摘要
该研究探讨了硫化氢(H2S)延缓大鼠主动脉内皮细胞(rat aortic endothelial cells,RAECs)老化的机制。原代RAECs传代培养至第4代为年轻组,传代至第12代为老年组。从第4代起,给予H2S的供体Na HS培养至第12代为Na HS组;从第4代起培养至第12代,在提取处理细胞前6 h加入Ca MKKβ(Ca2+/calmodulin-dependent protein kinase kinaseβ)特异性抑制剂STO-609共培养,即STO-609组;从第4代起,给予H2S的供体Na HS培养至第12代,在提取处理细胞前6 h加入STO-609共培养,即Na HS+STO-609组。采用SA-β-gal(senescence-associatedβ-galactosidase)染色法和DAPI荧光染色法检测细胞的老化程度和老化相关异染色质(senescence-associated heterochromatin foci,SAHF)的表达变化。通过化学比色法检测RAECs的NO(nitric oxide)含量。应用Western blot方法检测细胞Ca MKKβ、AMPK(AMP-activated protein kinase)和p-AMPK蛋白的表达。结果显示,与年轻组相比,老年组细胞老化程度明显增加,细胞中SA-β-gal阳性率和SAHF形成率显著增加(P<0.01),NO含量明显下降(P<0.01)。与老年组相比,Na HS处理组的SA-β-gal阳性率和SAHF形成率明显下降,同时NO含量上升(P<0.01)。与年轻组相比,老年组Ca MKKβ、AMPK和p-AMPK蛋白质水平明显降低(P<0.01),而给予Na HS处理后,Ca MKKβ、AMPK和p-AMPK增加(P<0.05)。给予STO-609阻断Ca MKKβ信号通路,能够降低或消除Na HS的细胞保护作用(P<0.01)。结果表明,外源性H2S通过影响Ca MKKβ-AMPK信号通路减轻RAECs的衰老过程,延缓了细胞老化的进程。
To investigate the effect of the exogenous H2S on RAECs （rat aortic endothelial cells） senescence and the underlying mechanisms, RAECs were cultured at fourth and twelfth passages as young and old groups, respectively. Cultured RAECs from the fourth to twelfth passage with NariS （a H2S donor） was taken as NariS group. STO-609 [a specific inhibitor of CaMKKβ （Ca2＋/calmodulin-dependent protein kinase kinase β）] added into the old group and NariS group for 6 h before extracting the protein from the two groups as STO-609 and NaHS＋STO-609 groups, respectively. The status of senescence of RAECs was measured by SA-β-gal （senescence- associated β-galactosidase） staining, and SAHF （senescence-associated heterochromatin foci） formation was detected by DAPI fluorescent dying. Colorimetric methods were used to evaluate NO content of RAECs. The protein levels of CaMKKβ and AMPK （AMP-activated protein kinase） and p-AMPK （phosphorylation AMP- activated protein kinase） were analyzed by Western blot in RAECs. The results showed that the levels of SA-β-gal positive cell numbers and SAHF formation rates in NariS group were both lower than those in old group （P〈0.01）, but higher than those in young group （P〈0.01）, and the levels of NO were elevated in NariS group compared to the old group （P〈0.01）, but lower than that in young group （P〈0.01）. STO-609 inhibited the effects of NariS on the RAECs. Moreover, the protein levels of CaMKKβ, AMPK and p-AMPK in NariS group were increased compared to the old group, but lower than that in young group （P〈0.05）, and STO-609 reversed the protective effects of NariS on RAECs. These results indicate that H2S delays senescence of RAECs via CaMKKβ and AMPK signaling pathway.
出处
《中国细胞生物学学报》
CAS
CSCD
2016年第5期566-575,共10页
Chinese Journal of Cell Biology
基金
江苏省自然科学基金(批准号:BK20131119)
国家自然科学基金(批准号:81200250)
江苏省研究生创新计划项目(批准号:KYLX14-1444)资助的课题~~
