益气活血中药组分配伍调节线粒体自噬改善心肌缺血再灌注损伤的机制研究

Mitophagy can inhibit the opening of mitochondrial membrane permeability transition pore (mPTP) and apoptosis, which is an important mechanism in reducing ischemia-reperfusion (I/R) injury after acute myocardial infarction (AMI). HIF-1α/BNIP3 is the key pathway of mitochondrial autophagy, which is closely related to myocardial I/R injury. Previous studies have confirmed that the component of qi-tonifying herbal medicine—Panax Quinquefolium Saponins (PQS) and that of blood-circulation-invigorating herbal medicine—Panax Notoginseng Saponins (PNS) can reduce I/R injury by inhibiting the opening of mPTP, but there still lacks studies about whether the mechanism has relation to the mitophagy pathway HIF-1α/BNIP3. Accordingly, the hypothesis is put forward: The compatibility of Qi-tonifying and blood-circulation-invigorating herbal medicine components (PQS+PNS) can remove damaged mitochondria and reduce ROS content through HIF-1α/BNIP3 mitochondrial autophagy pathway, thereby inhibiting mPTP opening and cell apoptosis, and reducing I/R injury. Therefore, the in vivo experiment is set up to verify the correlation between the reduction on I/R injury after using PQS+PNS and mitophagy in rats. In vitro experiment, we will further explore the regulation effect of PQS+PNS on ROS, mPTP, apoptosis and autophagy flow in hypoxia reoxygenation rat cardiomyocytes via HIF-1α/BNIP3 pathway, which can provide a new experimental basis for the clinical prevention and treatment of I/R injury using qi-tonifying medicine and blood-circulation-invigorating herbal medicine.

线粒体自噬能抑制线粒体膜通透性转换孔(mPTP)开放及细胞凋亡，是减轻心肌缺血再灌注(I/R)损伤的重要机制。HIF-1α/BNIP3是线粒体自噬关键通路，与心肌I/R损伤关系密切。前期研究证实益气中药组分西洋参茎叶总皂苷(PQS)、活血中药组分三七总皂苷(PNS)能通过抑制mPTP开放减轻心肌I/R损伤，但其机制是否与线粒体自噬相关尚缺乏报道。据此提出“益气活血中药组分配伍(PQS+PNS)可通过HIF-1α/BNIP3线粒体自噬通路清除受损线粒体，降低ROS含量，从而抑制mPTP开放及细胞凋亡，减轻心肌I/R损伤”的假说。本研究拟通过在体实验明确PQS+PNS减轻大鼠心肌I/R损伤作用与线粒体自噬的相关性，并通过离体实验进一步探讨PQS+PNS通过HIF-1α/BNIP3通路对缺氧复氧心肌细胞ROS、mPTP、凋亡及自噬的调控作用，为益气活血中药用于临床防治I/R损伤提供新的实验依据。

再灌注是临床治疗急性心肌梗死（AMI）的最有效策略。但再灌注引起的心肌缺血再灌注（I/R）损伤会进一步加重缺血心肌组织受损，影响心功能恢复，是AMI防治领域的难点。HIF-1α/BNIP3是线粒体自噬关键通路，与心肌I/R损伤关系密切。前期研究证实，益气中药组分西洋参茎叶总皂苷(PQS)、活血中药组分三七总皂苷(PNS)能通过抑制线粒体膜通透性转换孔（mPTP）开放减轻心肌I/R损伤，但其机制是否与线粒体自噬相关，尚缺乏报道。有研究表明，益气活血中药及其复方对再灌注心肌的保护作用优于单用益气药或活血药，且具有多组分多靶点的协同作用。据此提出“益气活血中药组分配伍（PQS+PNS）可通过HIF-1α/BNIP3线粒体自噬通路清除受损线粒体，减少活性氧（ROS）产生，从而抑制mPTP开放及细胞凋亡，减轻心肌I/R损伤”的假说。本研究通过建立大鼠心肌I/R损伤模型及H9C2细胞缺氧/复氧（H/R）损伤模型，从体内和体外两个途径，给予PQS+PNS进行干预，并加入自噬抑制剂或通过沉默目标基因作为对照，采用分子生物学、质粒构建与转染等技术，利用透射电镜、激光共聚焦显微镜等进行研究，为益气活血中药减轻心肌I/R损伤提供科学依据。通过在体实验发现：与模型组相比，益气活血中药组大鼠心梗面积显著减少，血清LDH、CK-MB含量明显下降，心肌细胞结构相对完整且水肿减轻，心肌细胞凋亡数目显著减少，心肌cleaved caspase-3、Cyt c、Bax蛋白表达水平显著降低，Bcl-2、HIF-1a、BNIP3蛋白表达水平增高，线粒体膜结构更为完整，明确了PQS+PNS减轻心肌I/R损伤与线粒体自噬的相关性，证实其I/R心肌保护作用与介导线粒体自噬、抑制线粒体凋亡通路激活有关。通过离体实验发现：与模型组相比，益气活血中药组明显减轻H/R心肌细胞损伤，且效果优于单用PQS或PNS，心肌细胞凋亡率显著降低，胞内及线粒体内ROS显著减少，自噬溶酶体数量显著升高，线粒体超微结构改善，HIF-1a与BNIP3的mRNA表达量显著下降。证实PNS+PQS对 H/R心肌细胞的保护机制可能与调控HIF-1α/BNIP3线粒体自噬通路、抑制过度自噬相关。本研究揭示了益气活血中药减轻心肌I/R损伤的新的分子机制，为益气活血中药及治法用于临床防治AMI患者I/R损伤提供了新的实验依据。