瑞马唑仑通过调节肺泡巨噬细胞极化减轻脂多糖诱导的急性肺损伤

Remimazolam alleviates LPS⁃induced acute lung injury by regulating macrophage polarization

目的探讨瑞马唑仑对急性肺损伤(ALI)的作用及潜在分子机制。方法将C57BL/6雄性小鼠随机分为正常对照组(Control组)、模型组(LPS组)、瑞马唑仑干预组(RM+LPS组)和瑞马唑仑对照组(RM组),12 h后处死小鼠取材,收集肺泡灌洗液(BALF)、血清和双侧肺组织。通过苏木素⁃伊红(HE)染色、肺组织的湿干质量比(W/D)、BCA法检测BALF中总蛋白含量评价肺损伤情况;采用酶联免疫吸附实验(ELISA)检测BALF和血清中肿瘤坏死因子⁃α(TNF⁃α)、白细胞介素(IL⁃6、IL⁃1β、IL⁃10)的含量;通过流式细胞术检测F4/80+标记的巨噬细胞中M1巨噬细胞阳性标志物iNOS+和M2巨噬细胞标志物CD206+的细胞比例;通过实时荧光定量PCR和蛋白质免疫印迹分析检测肺组织中TNF⁃α、iNOS、CD206、Arg⁃1的mRNA和蛋白水平的表达量。结果LPS滴注12 h后小鼠出现明显肺损伤;BALF和血清中TNF⁃α、IL⁃6、IL⁃1β含量升高;流式细胞术检测iNOS+细胞数量显著增加;肺组织中TNF⁃α、iNOS mRNA含量及蛋白水平均显著增加。给予瑞马唑仑干预后,与LPS组相比,RM+LPS组小鼠肺损伤明显改善;BALF和血清中TNF⁃α、IL⁃6、IL⁃1β含量显著降低,IL⁃10含量显著增加;流式细胞术检测iNOS+细胞数量显著降低,CD206+细胞显著增加;肺组织中TNF⁃α、iNOS mRNA含量及蛋白水平均显著降低,CD206、Arg⁃1 mRNA含量及蛋白水平均显著增加。结论瑞马唑仑可通过抑制肺泡巨噬细胞向M1型极化,促进其向M2型极化,减轻炎症反应,缓解脂多糖诱导的ALI。

Objective To investigate the effect and molecular mechanism of remimazolam on LPS⁃induced acute lung injury.Methods C57BL/6 mice were divided into normal control group,acute lung injury group,remimazolam pretreatment group and remimazolam control group,After 12 hours,the mice were sacrificed for BALF,serum and lung tissue collection.Lung injury was evaluated by HE,W/D ratio and total protein concentra⁃tion in BALF.ELISA was used to detected the contents of TNF⁃α,IL⁃6,IL⁃1β,IL⁃10.The M1 macrophage marker iNOS+and the M2 macrophage marker CD206+in F4/80+macrophages were detected by flow cytometry and the mRNA and protein expressions of TNF⁃α,iNOS,CD206 and Arg1 were detected by real⁃time quantitative PCR and Western blotting.Results Twelve hours after intratracheal injection of LPSA,the lung injury score,W/D ra⁃tio and total protein concentration in BALF were significantly higher,the TNF⁃α,IL⁃6,IL⁃1βin BALF and serum was significantly increased,iNOS+macrophage was significantly increased,the mRNA and protein expressions of TNF⁃αand iNOS were significantly increased,as compared with the control group.After activated by remimazol⁃am,the lung injury score,W/D ratio and total protein concentration in BALF were significantly decreased,the TNF⁃α,IL⁃6,IL⁃1βin BALF and serum was significantly decreased,IL⁃10 was significantly increased,iNOS+macrophage was significantly decreased,CD206+macrophage was significantly increased,the mRNA and protein expressions of TNF⁃αand iNOS were significantly decreased,the mRNA and protein expressions of CD206 and Arg⁃1 were significantly increased,as compared with the LPS group.Conclusion Remimazolam alleviates LPS⁃in⁃duced acute lung injury by inhibiting macrophage polarization to M1 phenotype and promoting the macrophage po⁃larization to M2 phenotype.