目的 探讨缺氧复氧（hypoxia reoxygenation, HR）对H9C2心肌细胞凋亡、自噬和焦亡3种细胞死亡方式的影响。 方法 将培养的H9C2心肌细胞按随机数字表法分为正常对照组（Ctrl组）和HR组，其中HR组H9C2心肌细胞在缺氧培养箱中进行氧糖剥夺8 h，然后复氧12 h。通过检测细胞培养液中的乳酸脱氢酶（lactate dehydrogenase, LDH）含量及四甲基偶氮唑盐［3‑（4,5‑dimethylthiazol‑2‑yl）‑2,5 diphenyl tetrazolium bromide, MTT］比色法检测细胞活力来评估细胞损伤情况（每组6孔），Western blot检测（每组9孔）细胞凋亡相关蛋白［天门冬氨酸特异性半胱氨酸蛋白酶（cysteinyl aspartate‑specific proteinase, caspase）‑3、B细胞淋巴瘤/白血病‑2（B‑cell lymphoma/leukemia 2, Bcl‑2）、Bcl相关蛋白（bcl‑associate x protein, Bax）］、自噬相关蛋白［（轻链蛋白3（light chain 3, LC3）Ⅱ/Ⅰ、p62、Beclin‑1、磷酸化哺乳动物雷帕霉素靶蛋白（phosphorylated mammalian target of rapamycin, p‑mTOR）］、焦亡相关蛋白［Nod样受体蛋白‑3（nod‑like receptor pyrin domain3, NLRP3）、凋亡相关斑点样蛋白（apoptosis associated speck‑like protein, ASC）、caspase‑1p20、IL‑1β、IL‑18］。通过免疫荧光染色进一步评估细胞凋亡、自噬及焦亡情况（每组6孔）。 结果 与Ctrl组比较，HR组H9C2心肌细胞HR后细胞活力降低（P<0.05），LDH释放增加（P<0.05），活化的caspase‑3及Bax的表达上调（P<0.05），Bcl‑2表达下降（P<0.05），TUNEL染色显示HR后细胞凋亡显著增加（P<0.05），提示HR后细胞凋亡及损伤增加；而p‑mTOR、p62均表达增加（P<0.05），但LC3Ⅱ/Ⅰ和Beclin‑1蛋白降低（P<0.05），LC3Ⅱ/Ⅰ免疫荧光染色显示HR后细胞内的自噬小体增加（P<0.05），表明HR后H9C2心肌细胞自噬受到明显抑制；同时炎性小体NLRP3、ASC、caspase‑1p20蛋白均显著上调（P<0.05），活化的炎症细胞因子IL‑1β、IL‑18表达增加（P<0.05），提示HR后NLRP3炎性小体活化，细胞焦亡增加。 结论 H9C2心肌细胞在HR损伤过程中自噬被抑制，细胞焦亡及凋亡增加。
Objective To investigate the effects of hypoxia/reoxygenation (HR) on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes. Methods The cultured H9C2 cardiomyocytes were divided into two groups according to the random number table method: a normal control (Ctrl) group and an HR group. The H9C2 cardiomyocytes in the HR group were deprived of oxygen and glucose for 8 h in a hypoxic incubator, followed by reoxygenation for 12 h. Cell damage was assessed through detection of lactate dehydrogenase (LDH) content in culture medium and determination of cell viability by 3‑[4,5‑dimethylthiazol‑2‑yl]‑2,5 diphenyl tetrazolium bromide (MTT) method (n=6). Apoptosis related proteins ［cysteinyl aspartate‑specific proteinase (caspase)‑3, B‑cell lymphoma/leukemia 2 (Bcl‑2) and Bcl‑associate x protein (Bax)］, autophagy related proteins ［(light chain 3 (LC3) Ⅱ/Ⅰ, p62, Beclin‑1, and phosphorylated mammalian target of rapamycin (p‑mTOR)］and pyroptosis related proteins ［(nod‑like receptor pyrin domain 3 (NLRP3), apoptosis associated speck‑like protein (ASC), caspase‑1p20, interleukin (IL)‑1β, and IL‑18］were detected by Western blot (n=9). Cell apoptosis, autophagy and pyroptosis were further assessed by immunofluorescence staining (n=6) Results Compared with the Ctrl group, the HR group presented reduced viability of H9C2 cardiomyocytes (P<0.05), increased LDH release (P<0.05), and up‑regulated expression of activated caspase‑3 and Bax (P<0.05), as well as decreased expression of Bcl‑2 (P<0.05). TUNEL staining showed that apoptosis significantly was enhanced in the cells after HR (P<0.05). These results indicated that apoptosis and cell damage were enhanced after HR. In contrast, the expression of p‑mTOR and p62 increased (P<0.05), but the expression of LC3 Ⅱ/Ⅰ and Beclin‑1 protein decreased (P<0.05). The immunofluorescence staining of LC3 Ⅱ/Ⅰ showed that the number of autophagosome within the cells increased after HR (P<0.05), indicating that autophagy in H9C2 myocardial cells was significantly inhibited. Meanwhile, the expression of NLRP3 inflammasome, ASC and caspase‑1p20 protein was remarkably up‑regulated (P<0.05), and the expression of activated inflammatory cellular factors IL‑1β and IL‑18 increased (P<0.05), suggesting that NLRP3 inflammasomes were activated and pyroptosis enhanced after HR. Conclusions Autophagy is inhibited in H9C2 cardiomyocytes during HR injury, but pyroptosis and apoptosis are enhanced.
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