RESUMEN
Objective To explore the effect of endogenous sulfur dioxide (SO2)on the apoptosis induced by cobalt chloride (CoCl2)in the human pulmonary arterial endothelial cells (HPAECs).Methods CoCl2was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1),and the key enzyme generating endogenous SO2 were over -expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups:vehicle group,vehicle + CoCl2 group,AAT1 group and AAT1 + CoCl2 group.The expressions of AAT1,B-cell lymphoma-2 (bcl-2),bcl-associated X protein (bax),Caspase-3 and activated Caspase-3 (cleaved Caspase-3)in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)assay.Results There were significant differences in the endogenous SO2 content,the expre-ssions of AAT1 and bcl-2,and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F＝147.364,23.738,6.521,64.884,all P＜0.05).However,there was no difference in the expression of bax among 4 groups of HPAECs (F＝1.620,P＞0.05).Compared with vehicle group,AAT activity [(0.96 ± 0.24)Carmen's unit/μg vs.(2.21 ± 0. 60)Carmen's unit/μg],endogenous SO2 content (40.71 ± 7.72 vs.105.60 ± 16.20)and bcl-2 expression (0.59 ± 0.19 vs.1.02 ± 0.20)in the HPAECs of vehicle +CoCl2 group were significantly de-creased,while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56 ± 0.25 vs.0.95 ± 0.13)were significantly increased (all P＜0.05).However,there were no differences in the expression of AAT1 (0. 50 ± 0.12 vs.0.53 ± 0.11)in the HPAECs between vehicle group and vehicle+CoCl2 group (P＞0.05). The SO2 content (351.50 ± 42.43 vs.105.60 ± 16.20)and AAT1 expression (1.22 ± 0.33 vs.0.53 ± 0.11)in the HPAECs of AAT1 group were higher than those of vehicle group (all P ＜0. 05 ). Compared with AAT1 group, endogenous SO2content (333.50 ± 46.22 vs.351.50 ± 42.43)and the expression of AAT1 (1.26 ± 0.36 vs.1.22 ± 0.33)and bcl-2 (1.14 ± 0.38 vs.1.03 ± 0.27)in the HPAECs of AAT1 +CoCl2group did not change (all P＞0. 05).Moreover,no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51 ± 0.17 vs.0.50 ± 0.11)between the two AAT1 -overexpressed groups (all P ＞0. 05).Conclusion Endo-genous SO2inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2.
RESUMEN
Objective To investigate the effects of endogenous sulfur dioxide (SO2) on the oxidative stress induced by cobalt chloride (CoCl2) in the rat pulmonary artery smooth muscle cells (PASMCs).Methods Rat PASMCs were treated with 200 μ mol/L CoCl2 to mimic the hypoxia insult.Endogenous SO2 generating enzyme aspartate aminotransferase 1 (AAT1) expression was upregulated or downregulated (AAT1 sh) by transfection with lentivirus.Rat PASMCs were randomly divided into 8 groups:vehicle group,vehicle + CoCl2 group,AAT1 group,AAT1 + CoCl2 group,scramble group,scramble + SO2 group,AAT1 sh group and AAT1 sh + SO2 group.SO2 donor Na2 SO3/NaHSO3 at concentration of 100 μ mol/L were added in scramble + SO2 group and AAT1sh + SO2 group.The expressions of AAT1,superoxide dismutase 1 (SOD1) and SOD2 in PASMCs were detected by Western blot method.In situ SO2 content in PASMCs was detected by fluorescent probe.The superoxide anions in PASMCs were labeled by dihydroethidium (DHE) probe under fluorescent microscope.Results Compared with the vehicle group,the levels of SO2 and the expressions of AAT1 (0.221 ± 0.002 vs.0.446 ± 0.004),SOD1 (0.076 ± 0.028 vs.0.171 ± 0.019) and SOD2 (0.080 ± 0.031 vs.0.196 ± 0.018) significantly decreased (all P ＜ 0.01),and superoxide anion increased in rat PASMCs of vehicle + CoCl2 group.Meanwhile,compared with vehicle + CoCl2 group,the levels of SO2 and the expressions of AAT1 (0.839 ± 0.056 vs.0.221 ± 0.002),SOD1 (0.177 ± 0.020 vs.0.076 ± 0.028) and SOD2 (0.195 ±0.018 vs.0.080-± 0.031) markedly increased (all P ＜ 0.01),and superoxide anion decreased in rat PASMCs of AAT1 + CoCl2 group.On the contrary,compared with the scramble group,the levels of SO2 and the expressions of AAT1 (0.062 ±0.017 vs.0.354 ±0.034),SOD1 (0.054 ±0.029 vs.0.157 ±0.023) and SOD2(0.180 ±0.100 vs.0.586 ± 0.176)significantly decreased (all P ＜ 0.01),and superoxide anion increased in rat PASMCs of AAT1sh group.Furthermore,compared with the AAT1 sh group,the levels of SO2 and the expressions of SOD1 (0.155 ± 0.022vs.0.054 ± 0.029) and SOD2 (0.578 ± 0.200 vs.0.180 ± 0.100) significantly increased (all P ＜ 0.01),and superoxide anion decreased in rats PASMCs of AAT1sh + SO2 group.Conclusion Endogenous SO2/AAT1 inhibits CoCl2-induced oxidative stress in rat PASMCs.