Perilipin 5 Promotes Lipid Droplet Formation In Hepatic Stellate Cells And Inhibits Their Activation In Vitro

Objectives Non-alcoholic fatty liver disease(NAFLD)is a worldwide chronic liver disease that includes a series of continuous diseases,ranging from simple steatosis to non-alcoholic steatohepatitis(NASH),liver fibrosis,cirrhosis and even hepatocellular carcinoma(HCC).Liver fibrosis is an important pathophysiological process in the development of NAFLD.Because its specific pathogenesis is still unclear,there is still no specific treatment.The occurrence of hepatic fibrosis depends on the activation of hepatic stellate cells(HSCs).A large amount of LDs is stored in static HSCs.When chronic liver damage occurs,HSCs are activated,accompained by loss of a large amount of LDs and excessive production of ?-SMA and collagen,leading to the increase of extracellular matrix(ECM)and triggering liver fibrosis.Therefore,the loss of intracellular fat is considered as a hallmark of HSC activaton.Perilipin 5(Plin 5),as a newly discovered lipid drop(LDs)related protein of Perilipin family,is highly expressed in the oxidized tissues,such as liver,brown adipose tissue(BAT),heart and skeletal muscle.Previous studies have shown that Plin 5 plays an important role in regulating LDs metabolism and intracellular lipid balance,but its specific role and mechanism in the activation of HSCs is still unclear.In this study,the primary HSCs were isolated and cultured in vitro to analyze the effects of different fragments of Plin 5 on LDs formation,and to explore the relationship between full-length Plin 5 and LDs formation in HSCs and the activation of HSCs,providing a new target for the treatmet and even reversing liver fibrosis.Methods(1)Plin 5 c DNA was truncated into different fragments,amplied and loaded into Lenti-p FLRu-YFP vector to product lenti-virus with different fragments of Plin 5,the functions of each fragment were determined.Full-length Plin 5 was used in all the experiments;(2)Fatty liver with liver fibrosis in mice model induced by high-fat and high-fructose diet(HFF),real-time PCR was used to detect the contents of Plin 5 and collagen in HSCs.The contents of Plin 5 in HSCs cultured for 1 day and 7 days were detected by real-time PCR and western blot in vitro;(3)Recombinant lentivirus p FLRu-PLIN 5 or vector lentivirus p FLRu-YFP transducted activated HSCs,and the lipid contents in HSCs were detected by real-time PCR and western blot;(4)Leica DM 4000 B fluorescence microscophe was used to observe the effect of exogenous Plin 5 on the formation of LDs in HSCs;(5)Real-time PCR and western blot were used to detect the effect of exogenous Plin 5 on the expression of HSCs activation markers;(6)MTS technology was used to detect the effect of exogenous Plin 5 on the growth of HSCs,and western blot was used to detect the effect of exogenous Plin 5 on the expression of cell cycle-related genes and growth-related genes.Results(1)The domain of amino acid 1-116(aa1-116)of Plin 5 was shown to inhibit the activation of HSCs,promoted the formation of LDs,and increased the contents of intracellular TG and FA.The amino acids 117-463 of Plin 5(the expression of the domain aa117-463)did not show this function;(2)Plin 5 decreased in activated HSCs in vivo and in vitro(P < 0.05);(3)Exogenous Plin 5 increased the contents of FA and TG in HSCs(P < 0.05);(4)Plin 5 facilitated the formation of LDs in HSCs in vitro;(5)Exogenous Plin 5 decreased the expression of fibrosis-related genes ?-SMA and collagen I(P < 0.05)and increased the expression of PPAR?(P < 0.05);(6)Exogenous Plin 5 inhibited the growth of HSCs and significantly decreased the expression of cell cycle-related gene Cyclin D1 and growth-related genes PDGF-?R,CTGF,TGF?-RI and TGF?-RII.Conclusions(1)The main functional regions of Plin 5 are located in the structural domain of amino acid 1-116(aa1-116)of Plin 5;(2)Plin 5 can promote the formation of LDs in HSCs,increase the cell fat cotents and inhibit the activation and growth of HSCs.