缺氧复氧对H9C2心肌细胞凋亡、自噬和焦亡的影响

Effects of hypoxia/reoxygenation on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes

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8h,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 ra-pamycin,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(apopto-sis 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cas-pase-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、cas-pase-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 dehy-drogenase(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/leuke-mia 2(Bcl-2)and Bcl-associate x protein(Bax)],autophagy related proteins[(light chain 3(LC3)Ⅱ/Ⅰ,p62,Beclin-1,and phosphory-lated 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 apopto-sis,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 expres-sion 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 LC3Ⅱ/Ⅰand Beclin-1 protein decreased(P<0.05).The immunofluorescence staining of LC3Ⅱ/Ⅰshowed 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 inflamma-some,ASC and caspase-1p20 protein was remarkably up-regulated(P<0.05),and the expression of activated inflammatory cellular factors IL-1βand 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.