The Mechanism Of Circadian Clock Gene Per2 Involved In Phenotypic Transformation Of Vascular Smooth Muscle Cells By Regulating Autophagy

Objective: To investigate the effect and mechanism of circadian clock gene Per2 involved in the transition of VSMCs from a contractile phenotype(VSMCs-C)into a synplastic phenotype(VSMCs-S)by constructing a phenotypic transformation model of vascular smooth muscle cells(VSMCs)in vitro.Methods:(1)Establish a phenotypic transformation model of VSMCs by Ang-Ⅱ(angiotensin Ⅱ)in vitro: the expression of α-smooth muscle actin(α-SMA)and osteopontin(OPN)were detected by western blotting in a set concentration and time gradient,so as to determine the optimal concentration and timing of Ang-Ⅱ stimulation and the success of the model.(2)VSMCs protein(α-SMA,OPN),Per2,autophagy markers(LC3 Ⅱ/LC3 I,p62)were detected: The cell models with phenotypic transformation were treated by silencing Per2 gene and overexpressing Per2 gene,respectively.The expression of α-SMA,OPN,Per2,LC3 Ⅱ/LC3 I and p62 in the cells was detected by western blotting.Results:(1)VSMCs were stimulated with different concentrations of AngⅡ for various time,the protein level of contractile marker α-SMA decreased,while the protein level of synthetic marker OPN increased.(2)Ang-Ⅱ-stimulated VSMCs was treated by silencing Per2(siRNA-Per2),the protein level of VSMCs synthetic marker OPN and autophagy marker LC3Ⅱ/LC3 I at the protein level was inhibited,and the inhibition of VSMCs systolic marker α-SMA and autophagy marker p62 at the protein level was reversed.While overexpression of Per2(pc DNA-Per2)in Ang-Ⅱ-stimulated VSMCs,VSMCs synthetic marker OPN and autophagy marker LC3 Ⅱ/LC3 I were more significantly increased in protein level,while VSMCs systolic marker α-SMA and autophagy marker p62 were more significantly decreased in protein level.Conclusion:(1)Ang-Ⅱ can effectively construct phenotypic transformation models of VSMCs in vitro.(2)The clock gene Per2 activates the expression of autophagy and promotes phenotypic transformation of VSMCs.(3)In addition,autophagy may be the key in regulating phenotypic transition of VSMCs by the clock gene Per2.