益气温阳护卫汤调控PI3K/Akt/mTOR自噬途径治疗支气管哮喘大鼠机制

Yiqi Wenyang Huwei Decoction regulates PI3K/Akt/mTOR autophagic pathway for treatment of bronchial asthma in rats

目的:观察益气温阳护卫汤(YWHD)对哮喘大鼠及支气管上皮细胞(16HBE)自噬和磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的调控,探讨YWHD治疗支气管哮喘(BA)的作用机制。方法:48只大鼠随机分为正常组、模型组、地塞米松组及YWHD低、中、高剂量组,每组8只。大鼠腹腔注射卵清蛋白(OVA)+氢氧化铝混悬液和OVA雾化吸入2周,建立BA动物实验模型,正常组给予等体积生理盐水。动物肺功能仪检测乙酰甲基胆碱(Mch)激发的大鼠支气管最大气道阻力(Max Rrs);酶联免疫吸附测定法(ELISA)检测大鼠肺泡灌洗液中炎症因子白细胞介素(IL)-4、IL-13、IL-6、IL-33、IL-25、肿瘤坏死因子-α(TNF-α)、免疫球蛋白E(IgE)水平;苏木素-伊红(HE)及马松(Masson)染色法观察大鼠肺组织支气管病理变化;免疫荧光法检测支气管自噬蛋白微管相关蛋白1轻链3B(LC3B)、Beclin1水平,实验观察YWHD含药血清对IL-13诱导的16HBE细胞自噬作用,通过mRFP-GFP-LC3腺病毒感染示踪细胞自噬水平;蛋白免疫印迹法(Western blot)检测细胞中LC3Ⅱ/Ⅰ、磷酸化(p)-PI3K、p-Akt、p-mTOR蛋白表达。结果:与正常组比较,OVA诱导的模型组大鼠Max Rrs显著增大(P<0.01);肺泡灌洗液中IL-4、IL-13、IL-6、IL-33、IL-25、TNF-α、IgE的水平明显升高(P<0.05,P<0.01);支气管及血管周围伴有炎性细胞和淋巴细胞呈灶性浸润及上皮杯状细胞化生和上皮下胶原沉积增加;支气管自噬蛋白LC3B、Beclin1表达显著升高(P<0.01);IL-13诱导的16HBE细胞自噬流GFP向mRFP转变水平显著升高(P<0.01);p-PI3K、p-Akt、p-mTOR蛋白表达显著下降,LC3Ⅱ/Ⅰ值显著升高(P<0.01)。与模型组比较,YWHD中、高剂量组大鼠Max Rrs显著减小(P<0.01);肺泡灌洗液中IL-4、IL-13、IL-6、IL-33、IL-25、TNF-α、IgE的水平明显降低(P<0.05,P<0.01);经YWHD中、高剂量给药后大鼠肺组织支气管少见淋巴细胞和粒细胞浸润,上皮杯状细胞化生和上皮下胶原沉积减少;肺组织支气管LC3B、Beclin1表达水平明显降低(P<0.05,P<0.01);YWHD含药血清组GFP向mRFP转变的自噬流水平降低;p-PI3K、p-Akt、p-mTOR蛋白表达明显升高,LC3Ⅱ/Ⅰ值明显降低(P<0.05,P<0.01)。结论:YWHD可以改善BA大鼠气道高反应性和气道炎症,抑制肺组织中气道上皮细胞的自噬水平,其机制可能与PI3K/Akt/mTOR信号通路的激活有关。

Objective:To observe the regulation of Yiqi Wenyang Huwei Decoction(YWHD)on autophagy and phosphatidyl inositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway in asthmatic rats and bronchial epithelial cells(16HBE).To investigate the mechanism of YWHD in treating bronchial asthma(BA).Methods:48 rats were randomly divided into normal group,model group,dexamethasone group and YWHD low-dose,medium-dose and high-dose groups,with 8 rats in each group.Rats were intritoneally injected with OVA+aluminum hydroxide suspension and inhaled with OVA atomization for 2 weeks to establish an animal experimental model of BA.Normal group was given equal dose of normal saline.Rat bronchial maximum airway resistance(Max Rrs)induced by acetylmethylcholine(Mch)was determined by animal lung function apparatus.Enzyme linked immunosorbent assay(ELISA)ELISA)were used to detect the levels of inflammatory factors interleukin4(IL-4),interleukin13(IL-13),interleukin6(IL-6),interleukin33(IL-33),interleukin25(IL-25),tumor necrosis factor-α(TNF-α),and immunoglobulin E(IgE)in rat alveolar lavage fluid;Hematoxylin-eosin(HE)and Masson staining were used to observe the pathological changes of bronchus in lung tissue of rats.The levels of bronchial autophagy protein LC3B and Beclin-1 were detected by immunofluorescence assay.The autophagy of 16HBE cells induced by IL-13 was observed by YWHD drug-containing serum,and the autophagy level was traced by mRFP-GFP-LC3 adenovirus infection.The protein expressions of LC3Ⅱ/Ⅰ,p-PI3K,p-Akt and p-mTOR were detected by Western blot.Results:Compared with normal group,Max Rrs in OVA-induced model group was significantly increased(P<0.01).The levels of IL-4,IL-13,IL-6,IL-33,IL-25,TNF-αand IgE in alveolar lavage fluid were significantly increased(P<0.01).There were focal infiltration of inflammatory cells and lymphocytes around bronchus and blood vessels,epithelial goblet cell metaplasia and increased subepithelial collagen deposition.The expressions of bronchial autophagy protein LC3B and Beclin-1 were significantly increased(P<0.01);The level of autophagic flow GFP to mRFP in 16HBE cells induced by IL-13 was significantly increased.The protein expressions of PPI3K,P-Akt and P-mtor were significantly decreased,while the ratio of LC3II/I was significantly increased(P<0.01).Compared with model group,Max Rrs in medium and high dose YWHD groups was significantly decreased(P<0.01);The levels of IL-4,IL-13,IL-6,IL-33,IL-25,TNF-αand IgE in alveolar lavage fluid were decreased(P<0.05;P<0.01);Lymphocyte and granulocyte infiltration were rare in bronchus of lung tissue,epithelial goblet cell metaplasia and subepithelial collagen deposition were reduced after YWHD administration in medium and high doses.The expression levels of LC3B and Beclin-1 in bronchus of lung tissue were significantly decreased(P<0.05;P<0.01);The autophagy flow level of GFP to mRFP was decreased in YWHD drug-containing serum group.The protein expressions of P-PI3K,p-Akt and p-MTOR were significantly increased,while the LC3II/I ratio was significantly decreased(P<0.05;P<0.01).Conclusion:YWHD ameliorates airway hyperresponsiveness and airway inflammation in BA rats and inhibits the level of autophagy in airway epithelial cells in lung tissue,and the mechanism may be related to the activation of PI3K/Akt/mTOR signaling pathway.