二氢杨梅素通过促进AMPK活化抑制低氧诱导的H2c9心肌细胞凋亡

Dihydromyricetin inhibits hypoxia-induced apoptosis of H2c9 cells through activating AMPK

目的研究二氢杨梅素(dihydromyricelin,DHM)对低氧诱导的H2c9心肌细胞凋亡的影响及其与磷酸腺苷活化蛋白激酶(AMPK)活性的关系。方法 H2c9细胞分为对照组(20%O2)、10%O2组、5%O2组和2.5%O2组,然后检测细胞活力和凋亡,观察低氧对H2c9细胞活力和凋亡的影响。H2c9细胞分为对照组(20%O2)、20%O2+40μmol/L DHM组、5%O2组、5%O2+20μmol/L DHM组、5%O2+40μmol/L DHM组和5%O2+80μmol/L DHM组,然后检测细胞活力和凋亡,观察二氢杨梅素对低氧诱导的H2c9细胞凋亡的拮抗作用。H2c9细胞分为20%O2(对照组)、5%O2组、5%O2+AICAR组、5%O2+DHM组、5%O2+Dorsomorphin组、5%O2+DHM+AICAR组、5%O2+DHM+Dorsomorphin组、5%O2+AICAR+Dorsomorphin组,然后检测AMPK和p-AMPK蛋白水平,观察DHM拮抗低氧诱导的H2c9细胞是否与AMPK活性有关。使用CCK-8检测H2c9细胞活力,AnnexinⅤ-FITC/PI双标记流式细胞仪检测H2c9细胞凋亡,蛋白质免疫印迹法检测H2c9细胞AMPK和p-AMPK蛋白水平。结果与20%O2组比较,5%O2组的H2c9细胞活力显著降低、细胞凋亡率明显增加(均P<0.05)。与5%O2组对比,5%O2+20μmol/L DHM组、5%O2+40μmol/L DHM组和5%O2+80μmol/L DHM组的H2c9细胞活力明显增加及凋亡率明显降低(均P<0.05)。与20%O2组比较,5%O2组H2c9细胞内的活性形式AMPK即磷酸化的AMPK(p-AMPK)蛋白水平显著降低(P<0.05)。与5%O2组比较,5%O2+AICAR组、5%O2+DHM组、5%O2+DHM+AICAR组中H2c9细胞中p-AMPK蛋白水平明显增加(均P<0.05),而5%O2+DHM+Dorsomorphin组和5%O2+AICAR+Dorsomorphin组中H2c9细胞中p-AMPK无明显增加。结论 DHM通过促进AMPK活化拮抗低氧诱导的AMPK活性下降,进而抑制低氧诱导的H2c9细胞凋亡。

Objective To investigate the effect of dihydromyricetin(DHM)on the apoptosis of H2c9 cardiomyocytes induced by hypoxia and its association with AMP-activated protein kinase(AMPK).Methods H2c9 cells were divided into control group(20%O 2),10%O 2 group,5%O 2 group and 2.5%O 2 group,and then the cell viability and the apoptosis were detected to observe the effect of hypoxia on the viability and the apoptosis of H2c9 cells.H2c9 cells were divided into control group(20%O 2),20%O 2+40 mol/L DHM group,5%O 2 group,5%O 2+20 mol/L DHM group,5%O 2+40 mol/L DHM group and 5%O 2+80 mol/L DHM group,and then the cell viability and the apoptosis were detected to observe the effect of dihydromyricetin against hypoxic-induced apoptosis of H2c9 cells.H2c9 cells were divided into 20%O 2(control group),5%O 2 group,5%O 2+AICAR group,5%O 2+DHM group,5%O 2+Dorsomorphin group,5%O 2+DHM+AICAR group,5%O 2+DHM+Dorsomorphin group,and 5%O 2+AICAR+Dorsomorphin group,and then AMPK and p-AMPK protein levels were detected to observe whether DHM antagonized hypoxic-induced H2c9 cells and AMPK activity.The cell viability of H2c9 cells was assessed by CCK-8 assay.The apoptotic rate of H2c9 cells was measured after AnnexinⅤ/PI staining by flow cytometry(FCM).The levels of AMPK and p-AMPK in H2c9 cells were detected by Western blotting assay.Results Compared with 20%O 2 group,H2c9 cell activity in 5%O 2 group was significantly reduced and the apoptosis rate was significantly increased(all P<0.05).Compared with 5%O 2 group,the activity was significantly increased and the apoptosis rate was significantly decreased in H2c9 cells in 5%O 2+20 mol/L DHM group,5%O 2+40 mol/L DHM group and 5%O 2+80 mol/L DHM group(all P<0.05).Compared with 20%O 2 group,the level of phosphorylated AMPK(p-AMPK)protein in H2c9 cells was significantly reduced in 5%O 2 group(P<0.05).Compared with 5%O 2 group,the levels of p-AMPK protein in H2c9 cells increased significantly in 5%O 2+AICAR group,5%O 2+DHM group,and 5%O 2+DHM+AICAR group(all P<0.05).However,there was no significant increase in p-AMPK in H2c9 cells in 5%O 2+DHM+Dorsomorphin treatment group and 5%O 2+AICAR+Dorsomorphin group compared with 5%O 2 group.Conclusion DHM may inhibit the apoptosis of H2c9 cardiomyocytes induced by hypoxia through activating AMPK.