Metformin Inhibits The Proliferation And The Migration Of Primary Human Aortic Smooth Muscle Cells Through The P53-IFI16-AMPK Pathway

Objective: The proliferation and migration of vascular smooth muscle cells are significant in the development and progression of atherosclerosis and plaque rupture.Metformin,a widely used anti-diabetic drug,has been shown to inhibit cell growth and migration.The purpose of the present study was to investigate the effects of metformin on the proliferative and migratory capabilities of primary human aortic smooth muscle cells(HASMC)in vitro and to clarify the underlying mechanism.Methods: Senescence-associated ?-galactosidase staining,cell counting,cell counting kit-8(CCK8),cell cycle and cell transwell analysis were performed to detect the senescent,proliferative and migratory capabilities of HASMC at different passage numbers,including passage number 3 and passage number 7.Western blot was performed to detect the protein levels of P53,IFI16,AMPK and P-AMPK in the two groups.Immunofluorescence was performed to detect the endogenous P53 and IFI16 expression in the two groups.Following treatment with various concentrations of metformin(0,2,5,10 mmol/l)for 24 h,RT-PCR was performed to examine the mRNA levels of P53 and IFI16 in the various metformin treated HASMC(passage 3),western blot was performed to examine the protein levels of P53,P-P53(Ser-15),IFI16,AMPK and P-AMPK in the various metformin treated cells(passage 3).Cell counting,CCK8,cell cycle and cell transwell analysis were performed to detect theproliferative and migratory capabilities in various metformin treated HASMC.Following transfection with control small interfering RNA(siRNA)and P53 siRNA,RT-PCR and western blot were performed to detect the expression levels of P53,IFI16,AMPK and P-AMPK in the untreated group,control siRNA group and P53 siRNA group.Cell counting,CCK8,cell cycle and cell transwell analysis were performed to reassess the effects of metformin on P53-knockdown HASMC.Following transfection with control siRNA and IFI16 siRNA,RT-PCR and western blot were performed to detect the expression levels of P53,IFI16,AMPK and P-AMPK in the untreated group,control siRNA group and IFI16 siRNA group.Cell counting,CCK8,cell cycle and cell transwell analysis were performed to reassess the effects of metformin on IFI16-knockdown HASMC.Western blot was performed to detect the protein levels of P53,P-P53(Ser-15),IFI16,AMPK and P-AMPK in the P53-knockdown and IFI16-knockdown HASMC following by treatment with metformin(10mmol/l)for 24 h.Results: The present study observed that up-regulation of P53,interferon-inducible protein 16(IFI16)and AMPK in the senescent primary human aortic smooth muscle cells(passage 7),which exhibited a decrease in proliferation and migration.Metformin could activate P53,IFI16 and AMPK to inhibit the proliferation and the migration of the HASMC.Furthermore,the siRNA-mediated of P53 and IFI16 attenuated the AMPK expression and powerfully reversed the suppressive effects of metformin on HASMC.Interestingly,in response to metformin,the activation of AMPK was not observed in the P53 and IFI16 silenced HASMC.Conclusion: These results indicate that metformin suppresses theproliferation and migration of HASMC through the P53-IFI16-AMPK pathway.These findings suggest metformin has the potential for use in atherosclerosis therapy.