平喘颗粒通过PERK信号通路介导的内质网应激抑制细胞自噬改善哮喘小鼠气道重塑的机制研究

Mechanism research of Pingchuan Granules ameliorate airway remodeling in asthmatic mice by inhibiting autophagy through endoplasmic reticulum stress mediated by PERK signal pathway

目的:观察平喘颗粒对PERK信号通路介导的内质网应激的影响,明确其抑制细胞自噬改善气道重塑的机制。方法:通过卵清蛋白致敏-激发构建哮喘小鼠模型并给予平喘颗粒、PERK抑制剂进行干预,分别以空白组及模型组小鼠作为对照。采用无束缚全身体积描记系统检测小鼠气道高反应性,ELISA法检测肺泡灌洗液中炎症因子及总IgE的含量,HE、PAS和Masson染色观察各组小鼠肺组织病理形态,透射电镜观察肺组织自噬体数量,Western blot检测自噬及内质网应激相关蛋白的表达水平。结果:与模型组比较,平喘颗粒组与抑制剂组治疗后气道阻力明显改善,肺泡灌洗液中IL-4、IL-5、IL-13及总IgE的含量显著降低(P<0.01),气道炎症评分、PAS评分及胶原染色面积显著降低(P<0.01),Beclin-1、PERK、ATF4、CHOP蛋白表达和LC3Ⅱ/LC3Ⅰ、p-eIF2α/totaleIF2α的比值显著降低(P<0.01),p62蛋白的表达显著增加(P<0.01)。结论:平喘颗粒通过抑制PERK/eIF2α/ATF4/CHOP信号通路的激活介导的内质网应激抑制细胞自噬水平,从而改善哮喘的气道重塑。

Objective: To observe the effects of Pingchuan Granules on endoplasmic reticulum stress mediated by PERK signal pathway, and to clarify its mechanism of inhibiting autophagy and improving airway remodeling. Methods: Asthma model was established by ovalbumin sensitization and stimulation, and intervened by Pingchuan Granules and PERK inhibitor.Mice in the control group and model group were used as controls respectively. The airway hyperresponsiveness was detected by unbound whole-body plethysmography system, the contents of inflammatory factors and total IgE in bronchoalveolar lavage fluid were detected by ELISA, the pathological morphology were observed by HE, PAS and Masson staining, and autophagy of lung tissue was observed by optical microscope and transmission electron microscope, and the expression levels of autophagy and endoplasmic reticulum stress-related proteins were detected by Western blot. Results: Compared with the model group, the airway resistance in Pingchuan Granules, group and inhibitor group was significantly reduced, and the contents of IL-4, IL-5, IL-13 and total IgE in bronchoalveolar lavage fluid were significantly reduced(P<0.01), the airway inflammation score, PAS score and collagen staining area in Pingchuan Granules group and inhibitor group were significantly lower(P<0.01), Beclin-1, PERK, ATF4,CHOP protein expressions and the ratio LC3Ⅱ/LC3Ⅰ and p-eIF2α/total-eIF2α in Pingchuan Granules group and inhibitor group decreased significantly(P<0.01), while the expression of p62 protein in Pingchuan Granules group increased significantly(P<0.01). Conclusion: Pingchuan Granules inhibits endoplasmic reticulum stress by inhibiting the activation of PERK/eIF2α/ATF4/CHOP signal pathway, further inhibits the level of autophagy and improves airway remodeling in asthma.