激活Notch1通路减轻高温高湿条件下H9C2心肌细胞缺氧/复氧损伤

Activation of Notch1 pathway alleviates hypoxia/reoxygenation injury of H9C2 cardiomyocytes in high temperature and humidity

目的明确Notch1通路在高温高湿条件下H9C2心肌细胞缺氧/复氧(Hypoxia/Reoxygenation,H/R)损伤中的作用及其潜在机制。方法常规培养H9C2心肌细胞并将其分6组即对照组;H/R组;高温高湿组;高温高湿+H/R组;高温高湿+Jagged1(Notch1激动剂)+H/R组;高温高湿+溶剂+H/R组。TUNEL法检测细胞凋亡,荧光探针JC-1检测线粒体膜电位,ATP检测试剂盒检测ATP含量,Western blot检测Notch1细胞内段(Notch1 intracellular domain,Notch1 ICD)、Hairy和分裂增强子(Hairy and enhancer of split,Hes1)、微管相关蛋白1轻链3(microtubuleassociated protein1 light chain 3,LC3)和p62的蛋白表达水平。结果与对照组相比,急性损伤H/R后,细胞凋亡增加(P<0.05),线粒体膜电位降低(P<0.05),ATP含量减少(P<0.05),Notch1 ICD、Hes1、LC3-II/I(p62相应降低)表达升高(P<0.05),而慢性损伤高温高湿后,细胞凋亡增加(P<0.05),线粒体膜电位降低(P<0.05),ATP含量减少(P<0.05),Notch1 ICD、Hes1、LC3-II/I表达降低(p62相应升高)(P<0.05);和H/R组或高温高湿组对比,高温高湿+H/R组中细胞凋亡进一步增加(P<0.05),线粒体膜电位和ATP含量进一步降低(P<0.05),Notch1ICD、Hes1、LC3-II/I表达进一步降低(p62进一步升高)(P<0.05);和高温高湿+H/R组对比,加入Notch1激动剂Jagged1后,细胞凋亡减少(P<0.05),线粒体膜电位和ATP含量增高(P<0.05),Notch1 ICD、Hes1、LC3-II/I表达升高(p62相应降低)(P<0.05)。结论激活Notch1通路通过促进自噬从而缓解高温高湿条件下H9C2心肌细胞缺氧/复氧损伤。

AIM To investigate the role of Notch1 pathway and its underlying mechanisms in H9C2 cardiomyocytes subjected to hypoxia/reoxygenation （H/R） injury in high temperature and humidity. METHODS H9C2 cardiomyocytes were cultured and divided into six groups： control group, H/R group, high temperature and humidity group, high temperature and humidity＋H/R group, high temperature and humidity＋Jagged1 （an activator of Notch1）＋H/R group and high temperature and humidity＋vehicle＋H/R group. Cell apoptosis was detected by TUNEL, mitochondrial membrane potential was assessed by JC-1 and ATP content was measured by an ATP bioluminescent assay kit. The expression of Notch1 intracellular domain （Notch1 ICD）, Hairy and enhancer of split （Hes1）, microtubule-associated protein1 light chain 3 （LC3） and p62 were analyzed by Western blot. RESULTS Compared with those in control group, H/R injury significantly increased cell apoptosis index （P〈0.05）, reduced mitochondrial membrane potential （P〈0.05） and ATP content （P〈0.05）, and increased expressions of Notch1 ICD, Hes1 and LC3-II/I （except p62） （P〈0.05）. Although high temperature and humidity also increased cell apoptosis index （P〈0.05）, reduced mitochondrial membrane potential （P〈0.05） and ATP content （P〈0.05）, it reduced expressions of Notch1 ICD, Hes1 and LC3-II/I （except p62） （P〈0.05） compared with those in control group. In addition, compared with those in H/R group or high temperature and humidity group, cell apoptosis index further increased （P〈0.05）, mitochondrial membrane potential （P〈0.05） and ATP content reduced （P〈0.05）, and the expressions of Notch1 ICD, Hes1 and LC3-II/I（except p62） decreased （P〈0.05） in high temperature and humidity＋H/R group. Moreover, compared with those in high temperature and humidity＋H/R group, adding Jagged1 （an activator of Notch1） reduced cell apoptosis index （P〈0.05） and increased the mitochondrial membrane potential （P〈0.05） and ATP content （P〈0.05） as well as the expressions of Notch1 ICD, Hes1 and LC3-II/I （except p62） （P〈0.05）. CONCLUSION Activation of Notch1 pathway can alleviate hypoxia/reoxygenation injury of H9C2 cardiomyocytes in high temperature and humidity through accelerating autophagy.