法舒地尔减轻脂多糖诱导急性肺损伤的作用及其机制

Effect of fasudil attenuates acute lung injury induced by lipopolysaccharide and its mechanism

目的探讨Rho激酶(ROCK)抑制剂法舒地尔(FS)对脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)的作用及其可能的机制。方法将40只雄性C57BL/6小鼠随机分为对照组、ALI组、FS组和ALI+FS组,每组10只,分别给予相应处理后,评估各组小鼠的肺组织病理变化、湿/干重比和髓过氧化物酶(MPO)活性,以及炎性因子水平、ROCK和细胞焦亡相关蛋白(NLRP3、ASC、caspase-1、cleaved caspase-1、GSDMD、cleaved GSDMD、IL-1β)的表达水平。将培养的人脐静脉内皮细胞(HUVECs)分为对照组、ALI组、FS组和ALI+FS组,分别给予相应处理后,测定各组细胞活性、乳酸脱氢酶(LDH)释放率、炎性因子水平及上述细胞焦亡相关蛋白的表达水平。结果小鼠实验显示,与对照组比较,ALI组小鼠肺泡出血、肺泡间隔增厚和肺组织水肿更加明显;FS预处理后,ALI+FS组上述肺组织病理改变明显减轻。与对照组比较,ALI组小鼠肺湿/干重比值增高,支气管肺泡灌洗液(BALF)中总细胞数增多、总蛋白浓度升高,髓过氧化物酶(MPO)活性升高,炎性因子肿瘤坏死因子-α(TNF-α)、IL-6、IL-18和IL-1β水平上升,肺组织中ROCK1、ROCK2、NLRP3、ASC、cleavedcaspase-1、cleavedGSDMD和IL-1β表达上调,差异均有统计学意义(P<0.05);与ALI组小鼠比较,ALI+FS组小鼠肺湿/干重比值降低,BALF中总细胞数减少、总蛋白浓度降低,MPO活性降低,炎性因子水平降低,肺组织中ROCK1、ROCK2以及焦亡相关蛋白表达水平均明显降低(P<0.05)。HUVECs细胞实验显示,与对照组比较,ALI组细胞活性明显下降,LDH释放率明显升高,细胞死亡率明显增高,炎性因子水平升高,焦亡相关蛋白表达水平明显增高(P<0.05);与ALI组比较,ALI+FS组细胞活性增加,LDH释放率降低,细胞死亡率降低,炎性因子及焦亡相关蛋白表达水平降低,差异均有统计学意义(P<0.05)。结论FS可能通过抑制内皮细胞焦亡缓解LPS诱导的ALI。

Objective To investigate the effect of ROCK inhibitor fasudil(FS)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice and its possible mechanisms.Methods Forty male C57BL/6 mice were randomly divided into four groups(n=10):control group,ALI group,FS group,and ALI+FS group.After administration of LPS or FS,lung histopathological changes,inflammatory cytokine levels,expression levels of Rho kinase(ROCK),and cell pyroptosis-related proteins,including NLRP3,ASC,caspase-1,cleaved caspase-1,GSDMD,cleaved GSDMD,and IL-1β,were assessed.In vitro,human umbilical vein endothelial cells(HUVECs)were cultured and the cell activity,LDH release rate,inflammatory factor levels,and the expression levels of cell pyroptosis-related proteins were measured after administration of LPS+ATP and FS.Results In vivo experiments on mice demonstrated that the alveolar hemorrhage,alveolar septum thickening,and lung tissue edema were more pronounced in ALI group compared to the control group,and FS pretreatment significantly reduced these histopathological changes.Compared with the control group,mice in ALI group showed increased lung wet/dry weight ratio,increased total cell count and total protein concentration in alveolar lavage fluid,increased levels of TNF-α,IL-6,IL-18,and IL-1β,and increased expression of ROCK1,ROCK2,and pyroptosis-related proteins including NLRP3,ASC,cleaved caspase 1,cleaved GSDMD,and IL-1βin pulmonary tissues(P<0.05);compared with mice in ALI group,mice in ALI+FS group had a lower lung wet/dry weight ratio,a lower total cell count and total protein concentration in alveolar lavage fluid,lower levels of inflammatory factors and lower expression levsls of ROCK1,ROCK2,and pyroptosis-related proteins in lung tissue(P<0.05).In vitro experiments on HUVECs cells confirmed that compared with the control group,ALI group showed a significant decrease in cell viability,an increase in LDH release rate,cell death rate,and inflammatory factor levels,and an upregulation of the expression of the pyroptosis-related proteins(P<0.05);compared with ALI group,ALI+FS group showed an increase in cell viability,a significant decrease in LDH release rate,cell death rate,and inflammatory factor levels,and downregulation of the expression of pyroptosis proteins(P<0.05).Conclusion FS may alleviate LPS-induced ALI by inhibiting endothelial cell pyroptosis.