基于iNOS/NO-cGMP-PKG信号通路探讨蝉芩颗粒抑制TRPV1通道减轻感染后咳嗽气道神经源性炎症的机制研究

Post-infectious cough affects the physical and mental health of the patients seriously,and still lacks of effective treatment.How to inhibit neurogenic inflammation is a key problem in the treatment of post-infectious cough.The activation of TRPV1 channel induced by iNOS/NO-cGMP-PKG signal pathway can release the tachykinin, which plays an important role in neurogenic inflammation. Previous research showed Chanqin granules can reduce tachykinin expression and inhibit the airway neurogenic inflammation. Systems pharmacology studies have shown that iNOS may be the target of Chanqin granules in the treatment of post-infectious cough. According to this, a hypothesis of"Chanqin granules can down regulate iNOS expression in neurons, inhibit TRPV1 channel activity through iNOS/NO-cGMP-PKG signal pathway, reduce tachykinin release and alleviate airway neurogenic inflammation after infection."is put forward. We intend to develop models of PIV3 infected guinea pigs and guinea pig's superior cervical ganglion cell. We will observe the effect of Chanqin granules on iNOS/NO-cGMP-PKG signal pathway, TRPV1 channel, airway neurogenic inflammation and cough sensitivity by using cell patch clamp, retrograde fluorescent labeling,etc. From the perspective of "airway neurogenic inflammation", the mechanism of "Wind Evil Encroach Lung"will be discussed, and the mechanism of Chanqin granules in treating post-infectious cough will be expounded.This study will provide scientific basis for the clinical application of Chanqin granules.

感染后咳嗽严重影响患者身心健康，尚缺乏有效治疗手段。如何抑制神经源性炎症是治疗感染后咳嗽的关键问题，iNOS/NO-cGMP-PKG通路诱导TRPV1通道活化，导致速激肽释放在神经源性炎症中起重要作用。前期研究发现蝉芩颗粒可下调速激肽蛋白表达，抑制神经源性炎症。网络药理学显示iNOS可能是蝉芩颗粒治疗感染后咳嗽的作用靶点。据此提出“蝉芩颗粒可下调神经元iNOS表达，通过iNOS/NO-cGMP-PKG途径抑制TRPV1通道活性，减少速激肽释放，减轻感染后咳嗽气道神经源性炎症”假说。拟构建PIV3感染豚鼠与感染后豚鼠颈神经元模型，采用全细胞膜片钳、逆行荧光标记等技术，观察蝉芩颗粒对iNOS/NO-cGMP-PKG通路、TRPV1通道、气道神经源性炎症、咳嗽敏感性的影响，从“气道神经源性炎症”角度讨论感染后咳嗽“风邪恋肺”病机，阐述蝉芩颗粒治疗感染后咳嗽的作用机制，为该方临床应用提供科学依据。

感染后咳嗽严重影响患者身心健康，尚缺乏有效治疗手段。如何抑制神经源性炎症是治疗感染后咳嗽的关键问题，iNOS/NO-cGMP-PKG通路诱导TRPV1通道活化，导致速激肽释放在神经源性炎症中起重要作用。前期研究发现蝉芩颗粒可下调速激肽蛋白表达，抑制神经源性炎症，本课题围绕“iNOS/NO-cGMP-PKG-TRPV1-神经源性炎症-感染后咳嗽”这一主线，展开研究。. 研究进展顺利，体内研究采用豚鼠H1N1病毒鼻腔滴注染毒模型，研究结果显示，治疗后蝉芩颗粒高剂量组血清SP、IL-6、IL-1β，TNF-α，ICAM-1水平下降，蝉芩颗粒高剂量可有效抑制豚鼠背根神经节iNOS、sGC（cGMP上游分子）、PKG mRNA 和TRPV1蛋白表达水平的升高，并可改善肺部损伤，减轻炎性细胞浸润。体外实验采用LPS模拟革兰氏阴性菌感染的方法诱导全反式维甲酸分化后人神经母细胞瘤细胞及人支气管上皮细胞神经源性炎症模型。LPS诱导人神经母细胞瘤细胞实验结果显示，高剂量蝉芩颗粒含药血清可有效降低iNOS、sGC、cGMP、PKG、SP、IL-6、IL-1β、ICAM-1 mRNA表达水平，降低iNOS、sGC、PKG蛋白表达，并可抑制［Ca2＋］i水平升高，由本实验结果可推测，蝉芩颗粒抑制神经源性炎症似无需依赖TRPV1活化，其机制值得进一步探索。LPS诱导人支气管上皮细胞实验结果显示：在高剂量蝉芩颗粒含药血清干预后，炎症因子 IL-6、IL-1β、ICAM-1 及速激肽 SP mRNA 水平下降，信号分子iNOS、sGC、cGMP、PKG与TRPV1 mRNA（或蛋白）表达水平也同步下降，且标志TRPV1通道活化的Ca2+内流减少，与iNOS抑制剂L-NAME作用趋势相似。. 研究结果表明：蝉芩颗粒可能通过下调iNOS/NO-cGMP-PKG信号通路、抑制TRPV1通道活化，减少速激肽及炎症因子释放，从而发挥减轻神经源性炎症的作用。. 本研究为进一步深入探索蝉芩颗粒抗气道神经源性炎症的靶点，阐明该方治疗感染后咳嗽的机制提供了科学的依据和基础。