The Role Of SGK2 In The Occurrence And Development Of Atherosclerosis By Regulating The Autophagy Of Mononuclear Macrophage

Background:Acute coronary syndrome is a cardiovascular disease with a very high risk of death.Vulnerable plaques are crucial in the pathogenesis of acute coronary syndromes.Enlarged necrotic core is one of the features of vulnerable plaque,mainly caused by monocytes that phagocytose oxidized low density lipoprotein（ox-LDL）in the blood and turned into foam cells.Recent studies have shown that autophagy is closely related to atherosclerosis（AS）.It is a special form of autophagy,called lipophagy,which can transport cholesterol ester（CE）from macrophages to lysosomes,where it is degraded by lysosomal lipases.The lipolysis of acid lipase as a critical rate-limiting step of reverse cholesterol transportation（RCT）,will block the formation of foam cells.Our group found that SGK2 and autophagy marker LC3 were upregulated in peripheral blood monocytes of patients with coronary heart diseases.It was reported that inhibition of SGK2 expression in monocytes caused blockage of autophagic flow.Thus SGK2 promotion of autophagy may play a protective role in the development of atherosclerosis.Objective:This study was designed to investigate the effects of SGK2 on the development of atherosclerosis through regulation of autophagy in vivo and in vitro,and to identify new targets for the prevention and treatment of atherosclerosis.Methods:（1）To detect SGK2 expression in peripheral blood mononuclear macrophage of patients with coronary artery disease by Western blot（WB）;To evalute its relationship with lesion severity and autophagy-related protein expression.（2）THP-1 cell lines with SGK2 knockdown and overexpression were constructed with lentivirus;To establish a THP-1-derived macrophage foamy model with ox-LDL;The expression of LC3 and P62 were detected by WB method;Oil red O staining was used to evaluate cellular foaming;autophagy flux were observed by WB,m RFP-GFP-LC3 adenovirus immunofluorescence,and transmission electron microscopy（TEM）.（3）LDLR-SGK2-Lyz2-Cre（-/-,f/f,+）mice were bred with high-fat diet for 12W and 16W to construct atherosclerosis models,and plaques were assessed by oil red staining,immunohistochemistry,and Masson staining,and autophagy-related protein expression within plaques was assessed by immunofluorescence methods.Results:（1）Compared with CON and SA groups,the expressions of SGK2,LC3II/I and P62 in peripheral blood mononuclear cells of NSTE-ACS patients were increased,and SGK2 was positively correlated with the expression of LC3II/I、P62 and Gensini scores.The top 10 Hub genes analysed by PPI network in CAD patients were GRB2,ERBB2,EGF,JUN,FGFR1,HGF,NGF,FLT4,FGF7,IL-1β.JUN and FGFR1 expressions were decreased in SA and NSTE-ACS patients,and JUN was further decreased in NSTE-ACS group compared with SA group.The expression of GRB2 in NSTE-ACS patients was higher than that in CON and SA groups,but there was no significant difference between CON and SA groups.The expression of IL-1βincreased in SA and NSTE-ACS patients,and the expression of IL-1βin NSTE-ACS group was higher than that in SA group.In NSTE-ACS patients,the expression of SGK2 in peripheral blood mononuclear cells was negatively correlated with plaque load,while the expression of LC3II/I and P62 was positively correlated with plaque load,and the expression of SGK2 was significantly increased in carotid artery plaques.（2）Knockdown of SGK2 promoted ox-LDL-induced foaming of THP-1-derived macrophages,inhibited cholesterol efflux,and elevated the expression of both LC3II/I and P62;（3）Overexpression of SGK2 inhibited ox-LDL-induced THP-1-derived macrophage foaminess,promoted cholesterol efflux,decreased LC3II/I and P62 expression.More autophagic lysosomes was observed under electron microscopy in ox-LDL+oe-SGK2group than ox-LDLgroup,while in ox-LDL+sh-SGK2group shows more autophagosome,the above effects were reversed by the V-ATPase inhibitor(Bafilo mycin A1（Baf-A1）;（4）SGK2-Lyz2-Cre（f/f,+）(SGK2^CKO)mice,LDLR-SGK2-Lyz2-Cre（-/-,f/f,+）(LS^{c DKO})mice were successfully constructed;after 16 weeks of high-fat feeding,no atherosclerotic plaque formation was seen in the WT and SGK2^CKOgroups;after 16 weeks of high-fat feeding,the After16 weeks of high-fat feeding,atherosclerotic plaques were seen throughout the aorta in LS^{c DKO}mice and LDLR^-/-mice,and there was no difference in the area of positive oil red O staining,but the plaque fragility index was higher in LS^{c DKO}mice than in LDLR^-/-mice,and the expression of P62 was increased at the macrophage infiltration in atherosclerotic plaques in LS^{c DKO}aortic sinus;after 12 weeks of high-fat feeding,atherosclerosis was more severe in LS^{c DKO}mice than in LDLR^-/-mice.Plaque load was significantly greater in LS^{c DKO}mice compared with LDLR^-/-mice after 12 weeks of high-fat feeding.Conclusions:（1）SGK2 expression and autophagy in peripheral blood mononuclear cells were associated with the severity of coronary atherosclerosis.In NSTE-ACS patients,atherosclerotic plaque load was negatively correlated with SGK2 expression in peripheral blood mononuclear cells,and positively correlated with LC3II/I and P62 expression.（2）SGK2 regulates autophagy through V-ATPase,promotes cholesterol efflux,and inhibits THP-1-derived macrophages from foaming;（3）Conditional knockout of SGK2 in Macrophage can block autophagy flow and promote the development of atherosclerosis in LDLR^-/-mice.