The Study Of Homocysteine-induced Apoptosis And Death Receptor Pathway In Human Vascular Smooth Muscle Cells

Objective To investigate the mechanism of apoptosis in human umbilical arterial smooth muscle cells induced by homocysteine.Methods HVSMCs were cultured in vitro.The rate of apoptosis in HVSMCs was detected by flow cytometry and cell genomic DNA Ladder.Immunocytochemisty and western blot was undertaken to determine the expression of death receptor Fas and TNFR1,cytosolic caspase-8 and nuclear protein NF-κB.Caspase-8 was detected by spectrophotometry and NF-κB was detected by the elect rophoretic mobility shift assay respectively.The expression of Fas and TNFR1 mRNA in HVSMCs was detected by reverse- transcription polymerase chain reaction (RT- PCR).Data are expressed as a mean±S.D.Measurement data use analysis of variance and enumeration data use chi-square test.SPSS16.0 software for statistical analysis.P values of <0.05 were considered statistically significant.Results (1)The rate of apoptosis in homocysteine induced HVSMCs detected by flow cytometry and cell genomic DNA Ladder was increasing in a concentration and time dependent manner.(2)Immunocytochemical localization of Fas and TNFR1 protein was observed mainly in the cytoplasm,few was observed in the membrane,casepase-8 protein localization was in the cytoplasm,NF-κB proteins located in the cytoplasm and nucleus,the positive expression of four kinds of protein were all brown particles.The optical density of the four proteins were analyzed by professional image analysis system.The expression of four kinds of protein in HVSMCs induced by homocysteine was increasing in a concentration and time dependent manner(P<0.05).The result detected by western blot was the same with that.(3)Spectrophotometry suggested that the homocysteine induced the activity of caspase-8 in HVSMCs in a concentration and time-dependent manner; EMSA alao suggested that the homocysteine induced the activity of NF-κB in a concentration and time-dependent manner.RT-PCR suggested that the homocysteine induced the expression of Fas and TNFR1 mRNA in HVSMCs in a concentration and time-dependent manner.Conclusions 1,The rate of apoptosis in homocysteine induced HVSMCs was increasing in a concentration and time dependent manner.2,The pathway of apoptosis in HVSMCs induced by homocysteine maybe the way to death receptor (Fas and TNFR1)-induced,stimulates caspase-8 activation and NF-κB nuclear transcription process. Objective To investigate the effect and mechanism of folic acid to apoptosis in homocysteine-induced human vascular smooth muscle cells at the level of the molecular biology.Methods HVSMCs were cultured in vitro.Apoptosis were evaluated by flow cytometry and cell genomic DNA Ladder 24h after the addition of Hcy at the concentrations of 500μmol/L.In the second batch of cultures,we studied the effects of Hcy at the concentrations of 500μmol/L and folic acid at the concentration of 750ng/L.Immunocytochemisty was undertaken to determine the expression of death receptor Fas and TNFR1,cytosolic caspase-8 and nuclear protein NF-κB in two batchs. Caspase-8 was detected by spectrophotometry and NF-κB was detected by the elect rophoretic mobility shift assay in two batchs respectively.The expression of death receptor Fas and TNFR1 mRNA was detected by RT-PCR. Data are expressed as a mean±S.D.Measurement data use analysis of variance and enumeration data use chi-square test.SPSS16.0 software for statistical analysis.P values of <0.05 were considered statistically significant.Results (1)The rate of apoptosis in homocysteine induced HVSMCs was decreased after the intervention of folic acid(P<0.05).(2)The positive expression of four kinds of proteins was decreased after the intervention of folic acid detected by immunocytochemical method(P<0.05).(3)The expression of caspase-8 was was decreased after the intervention of folic acid detected by spectrophotometry(P<0.05).(4)EMSA show that after the intervention of folic acid the expression of NF-κB was decreased than before(P<0.01).(5)The expression of death receptor Fas and TNFR1mRNA was decreased after the intervention of folic acid detected by RT-PCR(P<0.01). Conclusions 1,Folic acid can reduce the rate of apoptosis in HVSMCs induced by homocysteine.2,Folic acid can decrease the expression of death receptor Fas,TNFR1,reduce the activities of caspase-8 and NF-κB nuclear transcription process.