TRPV1-NKA/NKB在七氟烷致发育期大鼠AHR中的作用机制研究

Sevoflurane is a major agent in pediatric anesthesia. Bidirectional effects on airway smooth muscles, namely spasm and relaxation of airway smooth muscles, induced by sevoflurane have become a hot research topic. Previous reports and previous research found that sevoflurane tended to play a spasm role in airway at development phase. It can induce the occurrence of neonatal severe airway hyperresponsiveness (AHR), but the mechanism is unknown and needs to be studied. We found in further pre-experiment that activation of proteins of transient receptor potential vanilloid -1 (TRPV1) pathway might be a key event in the process during which sevoflurane induced AHR, which is worth of design of target for prevention and treatment and clinical transformation. Considering this, this project intends to construct AHR model in neonatal rats, TRPV1 gene knock-out mice model and in vitro culture model of airway smooth muscle cells; using biological technologies including up-regulation/down-regulation of channel activity, detection on protein activation, cell biomechanics and airway mechanics, this study studied the correlations between the occurrence and development of AHR with the changes of expressions and activities of TRPV1 pathway and its important downstream genes and proteins when the airway at development phase was exposed to sevoflurane, to enhance the understanding on the mechanism of AHR at development phase and clarify the mechanism of TRPV1-NKA/NKB pathway on rats with AHR at development phase induced by sevoflurane, so as to provide a certain theoretical basis for finding a new way for clinical prevention and treatment.

七氟烷是儿科麻醉的主要用药，其诱发气道平滑肌痉挛/舒张的“双相”作用已成为研究热点。既往报道和我们前期研究发现，七氟烷在“发育期气道”中更倾向为痉挛作用，诱发新生儿严重气道高反应性（AHR）发生，但机制不明，亟待研究。我们进一步预实验发现香草酸瞬时受体1 (TRPV1)通道蛋白激活可能是七氟烷致发育期气道AHR进程中的关键事件，具备防治靶点设计及临床转化价值。鉴于此，本项目拟构建新生大鼠AHR模型、TRPV1基因敲除小鼠模型及气道平滑肌细胞离体培养模型；利用通道活性上调/下调、蛋白活化检测、细胞生物力学及气道力学等生物学技术，研究发育期气道暴露于七氟烷时AHR的发生发展与TRPV1通路及其下游重要基因/蛋白表达/活化变化的相关性；增进对发育期气道AHR机制的认识，以期阐明TRPV1-NKA/NKB通路在七氟烷致发育期大鼠AHR中的作用机制；从而探寻一条临床防治的新途径并奠定一定的理论基础。

通过本研究初步揭示了发育期大鼠吸入七氟烷对呼吸系统的影响与肺组织TRPV1-神经源性炎症相关蛋白的关系，以及与神经细胞胆碱能通路的相关性。阐明了TRPV1通道蛋白在七氟烷麻醉致发育期气道高反应发生的直接关系，明确了TRPV1作为干预靶点防治七氟烷诱发发育期大鼠气道高反应的可行性。.首先通过检测不同浓度七氟烷对发育期和成年期普通SD大鼠呼吸系统TRPV1-NKA、NKB、SP、CGRP蛋白含量，发现发育期大鼠吸入低浓度七氟烷可激活呼吸系统伤害性感受器TRPV1，并调节感觉神经肽NKA、NKB、SP、CGRP的释放增加，高浓度七氟烷对发育期及成年大鼠未见变化。使用TRPV1拮抗剂预处理后对发育期和成年期普通SD大鼠进行七氟烷干预，发现拮抗TRPV1受体可减轻发育期大鼠 TRPV1蛋白表达和下游NKA、NKB、SP、CGRP释放。该部分的实验提示低浓度七氟烷对发育期大鼠呼吸系统存在潜在风险，抑制 TRPV1受体有望成为治疗靶点。.接下来我们成功建立了发育期SD大鼠卵清蛋白诱导的哮喘模型，并以不同浓度七氟烷对该模型干预。完成血气分析，肺组织白介素4,干扰素γ和血浆IgE含量，肺组织HE染色切片进行病理评分。显示低浓度七氟烷会使发育期哮喘大鼠血氧饱和度降低，血二氧化碳分压升高，并且会使血浆IgE、肺组织白介素4,干扰素γ升高，而高浓度七氟烷可以降低血浆IgE、肺组织白介素-4，干扰素-γ和肺组织HE染色切片的病理评分。提示低浓度七氟烷会加重发育期哮喘大鼠的气道炎症，发挥气道损伤作用；而高浓度七氟烷能降低发育期哮喘大鼠的气道炎症，发挥气道保护作用。.最后我们通过体外培养神经细胞建立七氟烷吸入模型，观察吸入高浓度七氟烷2h、4h、6h后在光学显微镜下的细胞形态变化，CCK-8法检测细胞活力，ELISA法检测胆碱能相关蛋白表达，RT-PCR法测定胆碱能蛋白相关mRNA表达。发现在高浓度七氟烷处理4h和6h后神经细胞生长状态不佳，活性降低，胆碱能相关蛋白a7nAChR 、AChE 、ChAT表达含量及其mRNA相对表达量减少。提示高浓度七氟烷可能通过抑制神经细胞活力，影响咳嗽反射的传导，从而发挥气道保护作用。