雷帕霉素对过氧化氢诱导血管内皮细胞衰老的干预研究

目的:探讨雷帕霉素(Rapa)对过氧化氢(H2O2)诱导的人脐静脉内皮细胞(HUVECs)衰老的影响及其可能的机制。方法:把HUVECs分为空白组、衰老组(H2O2诱导)、Rapa+H2O2组和3-甲基腺嘌呤(3-MA)+H2O2组。噻唑蓝(MTT)比色法检测各组细胞活力;衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老情况;透射电子显微镜观察细胞超微结构改变;Western blot检测Rb、磷酸化Rb(p-Rb)、p21、LC-3II和beclin-1的表达变化。结果:与空白组相比,衰老组细胞活力下降,SA-β-Gal染色阳性率增高,细胞结构紊乱,并伴有衰老蛋白表达显著增加(P<0.01);与衰老组相比,Rapa+H2O2组细胞活力增强,细胞衰老染色减少,p-Rb和p21表达显著减少(P<0.05),电镜下细胞结构趋于正常,并可见自噬小体,beclin-1和LC3-II表达显著增多(P<0.01);与衰老组相比,给予自噬抑制剂3-MA的细胞则表现为细胞活力进一步下降,SA-β-Gal染色阳性率进一步增高,p-Rb和p21表达增多,细胞结构破坏明显,beclin-1和LC3-II几乎不表达(P<0.05)。结论:Rapa可延缓H2O2诱导的血管内皮细胞衰老,这种抗衰老作用可能与促进自噬有关。

Effects of rapamycin on hydrogen peroxide-induced vascular endothelial cell senescence

AIM: To investigate the effects of rapamycin(Rapa) on hydrogen peroxide(H2O2)-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS: The human umbilical vascular endothelial cells(HUVECs) were divided into 4 groups: control group, senescence group, Rapa+H2O2 group and 3-methyladenine(3-MA)+H2O2 group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase(SA-β-Gal) staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope(TEM). The protein levels of phosphorylated Rb(p-Rb), Rb, p21, LC3-II and beclin-1 were determined by Western blot. RESULTS: Compared with control group, the cell viability in H2O2 group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells(P<0.05) and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group(P<0.05). However, the cells pre-treated with Rapa prior to stimulation with H2O2 showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H2O2 alone(P<0.05). Moreover, the TEM observation showed that the structure of the cells in Rapa+H2O2 group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-II were increased(P<0.05). Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected(P<0.05), poor subcellular structures were captured, and no beclin-1 and LC3-II was detected. CONCLUSION: Rapa may retard the senescence of HUVECs induced by H2O2, and promoting autophagy may be the underlying mechanism.