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

Abstract

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. 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 P2X ligand gated ion channel 3 (P2X3) which dominate PNECs in the lung tissue were detected by real-time fluorescence quantitative PCR and Western blot, respectively. 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 P2X3 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). 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.