新型组蛋白去乙酰化酶抑制剂对缺氧损伤心肌细胞的保护作用研究

Myocardial protection of a novel histone deacetylase inhibitor against hypoxia injury

目的利用化学发光方法和细胞筛选模型,检测新型组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor,HDACi）JZ005的抑制组蛋白去乙酰化酶（histone deacetylases,HDACs）的活性;建立氯化钴损伤的心肌细胞缺氧模型,初步探讨JZ005对缺氧损伤细胞的保护作用。方法采用脂质体转染法将含有p21启动子元件的荧光素酶报告基因真核表达载体p CI-p21-Luc转入到人胚肾细胞293中,用G418筛选获得稳定转染荧光素酶报告基因的单克隆细胞系;采用已报道的HDACi曲古抑菌素A（trichostatina A,TSA）为阳性对照,检测细胞筛选模型的稳定性;用HDACi化学发光检测试剂盒及上述细胞筛选模型测定JZ005抑制HDACs的活性;用不同浓度的JZ005处理氯化钴缺氧损伤的大鼠胚胎心肌细胞（H9c2）,MTT法检测JZ005对缺氧损伤细胞的保护作用。免疫印迹法检测JZ005处理后正常及缺氧损伤心肌细胞组蛋白H3的乙酰化水平变化。流式细胞术检测JZ005对H9c2细胞缺氧损伤后凋亡的影响。结果建立含p21启动子元件荧光素酶报告基因的HDACi细胞筛选模型;JZ005能够显著抑制HDACs的活性,浓度50~400μmol/L,抑制率〉50%。对缺氧损伤的心肌细胞具有明显保护作用,与对照组相比,细胞存活率提高38.33%、56.00%和35.20%,同时能够上调缺氧损伤心肌细胞组蛋白H3的乙酰化水平,拮抗缺氧损伤心肌细胞的凋亡,细胞凋亡数目从对照组的12.89%分别下降到给药组（25,50和100μmol/L）的6.63%、10.56%和8.89%。结论成功建立了HDACi的细胞筛选模型;JZ005作为一种新型的HDACi,具有明显的保护心肌细胞拮抗缺氧损伤的作用,提示JZ005有可能开发成一种治疗缺氧损伤的药物。

Objective To verify enzyme activity inhibition of a novel histone deacetylase inhibitor（HDACi） JZ005 using an HDACi chemiluminescence detection kit and a cell-based screening model.Methods The plasmid with p21 gene promoter elements and luciferase reporter gene was transfected into human embryonic kidney cells 293,and the stable transfectants were established by G418 screening.Enzyme activity inhibition of JZ005 on histone deacetylases（HDACs）was verified by the HDACi chemiluminescence detection kit and the cell-based screening model.A well-known HDACi,trichostatin A（TSA） was used as the positive control.MTT assay was used to detect the protection of rat H9c2 myocardial cells suffering from Co Cl2-induced hypoxia and treated with different concentrations of JZ005.The expression of acetylated histone H3 protein of normal and Co Cl2-induced hypoxia H9c2 cells before and after JZ005 treatment was assayed by Western blotting while the effect of drug administration on apoptosis was detected by flow cytometry（FCM）.Results An HDACi cell-based screening system targeting the p21 gene promoter was ranging established.The JZ005,a HDACi,markedly suppressed the activity of HDACs by more than 50% with the concentration ranging from 50 to 400 μmol / L.JZ005 significantly protected H9c2 cells from hypoxia injury.Cell viability was increased by 38.33%,56.00% and 35.20% compared with control,accompanied by an enhanced acetylation level of histone H3.JZ005（25,50 and 100 μmol / L） treatment significantly decreased the number of apoptotic cells（6.63%,10.56% and 8.89%） compared to control group（12.89%）.Conclusion An HDACi cell-based screening system is successfully established.JZ005 effectively protects myocardial cells against hypoxia injury while enhancing the acetylation level of histone H3.Our results indicate that JZ005 might be developed as a potential drug for hypoxia treatment.