Low Level Lead Regulates The VSMCs Proliferation And Migration Via AMPK/mTOR/FoxO3a Signaling Pathway

Objective:We have established an in vitro model which to evaluate the role of low level lead on VSMC proliferation?migration and the underlying mechanism.We aimed at clarifying the contribution of the AMPK/mTOR/FoxO3a signaling pathway.Methods:Male SD rats weighing 100-120 g were selected to isolate their abdominal aortas in a sterile environment,and primary VSMCs were cultured by tissue patch method.The experiment was divided into control group?low dose group(0.1 mmol/L)? high dose group(1.0mmol/L)? low dose + rapamycin group?rapamycin group,low dose + AMPK inhibitor group and AMPK inhibitor group.The ability of VSMCs to proliferate and migrate was detected by CCK8 test and scratch test;the localization of FoxO3a in the nucleus and the morphology of cytoskeleton were detected by fluorescence microscopy;the expression levels of PCNA?MMP-2?MMP-9?mTOR? p-mTOR?AMPK and p-AMPK proteins in VSMCs were detected by Western Blot method;the data were statistically analyzed by SPSS 21.0 software,P<0.05 was statistically significant.Result:1.CCK8 and scratch test showed that lead could induce VSMCs to proliferate and migrate,and the proliferation and migration were most obvious in the low dose group;2.Western Blot assay was used to detect PCNA,MMP-2 and MMP-9,which showed that the content of low-dose histone was significantly increased.3.Immunofluorescence results showed that low-dose lead could agglomerate and thicken F-actin;4.Immunofluorescence results showed that low-dose lead could cause nuclear translocation of FoxO3a from cytoplasm to nucleus;5.After the addition of rapamycin,FoxO3a was localized in the cytoplasm,nuclear translocation was inhibited,F-actin arranged loosely,myofilament was fine,no phenotypic transformation occurred,PCNA,MMP-2,MMP-9 protein expression content was significantly reduced,cell proliferation and migration were inhibited;6.After adding AMPK inhibitor,the content of p-mTOR increased,FoxO3a translocated into the nucleus,F-actin arranged in bundles,fluorescence intensity increased,PCNA and MMP-9 protein content increased,and cell proliferation and migration occurred.Conclusion: Low level lead induced cell proliferation and migration,the molecular regulation mechanism of this phenomenon may correlate with a reduced phosphorylation of AMPK.Lead inhibits AMPK phosphorylation and promote mTOR phosphorylation,and promoted the nuclear translocation of FoxO3a,induces redistribution of the actin cytoskeleton and formation of stress fibers,transformation occurs in cells,VSMCs switch from a contractile to a proliferative.The expression of the PCNA?MMP-2?MMP-9 protein was significantly elevated,this implies cell proliferation and migration.In summary,we demonstrated that lead induces the proliferation and metastasis of VSMCs via AMPK/mTOR/FoxO3a signaling pathway.