基于PI3K/Akt/mTOR信号通路探讨银杏叶提取物防治慢性阻塞性肺疾病的机制

Mechanism of Ginkgo biloba extract in the prevention and treatment of chronic obstructive pulmonary disease based on PI3K/Akt/mTOR signaling pathway

目的观察银杏叶提取物对慢性阻塞性肺疾病(COPD)大鼠磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的影响,探讨其防治COPD的机制。方法将90只SPF级雄性Wistar大鼠随机分为正常组、模型组、银杏叶提取物组、康士得组、雷帕霉素组、Taselisib组,每组15只。除正常组外,其余各组大鼠均采用香烟烟雾熏吸联合气管内注入脂多糖(LPS)的方法构建COPD模型,银杏叶提取物组于实验的第15~28天腹腔注射舒血宁注射液,其余组于实验的第29~42天分别予Akt抑制剂康士得、mTOR抑制剂雷帕霉素、PI3K抑制剂Taselisib干预。实验的第43天处死各组大鼠,HE染色观察肺泡病理改变及气道重塑情况,酶联免疫吸附法(ELISA)检测肺泡灌洗液与血清中白细胞介素-10(IL-10)和γ-干扰素(IFN-γ)水平,免疫组化法检测肺组织中LC3蛋白表达情况,Western blot法检测肺组织中PI3K p110α、Akt、p-Akt、mTOR、p-mTOR、LC3Ⅱ蛋白表达情况,实时荧光定量PCR法检测肺组织中PI3K、Akt、mTOR、Beclin1 mRNA表达情况。结果与模型组比较,银杏叶提取物组和各抑制剂组肺泡破坏、气道重塑、肺泡与支气管周围炎症浸润均减轻,肺泡灌洗液与血清中IL-10水平均明显升高(P均<0.05),IFN-γ水平均明显降低(P均<0.05);银杏叶提取物组与各抑制剂组肺组织中LC3蛋白表达光密度值、LC3Ⅱ蛋白表达量、Beclin1 mRNA表达量均明显高于模型组(P均<0.05),肺组织中PI3K p110α、Akt、p-Akt、mTOR、p-mTOR蛋白表达量和PI3K、Akt、mTOR mRNA表达量均明显低于模型组(P均<0.05)。结论银杏叶提取物能通过调控PI3K/Akt/mTOR信号通路,减轻COPD大鼠气道炎症与全身炎症反应,抑制肺泡破坏,改善气道重塑,从而发挥治疗COPD的作用。

Objective It is to observe the effect of Ginkgo biloba extract(GBE)on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target protein of rapamycin(mTOR)signaling pathway in the rats with chronic obstructive pulmonary disease(COPD),and to explore its mechanism in preventing and treating this disease.Methods Ninety SPF grade male Wistar rats were randomly divided into normal control group,COPD model group,GBE group,comrader group,rapamycin group and Taselisib group,with 15 rats in each group.Except for the normal control group,the rat models of COPD were established by smoking cigarette smoke and intratracheal injection of lipopolysaccharide(LPS)in the other groups.The GBE group was intraperitoneally injected with Shuxuening injection from the 15th to 28th day of the experiment,the other groups were respectively intervened with Akt inhibitor casodex,mTOR inhibitor rapamycin and PI3K inhibitor Taselisib on the 29th to 42nd days of the experiment.On the 43rd day of the experiment,the rats in each group were executed.The alveolar pathological changes and airway remodeling were observed by HE staining,the levels of interleukin-10(IL-10)andγ-interferon(IFN-γ)in alveolar lavage fluid and serum were detected by enzyme-linked immunosorbent assay(ELISA),the expression of LC3 protein in lung tissues was detected by immunohistochemistry,the expression of PI3K p110α,Akt,p-Akt,mTOR,p-mTOR,LC3Ⅱprotein in lung tissue was detected by Western blot,and the expression of PI3K,Akt,mTOR,Beclin1 mRNA in lung tissue was detected by by real-time fluorescence quantitative PCR.Results Compared with COPD model group,alveolar destruction,airway remodeling,alveolar and peribronchial inflammatory infiltration were reduced in the GBE group and each inhibitor group,the levels of IL-10 in alveolar lavage fluid and serum were significantly increased(all P<0.05),and the levels of IFN-γwere significantly decreased(all P<0.05).The optical density values of LC3 protein expression,LC3Ⅱprotein expression and Beclin 1 mRNA expression in lung tissues of GBE group and each inhibitor group were significantly higher than those of the COPD model group(all P<0.05),and the expression of PI3K p110α,Akt,p-Akt,mTOR,p-mTOR protein and PI3K,Akt,mTOR mRNA in lung tissues were significantly lower than those of the COPD model group(all P<0.05).Conclusion GBE can regulate PI3K/AKt/mTOR signal pathway,thus to reduce airway inflammation and systemic inflammatory response,inhibit alveolar destruction,and improve airway remodeling in COPD rats,so as to play a role in the treatment of COPD.