环孢素A抑制博来霉素诱导的小鼠肺间质病变

Effect of CsA bleomycin-induced interstitial pulmonary disease in mice

目的:观察环孢素A(CsA)对博来霉素(BLM)所致肺间质病变的治疗作用,并探讨其作用机制。方法:将120只C57BL/6小鼠随机分为5组,模型组、模型对照组、CsA30mg治疗组、CsA50 mg治疗组和治疗对照组,每组24只;模型组及治疗组小鼠均经气管滴入BLM建立肺间质病变模型,模型对照组小鼠则经气管滴入同体积生理盐水。于造模当日起,治疗组经腹腔注射CsA,治疗对照组经腹腔注射等体积生理盐水,模型组及模型对照组不予治疗。在实验第4、7和14天分别抽取小鼠外周血,用细胞计数板计数白细胞总数,以流式细胞术检测CD4+T细胞、CD14+单核细胞和CD19+B细胞的数量比例;同步取支气管肺泡灌洗液(BLAF)细胞计数及细胞涂片Giemsa染色分析;并进行肺组织病理学研究。结果:模型组外周血白细胞总数明显升高,细胞分类显示CD14+单核细胞和CD19+B细胞在造模4 d、7 d、14 d均较模型对照组显著增高。CsA治疗后,CD14+细胞比例在4 d时下降最为明显,显著低于同一时间点模型组;7 d、14 d亦低于同一时间点模型组;治疗组CD19+B细胞比例在7 d、14 d时均显著低于同一时间点模型组,各组间CD4+T细胞比较均未见明显统计学差异;BALF细胞分析显示,模型组4 d、7 d、14 d时细胞总数及分类计数均明显高于同一时间点模型对照组,CsA治疗后均显著减少;肺组织病理学观察可见,在第4～14天,模型组及治疗对照组小鼠肺间质内浸润细胞逐渐增多,其中以淋巴细胞浸润为主,可见少量单核-巨噬细胞浸润,同时见间质内胶原纤维沉积逐渐增多。CsA治疗组小鼠肺间质炎症明显减轻,间质内及小支气管周围胶原纤维沉积减少;免疫组化研究显示,模型组小鼠和治疗对照组肺内组织内浸润的单核-巨噬细胞、中性粒细胞和淋巴细胞均高表达CD147分子,治疗组肺内表达CD147的细胞减少。结论:环孢素A可能通过抑制CD147-CypA的相互作用,抑制炎性细胞向肺内的趋化聚集,减轻肺部炎症、减少胶原沉积。

AIM： To observe the therapeutic effect of cyclosporine A（CsA） on bleomycin（BLM） induced pulmonary fibrosis and to investigate its mechanism.METHODS： One hundred and twenty C57BL/6 female mice were divided randomly into five groups： BLM model group,control saline group,CsA30 mg treatment group,CsA50 mg treatment group and control treatment group.Treatment groups and model groups were administrated BLM intratracheally to induce interstitial pulmonary disease model,with control saline group administrated with equal volume of normal saline instead.Mice in treatment groups were intraperitoneal injected with CsA,while control treatment group were injected with equal volume of normal saline instead.On the 4th,7th and 14th day after administration,8 mice of each group were sacrificed,and the peripheral blood was obtained to count total leucocytes with counting chamber and quantify CD4＋ T cells,CD14＋ monocytes and CD19＋ B cells by flow cytometry（FCM）.Bronchoalveolar levage fluid was harvested for cell counting and Giemsa staining.Lung tissues were harvested for immunohistochemical staining and pathological examination.RESULTS： The quantity of total leucocyte was higher in BLM model group than those in control saline group.The proportion of CD14＋ T cells and CD19＋B cells in BLM model group were increased markedly than those in control saline group on the 4th,7th and 14th day post BLM.With CsA treatment,The proportion of CD14＋ T cells was lower than BLM model group at the same time point,especially on the 4th day.The proportion of CD19＋ B cells were significantly lower than those of BLM model group at the same time point（7 d,14 d）.The total and classification of cells of BLM model group were increased markedly than those in control saline group,and decreased obviously in the treatment groups at the same time point.Examination of lung tissues： With the prolonged time of BLM administration,it showed wider alveolar septum,more collagen deposition,as well as more infiltrating inflammatory cells which consisted of generous lymphocyte and few mononuclear macrophages than those in saline control group.With the prolonged time of CsA injection,the interstitial pulmonary inflammation was remissive,and there was less fibroblast infiltration and collagen deposition in pulmonary interstitium and periphery of bronchiole.Alveolar epithelial cells,bronchiolar epithelial cells,mononuclear macrophages,neutrophils and lymphocytes were demonstrated to express CD147,there was higher CD147 expression in BLM model group than those in CsA treatment groups.CONCLUSION： CsA may heal BLM induced interstitial pulmonary disease by blocking CD147-CypA interaction,then decreasing chemotaxis for the immunocyte,and reducing migration of immunocytes to the lung and collagen deposition in the lung.