| Objective:To observe the polarization direction of AM in COPD rats with pure smoke for twelve weeks;to evaluate the effect of electroacupuncture(EA)at bilateral "ST36" and "BL13" on the regulation of M1/M2 polarization balance of AM in COPD rats,so as to explore the possible mechanism.Methods:1.Grouping and Model replication: The rats were randomly divided into four groups of ten animals each,that were Normal,Normal+EA,COPD,COPD+EA group,separately.The rats in COPD group and the COPD+EA group were induced by simple cigarette smoking for 12 weeks.2.EA treatment: Acupuncture points of "ST36" and "BL13" were selected from each treatment group for EA.A dense wave with a stimulation frequency of4/20 Hz and an intensity of 1-2m A was selected once a day for 30 min each time,once every other day for two weeks3.Index detection: The lung function and the pathological morphology of the lung tissue in each group were observed to evaluate the model.The levels of TNF-?,iNOS secreted by M1 phenotype and IL-10 and Arg-1 secreted by M2 phenotype in BALF of rats in each group were measured by ELISA method.The expression of M1 markers CD86,iNOS,M2 markers CD206,Arg-1 and key factors of M1 signaling pathway(MyD88,NF-?B p65)and M2 signaling pathway(IL-4,IL-4R,STAT6)m RNA and protein in AM of rats in each group were detected by QT-PCR and Western blotting.The immunopositive expression of M1(CD86)and M2(CD206)in rats lung tissues were observed by immunohistochemistry,and the phagocytosis of AM was determined by neutral red phagocytosis test.Results:1.After 12 months of fumigation,the pulmonary ventilation function of COPDgroup was significantly lower than that of Normal group,such as forced vital capacity(FVC),forced inspiratory volume in 0.1 second(FEV0.1),forced inspiratory volume in 0.3 second(FEV0.3),FEV0.1/FVC and FEV0.3/FVC,and so on(P<0.01);the results of pathological sections showed that the structure of lung tissue in Normal group was complete,the alveoli were continuous and uniform in size,however the alveolar wall thickened,the alveolar cavity enlarged and the inflammatory cells infiltrated obviously in COPD group.After EA treatment,the pulmonary function related indexes of the COPD+EA group were significantly increased,the pulmonary ventilation function was improved(P<0.01),the inflammatory cell infiltration and the alveolar wall thickening were improved.2.Compared with Normal group,the expression of M1 cytokines TNF-? and iNOS in COPD group were significantly increased(P<0.01),on the contrary,the expression of M2 cytokines IL-10 was significantly decreased,while Arg-1 was significantly increased(P<0.01);the m RNA and protein contents of M1 polarization markers CD86,iNOS and key factors of M1 signaling pathway MyD88,NF-?B p65 in AM of rats were significantly increased(P<0.01),while the m RNA and protein contents of M2 polarization markers CD206,Arg-1 and key factors of M2 signaling pathway IL-4,IL-4R,STAT6 were significantly decreased(P<0.01).Compared with COPD group,the expression of TNF-? and iNOS in BALF of COPD+EA group were significantly reduced(P<0.01),while the level of IL-10 and Arg-1significantly increased(P<0.01);the contents of CD86,iNOS,MyD88,NF-?B p65 m RNA and protein in AM decreased significantly(P<0.01,P<0.05);the contents of CD206,Arg-1,IL-4,IL-4R,STAT6 m RNA and protein increased significantly(P<0.01,P<0.05).Correlation analysis showed that TNF-? in BALF of each group was negatively correlated with the expression of lung function indicators FEV0.1/FVC and FEV0.3/FVC(P<0.01),while IL-10 was positively correlated with the above mentioned main indicators of lung function(P<0.01,P<0.05).3.The positive expression of M1(CD86)in the lung tissue of COPD groupwas significantly higher than that of Normal group(P<0.01),while the positive expression of M2(CD206)was significantly lower than that of Normal group(P<0.01).Neutral red phagocytosis assay showed that the AM phagocytosis ability of COPD group was significantly higher than that of Normal group(P<0.01).Compared with the COPD group,the positive expression of CD86 in the lung tissue of the COPD+EA group decreased(P<0.01),while the positive expression of CD206 increased(P<0.01);the results of neutral red phagocytosis experiment showed that the phagocytic ability of AM in COPD+EA group was lower than that in COPD group(P<0.01).Conclusions:1.The method of cigarette fumigation 12 weeks alone can successfully establish COPD model rats,which accords with the main pathological and pulmonary function changes of clinical COPD patients.2.Cigarette cigarette fumigation for 12 weeks can induce AM polarization in the M1 direction of rats,accompanied by enhanced AM phagocytosis.3.EA therapy can enhance M2 polarization,inhibit M1 polarization,reduce AM phagocytosis,achieve the M1/M2 polarization balance of regulating AM in rats,and improve pulmonary inflammatory response,which may be related to EA inhibiting the activation of MyD88/NF-?B p65 signaling pathway and enhancing the activation of IL-4/STAT6 signaling pathway. |
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