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目的检测乙醛脱氢酶2(ALDH2)被daidzin抑制时对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响,验证乙醛脱氢酶2 (ALDH2)在此过程中所起的作用．方法在确定daidzin的最佳抑制浓度后,运用心肌细胞缺氧模型,比较大鼠心肌细胞在单独缺氧和经ALDH2特异性抑制剂daidzin预处理24 h后缺氧的凋亡和MAPK信号通路的改变。酶活性检测采用乙醛代谢法、ROS的检测用C400测定,凋亡的测定通过用Hoechest 33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测,MAPK信号通路酶(ERK1／2、JNK、P38)磷酸化的改变用Western印迹法检测。结果60μmol/Ldaidzin浓度是ALDH2酶活性被抑制而细胞无凋亡发生的最佳工作浓度。在daidzin (60μmol／L)预处理24 h后再经缺氧诱导,比单独缺氧引起的心肌细胞凋亡更为明显:表现为在Hoechest 33324染色中,细胞核溶解和核碎裂(P<0．05),在FACS和TUNEL的检测中,凋亡细胞明显增加(P<0．05),经凋亡后的死亡细胞有增加趋势但差异无统计学意义,同时与MAPK信号通路有关的磷酸化ERK1／2、JNX和P38的酶活性均表达增强。结论daidzin使ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性,其机制是与细胞ROS和凋亡的增加及MAPK信号通路的激活有关。ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
Objective To detect the effect of ALDH2 on apoptosis and signal transduction induced by daidzin in rat cardiomyocytes under hypoxia and to verify the role of ALDH2 in this process Play a role. Methods After determining the optimal inhibitory concentration of daidzin, the myocardial cell hypoxia model was used to compare the apoptosis and MAPK signaling pathway in rat cardiomyocytes exposed to hypoxia alone and pretreatment with daidzin, a specific inhibitor of ALDH2, for 24 h change. The enzymatic activity was determined by acetaldehyde metabolism. The detection of ROS was determined by C400. The apoptosis was determined by Hoechest 33324, immunofluorescence labeled flow cytometry and TUNEL kit. The activity of MAPK signal pathway enzymes (ERK1 / 2, JNK, P38) Phosphorylation changes were detected by Western blotting. Results The concentration of 60μmol / Ldaidzin was the optimal working concentration of ALDH2 enzyme without inhibition of cell apoptosis. Induced by hypoxia after daidzin (60μmol / L) pretreatment for 24 h, the apoptosis of cardiomyocytes was more obvious than hypoxia alone. The results showed that in Hoechest 33324 staining, nuclear lysis and nuclear fragmentation (P <0 .05). The apoptotic cells were significantly increased in FACS and TUNEL assay (P <0.05), and the number of apoptotic cells increased with the increase of apoptotic cells, but the difference was not statistically significant. Meanwhile, the phosphorylation of MAPK signaling pathway Enzymatic activity of ERK1 / 2, JNX and P38 were increased. Conclusions The decrease of ALDH2 activity induced by daidzin can increase the susceptibility of cardiomyocytes to hypoxia-induced apoptosis. The mechanism is related to the increase of ROS and apoptosis and the activation of MAPK signaling pathway. ALDH2 has a protective effect on hypoxia-induced apoptosis.