Effects Of Scutellarin On Proliferation And Migration Of A7r5 Cells And Regulation Of PI3K/Akt/mTOR Signaling Pathway

ObjectivesTo investigate the effects of scutellarin on proliferation and migration of A7r5 in rat thoracic aortic smooth muscle cells induced by oxidized low density lipoprotein(ox-LDL),to observe the effects of scutellarin on the mRNA and protein expression of PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR,Bax,BCL-2 and Caspase-3,and to explore the regulation of scutellarin on PI3K/Akt/mTOR signaling pathway,so as to provide evidence for clinical application of scutellarin in treatment of atherosclerosis.Methods1.Rat thoracic aortic smooth muscle cells(A7r5)were cultured in vitro.The effects of different concentrations of ox-LDL(25 mg/L、50 mg/L、75 mg/L 和 100 mg/L)on the A7r5 cells activity were investigated by CCK-8 assay to determine the optimal induction concentration and time of ox-LDL.2.The effects of scutellarin on the proliferation of ox-LDL-induced A7r5 cells were tested by CCK-8 assay.Experimental groups:normal control group,model group,simvastatin group(15 μmol/L),scutellarin group with different concentrations(50 μmol/L,75 μmol/L,100 μmol/L).3.The effects of scutellarin on the migration of ox-LDL-induced A7r5 cells were observed by Culture-Insert 2 well method.4.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to examine the effects of scutellarin on mRNA expressions of PI3K,Akt,mTOR,Bax,BCL-2 and Caspase-3 in ox-LDL-induced A7R5 cells.5.The effects of scutellarin on protein expressions of mTOR,Bcl-2,Bax and Caspase-3 in ox-LDL-induced A7R5 cells were detected by immunofluores cence.6.Western blot assay was used to determine the effects of scutellarin on protein expressions of PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR,Bax,BCL-2 and Caspase-3 in ox-LDL-induced A7R5 cells.Results1.The effects of different concentrations(25 mg/L、50 mg/L、75 mg/L 和 100 mg/L)of ox-LDL on the A7r5 cells activity:compared with the normal group,all groups significantly promoted the proliferation of A7r5 cells after 24h and 48h,and ox-LDL at 75mg/L had the best effect on the proliferation of a7r5 cells.Therefore,ox-LDL at 75mg/L was selected as the optimal induction concentration and 24h as the optimal induction time in following experiment.2.The effects of different concentrations of scutellarin on the proliferation of ox-LDL-induced A7R5 cells:Compared with normal group,the proliferation rate of A7r5 cells in model group was significantly increased.Compared with the model group,the proliferation rate of A7r5 cells in simvastatin group and scutellarin groups of 50,75,and 100 μmol/L was decreased significantly after 48 hours.3.The effects of scutellarin on the migration of ox-LDL-induced A7R5 cells:Compared with normal group,migration distance of A7r5 cells in model group increased significantly.Compared with the model group,the migration distance of A7r5 cells in simvastatin group and scutellarin groups of 50,75,and 100 μmol/L decreased significantly.4.RT-qPCR results showed that compared with normal group,the mRNA expressions of PI3K,Akt,mTOR and BCL-2 in model group increased,while the mRNA expressions of Bax and Caspase-3 decreased.Compared with the model group,the mRNA expressions of PI3K,Akt,mTOR and BCL-2 in simvastatin group and scutellarin groups of 50,75,and 100 μmol/L decreased,while the mRNA expressions of Bax and Caspase-3 increased.5.The results of immunofluorescence showed that compared with normal group,the levels of protein expressions of mTOR and BCL-2 in A7r5 cells were markedly up-regulated in model group,while the protein expressions of Bax and Caspase-3 were down-regulated.Compared with the model group,the protein expressions of BCL-2 and mTOR in 100 μmol/L scutellarin group decreased,while the protein expressions of Bax and Caspase-3 increased.6.Western blot results showed that compared with normal group,the protein expressions of PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR and BCL-2 in model group increased,while the protein expressions of Bax and Caspase-3 decreased.Compared with the model group,the protein expressions of PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR and BCL-2 in simvastatin group and scutellarin groups of 50,75,and 100μmol/L decreased,while the protein expressions of Bax and Caspase-3 increased.Conclusions1.Scutellarin had obvious inhibitory effects on the proliferation and migration of ox-LDL-induced A7R5 cells.2.Scutellarin significantly down-regulate mRNA expressions of PI3K,Akt,mTOR and BCL-2 in ox-LDL-induced A7R5 cells,and up-regulate mRNA expressions of Bax and Caspase-3;scutellarin also down-regulate protein expressions of PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR and BCL-2 in ox-LDL-induced A7R5 cells,and up-regulate protein expressions of Bax and Caspase-3.3.The inhibitory effects of scutellarin on the proliferation and migration of ox-LDL-induced A7R5 cells may had close regulation of the PI3K/Akt/mTOR signaling pathway.