RGFP966 Inhibits Palmitic Acid Induced VSMCs Autophagy By Regulating ATGL

Metabolic disorders are the common pathological basis of several cardiovascular events,and lipid disorders play a key role in the pathology of atherosclerosis.Phenotypic transformation of vascular smooth muscle cells(VSMCs)causing abnormal cell proliferation and migration is an important pathological basis of atherosclerosis(AS),but the mechanism of phenotypic regulation remains to be elucidated.It has been shown that adipose triglyceride lipase(ATGL)is a rate-limiting enzyme of triglyceride catabolism and is closely related to the process of lipid droplet catabolism.RGFP966,a highly selective small molecule inhibitor of histone deacetylase 3(HDAC3),not only penetrates the blood-brain barrier,but also has other pharmacological effects to be developed.It regulates the activity of metabolic enzymes through the dynamic regulation of histone acetylation.This study aims to investigate the effect of RGFP966 on the functional changes of vascular smooth muscle cells(VSMCs)after palmitic acid(PA)treatment at the cellular level,and to suggest new research directions for the treatment of AS.Objectives:(1)To explore the role of RGFP966 on PA-induced phenotypic transformation of VSMCs;(2)to explore whether RGFP966 is involved in PA-induced phenotypic transformation of VSMCs through the regulation of ATGL;(3)to investigate whether the protective effect of RGFP966 on PA-induced disruption of lipid metabolism in VSMCs acts through the autophagy.Methods:(1)Human aortic smooth muscle cell line(HAVSMC)was used to establish a phenotypic transformation model of VSMCs using PA.Western blot was performed to detect the effect of RGFP966 on the acetylation level of histone H3 and the regulation of marker proteinsα-SMA and Vimentin in the phenotypic transformation model.Cell morphology was observed by immunofluorescence technology,CCK-8and Ed U assays were used to detect the proliferation ability of VSMCs.Scratch assay was used to detect the migration ability of VSMCs;(2)Western blot was used to detect the effect of PA or(and)RGFP966 on ATGL in VSMCs,and also oil red assay was used to detect the production of lipid droplets.The further intervention was performed using ATGL si RNA,and Western blot detected the effect of three ATGL silencing sequences on ATGL expression,and screened with the highest silencing efficiency for subsequent experiments.CCK-8 and Ed U assays were performed to detect the proliferation ability of VSMCs,scratch assay was used to detect the migration ability of VSMCs.Oil red assay was used to observe the generation of lipid droplets;(3)VSMCs were first treated with PA or(and)RGFP966,and the expression of p62 and LC3 was detected by Western blot,and then the autophagy inhibitor 3-MA was intervened to detect the effect on ATGL expression.Results:(1)Compared with normal VSMCs,PA was able to significantly induce VSMCs to undergo a phenotypic transformation,and down-regulate the expression of contractile marker protein α-SMA and Vimentin,induce abnormal proliferation and migration of VSMCs.It could also down-regulate the expression of ATGL,and lead to lipid accumulation.RGFP966 could up-regulate the acetylation level of histone H3,reverse PA-induced proliferation and migration.It could up-regulate the expression of ATGL,and promote lipid droplet degradation,but had no significant effect on normal cells;(2)the third silent sequence was selected for a follow-up experiments,and ATGL si RNA pre-intervention was able to promote the role of RGFP966 the inhibition of PA-induced phenotypic transformation of VSMCs,and the proliferation and migration of VSMCs,accompanying the lipid droplet accumuation;(3)RGFP966 could up-regulate PA-induced the expression of p62,and down-regulate the expression of LC3-II/LC3-I.PA significantly decreased the expression of ATGL,while 3-MA was able to reverse the effects of PA without affecting ATGL expression in normal VSMCs.Conclusions:(1)PA could induce aberrant proliferation and migration in VSMCs,and down-regulate ATGL expression.It could also lead to lipid accumulation.RGFP966 could reverse PA-induced proliferation and migration,and up-regulate ATGL expression.It could also promote lipid droplet degradation;(2)RGFP966 regulates ATGL against PA-induced lipid disorders in VSMCs as verified at the cellular level by pre-intervention treatment with ATGL si RNA;(3)the protective effect of RGFP966 against PA-induced lipid metabolism disorders of VSMCs acts through autophagy.