| [Objective]This study was to investigate the effect of hydroxysafflor yellow A and puerarin on the expression of EA.hy926-related apoptosis protein in human umbilical vein endothelial cells induced by hypoxia-reoxygenation,and explore the regulatory mechanism of the PI3K/Akt signaling pathway during this intervention.Providing a theoretical basis for the clinical treatment strategies of Chinese medicine safflower and Pueraria preventing myocardial ischemia-reperfusion injury[Methods]1.The HSYA(0,10-7,10-6,10-5,10-4mol/L),PUE(0,10-7,10-6,10-5,104mol/L),LY294002(10,15,20?mol/L)on the cell viability in different hypoxia(8h,12h),reoxygenation(4h,8h,12h)time were detected by MTT.Screening for the appropriate hypoxia-reoxygenation time and the concentration of HSYA,PUE and LY294002 for EA.hy926 endothelial cells.2.Western blot was used to detect the effects of HSYA and PUE pretreatment on the protein expression of B cell lymphoma/leukemia-2(Bcl-2),Bcl-2 related X protein(Bax),activated aspartate proteolytic enzyme-3(cleaved Caspase-3),and activated aspartate proteolytic enzyme-9(cleaved Caspase-9).3.Western blot was used to detect the effects of pretreated cells from HSYA and PUE on the expression of p-Akt and Akt proteins in the PI3K/Akt signaling pathway,and the effects of PI3K/Akt inhibitor LY294002 on the protein expression of p-Akt,Akt,Bcl-2,Bax,cleaved Caspase-3,cleaved Caspase-9.4.Real-time PCR was used to detect the gene expression of Bax and Bcl-2 after hypoxia for 12h and reoxygenation for 8h,and the effects of HSYA,PUE pretreatment on Bax and Bcl-2 expression.5.Hoechest staining was used to detect apoptosis after hypoxia for 12h and reoxygenation for 8h,and the effects of HSYA and PUE pretreatment on apoptosis.6.Flow cytometry was used to detect apoptosis after hypoxia for 12h and reoxygenation for 8h,and the effect of HSYA pretreatment on apoptosis.[Result]1.Hypoxia reoxygenation time and drug concentration screening.The results of MTT showed that compared with the control group,the activity of EA.hy926 cells decreased significantly after hypoxia for 8h,12h,and reoxygenation for 4h,8h,12h respectively.Among them,the decrease of cell viability was significantly decreased after hypoxia for 12h and reoxygenation for 8h(P<0.001).Therefore,the hypoxia 12h reoxygenation 8h was taken as the time point of hypoxia reoxygenation.For drug pretreatment,the concentrations of HSYA are 10-7,10-6,10-5,and 10-4mol/L respectively.The experimental results show that compared with the hypoxia-reoxygenation group,after pretreatment of cells with 10-5mol/L HSYA and 10-4mol/L HSYA,both drug concentrations can significantly improve cell viability after hypoxia-reoxygenation.10-5mol/L is particularly obvious(P<0.001),Therefore,10-5mol/L is the concentration of EA.hy926 in endothelial cells treated by HSYA.The PUE screening concentration is 10-7,10-6,10-5,10-4mol/L,Studies have shown that compared with the hypoxia-reoxygenation group,PUE 10-7mol/L pretreatment cells can significantly improve cell viability after hypoxia-reoxygenation(P<0.001).Therefore,10-7 mol/L was used as the concentration of EA.hy926 cells pretreated by PUE.The concentration of PI3K inhibitor LY294002 was screened at 10,15,20 ?mol/L.The results showed that compared with the control group,the concentration of LY294002 at 10 ?mol/L had no effect on cell viability.Therefore,10 ?mol/L was the concentration of LY294002 treated cells.2.Effects of Hypoxia and Reoxygenation on Apoptosis.Flow cytometry and Hoechest kit were used to detect the apoptosis of EA.hy926 endothelial cells after hypoxia-reoxygenation.Flow cytometry results showed that compared with the control group,apoptosis was significantly increased after hypoxia for 12h and reoxygenation for 8h(P<0.001).The Hoechest staining results showed that compared with the control group,the cells in the hypoxia for 12h and reoxygenation for 8h group exhibited a significantly reduced morphology of deep stained nuclei,indicating that apoptosis increased after hypoxia-reoxygenation.3.Effect of hypoxia and reoxygenation on the expression of apoptotic proteins.Western Blot showed that compared with the control group,the expressions of pro-apoptotic proteins cleaved Caspase-9,cleaved Caspase-3,and Bax were significantly increased,and the expression of anti-apoptotic protein Bcl-2 was significantly reduced after hypoxia for 12 hours and reoxygenation for 8 hours.Real-time PCR showed that compared with the control group,the expression of anti-apoptotic gene Bcl-2 decreased significantly after hypoxia for 12 hours and reoxygenation for 8 hours(P<0.001).Although Bax expression increased,it was not statistically significant.4.Effects of HSYA or PUE pretreatment on apoptosis of EA.hy926 cells after hypoxia and reoxygenation.Flow cytometry results showed that compared with the hypoxia-reoxygenation group,HSYA pretreatment significantly reduced apoptosis(P<0.01).In Hoechest staining,compared with the hypoxia-reoxygenation group,the nuclear deep staining morphology of the HSYA pretreatment group was significantly reduced.Hoechest results showed that compared with the hypoxia-reoxygenation group,the deep staining morphology of endothelial cells in the PUE pretreatment group was significantly reduced.The above experimental data show that pretreatment of cells with HSYA or PUE can reduce endothelial cell apoptosis after hypoxia-reoxygenation.5.Effects of HSYA or PUE pretreatment on the expression of apoptotic genes and proteins in EA.hy926 cells after hypoxia and reoxygenation.Western Blot showed that compared with the hypoxia-reoxygenation group,the expression levels of pro-apoptotic proteins Bax,cleaved Caspase-9,and cleaved Caspase-3 were significantly reduced and the expression of anti-apoptotic protein Bcl-2 was significantly increased after HSYA pretreatment.Real-time PCR showed that compared with the hypoxia-reoxygenation group,HSYA pretreatment decreased the expression of Bax(P<0.01)and increased the expression of Bcl-2(P<0.001).Western Blot tests showed that Bax,cleaved Caspase-9,and cleaved Caspase-3 protein expression were significantly reduced and Bcl-2 protein expression was significantly increased after PUE pretreatment compared with the hypoxia-reoxygenation group.Real-time PCR showed that compared with the hypoxia-reoxygenation group,PUE pretreatment reduced the expression of caspase-3(P<0.01)and increased the expression of Bcl-2(P<0.05).This proves that HSYA and PUE can inhibit the apoptosis of EA.hy926 cells induced by hypoxia and reoxygenation.6.Effects of HSYA or PUE pretreating on PI3K/Akt signal pathway.Western Blot showed that compared with the hypoxia-reoxygenation group,HSYA pretreatment up-regulated the expression of p-Akt.And compared with the hypoxia-reoxygenation group,the expression of p-Akt protein was significantly increased after PUE pretreatment.This indicates that the PI3K/Akt pathway mediates HSYA or PUE inhibition of EA.hy926 cell apoptosis after hypoxia-reoxygenation.7.Effects of PI3K inhibitor LY294002 on PI3K/Akt signaling pathway and apoptotic proteins.Western Blot results showed that compared with the HSYA pretreatment group,the level of p-Akt decreased after the PI3K/Akt inhibitor LY294002 was added,and the expression of anti-apoptotic protein Bcl-2 decreased,and the expression levels of pro-apoptotic proteins cleaved Caspase-3,cleaved Caspase-9,and Bax increased.The Western Blot results showed that compared with the PUE pretreatment group,the level of p-Akt decreased after the PI3K/Akt inhibitor LY294002 was added,and the expression of the anti-apoptotic protein Bcl-2 decreased.The expression of pro-apoptotic proteins cleaved Caspase-3,cleaved Caspase-9 and Bax increased.This indicates that LY294002 inhibits the PI3K/Akt signaling pathway mediated HSYA and PUE from exerting protective effects on hypoxia-reoxygenation-induced endothelial cell apoptosis.[Conclusions]1.Activity of EA.hy926 cells injured by hypoxia for 12h and reoxygenation for 8h.2.HSYA and PUE has protective effects on apoptosis of EA.hy926 cells induced by hypoxia-reoxygenation.3.The protective effect of HSYA and PUE on apoptosis of EA.hy926 cells induced by hypoxia-reoxygenation may be related to the regulation of PI3K/Akt signaling pathway. |
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