强心颗粒通过调控Rac-1/NOX/ROS轴抑制慢性心衰心气虚兼血瘀水肿证大鼠心肌细胞凋亡的分子机制

Abnormal activation of Rac-1/NOX/ROS axis plays an important role in the regulation of cardiomyocyte apoptosis in chronic heart failure.Previous experiments preliminarily confirmed that regulating the expression of myocardial Rac-1 protein and inhibiting ROS-induced cardiomyocyte apoptosis are important mechanisms for Qiangxin granule in the therapeutic effect of Qiangxin granule. In vivo and vitro experiments showed that AS-IV, an effective component of Astragalus membranaceus, could antagonize the apoptosis of cardiomyocytes induced by ROS by inhibiting the expression of NOX2 and NOX4, which downstream protein of Rac-1. Therefore, we propose the following scientific hypotheses: Qiangxin granule can inhibit cardiomyocyte apoptosis by regulating Rac-1/NOX/ROS axis in chronic heart failure rats with heart-qi deficiency leading to blood stasis and edema syndrome , but the specific molecular mechanism needs to be further investigated. This study will focus on ROS-induced cardiomyocyte apoptosis, and demonstrate that the drug can inhibit ROS-induced cardiomyocyte apoptosis by regulating myocardial Rac-1 protein phosphorylation, and further explore the mechanism of ROS-induced cardiomyocyte apoptosis. The key molecular signaling pathway downstream of Rac-1/NOX/ROS axis will be outlined, and the molecular mechanism of Qiangxin granule inhibiting cardiomyocyte apoptosis in chronic heart failure rats with heart-qi deficiency leading to blood stasis and edema syndrome by regulating Rac-1/NOX/ROS axis will be investigated in an all-round way.

Rac-1/NOX/ROS轴的异常激活在慢性心力衰竭心肌细胞凋亡的调控中发挥着至关重要的作用。前期实验初步证实调控心肌Rac-1蛋白表达以及抑制ROS诱导的心肌细胞凋亡是强心颗粒发挥疗效的重要机制。体内、外实验证实君药黄芪有效成分黄芪甲苷能够通过抑制Rac-1下游蛋白NOX2与NOX4表达拮抗ROS诱导的心肌细胞凋亡。因此，我们提出以下科学假说：强心颗粒可以通过调控Rac-1/NOX/ROS轴抑制慢性心衰心气虚兼血瘀水肿证大鼠心肌细胞凋亡，但具体分子机制有待进一步探明。本研究以ROS诱导的心肌细胞凋亡为着眼点，重点论证该药通过调控心肌Rac-1蛋白磷酸化抑制ROS诱导的心肌细胞凋亡，并深入挖掘、勾勒其干预调控Rac-1/NOX/ROS轴下游的关键分子信号通路，全面探明强心颗粒通过调控Rac-1/NOX/ROS轴抑制慢性心衰心气虚兼血瘀水肿证大鼠心肌细胞凋亡的分子机制。

强心颗粒可显著延缓慢性心衰心气虚兼血瘀水肿证大鼠心功能恶化，减少心肌ROS含量，降低心肌总Rac1、磷酸化Rac1以及NOX2、NOX4表达水平，减少DNA氧化损伤生物标志物8-OHdG核染（+）比率，减少心肌细胞凋亡指数，提示强心颗粒可通过调控Rac1蛋白磷酸化发挥抑制慢性心衰心气虚兼血瘀水肿证ROS诱导心肌细胞凋亡的作用。强心颗粒干预的大鼠在注射Rac1过表达腺病毒后，大鼠心功能持续恶化，心肌ROS含量、心肌总Rac1、磷酸化Rac1以及NOX2、NOX4表达水平、8-OHdG核染（+）比率、心肌细胞凋亡指数均显著升高，进一步反证了抑制Rac1蛋白磷酸化是强心颗粒拮抗ROS诱导心肌细胞凋亡的重要机制。研究通过网络药理学筛选、预测STAT1、TP53、RELA（p65-NF-κB）、IL-6等分子可能是强心颗粒调控Rac1/NOX/ROS轴的下游分子靶标，并通过进一步研究发现强心颗粒可显著减低慢性心衰心气虚兼血瘀水肿证大鼠心肌总p65-NF-κB、磷酸化p65-NF-κB、总IKK、磷酸化IKK表达水平，减低心肌组织TNF-α、IL-1β、IL-6含量，提示强心颗粒可通过调控p65-NF-κB磷酸化降低慢性心衰心气虚兼血瘀水肿证的心肌炎症反应，p65-NF-κB磷酸化是强心颗粒调控Rac1/NOX/ROS轴的重要下游调控靶标；而强心颗粒干预的大鼠在注射Rac1过表达腺病毒后，大鼠心肌总p65-NF-κB、磷酸化p65-NF-κB、总IKK、磷酸化IKK表达均显著升高，大鼠心肌TNF-α、IL-1β、IL-6含量亦显著升高，进一步反证了磷酸化p65-NF-κB是强心颗粒调控Rac1/NOX/ROS轴的重要下游调控分子，强心颗粒对慢性心衰心气虚兼血瘀水肿证心肌炎症的调控作用值得进一步挖掘。