间充质干细胞中血红素加氧酶-1的过表达抑制NOD样受体热蛋白结构域相关蛋白3炎症小体活化改善大鼠急性肺损伤

Mesenchymal stem cells overexpressing heme oxygenase-1 ameliorate rat acute lung injury by inhibiting NOD-like receptor family pyrin domain containing 3 inflammasome activation

目的探究过表达血红素加氧酶-1(HO-1)的骨髓间充质干细胞(BMSCs)对大鼠急性肺损伤(ALI)的作用及其可能机制。方法将40只SD大鼠随机分为对照组、模型组、BMSCs-NC组和BMSCs-HO-1组,每组各10只。应用腹腔注射脂多糖(LPS)建立ALI大鼠模型。BMSCs-NC组大鼠尾静脉注射阴性对照BMSCs,BMSCs-HO-1组大鼠尾静脉注射过表达HO-1的BMSCs,其余组大鼠尾静脉注射生理盐水。检测各组大鼠肺泡灌洗液(BALF)中炎症细胞数;采用酶联免疫吸附试验(ELISA)检测BALF中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β水平。采用苏木素-伊红(HE)染色检测肺组织病理学变化;测定大鼠肺组织湿/干重比(W/D);实时荧光定量聚合酶链反应(qRT-PCR)检测HO-1、表面活性蛋白C(SP-C)mRNA表达水平;蛋白质免疫印迹法(Western blot)检测HO-1、SP-C、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)和cleaved-Caspase-1蛋白表达水平。结果与对照组相比,模型组大鼠BALF中细胞总数、中性粒细胞数、巨噬细胞数、TNF-α、IL-6、IL-1β水平、肺损伤评分及W/D均明显升高,肺组织中HO-1和SP-C mRNA、蛋白表达水平均明显降低,NLPR3、ASC和cleaved-Caspase-1蛋白表达水平均明显升高(P<0.05)。与模型组相比,BMSCs-NC组大鼠BALF中细胞总数、中性粒细胞数、巨噬细胞数、TNF-α、IL-6、IL-1β水平、肺损伤评分及W/D均明显升高,肺组织中HO-1和SP-C mRNA、蛋白表达水平均明显升高,NLPR3、ASC和cleaved-Caspase-1蛋白表达水平均明显降低(P<0.05)。与BMSCs-NC组相比,BMSCs-HO-1组大鼠各项指标均得到进一步改善(P<0.05)。结论过表达HO-1的BMSCs可能通过抑制NLPR3炎症小体活化改善大鼠ALI。

Objective To explore the effect of bone marrow mesenchymal stem cells(BMSCs)overexpressing heme oxygenase-1(HO-1)on acute lung injury(ALI)in rats and its possible mechanism.Methods Forty SD rats were randomly divided into control group,model group,BMSCs-NC group and BMSCs-HO-1 group,with 10 rats in each group.Intraperitoneal injection of lipopolysaccharide(LPS)was used to establish a rat model of ALI.The rats in BMSCs-NC group were injected with negative control BMSCs(BMSCs-NC)through tail vein,rats in BMSCs-HO-1 group were injected with BMSCs overexpressing HO-1(BMSCs-HO-1)through tail vein,rats in other groups were injected with normal saline through tail vein.Number of inflammatory cells in alveolar lavage fluid(BALF)of rats in each group was measured.Enzyme-linked immunosorbent assay(ELISA)was used to detect tumor necrosis factor(TNF)-α,interleukin(IL)-6 and IL-1βin BALF.Hematoxylin-eosin(HE)staining was used to detect lung histopathological changes.Wet/dry weight ratio(W/D)of rat lung tissue was measured.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect expression of HO-1 and SP-C mRNA.Western blotting was used to detect expression of HO-1,SP-C,NOD-like receptor family pyrin domain containing 3(NLRP3),apoptosis-related dot-like protein(ASC)and cleaved-Caspase-1 protein.Results Compared with control group,total cells count,neutrophils count,macrophages count,levels of TNF-α,IL-6 and IL-1βin BALF,lung injury score and W/D in model group were significantly increased,expression levels of HO-1 and SP-C mRNA and protein in lung tissue were significantly reduced,and expression levels of NLPR3,ASC and cleaved-Caspase-1 protein were significantly increased(P<0.05).Compared with model group,total cells count,neutrophils count,macrophages count,levels of TNF-α,IL-6 and IL-1βin BALF,lung injury score and W/D of rats in BMSCs-NC group were significantly increased,expression levels of HO-1 and SP-C mRNA and protein in lung tissue were significantly increased,expression levels of NLPR3,ASC and cleaved-Caspase-1 protein were significantly reduced(P<0.05).Compared with BMSCs-NC group,all above indicators of BMSCs-HO-1 group were further improved(P<0.05).Conclusion BMSCs overexpressing HO-1 may ameliorate acute lung injury in rats by inhibiting NLPR3 inflammasome activation.