电针对慢性阻塞性肺疾病大鼠肺神经内分泌细胞及神经活性物质分泌的影响

Effect of electroacupuncture on pulmonary neuroendocrine cells and secretion of neuroactive substances in lung of COPD rats

目的:观察电针“足三里”和“肺俞”穴对慢性阻塞性肺疾病(COPD)大鼠肺神经内分泌细胞(PNECs)的激活及其分泌的神经活性物质降钙素基因相关肽(CGRP)、五羟色胺(5-HT)的影响,探讨电针治疗COPD的作用机制。方法:将雄性SD大鼠随机分为正常组、模型组和电针组,每组7只。采用香烟烟熏12周的方法复制COPD大鼠模型。电针组大鼠予电针双侧“足三里”和“肺俞”穴,每次30 min,每日1次,连续14 d。肺功能分析仪检测大鼠肺功能;HE染色法观察肺组织病理形态;Grimelius银染色法观察肺组织PNECs的免疫阳性反应;免疫组织化学法观察肺组织中CGRP、5-HT阳性表达;ELISA法检测支气管肺泡灌洗液(BALF)和肺组织中CGRP、5-HT及肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、转录生长因子(TGF)-β1的含量,并分析CGRP、5-HT含量分别与TNF-α、IL-1β含量的相关性;实时荧光定量PCR和Western blot法分别检测肺组织中CGRP、三磷酸腺苷受体P2X配体门控离子通道3(P2X3)的mRNA和蛋白表达。结果:与正常组比较,模型组用力肺活量(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和FVC比值(FEV0.1/FVC)、FEV0.3和FVC比值(FEV0.3/FVC)均呈不同程度下降(P<0.05,P<0.01);小支气管管壁及肺泡间隔增厚,炎性细胞浸润;肺组织中小气道上皮银染的PNECs阳性表达显著升高(P<0.01);肺组织中CGRP、5-HT阳性表达显著升高(P<0.01);BALF和肺组织中CGRP、5-HT、TNF-α、IL-1β、TGF-β1含量显著升高(P<0.01,P<0.05);肺组织中CGRP、P2X3 mRNA和蛋白表达显著升高(P<0.01)。与模型组比较,电针组FVC、FEV0.1、FEV0.1/FVC、FEV0.3/FVC均显著升高(P<0.05,P<0.01);肺组织病理变化改善;肺组织中PNECs阳性表达显著降低(P<0.01);肺组织中CGRP、5-HT阳性表达显著降低(P<0.01);BALF和肺组织中CGRP、5-HT、TNF-α、IL-1β、TGF-β1含量显著降低(P<0.05,P<0.01),其中CGRP、5-HT和TNF-α、IL-1β均分别呈两两正相关(P<0.01);肺组织中CGRP、P2X3 mRNA和蛋白表达显著降低(P<0.01)。结论:电针COPD大鼠“足三里”“肺俞”穴具有改善肺功能和抗炎的治疗作用,其作用机制可能与其抑制PNECs的激活及其神经活性物质的释放有关。

Objective To observe the effect of electroacupuncture(EA) at “Zusanli”(ST36) and “Feishu”(BL13) on the activation and secretion of calcitonin gene-related peptide(CGRP) and 5-hydroxytryptamine(5-HT) of pulmonary neuroendocrine cells(PNECs) and inflammatory response in rats with chronic obstructive pulmonary disease(COPD), so as to explore its underlying mechanisms in treating COPD. Methods Male SD rats were randomly divided into normal control, COPD model and EA groups, with 7 rats in each group. The COPD model was established by forced inhale of cigarette smoke for 1 h in a self-made box(1 m×1 m×1 m in volume), twice daily for 12 weeks. EA(4 Hz/20 Hz, 1―3 mA) was applied at bilateral ST36 and BL13 acupoints for 30 min, once a day for 14 consecutive days. The pulmonary function including the forced vital capacity(FVC), forced expiratory volume at 0.1 second(FEV0.1), FEV0.3, FEV0.1/FVC and FEV0.3/FVC was detected using a lung function analyzer for small animals. The lung tissue was sampled for observing histopathological changes by using H.E. staining, for observing expression and distribution of PNECs by Grimelius silver staining, and for detecting the immunoactivity(integrated optical density) of CGRP and 5-HT by using immunohistochemistry. The contents of CGRP, 5-HT, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and transforming growth factor-β1(TGF-β1) in the bronchoalveolar lavage fluid(BALF) and lung tissue were detected by ELISA, and the correlations between TNF-α and CGRP, IL-1β and CGRP, TNF-α and 5-HT, and IL-1β and 5-HT levels were analyzed. The mRNA and protein expression levels of nerve fiber markers of CGRP and purinergic receptor P2 X ligand gated ion channel 3(P2 X3) which dominate PNECs in the lung tissue were detected by real-time fluorescence quantitative PCR and Western blot, respectively. Results Compared with the normal control group, the levels of FVC, FEV0.1, FEV0.3, and the ratios of FEV0.1/FVC and FEV0.3/FVC were significantly decreased(P<0.05, P<0.01), while the immunoactivity of PNECs, CGRP and 5-HT, the contents of CGRP, 5-HT, TNF-α, IL-1β and TGF-β1 in the BALF and lung tissue, and the expression levels of CGRP and P2 X3 mRNAs and proteins in the lung tissue significantly increased in the COPD model group(P<0.01, P<0.05). Following EA intervention, both the increased and decreased levels of all the indexes mentioned above were reversed(P<0.05, P<0.01) except FEV0.3. H.E. staining showed severe deformed bronchial lumen with thickened wall and alveolar septum, and obvious inflammatory cell infiltration and reduced number of alveolar lumen fusion in the COPD model group, which was mild in the EA group. A positive correlation was found between TNF-α and CGRP, IL-1β and CGRP, TNF-α and 5-HT,IL-1β and 5-HT levels in both BALF and lung tissues(P<0.01). Conclusion EA at ST36 and BL13 can improve lung function and reduce inflammatory response in COPD rats, which may be related to its function in inhibiting the activation of PNECs and release of neuroactive substances.