缺氧复氧致心肌AC16细胞损伤途径的研究

目的:探讨缺氧复氧条件下不同复氧时间致心肌细胞损伤的途径。方法:培养人心肌AC16细胞,先置入1%O2、无糖无血清条件下培养24 h,再更换含10%胎牛血清的低糖DMEM联合21%O2进行不同时间的复氧培养,采用CCK-8法检测细胞活力,并通过Western blot法检测不同细胞损伤途径标记分子的蛋白水平,如细胞自噬相关蛋白LC3-II/-I、细胞焦亡相关蛋白caspase-1和gasdermin D及凋亡相关蛋白caspase-3、Bax和Bcl2。结果:与空白对照组相比,各缺氧复氧组细胞活力降低,且随复氧时间延长,细胞活力相应持续降低(P<0.05),同时缺氧组LC3-II/-I上调(P<0.05),而复氧后LC3-II/-I较缺氧组下降,但仍高于对照组;此外,cleaved caspase-1和cleaved gasdermin D蛋白在复氧6 h组和复氧12 h组表达水平上调(P<0.05);cleaved caspase-3水平和Bax/Bcl2在复氧12 h组上调(P<0.05)。结论:缺氧致心肌AC16细胞的自噬上调,并随复氧时间增加自噬水平减弱,且在复氧过程中细胞焦亡的激活早于细胞凋亡。

Injury pathways of hypoxia/reoxygenation in AC16 cardiomyocyte

AIM: To investigate the effects of hypoxia/reoxygenation(H/R) for different reoxygenation times on cardiomyocyte injury. METHODS: Human cardiomyocyte AC16 was cultured in glucose-free and serum-free DMEM with 1% O2 for 24 h, 10% fetal bovine serum and low glucose DMEM combined with 21% O2 were used to establish reoxygenation for 2 h, 6 h and 12 h, respectively. The cell viability was measured by CCK-8 assay. The protein levels of different cell injury pathway related molecules, such as LC3-II/-I(autophagy), caspase-1 and gasdermin D(pyroptosis) and caspase-3 and Bax/Bcl2(apoptosis), were determined by Western blot. RESULTS: Compared with blank control group, the cell viability in each H/R group was continuously decreased with the extension of reoxygenation time(P<0.05). The expression of LC3-II/-I was up-regulated in hypoxia group and H/R group compared with blank control group(P<0.05). In addition, the protein levels of cleaved caspase-1 and cleaved gasdermin D were increased in H/R groups for 6 h and 12 h, respectively(P<0.05). Cleaved caspase-3 and Bax/Bcl2 were increased after reoxygenation for 12 h(P<0.05). CONCLUSION: Autophagy in hypoxia-induced AC16 cells is up-regulated, and then decreased by reoxygenation. The cell pyroptosis is activated earlier than the apoptosis during reoxygenation.