针刺调节丝裂原活化蛋白激酶通路影响哮喘大鼠肺组织嗜酸性粒细胞凋亡的机制研究

Acupuncture affects the apoptosis of eosinophilic granulocytes in lung tissue of asthmatic rats by regulating the P38MAPK signaling pathway

目的:观察针刺对哮喘大鼠肺组织磷酸-P38丝裂原活化蛋白激酶(p-P38MAPK)、细胞间黏附分子-1(ICAM-1)、干扰素γ(IFN-γ)和嗜酸性粒细胞(EOS)的影响,探讨针刺对EOS凋亡的作用机制。方法:清洁级雄性Wistar大鼠随机分为正常组、模型组、地塞米松组与针刺组,每组8只。采用腹腔注射卵蛋白与氢氧化铝混悬液法复制哮喘大鼠模型。地塞米松组给予腹腔注射地塞米松0.9 mg/kg,每日1次,连续2周;针刺组予双侧“肺俞”“脾俞”“肾俞”“定喘”与“膻中”进行针刺治疗,平补平泻,留针30 min,隔日1次,连续2周。用HE染色法观察各组大鼠肺组织病理形态学变化;TUNEL法检测各组大鼠肺组织中EOS凋亡情况;Western blot法检测各组大鼠肺组织p-P38MAPK表达水平;免疫组织化学法和实时荧光定量PCR法检测各组大鼠肺组织ICAM-1、IFN-γ蛋白及mRNA的表达水平。结果:HE染色结果显示,模型组大鼠肺泡及周围组织中可见大量炎性物质渗出,肺间质增厚;地塞米松组与针刺组大鼠肺泡结构破损较少,气道周围只有少量炎性细胞。与正常组比较,模型组大鼠肺组织EOS凋亡率、IFN-γ蛋白及mRNA表达水平均明显降低(P<0.01),肺组织p-P38MAPK、ICAM-1蛋白及mRNA表达水平均明显升高(P<0.01)。治疗后与模型组比较,地塞米松组与针刺组大鼠肺组织EOS凋亡率、IFN-γ蛋白及mRNA表达水平均明显升高(P<0.05,P<0.01),肺组织p-P38MAPK、ICAM-1蛋白及mRNA表达水平均明显降低(P<0.01,P<0.05)。结论:针刺治疗哮喘可能是通过抑制P38MAPK信号通路下调ICAM-1表达、减少EOS聚集、上调IFN-γ表达、促进EOS凋亡来缓解哮喘气道炎性反应。

Objective To investigate the effects of acupuncture on phosphorylated P38 mitogen-activated protein kinase(p-P38 MAPK),intercellular adhesion molecule-1(ICAM-1),interferonγ(IFN-γ),and eosinophilic granulocytes(EOS)in lung tissue of asthmatic rats,and to explore the mechanism of acupuncture in regulating the apoptosis of EOS.Methods Clean-grade male Wistar rats were randomly divided into normal,model,dexamethasone and acupuncture groups,8 rats in each group.The asthmatic model was established by intraperitoneal injection of mixture suspension(1 mL)of 10%ovalbumin and 10%Al(OH)+normal saline,followed by inhalation of atomized 1%ovalbumin solution for 30 min,once daily for 2 weeks to trigger occurrence of asthmatic symptoms.The rats in dexamethasone group were intraperitoneally injected with 0.9 mg/kg dexamethasone since day 15 once a day for two consecutive weeks.In the acupuncture group,bilateral“Feishu”(BL13),“Pishu”(BL20),“Shenshu”(BL23),“Dingchuan”(EX-B1),and“Danzhong”(CV17)were selected for acupuncture treatment once every other day since day 15 for two consecutive weeks.Uniform reinforcing and reducing manipulation was carried out,and the needles were retained for 30 min.The pathological changes of the lung tissue were observed by hematoxylin-eosin(HE)staining.The apoptosis of EOS in the lung tissue of rats was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay.The expression of p-P38 MAPK in the lung tissue was detected by Western blot.The protein and mRNA levels of ICAM-1 and IFN-γin the lung tissue were determined by immunohistochemistry and real-time PCR,respectively.Results The results of HE staining showed that the pulmonary alveoli and surrounding tissues were intact with no inflammatory cell infiltration in the normal group.The model group showed massive exudation of inflammatory materials and thickened pulmonary interstitium.The dexamethasone group and acupuncture group showed less damage of the alveolar structure and only a small number of inflammatory cells around the airway.Compared with the normal group,the apoptosis rate of EOS in lung tissue was decreased(P<0.01),the expression levels of p-P38 MAPK and ICAM-1 proteins and mRNAs in the lung tissue were up-regulated(P<0.01),while the expression levels of IFN-γprotein and mRNA in the lung tissue were down-regulated(P<0.01)in the model group.Compared with the model group,the apoptosis rate of EOS in the lung tissue was increased(P<0.05),the expression levels of p-P38 MAPK and ICAM-1 proteins and mRNAs in the lung tissue were down-regulated(P<0.01,P<0.05),while the expression levels of IFN-γprotein and mRNA were up-regulated(P<0.05,P<0.01)in the dexamethasone and acupuncture groups.Conclusion Acupuncture may inhibit the P38 MAPK signaling pathway,down-regulate ICAM-1 expression,and up-regulate IFN-γexpression to promote the apoptosis of EOS and reduce EOS aggregation,thus alleviating the inflammatory response of airway in asthma.