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目的:探讨APPL1在脂联素(adiponectin,ANP)拮抗SD乳鼠心肌细胞(neonatal cardiomyocytes)缺氧/复氧(H/R)损伤中的作用。方法:分离SD乳鼠心肌细胞并培养。通过对培养的心肌细胞H/R损伤模拟缺血/再灌注(simulated ischemia reperfusion,SI/R)后,随机分为对照组、H/R组、H/R+APN组及H/R+APN+APPL1 RNAi组。采用四甲基偶氮唑蓝(MTT)比色法检测细胞的生存率,原位缺口末端标记(TUNEL)法检测细胞的凋亡,Westernblot检测APPL1蛋白的表达。结果:与对照组相比,H/R组吸光度值明显降低(P<0.01),凋亡指数(AI)显著上升(P<0.01)。与对照组和H/R组相比,H/R+APN组中APPL1的表达明显上升(P<0.05)。以RNAi抑制APPL1表达后,与H/R+APN组相比,H/R+APN+APPL1 RNAi组凋亡指数率(%)明显上升[(28.32±4.13)%vs.(9.78±2.16)%,P<0.01]。结论:APN可显著抑制H/R损伤诱导的心肌细胞凋亡,促进心肌细胞存活,其拮抗作用与上调APPL1蛋白的表达相关。
Objective: To investigate the effect of APPL1 on adiponectin (ANP) -mediated hypoxia / reoxygenation (H / R) injury in neonatal cardiomyocytes. Methods: SD neonatal rat cardiomyocytes were isolated and cultured. H / R group, H / R + APN group and H / R + APN group were randomly divided into control group, H / R group, APN group + APPL1 RNAi group. Cell viability was detected by MTT colorimetric assay. Cell apoptosis was detected by TUNEL method. APPL1 protein was detected by Western blot. Results: Compared with the control group, the absorbance of H / R group was significantly decreased (P <0.01) and the AI was significantly increased (P <0.01). Compared with the control group and the H / R group, the expression of APPL1 in H / R + APN group was significantly increased (P <0.05). Compared with H / R + APN group, the apoptotic index rate (%) of H / R + APN + APPL1 RNAi group significantly increased after RNAi inhibited APPL1 expression [(28.32 ± 4.13)% vs (9.78 ± 2.16)% , P <0.01]. CONCLUSION: APN can significantly inhibit the apoptosis of cardiomyocytes induced by H / R injury and promote the survival of cardiomyocytes. The antagonism of APN is related to the up-regulation of APPL1 protein expression.