Cigarette Smoke Extracts Promote Apoptosis Of Human Pulmonary Artery Endothelial Cells By Endoplasmic Reticulum Stress Chop Signaling Pathway

Objective:To determine whether cigarette smoke extract can promote apoptosis of human pulmonary artery endothelial cells via endoplasmic reticulum stress CHOP signaling pathway,and whether the addition of phenylbutyric acid(PBA)can inhibit apoptosis.2.Methods:The expression of CHOP gene in HPAEC human pulmonary artery endothelial cells was silenced by lentiviral transfection with recombinant RNA.HPAEC human pulmonary artery endothelial cells without CHOP gene intervention and CHOP genes will be silenced HPAEC~-CHOPCHOP human pulmonary artery endothelial cells respectively were divided into control group(Ctrl group,conventional culture do not make special treatment)group,cigarette smoke extract group(CSE group,adding 10%of cigarette smoke extract medium),phenyl butyric acid group(PBA group,medium to join 5mmol/L phenylbutyric acid)and cigarette smoke joint phenyl butyric acid group(CSE+PBA group,medium with 10%concentration of cigarette smoke extract as well as the tendency for 5mmol/L PBA),each cell respectively deal with 6 h,12 h,24 h,Ctrllecting cells for subsequent experimental observation.Morphological changes of the endoplasmic reticulum were observed by transmission electron microscopy,apoptosis ratio was detected by flow cytometry,expression levels of CHOP protein were detected by Western-blot,and expression levels of CHOP mRNA were detected by Realtime-PCR.P<0.05 was considered statistically significant.Results:1.The expression level of CHOP protein in HPAEC~-CHOPCHOP cells was lower than that of HPAEC cells by more than 80%,indicating that CHOP gene silencing achieved the desired effect.2.Endoplasmic reticulum morphology was observed by transmission electron microscopy.The shape of endoplasmic reticulum of HPAEC cells in Ctrl group and PBA group was normal,and the endoplasmic reticulum of HPAEC cells in CSE group was swollen(manifested as increased volume and flat and shallow folding),while CSE+PBA group was less severe than that in the CSE group.The morphologies of HPAEC~-CHOPCHOP cells endoplasmic reticulum in Ctrl group,PBA group and CSE+PBA group were basically normal.In CSE group,HPAEC~-CHOPCHOP cells had no obvious swelling of endoplasmic reticulum,but the electron density of endoplasmic reticulum increased.3.Flow cytometry was used to detect the proportion of apoptosis.Comparison of the same treatment time of HPAEC cells in different treatment groups:there was no difference in apoptosis ratio between PBA group and Ctrl group.The proportion of apoptosis in CSE group was higher than that in Ctrl group and PBA group.The apoptosis rate in the CSE+PBA group was higher than that in the Ctrl group and the PBA group,but lower than that in the CSE group.Compared with each group with different CSE treatment time of HPAEC cells,the apoptosis rate at24h was higher than that at 12h,and the apoptosis rate at 12h was higher than that at 6h.Comparison of the same treatment time of HPAEC~-CHOPCHOP cells in different treatment groups:there was no difference in apoptosis ratio between PBA group and Ctrl group.The proportion of apoptosis in CSE group was higher than that in Ctrl group and PBA group.The apoptosis rate in the CSE+PBA group was higher than that in the Ctrl group and the PBA group,but lower than that in the CSE group.HPAEC~-CHOPCHOP cells was no significant difference in apoptosis rate among the groups with different CSE treatment time.Comparison of HPAEC~-CHOPCHOP cells with HPAEC cells in the same processing time:the apoptosis rate of the HPAEC~-CHOPCHOP cells CSE group was lower than that of the HPAEC cells CSE group.The apoptosis rate of the HPAEC~-CHOPCHOP cells CSE+PBA group was lower than that of the HPAEC cells CSE+PBA group.The apoptosis rate of the HPAEC~-CHOPCHOP cells CSE+PBA group was lower than that of the HPAEC cells CSE+PBA group.4.CHOP protein expression level detection.Comparison of the same treatment time of HPAEC cells in different treatment groups:there was no difference in the expression level of CHOP protein between PBA group and Ctrl group.The expression level of CHOP protein in CSE group was higher than that in Ctrl group and PBA group.The expression level of CHOP protein in CSE+PBA group was higher than that in Ctrl group and PBA group,but lower than that in CSE group.The expression level of CHOP protein in HPAEC cells at 12h was higher than that at 6h,and the expression level of CHOP protein in cells at 24h was higher than that at 12h.Comparison of the same processing time of HPAEC~-CHOPCHOP cells in different treatment groups:expression level of CHOP protein in PBA group was not different from that in Ctrl group.The expression level of CHOP protein in CSE group was higher than that in Ctrl group and PBA group.The expression level of CHOP protein in CSE+PBA group was higher than that in Ctrl group and PBA group,but lower than that in CSE group.The expression levels of CHOP protein in HPAEC~-CHOPCHOP cells were not significantly different among the groups with different CSE processing times.Comparison of HPAEC~-CHOPCHOP cells with HPAEC cells in the same processing time:expression levels of CHOP protein of the HPAEC~-CHOPCHOP cells CSE group was lower than that of the HPAEC cells CSE group.expression levels of CHOP protein of the HPAEC~-CHOPCHOP cells CSE+PBA group was lower than that of the HPAEC cells CSE+PBA group.The expression levels of CHOP protein of the HPAEC~-CHOPCHOP cells CSE+PBA group was lower than that of the HPAEC cells CSE+PBA group.5.CHOP mRNA expression level detection.Comparison of the same treatment time of HPAEC cells in different treatment groups:there was no difference in the expression level of CHOP mRNA between PBA group and Ctrl group.The expression level of CHOP mRNA in CSE group was higher than that in Ctrl group and PBA group.The expression level of CHOP mRNA in CSE+PBA group was higher than that in Ctrl group and PBA group,but lower than that in CSE group.The expression levels of CHOP mRNA in HPAEC cells were not significantly different among the groups with different CSE treatment times.Comparison of the same processing time of HPAEC~-CHOPCHOP cells in different treatment groups:expression level of CHOP mRNA in PBA group was not different from that in Ctrl group.The expression level of CHOP mRNA in CSE group was higher than that in Ctrl group and PBA group.The expression level of CHOP mRNA in CSE+PBA group was higher than that in Ctrl group and PBA group,but lower than that in CSE group.The expression level of CHOP mRNA in HPAEC~-CHOPCHOP cells at 12h was higher than that at6h,and the expression level of CHOP mRNA in cells at 24h was higher than that at 12h.Comparison of HPAEC~-CHOPCHOP cells with HPAEC cells in the same processing time:expression levels of CHOP mRNA of the HPAEC~-CHOPCHOP cells CSE group was lower than that of the HPAEC cells CSE group.expression levels of CHOP mRNA of the HPAEC~-CHOPCHOP cells CSE+PBA group was lower than that of the HPAEC cells CSE+PBA group.The expression levels of CHOP mRNA of the HPAEC~-CHOPCHOP cells CSE+PBA group was lower than that of the HPAEC cells CSE+PBA group.Conclusion:1.CSE can induce apoptosis of human pulmonary artery endothelial cells,and its mechanism is related to endoplasmic reticulum stress CHOP apoptosis signaling pathway.Endoplasmic reticulum stress inhibitor PBA can alleviate CSE induced apoptosis of human pulmonary artery endothelial cells.