Celasterol Ameliorates NAFLD As An Allosteric SIRT6 Activator

Background:Non-alcoholic fatty liver disease(NAFLD)is a common chronic disease,which is one of the main factors contributor to hepatic steatosis in addition to alcohol,drugs and other definite liver risk factors.NAFLD is closely related to and interact with insulin resistance,obesity,type 2 diabetes mellitus and dyslipidemia,etc..Currently,there are no effective drugs for NAFLD,To find safe and effective drugs are urgently desired.SIRT6 is a member of highly conserved NAD+ dependent deacetylase and ADP-ribosyltransferase family.It plays an important role in regulating life cycle,cancer,obesity,inflammatory response,insulin resistance and energy metabolism.SIRT6 maintains the homeostasis of glucose and lipid by participating in regulation of glucose and lipid metabolism,insulin secretion and other metabolic pathways.Therefore,activation of SIRT6 may be a potential target for the treatment of nonalcoholic fatty liver disease.Celastrol is one of the main active ingredients of the Chinese medicincal herb--Tripterygium wilfordii.It has strong anti-obesity and hypoglycemic effects,but the underlying mechanism has not been fully elucidated,which needed further study.Objective:In the present study,we will screen celastrol and its derivatives based on the SIRT6 deacetylase activity assay to identify potential SIRT6 agonists and study the pharmacological effects as well as molecular mechanisms of SIRT6 allosteric agonists in the treatment of NAFLD and metabolic syndrome,and reveal new targets of Celastrol in the treatment of NAFLD.This research might provide data for the development of therapeutic drugs for NAFLD.Methods:1.Based on in vitro SIRT6 deacetylase activity assay system,celastrol and its derivatives were screened for activation of SIRT6 deacetylase activity.2.The effects of celastrol on lipid accumulation in hepatocytes in vitro and on fat deposition of Caenorhabditis elegans(C.elegans)were evaluated.3.High-fat diet-induced(DIO)obese C57BL/6 mice,leptin receptor deficient db/db mice,leptin deficient ob/ob mice,and MCD died-induced non-alcoholic steatohepatitis(NASH)mice were used to evaluate the efficacy of Celastrol in the treatment of glycolipid metabolic disorders and hepatic steatosis.Combined with physiology monitoring and morphalogical asaay,the relationship between the pharmacodynamics and the activation of SIRT6 was also studied.And normalC57BL/6 mice were administered to determine whether celastrol had toxic and side effects on the liver of mice.4.Liver-specific SIRT6 knockout mice were used to validate the efficacy of celastrol on the treatment of glycolipid metabolic disorders and hepatic steatosis.5.The effects of celastrol on VLDL secretion,oxidative stress and endoplasmic reticulum stress,etc were tested in the hepatic steatosis mice.Results:1.Celastrol and one of its derivatives were found to activate SIRT6 deacetylase in vitro.Moreover,celastrol binds to SIRT6 allosteric sites and works as a SIRT6 allosteric activator.2.Celastrol could improve lipid accumulation in hepatocytes in vitro and significantly reduce fat deposition of C.elegans,suggesting that celastrol could regulate lipid metabolism.3.Animal experiments proved that celastrol could ameliorate glucose and lipid metabolism disorder,hepatic steatosis caused by a high-fat diet and genetic defects,and NASH caused by malnutrition.The celastrol increased the activity of SIRT6 in liver of these models.Moreover,no obvious toxic and side effects on normal mice were observed.4.The therapeutic effect of celastrol on NAFLD weakened in liver-specific knockout SIRT6 mice.5.Celastrol promoted the excretion of triglycerides from the liver via the secretion rate of VLDL.6.The improvement of steatosis by celastrol was not related to oxidative stress and endoplasmic reticulum stress.Conclusions:Celastrol is a SIRT6 allosteric agonist.Celastrol may exert the modulation effects on glycolipid metabolism disorder and NAFLD via the activation of SIRT6 deacetylase.