The Mechanism Of Salidroside Alleviated Lipid Accumulation Via Activating Autophagy In HepG2 Cells

Lipid metabolism includes the biosynthesis and degradation of lipids such as fatty acids,triglyceride and cholesterol.Liver is the main organ that regulates lipid homeostasis,providing the body with energy by regulating lipid metabolism and storing excess lipids in the form of lipid droplets(LDs).Excessive accumulation of LDs in the liver is known as hepatic steatosis,it will further result in lipid toxicity to hepatocytes.Autophagy is a process of cell self-degradation,it plays a vital role in maintaining energy balance and homeostasis during the critical period of growth and development and in coping with nutritional stress.The autophagic degradation process of LDs is called lipophagy,and lipophagy is not only essential for lipid metabolism in cells,but also closely related to a variety of metabolic abnormalities(such as obesity,hepatic steatosis,arteriosclerosis,etc).Salidroside,a compound of p-hydroxyphenylethyl-?-glucoside,is the main active ingredient in Rhodiola rosea,which has anti-aging,anti-cancer,liver protection,anti-virus,anti-oxidation,anti-inflammatory,lipid-lowering and other functions.The researches on salidroside domestic and overseas mainly focused on its anti-oxidation and anti-inflammation,and most studies on its lipid-lowering function were the elaboration of the effect,including salidroside could reduce the accumulation of LDs and triglycerides and exert lipid-lowering function through AMPK signaling pathway,etc.However,the mechanism of salidroside's lipid-lowering function still needs to be further explored,especially the role of autophagy in salidroside's lipid-lowering process.Therefore,the purpose of this study was to investigate the effects of salidroside on lipid accumulation and autophagy in HepG2 cells,and investigated the mechanisms further.The main research contents of this research are as follows:(1)Salidroside could reduce the lipid accumulation of palmitic acid-induced HepG2 cells.HepG2 cells were induced with 200 ?M palmitic acid to establish hyperlipidemic model.The non-toxic dose ranges of salidroside were 0-40 ?M by MTT assay.The LDs were stained by BODIPY and Oil Red O staining to observe the effect of salidroside on the number of LDs in cells.The intracellular TC and TG contents were measured by the intracellular total cholesterol(TC)assay kit and the intracellular triglyceride(TG)assay kit.The results showed that the number of LDs and the TC and TG contents increased significantly after treatment with palmitic acid,while after treatment with salidroside(2.5,5,10 ?M),the number of LDs and the contents of TC and TG were significantly lower than those treated with palmitic acid,which indicated that salidroside had lipid-lowering effect.The expression of proteins related to fatty acid and cholesterol synthesis were detected by Western blot,it was found that salidroside(2.5,5,10 ?M)significantly decreased the expression of sterol regulatory element binding protein 1(SREBP-1),sterol regulatory element binding protein 2(SREBP-2),fatty acid synthase(FAS)and 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR).(2)Salidroside activated palmitic acid-induced autophagy in HepG2 cells via SIRT1/mTOR signaling pathway.MDC staining was used to observe the number of autophagosomes,and adenovirus transfection was used to observe the autophagy flux,the expression of microtubule-associated protein 1 light chain 3(LC3),p62/SQSTM1 and transcription factor EB(TFEB)protein were detected by Western blot,and lysosomal p H which was detected by Lyso-Tracer Red probe and AO staining showed that palmitic acid could inhibit the autophagy in HepG2 cells,while salidroside could activate the palmitic acid-inhibited autophagy in HepG2 cells and decrease the lysosomal p H.Pretreatment with SIRT1 inhibitor EX-527 could significantly inhibit the activation of autophagy by salidroside,the results showed that salidroside activated palmitic acid-inhibited autophagy in HepG2 cells via SIRT1/mTOR signaling pathway.(3)Salidroside activated autophagy via SIRT1/mTOR signaling pathway to alleviate palmitic acid-induced lipid accumulation in HepG2 cells.HepG2 cells were treated with chloroquine(CQ),an autophagy inhibitor,and EX-527,an SIRT1 inhibitor,respectively.The lipid metabolism-related indexes and protein levels were determined to explore the effects of CQ on salidroside activated autophagy and lipid-lowering function and whether SIRT1/mTOR signaling pathway was involved in this process.The results showed that salidroside activated autophagy through SIRT1/mTOR signaling pathway and then decreased lipid accumulation.Therefore,we demonstrated that salidroside alleviated lipid accumulation in palmitic acid-induced HepG2 cells by activating autophagy through the SIRT1/mTOR pathway.In conclusion,this study demonstrated that salidroside could alleviate palmitic acid-induced lipid accumulation in HepG2 cells,and salidroside could activate palmitic acid-inhibited autophagy in HepG2 cells via SIRT1/mTOR signaling pathway,it was further demonstrated that salidroside could improve palmitic acid-induced lipid accumulation in HepG2 cells by activating autophagy via SIRT1/mTOR signaling pathway.This study provid an experimental basis for salidroside to intervene lipid accumulation in cells and develop healthy food,which has certain application value.