中国全科医学 ›› 2023, Vol. 26 ›› Issue (03): 293-303.DOI: 10.12114/j.issn.1007-9572.2022.0558

• 论著 • 上一篇    下一篇

银杏叶提取物防治慢性阻塞性肺疾病的机制研究:基于PI3K/Akt/mTOR信号通路调控肺泡巨噬细胞自噬

郭栋伟1, 张鹏飞1,*(), 任明君2, 廖丽君1, 黄茹妍2, 罗湘蓉2   

  1. 1.545000 广西壮族自治区柳州市中医医院呼吸与危重症医学科
    2.530200 广西壮族自治区南宁市,广西中医药大学
  • 收稿日期:2022-07-26 修回日期:2022-08-22 出版日期:2023-01-20 发布日期:2022-09-30
  • 通讯作者: 张鹏飞
  • 郭栋伟,张鹏飞,任明君,等.银杏叶提取物防治慢性阻塞性肺疾病的机制研究:基于PI3K/Akt/mTOR信号通路调控肺泡巨噬细胞自噬[J].中国全科医学,2023,26(3):293-303. [www.chinagp.net]
    作者贡献:郭栋伟负责实验设计、文章的构思与设计,并对文章负责;张鹏飞组织课题实施、控制实验质量,负责论文起草、论文修订与审校;任明君、廖丽君进行动物实验与细胞实验以及实验相关指标检测;黄茹妍、罗湘蓉负责论文检索与查重。
  • 基金资助:
    国家自然科学基金青年基金资助项目(81904111); 广西自然科学基金青年基金资助项目(2020GXNSFBA297022)

Mechanistic of Ginkgo Biloba Extract in the Prevention and Treatment of COPD: Regulating Autophagy in Alveolar Macrophages via PI3K/Akt/mTOR Signaling Pathways

GUO Dongwei1, ZHANG Pengfei1,*(), REN Mingjun2, LIAO Lijun1, HUANG Ruyan2, LUO Xiangrong2   

  1. 1.Department of Respiratory and Critical Care Medicine, Liuzhou Traditional Chinese Medical Hospital, Liuzhou 545000, China
    2.Guangxi University of Chinese Medicine, Nanning 530200, China
  • Received:2022-07-26 Revised:2022-08-22 Published:2023-01-20 Online:2022-09-30
  • Contact: ZHANG Pengfei
  • About author:
    GUO D W, ZHANG P F, REN M J, et al. Mechanistic of ginkgo biloba extract in the prevention and treatment of COPD: regulating autophagy in alveolar macrophages via PI3K/Akt/mTOR signaling pathways [J] . Chinese General Practice, 2023, 26 (3) : 293-303.

摘要: 背景 银杏叶提取物(GBE)能抑制慢性阻塞性肺疾病(COPD)大鼠气道及全身炎性反应,改善气道重塑,但其具体作用机制尚不清楚。 目的 探讨GBE通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调控肺泡巨噬细胞自噬防治COPD的作用机制。 方法 于2020年11月至2022年3月,选取90只SPF级雄性Wistar大鼠随机分为正常对照组、COPD模型组、GBE组、比卡鲁胺组、雷帕霉素组、Taselisib组,每组15只。正常对照组第1 、14天向气管内注入0.9%氯化钠溶液,其余时间进行正常饲养。COPD模型组、GBE组、比卡鲁胺组、雷帕霉素组和Taselisib组采用香烟熏吸联合气管内注入脂多糖(LPS)的方法构建COPD大鼠模型。GBE组于实验的第15~28天腹腔注射舒血宁注射液,比卡鲁胺组、雷帕霉素组、Taselisib组于实验的第29~42天分别给予Akt抑制剂比卡鲁胺、mTOR抑制剂雷帕霉素、PI3K抑制剂Taselisib进行干预。HE染色观察各组大鼠肺泡病理改变及气道重塑情况;酶联免疫吸附法(ELISA)检测大鼠肺泡灌洗液(BALF)与血清白介素6(IL-6)和白介素-8(IL-8)水平;显微镜下计数各组大鼠肺泡巨噬细胞数量;透射电镜观察各组大鼠肺泡巨噬细胞的超微结构;蛋白免疫印迹法(WB)检测各组大鼠肺泡巨噬细胞自噬相关蛋白表达水平并计算微管相关蛋白轻链3(LC3)-Ⅱ/LC3-Ⅰ比值。 结果 造模结束后各组大鼠数量为:正常对照组12只、COPD模型组11只、GBE组12只、比卡鲁胺组12只、雷帕霉素组12只、Taselisib组11只。HE染色结果显示,GBE组与各抑制剂组大鼠肺泡损伤程度较COPD模型组减轻;GBE组、比卡鲁胺组、雷帕霉素组、Taselisib组肺平均内衬间隔(MLI)与平均肺泡面积(MAA)小于COPD模型组,平均肺泡数(MAN)大于COPD模型组(P<0.05);GBE组、比卡鲁胺组、雷帕霉素组、Taselisib组支气管壁结构较COPD模型组完整。COPD模型组BALF与血清IL-6、IL-8水平高于其他各组(P<0.05)。正常对照组巨噬细胞数量低于其他各组(P<0.05),COPD模型组巨噬细胞数量高于其他各组(P<0.05)。透射电镜观察显示GBE组、比卡鲁胺组、雷帕霉素组、Taselisib组肺泡巨噬细胞的自噬溶酶体较COPD模型组多。正常对照组的PI3Kp110α、Akt、p-Ak、mTOR、p-mTOR表达水平高于其他各组,LC3-Ⅱ/LC3-Ⅰ比值低于其他各组(P<0.05);COPD模型组与GBE组、比卡鲁胺组、雷帕霉素组、Taselisib组比较,PI3Kp110α、Akt、p-Akt、mTOR、p-mTOR表达水平升高,LC3-Ⅱ/LC3-Ⅰ比值降低(P<0.05)。 结论 GBE能通过调控PI3K/Akt/mTOR信号通路,维持COPD大鼠肺泡巨噬细胞的自噬功能,减轻巨噬细胞浸润,抑制炎性反应及肺泡破坏,改善气道重塑。

关键词: 肺疾病,慢性阻塞性, 银杏叶提取物, PI3K/Akt/mTOR信号通路, 肺泡巨噬细胞, 自噬

Abstract:

Background

Ginkgo biloba extract (GBE) has been found to be effective in inhibiting the airway and systemic inflammatory response and improve airway remodeling in rat models of chronic obstructive pulmonary disease (COPD) , but the mechanism remains unclear.

Objective

To discuss the mechanism of GBE regulating alveolar macrophage autophagy through phosphatidylinositol 3-kinase (PI3K) /protein kinase B (Akt) /mammalian target of rapamycin (mTOR) signaling pathways to prevent and treat COPD.

Methods

A total of 90 SPF male Wistar rats were equally randomized into normal control group, COPD model group, GBE group, bicalutamide group, rapamycin group, and Taselisib group. The normal control group were normally fed except that normal saline was injected into their trachea on the 1st and 14th days of intervention, the other 5 groups were treated with exposure to cigarette smoking combined with intratracheal injection of lipopolysaccharide (LPS) to establish rat models of COPD. The GBE group received intraperitoneal injection of Shuxuening injection from the 15th day to the 28th day of the experiment, while bicalutamide, rapamycin, and Taselisib groups were given bicalutamide, rapamycin, and taselisib, respectively, from the 29th day to the 42nd day of the experiment. HE staining was used to observe alveolar pathological changes and airway remodeling. ELISA was used to detect the levels of interleukin -6 (IL-6) and interleukin -8 (IL-8) in alveolar lavage fluid (BALF) and the serum. The number of alveolar macrophages was counted under microscope. The ultrastructure of alveolar macrophages was observed by transmission electron microscope. Western blotting was used to measure the expression levels of autophagy-related proteins in alveolar macrophages. The ratio of microtubule-associated protein light chain 3 (LC3) -Ⅱ/LC3-Ⅰwas calculated subsequently.

Results

As of the models being successfully established, the rats in normal control, COPD model, GBE, bicalutamide, rapamycin, and Taselisib groups numbered 12, 11, 12, 12, 12, and 11, respectively. H&E staining showed that the degree of alveolar injury in COPD model group was more severe than that of GBE, bicalutamide, rapamycin, or Taselisib group (P<0.05) . COPD model group had larger mean linear intercept and mean alveolar area as well as less mean alveolar number than GBE, bicalutamide, rapamycin, or Taselisib group (P<0.05) . Moreover, COPD model group had less complete bronchial wall structure than GBE, bicalutamide, rapamycin, or Taselisib group. The levels of BALF and serum IL-6 and IL-8 in COPD model group were higher than those of each of other five groups (P<0.05) . Among all groups, the number of macrophages in the normal control group was the lowest, while that of COPD model group was the highest (P<0.05) .Transmission electron microscopy showed that COPD model group had less autophagolysosomes in alveolar macrophages than GBE, bicalutamide, rapamycin, or Taselisib group. The normal control group had higher expression levels of PI3Kp110α, Akt, p-Ak, mTOR and p-mTOR and lower ratio of LC3-II/LC3-I than each of other five groups (P<0.05) . COPD model group had higher expression levels of PI3Kp110α, Akt, p-Akt, mTOR and p-mTOR, and lower ratio of LC3-Ⅱ/LC3-Ⅰ compared with GBE, bicalutamide, rapamycin or Taselisib group (P<0.05) .

Conclusion

GBE maintained the autophagy function of alveolar macrophages, reduced macrophage infiltration, inhibited the inflammatory response and alveolar damage, and improved airway remodeling in model rats of COPD through regulating the PI3K/Akt/mTOR signaling pathway.

Key words: Pulmonary disease, chronic obstructive, Ginkgo biloba extract, PI3K/Akt/mTOR signal pathway, Alveolar macrophages, Autophagy