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本文旨在观察硫化氢(hydrogen sulfide,H2S)供体硫氢化钠(NaHS)预处理对人脐静脉内皮细胞老化的影响及机制。原代人脐静脉内皮细胞株(HUVECs)传代培养到第12代为老年组;自第4代细胞(年轻组)起,给予NaHS孵育到第12代即为NaHS组。细胞衰老β-半乳糖苷酶(SAβ-gal)染色法检测细胞老化程度,DAPI荧光染色法检测细胞凋亡,免疫印迹法测定细胞黄嘌呤氧化酶(XOD)、锰-超氧化物歧化酶(Mn-SOD)和烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶亚单位蛋白表达,化学比色法检测HUVECs内SOD活性和过氧化氢(H2O2)含量。结果显示,与年轻组相比,老年组内皮细胞SAβ-gal阳性率和凋亡率显著增加(P<0.01),而NaHS预处理能明显减少细胞SAβ-gal阳性率和凋亡率(P<0.01)。与年轻组相比,老年组内皮细胞XOD和NADPH氧化酶亚单位p67phox蛋白表达增加,Mn-SOD蛋白表达减少;NaHS组与老年组相比,XOD和p67phox蛋白表达减少,Mn-SOD蛋白表达增加(P<0.05)。与年轻组相比,老年组SOD活性降低,H2O2含量增加;NaHS组与老年组相比,SOD活性提高,H2O2含量减少(P<0.01)。以上结果提示,NaHS预处理能减轻老化HUVECs氧化应激水平,起到延缓HUVECs老化的作用。
This article aims to observe the effects of hydrogen sulfide (H2S) donor sodium hydrosulfide (NaHS) pretreatment on the aging of human umbilical vein endothelial cells and its mechanism. The primary human umbilical vein endothelial cell line (HUVECs) was subcultured to the 12th generation in the elder group, and from the 4th generation to the 12th generation NaHS in the NaHS group. Cell senescence was detected by SAβ-gal staining, apoptosis was detected by DAPI staining, and XRD and Mn-SOD were measured by immunoblotting Mn-SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit protein expression were detected by enzyme-linked immunosorbent assay (ELISA). The SOD activity and H2O2 content in HUVECs were detected by chemical colorimetry. The results showed that compared with the young group, the positive rate of SAβ-gal and the apoptosis rate of endothelial cells in the aged group were significantly increased (P <0.01), while NaHS pretreatment significantly decreased the positive rate of SAβ-gal and the apoptosis rate (P < 0.01). Compared with the young group, the expressions of XOD and NADPH oxidase subunits p67phox protein and Mn-SOD protein were decreased in the aged group. Compared with the aged group, the expression of XOD and p67phox proteins and the expression of Mn-SOD protein were increased in the NaHS group (P <0.05). Compared with the young group, the activity of SOD and the content of H2O2 increased in the aged group. The activity of SOD and the content of H2O2 decreased in the NaHS group compared with the old group (P <0.01). These results suggest that NaHS pretreatment can reduce the level of oxidative stress in aged HUVECs and play a role in delaying the aging of HUVECs.