Vorapaxar Of β-Intervention Of Glycerophosphate Induced Calcification In Vascular Smooth Muscle Cells

Objective : To investigate the mechanism of calcification of vascular smooth muscle cells by inhibiting protease activated receptor 1(PAR-1)in vorapaxar.Methods: Human vascular smooth muscle cells(HVSMCs)were cultured.Construct cell experimental group: normal control group,β-Glycerophosphate group,β-Glycerophosphate(0mmol/l,5mmol/l,10mmol/l,20mmol/l)was induced for 14 days;vorapaxar intervention group,vorapaxar(20 μ mol/L、40 μ mol/L、80 μ The intervention was24 hours;In the overexpression group,the overexpression plasmid of PAR1 was transfected for 24 hours.Detection: 1.different concentrations were detected by CCK-8 staining experiment β-The survival rate of HVSMCs cells was induced by glycerophosphate(cell activity%)and the survival rate of HVSMCs cells was interfered by vorapaxar at different concentrations.2.the calcification and apoptosis of HVSMCs cells were detected by alizarin red staining,and the calcification level of HVSMCs cells was analyzed.3.Western blot test was used to detect PAR-1 in HVSMCs calcification group by different vorapaxar and each dosing group/ β-The expression of catenin/runx2 protein.Results: 1.the CCK-8 test was used to detect 0,5,10 and 20mmol/l in each group β-The absorbance value of glycerophosphate group was0.987,respectively ± 0.148,0.792 ± 0.136,0.461 ± 0.139 and 0.186± 0.135。 Cell activity(%)was 100 ± 0.00,75.33 ± 0.92,41.94 ±0.94,14.82 ± 0.92。 The results show that 10,20mmol/l β-The glycerophosphate group was significantly lower than that of the control group(P < 0.05).When it is at 10mmol/l β-Glycerophosphate group added 20,40,80 μ After mol/l vorapaxar,apoptosis decreased and cell activity increased,and the absorbance value was 0.982 ± 0.134;zero point four six nine ± 0.141;zero point four seven three ± 0.138;zero point five eight six ± 0.132 and 0.762 ± 0.142,cell activity(%)was 100 ±0.00;forty-two point five ± 1.05;forty-three point three ± 1.03;fifty-five point two ± 0.95 and 71.8 ± 1.06,the results show 40,80 μMol/l vorapaxar formation and β-There was significant difference between the glycerophosphate group and the glycerophosphate group(P <0.05).After transfer into the PAR-1 overexpression plasmid,apoptosis increased,cell activity decreased,and absorbance value was 0.481 ± The cell activity(%)was 48.9 ± The results showed that there was significant difference between the control group and the vorapaxar group(P < 0.05).2.the results of alizarin red cell staining microscope showed that:10mmol/l β-The calcium deposition in glycerophosphate cells increased,and the average optical density of field was 0.886 ± 0.073,0.332 compared with the normal control group ± There was significant difference between 0.076 and that of the two groups(P < 0.05).After joining vorapaxar,40,80 μ The calcium deposition in the vorapaxar cells decreased with the average optical density of 0.543 ± 0.039、0.418 ±0.053,0.332 compared with the normal control group ± Compared with0.076,there was significant difference(P < 0.05),and 80% of them were used in combination μ After mol/l vorapaxar and overexpression of PAR-1 plasmid,calcium salt deposition increased,and the average optical density of field of vision was 0.721 ± 0.075,and 40,80 μ There was significant difference between the two groups(P < 0.05).3.Western blot detection PAR-1 β-The protein expression levels of catenin and Runx2 were also observed.stay β-The gray ratio of PAR-1protein was 0.437 in glycerophosphate group by adding 0-80umol/l vorapaxar respectively ± 0.016、O.412 ± 0.022、O ± 0.000、O ±0.000,β-The gray ratio of catenin protein level analysis was 0.612,respectively ± 0.035、0.584 ± 0.042、0.344 ± 0.034、0.195 ± The gray ratio of Runx2 protein analysis was 0.066 and 0.537 respectively ±0.068、0.534 ± 0.075、0.367 ± 0.036、0.148 ± The results show that 40,80umol/l vorapaxar and β-There was significant difference in the glycerophosphate group(P < 0.05).The control group was set up by the next experiment.After overexpression of PAR-1,PAR-1 β-The expression of catenin and Runx2 protein was up regulated,and the gray ratio was 0.433,respectively ± 0.018、0.583 ± 0.052、0.346 ± There was significant difference between 0.046 and vorapaxar group(P < 0.05).Conclusion: vorapaxar of β-Glycerophosphate can protect vascular smooth muscle cells from calcification.vorapaxar affects PAR-1 in vascular smooth muscle by inhibiting protease activated receptor 1/ β-The protein expression level of catenin / Runx2.