急性肺栓塞时交感介导下PKA/p38信号通路及其钙通道调控肺血流的机制研究

The mortality rate of the acute pulmonary embolism combined shock was high, and the effect of clinical treatment was not very good. Our previous research found that the small area of pulmonary embolism also led to the attenuation of the pulmonary blood flow and circulation failure, and the pulmonary vasospasm in none pulmonary embolism tissues may play an important role in the process of shock caused by acute pulmonary embolism. Our preliminary experiment indicated the over-expression of sympathetic mediator such as TH and α1 receptor in pulmonary tissues was synchronous with the blood flow attenuation degree of none pulmonary embolism area. The detail mechanism about the sympathetic activity activated the the contraction of the pulmonary artery smooth muscle cells was still unclear. This research project aimed at ① Detecting the sympathetic activity expression of none pulmonary embolism area in vivo to observe the mechanism effect of the higher activation of PKA/p38 signaling pathways mediating ET-1/NO imbalance and the activation of calcium influx promoting the MLC phosphorylation to increase pulmonary artery smooth muscle cell constriction. ② α- blocker phentolamine was given to evaluate the regulation effects of sympathetic activity down-regulation on the PKA/p38 signaling pathways and Ca2+ channel activated MLC phosphorylation. And the cell experiment in vitro was performed to elucidate related mechanisms. Previous imaging results confirmed that the pulmonary blood flow attenuation, led the systemic failure. The tyrosine hydroxylase (TH), norepinephrine (NE), α1 receptor expression and neuropeptide Y (NPY) contents also increased significantly in the embolism and non-embolism lung tissue. Our follow-up experiments further aimed to obtain the regulation of pulmonary vascular endothelial ET-1/NO imbalance mediated by PKA/p38 signaling pathways and MLC phosphorylation with α1 receptor over- expression. Meanwhile, the pulmonary artery smooth muscle function would be evaluated by detecting PKA/p38 signaling pathways and MLC phosphorylation levels post blocking ofα1 receptor of pulmonary vessels. Our research was hopeful to explain the whole pulmonary blood flow loss, provide new theoretical basis for pulmonary failure and a new idea for its treatment and reducing the mortality in the acute pulmonary embolism combined shock.

急性肺栓塞合并休克的死亡率居高不下，目前临床治疗以溶栓为主，但其效果不佳，有效率不到50%。降低急性肺栓塞合并休克的高死亡率已 成为临床医学的重大课题。我们前期研究发现，小面积肺栓塞也可导致全肺血流减少和体循环衰竭非栓塞区肺血管痉挛在急性肺栓塞引起的休克过程中发挥重要作用。早期研究表明，在肺动脉高压、低氧和休克刺激下，交感神经系统被继发性激活。本项目预实验结果提示，急性肺栓塞合并休克家兔肺组织交感神经介质TH和α1受体高表达与非栓塞区血流衰减密切相关。然而交感递质如何介导肺血管平滑肌细胞收缩导致肺血管痉挛的具体机制尚不明确。. 本项目拟通过在体和离体实验论证：①栓塞后，非栓塞区交感活性上调，介导激活了PKA/p38信号通路，ET-1/NO失衡导致肺动脉平滑肌收缩以及交感介质的高表达激活钙通道，钙离子内流，与肌球蛋白轻链激酶（MLCK）结合后促使MLC磷酸化增加，ATP水解激动肺动脉平滑肌收缩的机制。②给予α受体阻断剂酚妥拉明，评估交感活性下调后对PKA/p38信号通路及钙通道下游MLC磷酸化的调控效应，以论证交感下调后是否弱化平滑肌收缩，改善肺血流，逆转循环衰竭. 前期影像学结果证实，全肺血流衰减，尤其是非栓塞区肺血流减少导致体循环衰竭。栓塞区和非栓塞区肺组织交感活性相关的酪氨酸羟化酶（TH）、去甲肾上腺素（NE）和α1受体表达上调，神经肽Y（NPY）含量也明显增加。后续实验将进一步获得在交感α1受体高表达下，PKA/p38信号通路介导的肺血管内皮ET-1/NO失衡，及钙通道MLC磷酸化状态。同时阻断肺血管α1受体后，PKA/p38信号通路的表达和MLC磷酸化水平，评估肺动脉平滑肌功能状态。. 本课题有望为解释急性肺栓塞合并休克的全肺血流丢失，肺循环衰竭提供新的理论依据，为指导临床急性肺栓塞合并休克治疗、降低死亡率提供新思路。

急性肺栓塞（APE）合并休克时，肺动脉血流丢失导致了体循环衰竭。我们前期研究已证实非栓塞区肺动脉痉挛是发生APE休克的重要机制之一。为拓展治疗方法学，本课题在构建家兔APE休克模型基础上，检测非栓塞区肺动脉交感α1、α2受体活性表达，论证α1/α2受体经蛋白激酶A（PKA）信号通路和/或钙通道并行介导下实现肺血流的调控；给予α受体阻滞剂酚妥拉明（PTL），评估交感α受体阻滞对下游PKA信号通路及钙通道肌球蛋白轻链（MLC）磷酸化的影响，逆向解析α受体介导下肺血流衰减的发生机制。. 结果表明：（1）APE的急性缺氧和炎症爆发将诱导非栓塞区肺动脉α1、α2受体活性上调。α1受体过度激活使磷脂酶（PLC）活化，三磷酸肌醇（IP3）生成增加，平滑肌胞质内Ca2+增多；α2受体上调并与G蛋白偶联受体结合，阻断了腺苷酸环化酶（AC）活化，抑制PKA生成，下调p38蛋白表达，阻滞Ca2+向肌浆网转移，胞内Ca2+水平增高。胞内增加的游离Ca2+与钙调蛋白（CaM）结合形成复合物刺激MLCK活化，介导肌丝运动速度加快，肺动脉发生痉挛。痉挛后的肺动脉造影及全身血流动力学数据显示：平均肺动脉压（MPAP）显著上升2倍，肺血流衰减；平均动脉压（MAP）急剧下降至40~50 mmHg。（2）APE合并休克后，经肺动脉注射PTL 0.4 mg/kg，能有效降低MPAP，增加非栓塞区的肺血流量，缩短肺循环时；MAP升高至约70 mmHg，休克逆转率为71.43%。Western-blot和免疫组化实验结果显示，PTL抑制了非栓塞区的肺组织α1、α2受体表达。α1受体活性下调使得PLC活化和IP3抑制，肌浆网释放Ca2+减少；α2受体表达下调，p-PKA水平升高，p38蛋白表达上调，PASMCs内Ca2+浓度降低。MLCK、α-SMA表达均下调，平滑肌舒张。细胞实验进一步证实PTL抑制α受体活性，PLC下调并阻断Ca2+通道，促进eNOS释放。 . 本研究尝试建立APE休克的发病新机制。实验数据表明：除栓子堵塞外，交感α受体激活参与了全肺血流的丢失。依从机制的导引，应用PTL抑制α受体活性，下调平滑肌的胞内Ca2+负载，通过肺动脉舒张修复非栓塞区的肺血流，逆转肺、体循环衰竭。基于发病机制的认知创新，本研究探索了一种APE休克治疗的新方法。