Study On The Circadian Clock Mechanism Of Ang? Induced Proliferation Of Vascular Smooth Muscle Cells Through RAS/ERK Pathway

Objective:To explored Ang?-induced expression of circadian clock genes Per1 and Per2 in VSMCs,thereby affecting the abnormal proliferation of VSMCs by activating the RAS/ERK pathway,which in turn affects the circadian rhythm of blood pressure.Methods:Tissue adherence method was used to extract primary vascular smooth muscle cells of SD rats,stimulated with 50%FBS for 2 hours for cell timing,which was recorded as Control group,add 100 nM Ang?and record it as the Ang?group,add the inhibitors VAL,U0126 and SCH772984 as the inhibitor group.sampled at CT3,CT 7,CT 11,CT 15,CT 19,CT 23.RT-PCR was used to detected the expression of Per1,Per2,and Cyclin E genes in each group;WB was used to detected the expression of Per1,Per2,RAS,MEK,p-MEK,ERK,p-ERK and Cyclin E proteins in each group.Flow cytometry was used to detected cell cycle distribution,MTT method was used to detected cell viability.The expression of RAS,MEK,p-MEK,ERK,p-ERK and Cyclin E protein was detected by WB and the expression of Cyclin E gene was detected by RT-PCR after silencing Per1 and Per2 genes by SiRNA transfection in each group.Results:?1?Ang?can induce abnormal proliferation of VSMCs,and there is a dose effect,which is related to the induction of high expression of Cyclin E gene.VAL can reverse the high expression of Cyclin E gene induced by Ang?.?2?The expression of Per1,Per2,and Cyclin E genes in VSMCs induced by Ang?was significantly increased during short-term stimulation,and decreased significantly during long-term stimulation.There was a 12-hour phase difference between the expression of circadian protein Per1,Per2 and gene expression.VAL can blocked the high expression of Per1,Per2 and Cyclin E gene induced by Ang?and regulated the abnormal proliferation of VSMCs.?3?After silencing Per1 and Per2 genes,VSMCs showed G₁/S phase block,the expression of Cyclin E gene and protein was significantly down-regulated.?4?Ang?-induced RAS protein expression,MEK and ERK phosphorylation levels in VSMCs were consistent with Per1,Per2 protein expression within 24 h.Silencing Per1 and Per2 genes significantly inhibited RAS protein expression,MEK and ERK phosphorylation levels.?5?Added RAS/ERK pathway inhibitor U0126 and SCH772984,VSMCs cells showed G₁/S phase block,and the expression of Cyclin E gene and protein were decreased significantly.Conclusion:AngII induces high expression of the circadian clock genes Per1 and Per2 in VSMCs,thereby activating the RAS/ERK pathway,which in turn affects the expression of Cyclin E genes and proteins,and ultimately affects the proliferation of VSMCs.At the same time,VAL inhibits the expression of the circadian clock genes Per1 and Per2 by blocking the binding of AngII to the AT1 receptor,and inhibits cell proliferation through the RAS/ERK pathway.