ERK1/2通路对哮喘大鼠气道重塑、气道炎症的调控及针刺干预的分子机制

The asthma model of SD male rats are used as research objects with three points, Dazhui(GV14), bilateral Fengmen(BL12) and bilateral Feishu(BL13), punctured by filiform needle. The detection indexes include the airway function and the pathological changes of the lung tissue in the asthma rats. The airway smooth muscle cell (ASMC) will be administrated with acupuncture serum to observe the pathological changes by electron microscopy and the migration activity of ASMC through the tests of horizontal migration and transmembrane migration. Dectation the expression of ERK1/2 signaling pathway in the lung tissue and ASMC by the techniques of western blot, immunohistochemistry, immunofluorescence and RT-PCR in different groups. Test the contents of effectors, including Eotaxin, RANTES, TGF-β1, VEGF, Fibronectin and Collagen Ⅰin the lung tissue and ASMC by ELISA tests. Based on the experiments in vitro and in vivo, the function of ERK1/2 signaling pathway will be analyzed on airway remodeling and inflammation, and the effect of acupuncture and acupuncture serum will be certain on ERK1/2 signaling pathway.It will provide experimental basis for molecular targets of acupuncture in asthma prevention and treatment, and provide new idea for biology mechanism research to explore the modern biology research of integral regulation of acupuncture.

本研究以哮喘模型大鼠为研究对象，采用"三穴五针法"干预，检测大鼠气道功能，观察大鼠肺组织的病理学改变；进而培养哮喘大鼠气道平滑肌细胞（ASMC），给予针刺血清干预，利用电镜观察ASMC的病理学改变，通过平面迁移、跨膜迁移评估针刺血清对ASMC迁移功能的影响；用Western blot、免疫组化、免疫荧光及RT-PCR技术检测大鼠肺组织和大鼠ASMC中ERK1/2信号通路表达差异，用ELISA技术检测肺组织和大鼠ASMC中Eotaxin、RANTES、TGF-β1、VEGF、Fibronectin和Collagen Ⅰ效应因子的含量。通过动物整体实验和细胞实验两方面的研究，明确ERK1/2信号通路对哮喘大鼠气道重塑和气道炎症的调控，明确针刺干预和针刺血清对哮喘大鼠ERK1/2通路的影响，为确立针刺抗哮喘和哮喘防治的分子靶标提供实验依据，为进一步探讨针灸整体调节的现代生物学研究机制提供思路。

哮喘的主要病理特征是气道慢性炎症及气道重塑，而气道重塑的关键环节是气道平滑肌细胞的增殖。研究表明，ERK1/2信号通路在哮喘气道重塑中起作用并与哮喘的气道炎症密切相关。本研究从动物整体实验、细胞实验两方面入手，通过细胞实验，观察ERK1/2信号通路对哮喘模型大鼠ASMC的影响以及针刺血清的调控作用，探讨ERK1/2信号通路与针刺抗哮喘的关系；通过动物实验，验证ERK1/2信号通路对哮喘模型大鼠气道重塑及气道炎症的影响以及针刺的调控作用，为确立针刺抗哮喘和哮喘防治的分子靶标提供实验依据，为进一步探讨针灸整体调节作用的现代生物学机制提供研究思路。.ELISA法检测显示，针刺能明显降低哮喘模型大鼠支气管肺泡灌洗液（BALF）、血清及肺组织匀浆中炎症因子表达（p<0.05）；免疫组化、免疫荧光、Western blot及RT- PCR检测均显示，哮喘模型组（M）的ERK1/2、p-ERK1/2、pan-Ras、c-fos、p-c-fos以及AKT、p38MAPK蛋白、基因表达量均大于空白组（C）（P＜0.05），而针刺组（A）及阻断剂组（P）的表达量小于M组（P＜0.05）；流式细胞仪分析显示，针刺可改善哮喘模型组大鼠气道平滑肌细胞（ASMC）的细胞增殖异常现象；通过免疫组化、免疫荧光、Western blot及RT- PCR法检测各组ASMC，哮喘模型各组细胞的ERK1/2、p-ERK1/2、pan-Ras、c-fos、p-c-fos以及AKT、p38MAPK蛋白、基因表达量均大于空白组，差异有统计学意义（P＜0.05），而针刺血清可降低哮喘大鼠ASMC中ERK1/2、p-ERK1/2、pan-Ras、c-fos、p-c-fos以及AKT、p38MAPK蛋白、基因的表达；超微电镜观察示，M组大鼠ASMC细胞器出现空洞，溶酶体大量自噬，针刺血清干预后有所改善。.通过细胞实验，证实了ERK1/2信号通路参与了哮喘模型大鼠ASMC的迁移、增殖与炎症因子表达，而针刺血清对其有显著的调控作用。通过动物实验，验证了ERK1/2信号通路参与了哮喘模型大鼠气道重塑及气道炎症的过程并且针刺对其有显著的调控作用。