Study On The Mechanism Of Pim-2 Regulating MTOR Signal In Atherosclerotic Inflammation

Background:Atherosclerosis（AS）is the main cause of cardiovascular and cerebrovascular diseases such as coronary heart disease and ischemic stroke.Further study of the pathogenesis of AS has positive significance.AS is a chronic inflammatory disease.This study hypothesized that Pim-2 can reverse the occurrence and development of AS by inhibiting inflammation.The mTOR signal may be its pathway to exert the anti-inflammatory effect of AS.Objective:Establish the atherosclerosis model in vivo and in vitro.Determine the expression of Pim-2,mTOR and related inflammatory factors under AS conditions.Overexpression or knockdown of Pim-2 to elucidate the role of Pim-2 in the inflammatory mechanism of AS.To investigate whether anti-AS inflammation of Pim-2 is related to mTOR signaling.Methods:In vitro,THP-1 cell-derived macrophages were treated with oxidized low-density lipoprotein（ox-LDL）and transformed into foam cells.Pim-2over-expressing cell line and Pim-2 knock-out cell line of THP-1 cells were constructed with lentivirus.Oil red O staining was used to detect the lipid droplets in foam cells.The cell TC/FC detection kit was used to detect the total cholesterol（TC）,free cholesterol（FC）and cholesteryl ester（CE）content of cells.Western blot was used to detect the Pim-2,mTOR,4EBP1,S6K1 and phosphorylation levels of foam cells.The levels of IL-6,MCP-1,TNF-αand TLR4 m RNA were detected by RT-PCR.In vivo,Apo E^-/-mice were injected with adeno-associated virus（AAV）2/8via the tail vein to construct Pim-2 overexpression and Pim-2 knockdown Apo E^-/-mice.Apo E^-/-mice were fed on a high-fat diet for 12 weeks to establish atherosclerosis model.Weight,blood glucose,and total cholesterol,low-density lipoprotein,and triglyceride levels were measured;Oil red O staining was used to detect plaques in the longitudinal and transverse sections of the aorta,then calculated the plaque area;Intra-arterial macrophages infiltration were detected by Mac-2 immunohistochemical;Western blot detected the protein expression levels of Pim-2,mTOR,4EBP1,S6K1 and its phosphorylation levels in aortic root plaques;Serum CRP,IL-6,TNF-αlevels detected by ELISA;The levels of IL-6,MCP-1,TNF-α,and TLR4 m RNA in the aortic root plaque were detected by RT-PCR.Results:（1）Western blotting analysis showed that Pim-2,p-mTOR（Ser2448）,p-S6K1（Thr398）and p-4EBP1（Thr37/46）were upregulated in high fat induced Apo E^-/-mice and foam cells.（2）In vitro,overexpression of Pim-2 can reduce the deposition of lipid droplets in foam cells,reduce the m RNA expression levels of inflammatory factors IL-6,MCP-1,TLR4,TNF-αand the content of TC,FC,CE in cells.In vivo,compared with the negative control group,mice with Pim-2overexpression had reduced aortic plaques,reduced macrophage aggregation,IL-6,TNF-α,TLR4,MCP-1 m RNA expression in the aortic root and serum levels of CRP,IL-6 and TNF-αdecreased;Western Blot analysis showed that p-mTOR,p-S6K1and p-4EBP1 were dephosphorylated after Pim-2 overexpression.（3）Knockdown of Pim-2 produced the opposite result,either in vivo or in vitro.Conclusions:Pim-2 has anti-AS inflammatory effect,which may be related to the dephosphorylation of mTOR signaling pathway,suggesting that Pim-2 may be the therapeutic target of AS.