磷酸二酯酶9A在心肌细胞缺血缺氧再灌注损伤的表达变化及意义

Expression and significance of phosphodiesterase 9A in myocardial ischemia-anoxic reperfusion injury

目的 磷酸二酯酶9A(PDE9A)在缺血缺氧再灌注中的作用尚不明确。文中旨在探讨PDE9A对缺血缺氧再灌注所致乳鼠心肌细胞的作用。方法 培养原代乳鼠的心肌细胞鉴定后,采用氧糖剥夺/再灌注法(OGD/R)构建心肌细胞缺氧的模型;磷酸二酯酶9A (PDE9A)的干扰质粒转染入OGD/R细胞,并向细胞中分别加入可溶性鸟苷酸环化酶(sGC)和受体型鸟苷酸环化酶(rGC)的抑制剂处理。实验分为对照组(心肌细胞不干预)、OGD/R组(构建心肌OGD/R模型)、siPDE9A组(构建PDE9A的干扰质粒转染OGD/R细胞)、sGC组(正常细胞加入sGC抑制剂)、sGC+siPDE9A组(PDE9A的干扰质粒转染OGD/R细胞,并加sGC抑制剂)、sGC+rGC+siPDE9A组(sGC抑制剂+rGC抑制剂+siPDE9A)。采用Western blot检测PDE9A和环磷酸鸟苷(cGMP)的表达,流式细胞术检测细胞凋亡水平。向OGD/R的心肌细胞中加入PDE9的抑制剂BAY 73-6691,并按不同时间分为30 min组、60 min组、120 min组,180 min组及240 min组,采用CCK-8观察细胞活性,流式水平检测细胞凋亡情况和ATP检测细胞线粒体活性,采用Western blot检测PDE9A和cGMP及其下游相关蛋白的级联变化。结果 OGD/R组PDE9A表达量(1.00±0.21)显著高于对照组(2.13±0.26),差异有统计学意义(P<0.01)。与对照组比较,转染siPDE9至大鼠心肌细胞时,siPDE9A组的PDE9A蛋白被显著抑制(P<0.001)。与对照组比较,OGD/R组cGMP蛋白表达显著抑制,PDE9A蛋白表达显著增加(P<0.001);与OGD/R组比较,sGC组的cGMP蛋白表达显著抑制,siPDE9A组、siPDE9A+sGC组、 siPDE9A+sGC+rGC组的PDE9A蛋白表达显著抑制(P<0.001),cGMP蛋白表达显著增加(P<0.001)。流式细胞检测结果提示,与OGD/R组比较,对照组、siPDE9A组、siPDE9A+sGC组细胞凋亡率显著抑制(P<0.05)。CCK-8检测结果表明,对照组、60 min组、120 min组、180 min组、240 min组细胞增殖较OGD/R组显著上升(P<0.05)。流式细胞检测结果表明,对照组、60 min组、120 min组、180 min组的细胞凋亡率较OGD/R组显著抑制(P<0.05)。ATP检测结果表明,对照组、60 min组、120 min组、180 min组的ATP含量较OGD/R组显著上升(P<0.05)。结论 PDE9A参与心肌细胞缺血缺氧再灌注损伤,抑制PDE9A能降低因缺血缺氧再灌注损伤所致的细胞凋亡,且该作用机制可能与抑制cGMP水平有关,并且避开了NO信号通路。

Objective The role of phosphodiesterase 9 A(PDE9 A) in ischemia-anoxic reperfusion remains unclear. This paper aims to investigate the effects of PDE9 A on neonatal rats’ myocardial cells induced by ischemia-anoxic reperfusion. Methods After isolation and culture of primary neonatal rat cardiomyocytes, the oxygen glucose deprivation/reperfusion(OGD/R) method was performed to construct the OGD/R model of cardiomyocytes. The interference plasmid of PDE9 A was constructed and transfected into OGD/R model. The cells were then treated with soluble guanylyl cyclase(sGC) inhibitor, receptor guanylyl cyclase(rGC) inhibitor. The experiment was divided into control group(without intervention of cardiomyocytes), OGD/R group(myocardial OGD/R model was constructed), siPDE9 A group(OGD/R cells were transfected with PDE9 A interfering plasmid), sGC group(normal cells were added with sGC inhibitor), sGC+siPDE9 A group(OGD/R cells were transfected with PDE9 A interfering plasmid), and SGC +rGC+siPDE9 A group(sGC inhibitor +rGC inhibitor +siPDE9 A). Western blot was used to detect the expression of PDE9 A and cGMP, and flow cytometry was used to detect cell apoptosis. Cardiomyocytes with PDE9 inhibitor BAY 73-6691 were added into OGD/R cardiomyocytes and divided into 30 min, 60 min, 120 min, 180 min and 240 min groups according to different time. Cell activity was observed by CCK-8, cell apoptosis was detected by flow cytometry and mitochondrial activity was detected by ATP. Western blot was used to detect the cascade changes of PDE9 A and cGMP and their downstream related proteins. Results Compared with the control group, the expression of cGMP protein in OGD/R group was significantly inhibited, and the expression of PDE9 A protein was significantly increased(P<0.001). Compared with OGD/R group, cGMP protein expression was significantly inhibited in sGC group, PDE9 A protein expression was significantly inhibited in siPDE9 A group, siPDE9 A+sGC group and siPDE9 A+sGC+rGC group(P<0.001), and cGMP protein expression was significantly increased(P<0.001). Flow cytometry results showed that compared with OGD/R group, the apoptosis rate of control group, siPDE9 A group and siPDE9 A+sGC group was significantly inhibited(P<0.05). CCK-8 showed that cell proliferation in control group, 60 min group, 120 min group, 180 min group and 240 min group was significantly increased compared with OGD/R group(P<0.05). Flow cytometry results showed that the apoptosis rate of control group, 60 min group, 120 min group and 180 min group was significantly inhibited compared with OGD/R group(P<0.05). ATP detection results showed that ATP content in control group, 60 min group, 120 min group and 180 min group was significantly increased compared with OGD/R group(P<0.05). Conclusion PDE9 A is involved in myocardial ischemia-hypoxic-reperfusion injury. Inhibition of PDE9 A may reduce apoptosis caused by ischemia-hypoxic-reperfusion injury, and the mechanism of action may be related to the inhibition of cGMP levels without the involvement of NO signal pathway.