骨形态构建蛋白 2型受体下调单核细胞趋化蛋白 1/CC类趋化因子受体 2通路对哮喘小鼠气道炎症和 Th1/Th2平衡的影响

目的探究骨形态构建蛋白 2型受体( BMPR2)对小鼠哮喘模型气道炎症和 Th1/Th2平衡的影响及其机制。方法2021年 9月至 2022年 11月选用 24只 C57BL/6小鼠尾静脉注射 BMPR2腺病毒，随后卵清蛋白( OVA)致敏和激发建立哮喘小鼠于模型；采用随机数字表法分为对照组(生理盐水替代 OVA造模)、 OVA模型组( OVA诱导小鼠哮喘模型)、 OVA+载体( Vector)组(空载慢病毒处理的 OVA哮喘模型小鼠)OVA+BMPR2组( BMPR2过表达慢病毒处理的 OVA哮喘模型小鼠)，每组 6只。收集支气管肺泡灌洗液( BALF)和肺组织。免疫、组织化学检测肺组织 BMPR2表达水平；苏木精 -伊红染色观察肺组织病理学改变；瑞氏染色后计数 BALF中各类炎症细胞数目；酶联免疫吸附测定( ELISA)试剂盒检测 BALF中白细胞介素( IL)-4，IL-5和 IL-13炎症因子水平；蛋白质印迹法检测肺组织和气道上皮细胞 16HBE中 BMPR2、单核细胞趋化蛋白 -1(MCP1)及其受体 CC类趋化因子受体 2(CCR2)蛋白表达水平。免疫共沉淀实验检测 BMPR2与 MCP1相互作用。结果与对照组相比， OVA模型组肺组织 BMPR2(0.36±0.05比 1.04±0.04)显著降低(P<0.01)；小鼠肺泡破坏程度严重，肺组织有大量的淋巴细胞浸润； BALF中炎症细胞总数［(93.25±9.32)×104个/毫升比( 4.79±0.41)×104个/毫升， P<0.001］、嗜酸性粒细胞、中性粒细胞和淋巴细胞均升高； Th1相关炎症因子 γ干扰素( IFN-γ)水平降低， Th2相关炎症因子 IL-4，IL-5和 IL-13水平升高。过表达 BMPR2可降低肺泡破坏程度和淋巴细胞浸润程度。与 OVA+Vector组相比， OVA+BMPR2组 BALF中炎症细胞总数、嗜酸性粒细胞、中性粒细胞和淋巴细胞均降低； ELISA结果表明， OVA+BMPR2组 BALF中 IFN-γ水平升高， IL-4，IL-5和 IL-13水平降低，提示过表达 BMPR2可上调 Th1百分比，下调 Th2细胞百分比。进一步研究表明， BMPR2过表达显著下调了 MCP1及其受体 CCR2的表达水平，且 BMPR2与MCP1存在相互作用。结论 BMPR2可通过下调 MCP1/CCR2通路降低哮喘小鼠气道炎症，并调节 Th1/Th2平衡。

Effect of BMPR2 on airway inflammation and the Th1/Th2 balance in asthmatic mice through downregulation of the MCP1/CCR2 pathway

Objective To investigate the effects and underlying mechanisms of bone morphogenetic protein receptor 2 (BMPR2) onairway inflammation and the Th1/Th2 balance in a mouse model of asthma.Methods From September 2021 to November 2022, 24C57BL/6 mice were selected and injected with BMPR2 adenovirus via the tail vein, followed by ovalbumin (OVA) sensitization and elicitation to establish an asthma mouse model; the mice were divided into a control group (saline alternative to OVA for modeling), anOVA model group (OVA-induced asthma model in mice), an OVA+Vector group (OVA asthma model mice treated with empty lentivirus), and an OVA+BMPR2 group (OVA asthma model mice treated with BMPR2-overexpressing lentivirus), with 6 mice in each group.Bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The expression level of BMPR2 in lung tissue was detected by immunohistochemistry; pathological changes in lung tissue were observed by hematoxylin-eosin staining; the number of various types ofinflammatory cells in BALF was counted after Riehl''s staining; the levels of interleukin (IL)-4, IL-5, and IL-13 inflammatory factors in BALF were detected by and enzyme-linked immunosorbent assay (ELISA) kit; and the levels of BMPR2, MCP1, and its receptor CC-like chemokine receptor 2 (CCR2) were detected by Western blotting in lung tissue and airway epithelial cells 16HBE. A coimmunoprecipitation assay was used to detect the interaction between BMPR2 and MCP1.Results Compared with those in the control group, BMPR2 (0.36±0.05 vs. 1.04±0.04) expression was significantly lower in the lung tissues of the OVA model group (P<0.01); alveolar destruction was severe in the mice, and there was a large amount of lymphocyte infiltration in the lung tissues; the total number of inflammatory cells in the BALF [(93.25±9.32) ×104 cells/mL vs. (4.79±0.41) ×104 cells/mL, P<0.001]; eosinophils, neutrophils and lympho cytes were elevated; the level of Th1-associated inflammatory factor interferon gamma (IFN-γ) was decreased; the levels of the Th2-as sociated inflammatory factors IL-4, IL-5 and IL-13 were increased. Overexpression of BMPR2 reduced the degree of alveolar destruction and lymphocytic infiltration. Compared to those in the OVA+Vector group, the total numbers of inflammatory cells, eosinophils,neutrophils and lymphocytes in the BALF were significantly lower in the OVA+BMPR2 group. ELISA results showed that IFN-γ levels were increased and IL-4, IL-5 and IL-13 levels were decreased in BALF of the OVA+BMPR2 group, suggesting that overexpression ofBMPR2 increased the percentage of Th1 cells and decreased the percentage of Th2 cells. Furthermore, BMPR2 overexpression significantly downregulated the expression levels of MCP1 and its receptor CCR2, and BMPR2 interacted with MCP1.Conclusion BMPR2 can reduce airway inflammation and regulate the Th1/Th2 balance by downregulating the MCP1/CCR2 pathway in asthmatic mice.