补肺颗粒通过调控Akt通路对慢阻肺大鼠肺组织自噬和凋亡的影响

Effects of Bufei Granule on autophagy and apoptosis of lung tissues in COPD rats by regulating Akt pathway

目的:探究补肺颗粒对慢性阻塞性肺疾病(COPD)大鼠肺组织自噬和凋亡的影响,并初步探究可能的作用机制。方法:将大鼠随机分为假手术组、模型组、Akt通路激活剂组(SC79)、补肺颗粒组、Akt激活剂+补肺颗粒组,每组12只大鼠,肺功能检测仪检测吸气量(IC),用力肺活量(FVC)、第1秒用力呼气容积(FEV1)、最大呼吸中段流量(MMF);采用HE染色观察大鼠肺组织病理形态学变化,酶联免疫吸附法检测支气管肺泡灌洗液(BALF)中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-8、IL-6水平;透射电镜观察肺组织细胞自噬情况;Tunel法检测肺组织细胞凋亡情况;免疫印迹法检测肺组织中微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、自噬相关蛋白(Beclin1)、Bcl-2相关X蛋白(Bax)、Caspase-3、Bcl-2、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)表达。结果:与假手术组相比,模型组IC、FVC、FEV1、MMF降低;BALF中TNF-α、IL-8、IL-1β水平升高,肺组织细胞凋亡率升高,肺组织LC3-Ⅱ、Beclin-1、Bcl-2蛋白表达降低,Bax、Caspase-3、p-PI3K、p-Akt、p-mTOR蛋白表达升高(P<0.05)。与模型组、Akt激活剂+补肺颗粒组相比,Akt激活剂组IC、FVC、FEV1、MMF降低,BALF中TNF-α、IL-8、IL-1β水平升高,肺组织细胞凋亡率升高,肺组织LC3-Ⅱ、Beclin-1、Bcl-2蛋白表达降低,Bax、Caspase-3、p-PI3K、p-Akt、p-mTOR蛋白表达升高(P<0.05);补肺颗粒组IC、FVC、FEV1、MMF升高,BALF中TNF-α、IL-8、IL-1β水平降低,Bax、Caspase-3、p-PI3K、p-Akt、p-mTOR蛋白表达降低(P<0.05)。结论:补肺颗粒可上调COPD大鼠肺组织自噬,降低组织细胞凋亡进而缓解肺组织病理损伤,保护肺功能,其机制可能与抑制PI3K/Akt/mTOR信号通路有关。

Objective:To explore the effects of Bufei Granule,a traditional Chinese medicine,on autophagy and apoptosis of lung tissues in rats with chronic obstructive pulmonary disease(COPD),and to explore the possible mechanism.Methods:Rats were randomly divided into sham operation group,model group,Akt pathway activator group(SC79),Bufei Granule group,and Akt activator+Bufei Granule group,with 12 rats in each group,the inspiratory capacity(IC),forced vital capacity(FVC),forced expiratory volume in the first second(FEV1),and maximum mid-respiratory flow(MMF)were measured by lung function detector;HE staining was used to observe the pathological changes of lung tissues in rats,the levels of TNF-α,IL-8,and IL-6 in bronchoalveolar lavage fluid(BALF)were detected by ELISA;the autophagy of lung tissue cells was observed by transmission electron microscopy;Tunel method was used to detect the apoptosis of lung cells;the expressions of microtubule associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ),autophagy associated protein 1(Beclin1),Bcl-2 related X protein(Bax),Caspase-3,Bcl-2,phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),and mammalian target of rapamycin(mTOR)were detected by Western Blot.Results:Compared with sham operation group,IC,FVC,FEV1,and MMF in model group were decreased;the levels of TNF-α,IL-8,and IL-1βin BALF increased,the apoptotic rate of lung tissues increased,the expressions of LC3-Ⅱ,Beclin-1,and Bcl-2 proteins decreased,and the expressions of Bax,Caspase-3,p-PI3K,p-Akt,and p-mTOR proteins increased(P<0.05).Compared with model group and Akt activator+Bufei Granule group,IC,FVC,FEV1,and MMF expression levels in Akt activator group decreased,TNF-α,IL-8,and IL-1βlevels in BALF increased,apoptotic rate in lung tissues increased,expressions of LC3-Ⅱ,Beclin-1,and Bcl-2 proteins decreased,expression of Bax,Caspase-3,p-PI3K,p-Akt,and p-mTOR proteins increased(P<0.05).IC,FVC,FEV1,and MMF increased,TNF-α,IL-8,and IL-1βlevels decreased in BALF,while Bax,Caspase-3,p-PI3K,p-Akt,and p-mTOR proteins expression decreased in Bufei Granule group(P<0.05).Conclusion:The tradional Chinese medicine Bufei Granule can up-regulate pulmonary autophagy in COPD rats,reduce apoptosis of lung tissue cells,alleviate pathological damage of lung tissues and protect pulmonary function,and its mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.