The Study Of Orexin On Central Respiratory Regulation In Chronic Obstructive Pulmonary Diseases Rat And Acupuncture Therapy

The study of orexin on central respiratory regulation in chronic obstructive pulmonary diseases rat and acupuncture therapyPurpose Chronic obstructive pulmonary disease (COPD) is a common respiratory disease worldwide. The death rate of COPD might move from 6th in 1990 to 3rd in 2020 in the rank order of deaths according to WHO report. It's largely unknown about the reason and mechanism of COPD development. The interaction of genetic susceptibility with environmental factors (environmental pollution, smoking, age, airway inflammation) may be the main. The irreversible airflow limitation may relate to the abnormal excitability of respiratory center. However, which factors in the development of COPD result in abnormal regulation of the respiratory center has not been addressed. Orexin from the neurons in the hypothalamus has established as important neuropeptide for feeding, energy metabolism, endocrine, cardiovascular activity, sleep-arousal cycle and other physiological function. Recently, orexin has been found to regulate respiratory activity in respiratory center. Further more, plasma orexinA levels was lower in COPD patients. The present study was to investigate the change of prepro-orexin (PPO), orexinA (OXA), orexin type 1 receptor (OX1R) and orexin type 2 receptor (OX2R) and to test the hypothesis that orexin system may have a neuromodulatory effect on respiratory activity in COPD rats. Acupuncture therapy is available in many diseases according to traditional medicine in China. In this study tested the hypothesis that electroacupuncture treatment is benefit to ventilation and change orexin system in COPD rat. It will determine orexin contribution in the regulation of respiration and effect of acupuncture treatment.Methods 1. The Sprague-Dawley rats were exposed to cigarette smoke in an incomplete airtight cage for 1h/twice/day lasting for 12 weeks. The pathological change in lung tissue and pulmonary resistance and compliance were used to evaluate the COPD model. The central respiratory drive parameters were observed.2. The orexinA content of tissue homogenate and in plasma, expression and distribution of PPO mRNA, OXA, OX1R and OX2R in the hypothalamus, medulla and lung in COPD rat were observed by radioimmunoassay, real time quantitative PCR, immunohistochemistry.3. In electrophysiology experiment, the rats were placed on a stereotaxic frame and phrenic nerve discharge was observed pre- and post- orexin and glutamate microinjection into pre-Botzinger complex (PBC).4. Electroacupuncture on COPD rats was performed for 30 min/day during 2 weeks via selecting acupoints of FEISHU and ZUSANLI. The lung ventilation function, orexinA content, the expression of PPO mRNA and orexin receptors were detected by the methods mentioned above.Results1. The lung pathological changes were found after 12 weeks smoke. The alveolar ectasia, surface area of alveolar enlarging, alveolar wall partly coalescing and bronchia wall thickening resulted in the pulmonary dynamic compliance decrease (P<0.05) and airway resistance increase (P<0.05). There was no significant change in the ratio of tidal volume to inspiratory time.2. OXA content of hypothalamus and medulla homogenate were much larger in COPD rats than that in control rats by radioimmunoassay (P<0.01, P<0.001). However, plasma OXA levels in COPD rats were lower than that in control rats (P<0.05). But OXA levels in lung homogenate were not significantly decreased in COPD rat compared with control.3. PPO mRNA expressed only in hypothalamus of rats, not in lung and medulla by real time quantitative PCR. The PPO mRNA expression was higher in COPD rats than in control rats (P<0.05). OX1R mRNA expression in medulla of COPD rats was also higher than that in control rats (P<0.05). But there was not significant change in OX2R mRNA expression in medulla of COPD rats compared with control rats. OX1R mRNA expression was no much change in lung in COPD rats compared with control rats.4. Immunohistochemistry experiments demonstrated that OXA immunoactivity positive neurons were located mainly in lateral hypothalamus and perifornical nucleus, but scattered throughout posterior hypothalamus and zona incerta. Relative optical density (ROD) of positive cells in hypothalamus of COPD rats was higher than that of control rats (P<0.01). Compared with control rats, COPD rats has no significant increase of OXA-like immunoreactive cells in lung tissue. The expression of OX1R was in PBC and nucleus ambiguous, but OX2R only in PBC, they were both in hypoglossal nucleus and the spinal cord C3-C5 including the phrenic nucleus. Statistic data showed that OX1R and OX2R immunoreactivity cells in ventrolateral medulla (including PBC) in COPD rats were both higher than that in control rats (P<0.01). 5. The intensity of phrenic nerve discharge (PND) was significantly increased after microinjecting OXA and OXB into PBC in the control rats (P<0.01, P<0.05), the effect of OXA was stronger than that of OXB. It inhibited the integral area of PND during microinjecting OX1R blocking agent (P<0.01). The exciting respiration induced by OXA in COPD rats was larger than that in control rats (P<0.01). Microinjecting glutamate into PBC in COPD rats could increase the integral area of PND (P<0.01). The respiratory excitation effects induced by OXA could be partly inhibited by the blocking agent of NMDA receptor (P<0.05). It is interesting that respiratory excitation effects induced by glutamate could be partly inhibited by OX1R blocking agent (P<0.05). The respiratory frequency, heart rate and mean arterial pressure were not affected by all the above drugs.6. Pulmonary ventilation function was increased significantly after electroacupuncture treatment for 2 weeks, pulmonary compliance increased (P<0.05) and airway resistance decreased (P<0.05). There was a decrease trend of OXA levels in hypothalamus and medulla even if no obviously change by radioimmunoassay and immunohistochemistry. Real time PCR results displayed that the PPO mRNA expression had significantly decrease in hypothalamus after electroacupuncture (P<0.01). The expression of two receptors was both decrease in ventrolateral medulla of COPD rats after electroacupuncture,OX2R was less only (P<0.01).Conclusions1. The COPD model was established successfully because it fit the characteristics of the pathologic change and lung ventilation function in COPD patients.2. OXA maybe the important role in central regulation on respiration in COPD rats with the experimental evidences of both OX receptors up-regulation in ventrolateral medulla and significant increase of OXA synthesis in hypothalamus and axonal transport to medulla.3. Endogenous OX in PBC could maintain the respiratory excitability. Exogenous OX only increased respiratory intensity in PBC, and the effect of OXA may be stronger than OXB in control rats.The exciting respiration induced by OXA in COPD rats was larger than that in control rats with the upregulation of OX1R.4. Microinjecting glutamate into PBC could increase respiratory intensity too. The respiratory excitation effects induced by OXA could be partly inhibited by the antagonist of NMDA receptor. The exciting respiration induced by glutamate could be partly inhibited by the antagonist of OX1R. That might suggest they acted on the same signal pathway.5. It was sure that acupuncture treatment on COPD rats can improve the lung ventilation function. We noted that the expression of PPO mRNA in hypothalamus, OX1R and OX2R decreased after acupuncture treatment 2 weeks. It may suggest acupuncture therapy is benefit to lung ventilation function via OX system.