1类多巴胺受体对细胞凋亡的线粒体信号通路的影响

Effect of dopamine receptor-1 on mitochondrial signal pathway of apoptosis

目的以细胞凋亡的线粒体途径为切入点,探讨1类多巴胺受体(DR1)活性变化对缺氧、复氧诱导的心肌细胞凋亡的影响及可能机制。方法乳鼠心肌细胞以缺氧、缺血清培养2小时,复氧、复血清培养24小时的方法建立心肌细胞缺氧、复氧损伤模型。RT-PCR和Western blotting分别检测Bcl-2、caspase-3、caspase-9和细胞色素C(Cyt c)mRNA和蛋白质的表达;TUNEL、流式细胞仪、MTT检测心肌细胞的凋亡情况;紫外分光光度计检测细胞培养液中LDH、SOD活性和MDA含量;透射电镜观察心肌细胞形态变化。结果与正常组比较,缺氧-复氧组细胞培养液中LDH活性和MDA含量增加,SOD活性降低;心肌细胞凋亡指数增加;心肌细胞超微结构损伤加重;促凋亡因子(Cyt c、caspase-3、caspase-9)表达增加,抑凋亡因子(Bcl-2)表达亦增加。与缺氧-复氧组比较,DR1激动剂SKF-38393进一步增加LDH活性和MDA含量,降低SOD活性,增加心肌细胞凋亡指数,加重心肌细胞损伤,上调促凋亡因子表达,下调抑凋亡因子表达;DR1抑制剂SCH-23390对上述指标影响不明显。结论 DR1激活可促进缺氧、复氧诱导的心肌细胞凋亡,其机制与上调线粒体途径有关。

Objective To investigate the effect and possible mechanism of dopamine receptor-1 （DR1） activation on cardiomyocytes apoptosis induced by anoxia-reoxygenation through mitochondria signal pathway of apoptosis. Meth- od The anoxia-reoxygenation（A/R） injury model was established using primarily cultured neonatal rat cardiomyo- cytes in ischemia-mimetic solution for 2h, and re-incubated cells in normal culture medium for 24h. mRNA and pro- tein expression of Bcl-2, caspase-3, caspase-9 and the release of cytochrome c（ Cyt e） were analyzed using RT-PCR and Western blotting, the apoptotic cell death was assayed by MTr assay,TUNEL staining and flow eytometer, LDH, SOD activities and MDA contents were determined eolorimetrically using a spectrophotometer, the morphological al- terations were observed with transmission electron microscope. Result Compared with control group, LDH activity and MDA contents were increased, SOD activity was decreased, cardiomyocytes apoptotic index was heightened, cardio- myocytes ultrastrncture injury was aggravated, the expression of Bcl-2, caspase-3, caspase-9 and the release of Cyt c were increased in A/R group. Compared with A/R group, SKF-38393 （DR1 agonist） significantly increased LDH activity ,decreased SOD activity, increased MDA contents in culture medium, heightened apoptotic index, aggravated cardiomyocytes injury, up-regulated promoting apoptosis factors expressions and down-regulated preventing apoptosis factor expression. However, SCH-23390 （ DR1 antagonist） had no marked effect on above indicators. Conclusion DR1 activation is involved in the apoptosis of cultured neonatal rat cardiomyocytes in anoxia-reoxygenation injury through mitochondrial pathway.