针刺研究

2021, v.46(04) 266-271+277

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电针对慢性阻塞性肺疾病大鼠肺组织自噬相关蛋白表达的影响
Effect of electroacupuncture on the expression of autophagy related protein in lung tissue of rats with chronic obstructive pulmonary disease

程晨;张新芳;苏景超;张毅;李尹;项水英;王彩云;王媛;苏婧婧;刘自兵;
CHENG Chen;ZHANG Xin-fang;SU Jing-chao;ZHANG Yi;LI Yin;XIANG Shui-ying;WANG Cai-yun;WANG Yuan;SU;Jing-Jing;LIU Zi-bing;Graduate School,Anhui University of Chinese Medicine;Department of Physiology,College of Integrated Traditional Chinese and Western Medicine,Anhui University of Chinese Medicine;Institute of Acupuncture and Meridian,College of Acupuncture-moxibustion and Massage,Anhui University of Chinese Medicine;Key Laboratory of Xin'an Medicine,Ministry of Education,Anhui University of Chine

摘要(Abstract):

目的:观察电针"足三里"和"肺俞"对慢性阻塞性肺疾病(COPD)大鼠肺组织自噬相关蛋白及炎性因子表达的影响,探讨电针在COPD发病过程中的作用机制。方法:将雄性SD大鼠随机分为正常组、模型组、电针组,每组10只。采用香烟烟熏联合脂多糖(LPS)法复制COPD大鼠模型。电针组给予电针双侧"足三里"和"肺俞",隔日治疗1次,每次30 min,持续2周。于治疗结束后检测大鼠肺功能;HE染色法观察大鼠气道及肺组织病理变化;电镜观察大鼠气道及肺组织内自噬小体;实时荧光定量PCR法检测大鼠肺组织内腺苷酸活化蛋白激酶(AMPK)、雷帕霉素靶蛋白激酶(mTOR)、Unc-51样自噬激活激酶1(ULK1)、酵母自噬相关基因6的哺乳动物同源体(Beclin1)mRNA的表达;Western blot法检测大鼠肺组织中AMPK、mTOR、ULK1、Beclin1及微管相关蛋白轻链3(LC3Ⅱ/LC3Ⅰ)的表达;ELISA法检测大鼠支气管肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达。结果:与正常组比较,模型组大鼠用力肺活量(FVC)、第0.1秒用力呼气量(FEV0.1)、第0.3秒用力呼气量(FEV0.3)、FEV0.1占FVC比值(FEV0.1/FVC)、FEV0.3占FVC比值(FEV0.3/FVC)均显著降低(P<0.01);气道和肺组织内炎性细胞浸润明显,自噬小体明显增加;肺组织内自噬相关蛋白AMPK、ULK1、Beclin1的mRNA和蛋白表达水平及LC3Ⅱ/LC3Ⅰ的水平均显著升高(P<0.01),mTOR的mRNA和蛋白表达水平显著降低(P<0.01);BALF内TNF-α、IL-6含量显著升高(P<0.01)。与模型组比较,电针组大鼠FVC、FEV0.1、FEV0.3、FEV0.1/FVC、FEV0.3/FVC均显著升高(P<0.01,P<0.05);气道和肺组织内炎性反应得到明显改善,自噬小体明显减少;肺组织内自噬相关蛋白AMPK、ULK1、Beclin1的mRNA和蛋白表达水平及LC3Ⅱ/LC3Ⅰ的水平均显著降低(P<0.01),mTOR的mRNA和蛋白表达水平显著升高(P<0.01);BALF内TNF-α、IL-6含量显著降低(P<0.01)。结论:电针"足三里""肺俞"可以抑制COPD大鼠肺组织自噬水平,降低肺部炎性反应,改善肺功能。
Objective To observe the effect of electroacupuncture(EA) at "Zusanli"(ST36) and "Feishu"(BL13) on the expression of autophagy related proteins in the lung tissue of rats with chronic obstructive pulmonary disease(COPD), so as to explore the mechanism of EA underlying improvement of COPD. Methods Thirty male SD rats were randomly divided into normal, model and EA groups(n=10 in each group). The COPD model was established by intratracheal infusion of Lipopolysaccharide(LPS, 1 mg/kg) and exposure in cigarette smoke. EA was applied to bilateral ST36 and BL13 for 30 min, once every other day for 2 weeks. The pulmonary function(forced vital capacity [FVC], forced expiratory volume in 0.1 s and 0.3 s [FEV0.1, FEV0.3], FEV0.1/FVC and FEV0.3/FVC) was detected by animal pulmonary function analysis system. Histopathological changes of the airway and lung were displayed by H.E. staining. Autophagosomes in the airway and lung tissues were observed by electron microscope. The expression of AMP activated protein kinase(AMPK), mammalian target of rapamycin(mTOR), Unc-51 like autophagy activating kinase 1(ULK1), autophagy related protein ATG6(Beclin1)mRNAs in lung tissue were examined by quantitative real-time PCR. The expression of AMPK, mTOR, ULK1, Beclin1 and microtubule-associated protein 1 light chain 3(LC3)proteins in lung tissue were examined by Western blot. The contents of tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) in the broncho alveolar lavage fluid(BALF) were assayed by ELISA. Results Following modeling, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased(P<0.01), the infiltration of inflammatory cells and the increase of autophagosomes were obvious in airway and lung tissue, the mRNA and protein expression of AMPK, ULK1, Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ were increased(P<0.01), while the mRNA and protein expression of mTOR were decreased(P<0.01), the contents of TNF-α and IL-6 in the BALF were increased in the model group compared with the normal group(P<0.01). After EA intervention, all the indexes mentioned above were completely reversed in the EA group relevant to the model group(P<0.01, P<0.05). Conclusion EA at ST36 and BL13 can improve the lung function of COPD rats, which may be related to its effects in inhibiting the autophagy level and reducing the inflammation response in the lung.

关键词(KeyWords): 电针;慢性阻塞性肺疾病;自噬;炎性反应
Electroacupuncture;Chronic obstructive pulmonary disease;Autophagy;Inflammatory response

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(No.81373743);; 安徽省自然科学基金项目(No.2008085MH267);; 安徽中医药大学自然科学基金项目(No.2020zrzd03)

作者(Author): 程晨;张新芳;苏景超;张毅;李尹;项水英;王彩云;王媛;苏婧婧;刘自兵;
CHENG Chen;ZHANG Xin-fang;SU Jing-chao;ZHANG Yi;LI Yin;XIANG Shui-ying;WANG Cai-yun;WANG Yuan;SU;Jing-Jing;LIU Zi-bing;Graduate School,Anhui University of Chinese Medicine;Department of Physiology,College of Integrated Traditional Chinese and Western Medicine,Anhui University of Chinese Medicine;Institute of Acupuncture and Meridian,College of Acupuncture-moxibustion and Massage,Anhui University of Chinese Medicine;Key Laboratory of Xin'an Medicine,Ministry of Education,Anhui University of Chine

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DOI: 10.13702/j.1000-0607.201155

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