Effect And Mechanism Of Squalene On Proliferation And Apoptosis Of A7r5 Cells Induced By Angiotensin Ⅱ

Objective:1.To study the effect of Squalene(SQ)on the proliferation of rat vascular smooth muscle cells A7r5 induced by angiotensin Ⅱ(Angiotensin Ⅱ,AngⅡ).2.To study the effect of SQ on the apoptosis of rat vascular smooth muscle cells A7r5 induced by AngⅡ.3.To study the mechanism of SQ on the proliferation and apoptosis of rat vascular smooth muscle cells A7r5 induced by AngⅡ.Methods:1.MTT and CCK-8 experiments were used to determine the toxicity of AngⅡ and SQ to A7r5 cells,and the effective concentration and time of AngⅡ and SQ were determined.2.AngⅡ-induced proliferation of A7r5 cells was used to construct a model of AngⅡ-induced cell overproliferation.3.The level of reactive oxygen species(ROS)was detected by fluorescent probe DCFH-DA method,the level of total superoxide dismutase(SOD)was detected by WST-8 method and the concentration of NO was detected by Griess Reagent method.4.Based on network pharmacology,possible targets and potential signaling pathways of SQ regulating cell proliferation and apoptosis were analyzed,and molecular docking of potential core target proteins was conducted.5.Hoechst staining was used to observe the apoptosis of cell overproliferation models treated with different concentrations of SQ;Annexin V/FITC flow cytometry was used to detect the effects of different concentrations of SQ on the cell cycle and apoptosis of the overproliferation model6.Western blotting was used to detect the expression of cyclic-related proteins Cdk2,PCNA and apoptosis-related proteins Bax,Bcl-2 and Caspase3 by SQ.Results:1.AngⅡ had no effect on the survival rate of A7r5 cells when the concentrations of AngⅡ were 0,1,2,4 and 8μg/mL by MTT assay and the time was 6,12,24,48h.CCK-8 assay had no effect on the survival rate of A7r5 cells when the concentrations of SQ were 0,8,16,32 and 64μg/mL by CCK-8 assay and the time was 0-48h.2.The model of AngⅡ induced A7r5 cell overproliferation was established.When the induction concentration was 2μg/mL and the induction time was 24h,the proliferation effect of A7r5 cells was the most significant,and the cell morphology was good and the state was stable.Therefore,A7r5 cells were treated with 2 μg/mL AngⅡ for 24 h to establish the cell overproliferation model.3.SQ can regulate proliferation,reduce ROS concentration in overproliferated model cells,and increase NO and SOD concentration in model cells.4.Network pharmacological analysis showed that the possible targets of SQ regulating cell proliferation were EP300,PPARA,RXRB,AR,CYP2E1,CAMK4,HMOX1,and CYCs,and the potential signaling pathways were MAPK/ERK(DAPK),VEGF(P300/CBP,HO-1),PPAR(RXR,PPARA),c AMP(CAM /Ca MKs,CBP,PPARA),Bax/Bcl-2/Caspase3(CYCs),etc.5.Flow cytometry showed that the cell cycle of the model group mainly stayed in the S phase,while the SQ concentrations of 32 and 64μg/mL acted on the overproliferating model cells,and the cells staying in the S phase were significantly reduced with statistical significance(P<0.05).The apoptosis rate of the overproliferating model group was lower than that of the normal group,and the apoptosis rate of the overproliferating model cells was increased with the increasing concentration of SQ in 16,32,64 μg/mL.6.SQ down-regulated the expression of cyclic-related proteins CDK2 and PCNA,and the difference was statistically significant(P<0.05).SQ up-regulated the expression of apoptosis-related protein Bax and Caspase3,and down-regulated the expression of apoptosis-related protein Bcl2,with statistical significance(P<0.05).Conclusion:SQ can effectively inhibit the proliferation of A7r5 cells induced by AngⅡ,and the inhibitory effect is best when 64μg/mL SQ is treated for 24h.SQ can inhibit AngⅡ-induced changes in the concentration of ROS,NO and SOD in A7r5 cells,and has a protective effect on cells.The mechanism by which SQ inhibits the proliferation of A7r5 cells induced by AngⅡ is achieved by down-regulating the expression of Cdk2 and PCNA and arresting the cells in S phase.SQ can effectively promote the apoptosis of A7r5 cells inhibited by AngⅡ.The mechanism is achieved by up-regulating the expression of BAX and Caspase3 and down-regulating the expression of Bcl-2.