Inhibitory Effect And Mechanism Of CurcuMin Niacin On The Apoptosis Of Human Umbilical Vein Endothelial Cells Induced By Low Shear Stress

Objective:To investigate the inhibitory effect of Curcumin Niacin on the apoptosis of human umbilical vein endothelial cells induced by low shear stress and its mechanism.Method:Firstly,CCK-8 method was used to detect the effect of different concentrations of CN-treated HUVECs cells on the activity of HUVECs cells for 24 h.Then,a model of apoptosis of HUVECs induced by LSS was established by parallel plate flow chamber device.Hoechst 33258 fluorescence staining and flow cytometry were used to detect the effect of CN on apoptosis of HUVECs induced by LSS.Real-time fluorescence quantitative PCR was used to detect the effect of CN on the expression of miR-224 in HUVECs induced by LSS.The effect of CN on the expression of proprotein convertase subtilisin/kexin type 9(PCSK9)protein in HUVECs induced by LSS was detected by Western blot.And the effect of miR-224-5P on PCSK9 protein expression.RNAi technology was used to transfect PCSK9 siRNA into HUVECs and Western blot was used to detect the effect of PCSK9 siRNA on the expression of Bcl-2,Bax and Caspase-3 proteins.Result:CCK-8 assay results showed that compared with the control group,2.5umol/L CN had no effect on the cell viability of HUVECs(P>0.05);5umol/L,lOumol/L,and 20umol/L CN could promote the activity of HUVECs cells.<0.05);40umol/L CN inhibited the activity of HUVECs cells(P<0.05);Hoechst 33258 fluorescence staining showed that compared with the static culture group,HUVECs with apoptotic morphology in LSS group(3dyne/cm2,12h)increased significantly.Compared with the LSS group,HUVECs with apoptotic morphology were significantly reduced in the CN+LSS group,and the number of HUVECs exhibiting apoptotic morphology was the lowest in the 20umol/L CN+LSS group.Flow cytometry results showed that compared with the static culture group,the apoptosis rate of HUVECs in LSS group increased significantly(P<0.01);Compared with LSS group,CN group dose-dependently reduced the apoptosis of HUVECs.The rate was statistically significant(P<0.01).Among them,the apoptosis rate of HUVECs with 20umol/L CN+LSS group was the most obvious,the difference was statistically significant(P<0.01).The results of real-time fluorescence quantitative PCR showed that compared with the static culture group,the expression level of miR-224 in LSS group was significantly decreased(P<0.01),and the expression level of miR-224 in CN+LSS group was gradually increased compared with LSS group.,And showed a concentration-dependent,the difference was statistically significant(P<0.05).Among them,the expression level of miR-224 in CN+LSS group was significantly up-regulated by 20umol/L,and the difference was statistically significant(P<0.01).Western blot results showed that compared with the static culture group,the PCSK9 protein expression in the LSS group was significantly increased(P<0.01);compared with the LSS group,the expression of the PCSK9 protein in the CN+LSS group was decreased,and the concentration was shown Dependency,the difference was statistically significant(P<0.05).Among them,the expression of PCSK9 protein was significantly decreased in 20 μ mol/L CN+LSS group(P<0.01).MiR-224-5P mimic was transfected.Western blot results showed that compared with LSS group,miR-224-5P mimic significantly inhibited PCSK9 expression in LSS,and the difference was statistically significant(P<0.01).Using RNAi technology to silence the expression of PCSK9 in HUVECs,the results showed that PCSK9 siRNA with a concentration of 100 nM silenced the expression of PCSK9 in HUVECs(P<0.01).Western blot results showed that compared with the blank control group,the expression of Bax and Caspase-3 protein in LSS group increased,and the expression of Bcl-2 protein decreased.The difference was statistically significant(P<0.01).Compared with LSS group,PCSK9 siRNA group Bax,Caspase The protein expression of-3 decreased,and the expression of Bcl-2 protein increased.The difference was statistically significant(P<0.01).Conclusion:1.5umol/L,10umol/L,20umol/L CN promoted the activity of HUVECs cells,and 40umol/L CN had inhibitory effect on the activity of HUVECs.2.CN could inhibit the apoptosis of HUVECs induced by LSS.The mechanism may be related to the up-regulation of miR-224 and the down-regulation of PCSK9 by CN.3.miR-224-5P inhibits PCSK9 expression.4.PCSK9 siRNA reduced the expression of Bax and Caspase-3 protein in HUVECs treated with low shear stress and increased the expression of Bcl-2 protein.