基于PI3K/Akt和HIF-1α/HO-1信号通路探讨理气活血滴丸对大鼠心肌缺血再灌注损伤的防护机制

Myocardial ischemia reperfusion injury (MIRI) is a common cause of sudden death in patients with coronary recanalization. How to prevent MIRI has become an urgent problem for the treatment of ischemic cardiomyopathy. In the modern Chinese medicine, hypofunction of yang-qi in chest, yin cold coagulation and qi stagnation and blood stasis are common pathological mechanism of MIRI. Liqi huoxue dripping pills have the effect of warming yang, kuan xiong, manage qi and activating blood. Previous studies have found that the pretreatment of liqi huoxue dripping pills have protective effects on MIRI in rats, which is related to the activation of PI3K/Akt signaling pathway. The potential important targets of the network pharmacological analysis of liqi huoxue dripping pills are PI3K/Akt signaling pathway and HIF-1α/HO-1 signaling pathway. This project intends to adopt the rat MIRI and neonatal rat myocardial cell hypoxia/reoxygenation model, using the PI3K/Akt and HIF-1α/HO-1 signaling pathway inhibitors and Lentivirus mediated shRNA gene silencing technology, combine with traditional Chinese medicine pharmacology and molecular cell biology research methods, explore the role of PI3K/Akt and HIF-1α/HO-1 signaling pathway in the protection of myocardial ischemia reperfusion injury in rats with liqi huoxue dripping pills, to further clarify the drug targets, explain its pharmacodynamic mechanism, and provide theoretical basis for the prevention of MIRI with traditional Chinese medicine.

心肌缺血再灌注损伤（MIRI）是冠脉再通术患者猝死的常见原因。如何防治MIRI，已成为治疗缺血性心肌病亟待解决的问题。现代中医学认为胸阳不振、阴寒凝结、气滞血瘀是MIRI的常见病理机制。理气活血滴丸具有温阳宽胸、理气活血的功效。前期研究发现理气活血滴丸预处理对大鼠MIRI具有保护作用，与其激活PI3K/Akt信号通路有关；网络药理学分析发现理气活血滴丸的潜在重要靶标为PI3K/Akt信号通路和HIF-1α/HO-1信号通路。本项目拟采用大鼠MIRI及乳鼠心肌细胞缺氧/复氧模型，利用PI3K/Akt和HIF-1α/HO-1信号通路抑制剂、慢病毒介导的shRNA基因沉默技术，结合中药药理学和分子细胞生物学等研究方法，探究PI3K/Akt和HIF-1α/HO-1信号通路在理气活血滴丸防护大鼠MIRI中的作用，进一步明确该药作用靶点，阐释其药效作用机制，为中医药防治MIRI提供理论依据。

再灌注治疗是急性心肌梗死常用的治疗方案，然而大量实验研究和临床报道发现再灌注可引起心肌额外损伤，严重影响患者预后。如何防治心肌缺血再灌注损伤（MIRI）已成为治疗缺血性心肌病亟待解决的问题。现代中医家认为胸阳不振、阴寒凝滞、气滞血瘀是MIRI常见的病理机制。理气活血滴丸具有温阳宽胸、理气活血功效。本项目通过在在体和细胞实验中，添加PI3K/Akt和HIF-1α/HO-1信号通路特异性抑制剂LY294002、YC-1和ZnPP及腺病毒介导的RNA干涉技术，结合组织病理学技术、免疫印迹、免疫荧光、流式细胞术等方法，分析PI3K/Akt和HIF-1α/HO-1信号通路在理气活血滴丸保护大鼠心肌缺血再灌注损伤中的作用，明确该药作用机制和靶点。本研究结果发现理气活血滴丸能增强心肌缺血再灌注损伤大鼠心肌组织抗氧化能力，减轻线粒体损伤，改善心肌组织病理学变化，升高Bcl-2蛋白表达，降低Bax和Cleaved-Caspase-3蛋白表达，进一步升高Akt磷酸化表达，降低缺氧复氧损伤诱导的HIF-1α高表达，抑制大鼠心肌细胞凋亡，而PI3K抑制剂LY294002、HIF-1α抑制剂YC-1和HO-1抑制剂ZnPP，以及腺病毒干涉HIF-1α，能减弱理气活血滴丸对心肌缺血再灌注损伤大鼠的抗氧化能力和抑制细胞凋亡能力，降低p-Akt、HIF-1α、HO-1的表达，另外干涉HIF-1α表达能使大鼠心肌细胞缺氧复氧损伤更严重。这些研究说明理气活血滴丸对大鼠心肌缺血再灌注损伤和大鼠心肌细胞缺氧复氧损伤的保护作用，与其减轻大鼠心肌细胞氧化损伤、减轻线粒体损伤和抑制细胞凋亡有关；理气活血滴丸可激活PI3K/Akt信号通路，降低缺血再灌注后HIF-1α的高表达，稳定HIF-1α蛋白表达，HIF-1α从细胞质转移至细胞核内，调控HO-1基因表达，减轻心肌缺血再灌注损伤大鼠心肌细胞氧化损伤和抑制心肌细胞凋亡，进而保护大鼠心肌缺血再灌注损伤；阻断PI3K/Akt或HIF-1α/HO-1信号通路，将减轻理气活血滴丸对大鼠心肌细胞缺氧复氧损伤的保护作用，说明理气活血滴丸通过调控PI3K/Akt和HIF-1α/HO-1信号通路保护大鼠心肌缺血再灌注损伤。本研究为利用温阳宽胸、理气活血法防治心肌缺血再灌注损伤提供了实验依据，也为理气活血滴丸的潜在临床应用提供参考。

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