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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。
This study explored the mechanism by which hydrogen sulfide (H2S) delays the aging of rat aortic endothelial cells (RAECs). Primary RAECs were subcultured to the 4th generation for the young group and passaged to the 12th generation for the older group. From the 4th generation, the donor NaHS given H2S was cultured to the 12th generation in the Na HS group. From the 4th generation to the 12th generation, Ca MKKβ (Ca2 + / calmodulin-dependent protein STO-609, a group of STO-609-specific inhibitor; from the fourth passage, the donor HSS to which H2S was given was cultured to the 12th generation, and STO-609 Culture, ie Na HS + STO-609 group. The changes of cell aging and the expression of senescence-associated heterochromatin (SAHF) were detected by SA-β-gal staining and DAPI fluorescence staining. The contents of NO (nitric oxide) in RAECs were detected by chemical colorimetry. Western blot was used to detect the expression of CaKKβ, AMPK (AMPK) and p-AMPK protein. The results showed that compared with the young group, the aging degree of the aged group was significantly increased, the positive rate of SA-β-gal and the formation rate of SAHF were significantly increased (P <0.01), and the content of NO was significantly decreased (P <0.01). Compared with the aged group, the positive rate of SA-β-gal and the rate of SAHF formation in Na HS group were significantly decreased, while the content of NO was increased (P <0.01). Compared with the young group, the levels of Ca MKKβ, AMPK and p-AMPK were significantly decreased in the aged group (P <0.01), while the levels of Ca MKKβ, AMPK and p-AMPK were increased in the NaHS treated group (P <0.05). Administration of STO-609 to CaKKβ signaling pathway could reduce or eliminate the cytotoxicity of Na HS (P <0.01). The results showed that exogenous H2S delayed the process of cell aging by affecting Ca MKK|Â-AMPK signaling pathway.