem.The strategy of international treatment guidelines for early NAFLD mainly focus on lifestyle changes,the prevention and treatment of concomitant metabolic and cardiovascular complications.But with the development of the disease,we must use drugs for treatment of liver,to prevent the progression of liver disease,reduce or prevent liver cirrhosis,hepatocellular carcinoma and its complications.The lipid-lowering,hypoglycemic,antihypertensive drugs,such as statins,fibrates drugs,can reduce serum triglyceride,but are nonspecific to liver,also easily lead to liver toxicity,induce or aggravate liver injury.Insulin sensitizers metformin,had no obvious effect on the liver histological lesions.So the liver protecting drugs,especially specific treatment and reversal of hepatic steatosis,inflammation,fibrosis and cirrhosis is crucial to prevent the development of new drugs.NAFLD is the result of multiple factors,such as genetic environment metabolism stress,which is closely related to insulin resistance,hyperglycemia and hyperlipidemia.Previous studies have shown that,the key enzyme in Mevalonate pathway,Geranylgeranyl diphosphate synthase(GGPPS),can influence protein prenylation to,regulate hepatic glucolipids metabolism.Recently report revealed that silencing GGPPS in vivo can ameliorate obesity-induced hepatic steatosis,decrease the lipid contents in serum and tissues,and increase insulin sensitivity through altering the FPP/GGPP ratio.Here in,targeting GGPPS for protein prenylation related metabolism disorders has become an important research direction.In this study,we aimed at the conformation analysis of GGPPS protein,and in cooperation with the drug screening platform of Jiangsu Province Hospital on Integration of Chinese and Western Medicine,screened 46 compounds which may be combined with GGPPS specifically by computer simulation.Subsequently,we identified and developed a GGPPS inhibitor,4-Azidophlorizin,which has been reported as a SGLT2 inhibitor.It can inhibit renal reabsorption of glucose,but no studies have shown whether 4-Az can inhibit the formation of nonalcoholic fatty liver disease.Therefore,this study will focus on whether 4-Az can cause changes in expression through the combination with GGPPS,thereby inhibiting the accumulation of hepatic triglyceride and slowing down the development of NAFLD.First of all,we used highly expression GGPPS TSC2-null cell,as a pharmacological model,treated by the specific binding drug 4-Az at different concentrations and different times.The results showed that the expression of GGPPS was decreased in concentration and time dependent,but no change in transcription level.The small G protein RhoA were separated and enriched by immunoprecipitation,andthe organic and aqueous phase RhoA were separated by Tritton 114.The WB results showed that the level of isoprene G protein decreased after 4-Az treatment.Subsequently,we carried out the drug experiments at the cellular and animal levels respectively.We used 4-Az and DGBP to treat the liver lipid metabolism imbalance model-nonalcoholic fatty liver model mice.We found that the high fat diet induced wild type mice showed obviously fatty degeneration of the liver,CT scan showed increased lipid content in the liver,gross view of liver tissue showed pale and swelling appearance,HE staining showed bullous steatosis.While the phenotype in high fat diet mice treated with 4-Az were significantly get better,hepatic TG decreased and liver function improved.To explore the underlying mechanism,we proved that 4-Az can bind with GGPPS,promoted GGPPS degradation in a dose and time dependent manner via ubiquitin-proteasome pathway.Its pharmacological functions need to be further investigated.Thus,we found that 4-Az guide GGPPS toward the ubiquitin proteasome degradation.In FFA treated primary hepatocytes cell models and high-fat induced NAFLD mouse model,4-Az plays a significant role in reducing lipid accumulation.These results suggest that 4-Az may be an effective natural drug for NAFLD treatment.