摘要
目的:探究急性心肌梗死(AMI)过程中内皮细胞 tet甲基胞嘧啶双加氧酶 2(TET2)对趋化因子受体 4(CXCR4)DNA甲基化的影响以及对 AMI小鼠心肌组织自噬、炎症反应及组织细胞凋亡的影响机制,为临床探究 AMI发展的分子机制提供理论依据.方法:8周龄雄性 C57/BL6小鼠 50只,制备 AMI模型,尾部注射 TET2、CXCR4过表达质粒;蛋白免疫印迹(Western Blot)法检测心肌组织TET2、CXCR4、微管相关蛋白 3(LC3)、P62、B细胞淋巴瘤/白血病-2 基因(Bcl-2)关联 X蛋白(Bax)、半胱氨酸蛋白酶 3(Caspase-3)、Bcl-2表达;甲基化检测 CXCR4 DNA甲基化水平;酶联免疫吸附法(ELISA)检测心肌组织内炎性因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平;原位末端转移酶标记技术(TUNEL)检测各组心肌组织细胞凋亡指数.结果:与假手术组比较,模型组心肌组织内 TET2、CXCR4均表达上调,TET2、CXCR4均在心肌组织内过表达,TET2 过表达促进 CXCR4 表达,差异有统计学意义(P<0.05);与模型组比较,TET2 mimic组 CXCR4启动子区域 DNA甲基化程度降低,CXCR4 蛋白表达升高,差异有统计学意义(P<0.05);与假手术组比较,模型组小鼠心肌组织自噬蛋白 LC3、抑制细胞凋亡蛋白 Bcl-2 表达下调,炎性因子 IL-6、TNF-α、IL-1β水平、自噬蛋白 P62、促细胞凋亡蛋白 Bax、cleaved Caspase-3 表达上调,差异有统计学意义(P<0.05);TET2、CXCR4过表达进一步下调 LC3、Bcl-2 蛋白表达,上调炎性因子 IL-6、TNF-α、IL-1β水平,P62、Bax、cleaved Caspase-3 蛋白表达;TET2、CXCR4二者联合体现出最低 LC3、Bcl-2蛋白表达,最高炎性因子 IL-6、TNF-α、IL-1β水平以及 P62、Bax、cleaved Caspase-3 蛋白表达,差异有统计学意义(P<0.05).结论:AMI发展中,TET2 通过降低 CXCR4 DNA甲基化,促进 CXCR4 基因表达,进而抑制 AMI小鼠心肌组织自噬,上调炎症反应及细胞凋亡程度,促进疾病发展.
Abstract
Objective:To explore the effect of tet methylcytosine dioxygenase 2(TET2)on DNA methylation of C-X-C motif receptor 4(CXCR4)during acute myocardial infarction(AMI),and the mechanism of its effect on myocardial autophagy,inflammation and apoptosis in AMI mice.Methods:Fifty 8-week-old male C57/BL6 mice AMI model were prepared,TET2 and CXCR4 overexpressing plasmids were injected into the tail.The expressions of TET2,CXCR4,microtubule associated protein 3(LC3),P62,B-cell lymphoma/leukemia-2 gene(Bcl-2)associated X protein(Bax),Caspase-3,and Bcl-2 in cardiac tissue were detected by Western Blot.The methylation level of CXCR4 DNA was detected.The levels of inflammatory factors interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in myocardial tissue were detected by enzyme-linked immunosorbent assay(ELISA).Terminal deoxyribonucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)was used to detect apoptosis index of myocardial tissue in each group.Results:Compared with sham operation group,the expressions of TET2 and CXCR4 up-regulated in myocardial tissue of model group,and both TET2 and CXCR4 overexpressed in myocardial tissue,and overexpression of TET2 promoted CXCR4 expression(P<0.05).Compared with model group,DNA methylation of CXCR4 promoter region decreased and CXCR4 protein expression increased in TET2 mimic group(P<0.05).Compared with sham operation group,expressions of autophagy protein LC3 and apoptosis inhibiting protein Bcl-2 down-regulated in myocardial tissue of mice in model group,and levels of inflammatory factors IL-6,TNF-α,IL-1β,autophagy protein P62,pro-apoptotic protein Bax,cleaved Caspase-3 up-regulated(P<0.05).Overexpression of TET2 and CXCR4 further down-regulated the expression of LC3 and Bcl-2 proteins,up-regulated the levels of inflammatory factors IL-6,TNF-α,IL-1β,and expression of P62,Bax,cleaved Caspase-3 proteins.TET2 and CXCR4 showed the lowest LC3 and Bcl-2 protein expression,the highest inflammatory factors IL-6,TNF-α,IL-1βlevels,P62,Bax,cleaved Caspase-3 protein expression(P<0.05).Conclusion:In the development of AMI,TET2 can promote CXCR4 gene expression by reducing CXCR4 DNA methylation,thereby inhibiting myocardial autophagy in AMI mice,up-regulating inflammatory response and apoptosis,and promoting the development of the disease.
维普
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