Objective: To investigate the role of poly(ADP-ribose) polymerases-1 (PARP-1)-mediated blockade of autophagic flow in myocardial ischemia-reperfusion injury.
Methods: H9c2 cells, a rat cardiac myocyte line, were divided into control group, hypoxia/ reoxygenation model group (H/R group), PARP-1 inhibitor (PJ34) group, and PJ34 + H/R group. The total protein was extracted from the cells in each group to detect the expressions of pADPr, Bax, the DNA damage marker protein p-YH2ax, and autophagic flow-associated proteins LC3BⅡ/LC3Ⅰ, Beclin-1, and P62 using Western blotting.
Results: Compared with the control cells, the cells with H/R exhibited significantly increased expressions of pADPr, Bax and p-YH2ax (P < 0.05). The expressions of LC3B Ⅱ, beclin-1 and p62 were also increased significantly in the cells with H/R (P < 0.05), indicating the block of the autophagic flow. The application of PARP-1 inhibitor PJ34 in the cells with H/R significantly inhibited the expressions of pADPr (P < 0.05) and Bax (P < 0.01), and alleviated DNA damage in the cells. PJ34 treatment did not cause significant changes in the expressions of LC3B Ⅱ and beclin-1 but significantly decreased the expression of p62 (P < 0.05) in the cells with H/R.
Conclusions: Block of autophagic flow mediated by PARP-1 activation plays a role in myocardial ischemiareperfusion injury, and inhibition of PARP-1 activity can reverse autophagic flow block to reduce the injury.
目的: 探讨PARP-1介导的自噬流受阻在大鼠心肌缺血再灌注损伤（MIRI）中的作用。
方法: 采用大鼠心肌细胞H9c2制备缺血再灌注细胞模型。实验分为对照组、缺氧/复氧处理模型组、PARP-1抑制剂组、PARP-1抑制剂+模型组（PJ34+H/R组）。取各组处理后的6孔板H9c2细胞提取总蛋白后Western blot分别检测PARP-1活性蛋白pADPr，凋亡相关蛋白Bax，细胞DNA损伤标记蛋白p-γH2ax的表达，细胞自噬流相关蛋白：LC3BⅡ/LC3Ⅰ、Beclin-1、P62的表达量。通过GraphPad Prism 6统计软件对数据进行分析，比较各组间差异。
结果: 与对照组比较，MIRI模型组PARP-1活性标记物pADPr表达更高表示PARP-1激活增多，细胞凋亡损伤增加，DNA损伤（p-γH2ax）也增多（P < 0.05）。LC3B Ⅱ、beclin-1表达增高，同时p62表达也增高（P < 0.05），表示自噬流受阻。与模型组比较，加入PARP-1抑制剂后（H/R+PJ34组）可明显抑制心肌细胞内PARP-1活性（pADPr），细胞凋亡减少，细胞DNA损伤也减轻（P < 0.05）；自噬相关蛋白LC3B Ⅱ、beclin-1表达无明显变化，而p62表达降低（P < 0.05），表明自噬流受阻情况得到缓解（P < 0.05）。
结论: PARP-1激活介导的自噬流受阻在大鼠MIRI中发挥着作用，通过抑制PARP-1活性后可明显逆转自噬流受阻情况，减轻MIRI。
Keywords: autophagy; myocardial ischemia-reperfusion injury; poly(ADP-ribose) polymerases-1.