CTRP9对远端缺血预处理心肌梗死模型大鼠心肌缺血再灌注致脑损伤的保护作用及其机制研究

The protective effects and mechanisms of CTRP9 on cerebral injury induced by myocardial ischemia-reperfusion in rat models of myocardial infarction treated with remote ischemic postconditioning

摘要:

目的 探讨CTRP9对远端缺血预处理心肌梗死模型大鼠心肌缺血再灌注致脑损伤的保护作用。方法 选取40只雄性SD大鼠，随机分为4组：假手术组（NS组）、心肌缺血再灌注组（NIR组）、缺血预处理组（NIPost组）及缺血预处理+ CTRP9抑制剂组（NIPostI组），每组10只。采用苏木精-伊红染色观察大鼠脑组织病理学变化，免疫组织化学法检测大鼠脑组织凋亡因子、炎症因子的表达，TUNEL法检测大鼠脑组织细胞凋亡，Western blotting检测大鼠心肌组织p-AMPK/t-AMPK、LC3Ⅰ/Ⅱ及P62蛋白的表达。结果 ①HE染色结果显示，NS组皮层的神经元胞浆十分丰富，核呈圆形，碱染后呈蓝色，分布均匀且排列整齐；NIR组神经元结构受损，分布不均匀，胞浆出现空泡，胞核固缩，碱染后出现了淡红色；NIPost组细胞结构大多恢复正常，大部分神经元包膜完整，胞核清晰可见；NIPostI组经CTRP9抑制剂干预后，预处理的保护效应消失。②NIR组、NIPost组及NIPostI组脑组织Bax、IL-6、IL-8表达水平高于NS组（P <0.05），Bcl-2、IL-10表达水平低于NS组（P <0.05）；NIPost组较NIR组Bax、IL-6、IL-8表达水平降低（P <0.05），Bcl-2、IL-10表达水平升高（P <0.05）；NIPostI组较NIPost组Bax IL-6、IL-8表达水平升高（P <0.05），Bcl-2、IL-10表达水平降低（P <0.05）。③NIR组、NIPost组和NIPostI组脑组织凋亡细胞多于NS组（P <0.05）；NIPost组和NIPostI组较NIR组凋亡细胞减少（P <0.05）。NIPost组和NIPostI组比较，差异无统计学意义（P >0.05）。④NIR组、NIPost组及NIPostI组心肌组织p-AMPK/t-AMPK蛋白相对表达量低于NS组（P <0.05），LC3Ⅰ/Ⅱ和P62蛋白相对表达量高于NS组（P <0.05）；NIPost组较NIR组p-AMPK/t-AMPK蛋白相对表达量升高（P <0.05），LC3Ⅰ/Ⅱ和P62蛋白相对表达量降低（P <0.05）；NIPostI组较NIPost组p-AMPK/t-AMPK蛋白相对表达量降低（P <0.05），LC3Ⅰ/Ⅱ和P62蛋白相对表达量升高（P <0.05）。结论 远端缺血预处理可缓解大鼠心肌缺血再灌注所致脑损伤。CTRP9可通过激活AMPK信号通路，参与远端缺血预处理对心肌缺血再灌注所致脑损伤的保护过程。CTRP9水平升高有益于防治心肌梗死。

Abstract:

Objective To explore whether C1q/tumor necrosis factor-related protein 9 (CTRP9) is involved in protecting the cerebral injury induced by myocardial ischemia-reperfusion via regulating the adenosine 5'-monophosphate activated protein kinase (AMPK) pathway in rat models of myocardial infarction treated with remote ischemic postconditioning.Methods Forty male SD rats were selected and randomly divided into 4 groups, including sham operation group (NS group), myocardial ischemia-reperfusion group (NIR group), ischemic postconditioning group (NIPost group) and ischemic postconditioning + CTRP9 inhibitor group (NIPostI group), with 10 rats in each group. The histopathological changes of brain tissues were observed by HE staining. The expression levels of apoptosis and inflammatory factors were detected by immunohistochemistry. The apoptosis was measured by TUNEL method, and the protein expressions of p-AMPK/t-AMPK, LC3 Ⅰ/Ⅱ and P62 were measured by Western blotting.Results The histopathological examination showed that cortical neurons in NS group had abundant cytoplasm and round nuclei, which were stained blue by hematoxylin, distributed evenly and arranged neatly. In the NIR group, the structures of neurons were damaged, and the neurons were distributed unevenly, with cytoplasmic vacuolation and nuclear pyknosis, which were stained reddish by hematoxylin. In the NIPost group, the cell structures were basically restored to normal, and most of the neurons had intact membranes while the nuclei were clearly observed. In the NIPostI group, the protective effects of the postconditioning were counteracted by the intervention with CTRP9 inhibitor. The expression levels of Bax, IL-6, and IL-8 in the brain tissues in NIR group, NIPost group and NIPostI group were higher than those in NS group (P < 0.05), while the expression levels of Bal-2 and IL-10 were lower than those in NS group (P < 0.05). Compared with NIR group, the expression levels of Bax, IL-6 and IL-8 in NIPost group were decreased (P < 0.05), while the expression levels of Bal-2 and IL-10 were increased (P < 0.05). The expression levels of Bax, IL-6, and IL-8 in NIPostI group were higher than those in NIPost group (P < 0.05), and the expression levels of Bcl-2 and IL-10 in NIPostI group were lower than those in NIPost group (P < 0.05). Compared with NS group, the numbers of apoptotic cells in NIR group, NIPost group and NIPostI group were increased (P < 0.05). Compared with NIR group, the numbers of apoptotic cells in NIPost group and NIPostI group were decreased (P < 0.05). There was no difference in the number of apoptotic cells between NIPost group and NIPostI group (P > 0.05). Compared with NS group, the protein expressions of p-AMPK and t-AMPK in NIR group, NIPost group and NIPostI group were decreased (P < 0.05), while the protein expressions of LC3Ⅰ/Ⅱ and P62 in these groups were increased (P < 0.05). The protein expressions of p-AMPK and t-AMPK in NIPost group were higher than those in NIR group (P < 0.05), while the protein expressions of LC3 Ⅰ/Ⅱ and P62 in NIPost group were lower than those in NIR group (P < 0.05). Compared with NIPost group, the protein expressions of p-AMPK and t-AMPK in NIPostI group were lower (P < 0.05), but the protein expressions of LC3Ⅰ/Ⅱ and P62 in NIPostI group were higher (P < 0.05).Conclusions The remote ischemic postconditioning can alleviate the brain injury caused by myocardial ischemia-reperfusion in rats, where CTRP9 may participate in the process by activating the AMPK signaling pathway. Therefore, increasing the level of CTRP9 is beneficial to the prevention and treatment of myocardial infarction.