Study On The Role Of Macrophage Migration Inhibitory Factor In The Electro-acupuncture Therapy Of COPD Rats

Objectives:1.To study the changes of inflammatory factors,macrophage migration inhibitor?MIF?and its receptor complex CD74/CD44,receptor-mediated signaling pathway p38MAPK,AKT mRNA and protein in chronic obstructive pulmonary disease?COPD?rats,and to explore the possible mechanism of MIF in the pathophysiological process of COPD rats.2.In this experiment,the therapeutic effect of electroacupuncture on the two sides of"Zusanli"?BL13?and"Feishu"?ST36?points in COPD model rats from the aspects of pathological morphology and inflammatory mediators,and the relationship between electroacupuncture and MIF,its receptor complex,receptor-mediated signaling pathway,and explored the mechanism of MIF in electroacupuncture treatment of COPD rats.So as to provide a powerful basis for acupuncture treatment of COPD.3.To observe the expression of MIF-specific antagonist ISO-1/EAcombined with MIF specific antagonist ISO-1 on the pathological morphology,lung ventilation function,MIF and its receptor complex,and receptor-mediated signaling pathway in COPD rats,and to explore the possible role of ISO-1 in acupuncture treatment of COPD rats.Methods:1.Grouping:70 male Sprague-Dawley?SD?rats weighing 250-300g of SPF grade were randomly divided into seven groups?N=10?,which were Normal group,COPD group,COPD+EA group,COPD+ISO-1+EA group,COPD+DMSO+EA group?vehicle control group?,Normal+ISO-1+CS group,Normal+DMSO+CS group?vehicle control group?.2.Intervention:COPD rat model was established by cigarette smoking combined with lipopolysaccharide?LPS?intratracheal instillation.After one month of smoking,the pulmonary function of the rats was measured and the pathological morphology of the lung tissue was observed to determine whether the COPD rat model was successful or not.Rats in COPD+ISO-1+EA group were intraperitoneally injected with MIF-Antagonist ISO-1?120ug/kg?30 minutes before EA,and the control group was injected intraperitoneally with an equal amount of 1%DMSO solution for 7 days;Rats in Normal+ISO-1+CS group were intraperitoneally injected with ISO-1 30minutes before each smoking every other day,while rats in control group were intraperitoneally injected with an equal amount of 1%DMSO solution for one month.3.Electroacupuncture:The electroacupuncture group was selected from the two sides of"Zusanli"?BL13?and"Feishu"?ST36?.After acupuncture was injected into the bilateral acupoints of the rats,the two needle handles on the same side are respectively connected with the output end of the electroacupuncture therapeutic apparatus.The parameters were selected as 4/20 Hz alternating frequency,lasting 0.5ms,and the intensity was set to 1-3 mA,so as to cause slight shaking of lower limbs.EA was performed daily for 30 minutes and lasted for 7 days.4.Detection:The levels of MIF,TNF-?,IL-1?and IL-8 in serum,alveolar lavage fluid?BALF?and lung tissue of each group were detected by ELISA.Real-time quantitative PCR and Western blot were used to detect the relative expression of MIF,CD74,CD44,p38MAPK mRNA and protein in lung tissue of each group.The expression and distribution of CD74 and CD44 in lung tissue of each group were observed by immunohistochemistry.Results:1.After the intervention of smoke combined with LPS tracheal instillation,the pulmonary function was significantly decreased compared with the normal group,the forced vital capacity volume?FVC?,forced expiratory volume in 0.1Second(FEV_0.1),forced expiratory volume in 0.3 Second(FEV_0.3),FEV_0.1/FVC,and FEV_0.3/FVC were significantly decreased?P<0.01?;A large number of inflammatory cell infiltration occurred in lung tissue of COPD rats,alveolar cavity enlargement,alveolar structure Pathological changes occurred in accordance with the pathological changes of patients with clinical COPD;In addition,the levels of serum,BALF and inflammatory factors in lung tissue were significantly increased in COPD model group?P<0.01?,while the mRNA and protein expressions of MIF,CD74,CD44,p38MAPK,AKT and the positive expression of CD74,CD44 in lung tissue were increased in COPD model group?P<0.01?.2.One week after EA treatment,compared with COPD group,the pulmonary function of rats in COPD+EA group increased significantly?P<0.01?;The contents of MIF and IL-1?in serum of rats in COPD+EA group decreased significantly?P<0.01?,TNF-?and IL-8 decreased significantly?P<0.05?,and BALF and lung tissue inflammatory factors decreased significantly?P<0.01?;MIF,CD74,CD44,p38MAPK,AKT mRNA,protein content and CD74,CD44 positive expression levels decreased in lung tissue of rats?P<0.01?.3.Rats were intraperitoneally injected with MIF antagonist ISO-1 before EA. Compared with COPD+EA group,the levels of TNF-?,IL-1?and IL-8 in serum and BALF of COPD+ISO-1+EA group were decreased?P<0.01?,MIF content decreased?P<0.05?,MIF,IL-1?and IL-8 levels in lung tissue decreased?P<0.01?,TNF-?expression level decreased?P<0.05?;PCR and WB detection found the expression levels of mRNA and protein of MIF,CD74,CD44 and p38MAPK,AKT in lung tissue of COPD+ISO-1+EA group were significantly decreased?P<0.01?.Immunohistochemistry showed the positive expressions of CD74 and CD44 in lung tissue were decreased to different degrees?P<0.01;P<0.05?;There was significant difference between the COPD+ISO-1+EA group and the vehicle control group?P<0.01?.4.The rats were given ISO-1 pretreatment before smoking,compared with the COPD group,the levels of inflammatory factors in serum,BALF and lung tissues of the Normal+ISO-1+CS group were significantly decreased?P<0.01?,.The expressions of MIF,CD74,CD44,p38MAPK,AKT mRNA,protein and CD74,CD44 positive expression in lung tissue of rats were decreased?P<0.01?;There were significant differences between the Normal+ISO-1+CS group and the vehicle control group?P<0.01?.Conclusions:1.The combined method of CS and LPS intratracheal drip can successfully reproduce COPD model rats.The COPD model rats prepared in this experiment are consistent with the main pathological changes of COPD and the characteristics of pulmonary function.2.MIF is released in a large amount under the regulation of various factors such as CSE and LPS,and can regulate the expression of downstream inflammatory mediators through its receptor complex CD74/CD44 on p38MAPK and AKT signaling pathway.It is known that MIF participates in the pathophysiological process of COPD.3.EA treatment can effectively inhibit the formation of MIF,down-regulate or inhibit its receptor complex CD74/CD44 and post-receptor p38MAPK,AKT signaling pathway,and then inhibit the expression of inflammatory mediators,improve pulmonary inflammation and lung function.It suggests that EA has a significant therapeutic effect on COPD model rats,and its mechanism may be related to the inhibition of MIF and its receptor pathway.4.Pretreatment with ISO-1,a specific MIF antagonist,can effectively inhibit the expression of MIF in lung tissue of COPD rats,and block the p38MAPK and AKT signaling pathways through its receptor complex,thereby down-regulating the expression level of downstream inflammatory factors and play a protective role in COPD rats;In addition,EA combined with MIF specific antagonist ISO-1 in the treatment of COPD rats has a synergistic effect,which can significantly reduce the level of inflammation in rats and improve their lung ventilation function.It has a significant therapeutic effect on COPD rats.