摘要
目的 探讨杜鹃素对脂多糖(LPS)诱导小鼠急性肺损伤(ALI)的保护作用及机制.方法 ①ICR雄性小鼠随机分为正常对照组、模型组、模型+杜鹃素组.模型+杜鹃素组ig给予杜鹃素20和40 mg·kg-1,连续预处理4d,第5d模型组和模型+杜鹃素组均气管滴注LPS 3 mg·kg-1,诱导24h制备ALI小鼠模型.正常对照组气管滴注等体积生理盐水.苏木精-伊红染色(HE)观察肺部病理变化;湿干重比(W/D)评估肺水肿;伊文思蓝染色法(EBD)检测肺血管通透性;比色法检测肺组织中髓过氧化物酶(MPO)活性;血球计数板计数肺泡灌洗液(BALF)中白细胞数量;酶联免疫吸附法(ELISA)检测肺组织和BALF中白细胞介素1β(IL-1β)、IL-6、IL-10和肿瘤坏死因子α(TNF-α)含量;Western印迹法检测肺组织中诱导型一氧化氮合酶(iNOS)、精氨酸酶1(ARG1)、NOD样受体蛋白3(NLRP3)、胱天蛋白酶1(caspase1)和gasdermin家族成员(GSDMD-N)蛋白表达水平及NF-κB p65磷酸化水平.②将小鼠肺泡巨噬细胞(MH-S)随机分为细胞对照组、模型组、模型+杜鹃素组.模型组用LPS 100 μg·L-1和IFN-γ 40 μg·L-1 共孵育24 h,模型+杜鹃素组用杜鹃素5,10和20 μmol·L-1预处理24 h,然后用LPS 100 μg·L-1和IFN-γ 40 μg·L-1 共孵育24 h,建立体外炎症细胞模型.CCK-8测定细胞存活率;ELISA法检测细胞上清液中IL-1β,IL-6,IL-10和TNF-α含量;Western印迹法检测MH-S细胞中iNOS,ARG1,NLRP3,caspase1和GSDMD-N蛋白表达水平及NF-κB p65磷酸化水平.结果 ①与正常对照组相比,模型组小鼠肺组织病理损伤评分、W/D比值、肺血管通透性、MPO活性、白细胞数量和促炎因子IL-1β,TNF-α,IL-6含量及肺组织中iNOS,NLRP3,caspase1,GSDMD-N蛋白表达水平以及NF-κB p65磷酸化水平显著升高(P<0.05,P<0.01),抗炎因子IL-10和ARG1表达水平显著降低(P<0.05,P<0.01).与模型组相比,模型+杜鹃素组小鼠肺组织病理损伤评分、W/D比值、肺血管通透性、白细胞数量、MPO活性和IL-1β,TNF-α,IL-6含量及肺组织中iNOS,NLRP3,caspase1,GSDMD-N蛋白表达水平以及NF-κB p65磷酸化水平显著降低(P<0.05,P<0.01);IL-10和ARG1表达水平显著升高(P<0.05,P<0.01).②与细胞对照组相比,模型组IL-1β,TNF-α和IL-6含量和iNOS,NLRP3,caspase1,GSDMD-N蛋白表达水平及NF-κB p65磷酸化水平显著升高(P<0.05,P<0.01),IL-10和ARG1表达水平显著降低(P<0.01).与模型组相比,模型+杜鹃素组IL-1β,TNF-α和IL-6含量和iNOS,NLRP3,caspase1,GSDMD-N蛋白表达水平及NF-κB p65磷酸化水平显著降低(P<0.05,P<0.01),IL-10和ARG1表达水平升高(P<0.05,P<0.01).结论 杜鹃素对LPS诱导小鼠ALI具有保护作用,其作用机制可能与抑制NF-κB p65磷酸化、抑制NLRP3炎症小体的激活和减轻细胞焦亡及调节巨噬细胞极化有关.
Abstract
OBJECTIVE To investigate the protective effect of farrerol against lipopolysaccharide(LPS)induced acute lung injury(ALI)in mice and the mechanisms.METHODS ①ICR rats were randomly divided into the normal control group,model group and model+farrerol group,with 6 rats in each.The normal control group and model group were given an equal amount of normal saline intragaically for 4 d.The model+farrerol group was given 20 and 40 mg·kg-1 intragaically for 4 d,while the normal control group was given an equal amount of normal saline intragaically on the 5th day.The model group and model+farrerol group were injected with 3 mg·kg-1 LPS solution for 24 h to construct an animal model of ALI.② Mouse alveolar macrophages(MH-S)were cultured and randomly divided into the cell control group,model group and model+farrerol group.Cell control group(conventional culture for 24 h),model group(100 μg·L-1 LPS combined with 40 μg·L-1 IFN-γ co-incubation for 24 h),model+farrerol group(5,10 and 20 μmol·L-1 farrerol pretreatment of MH-S cells for 24 h,and then 100 μg·L-1 LPS combined with 40 μg·L-1 IFN-γ co-incubation for 24 h),an inflammatory cell model was established in vitro.HE was used to observe the pathological changes of the lungs.The wet-dry weight ratio(W/D)was used to assess pulmonary edema while Evans blue dye(EBD)was used to detect pulmonary vascular permea-bility.The activity of myeloperoxidase(MPO)was detected by colorimetry.The number of white blood cells in BALF was counted with a blood cell counting plate.Cell proliferation assay(CCK-8)was used to determine the cell viability.The levels of interleukin-1β(IL-1β),tumor necrosis factor α(TNF-α),IL-6 and IL-10 in lung tissue,bronchoalveolar lavage fluid(BALF)and cell supernatants were detected via enzyme-linked immunosorbent assay(ELISA).The protein levels of inducable nitric oxide synthase(iNOS),arginase 1(ARG1),phosphorylated NF-κB p65,NOD-like receptor protein 3(NLRP3),cysteinyl aspartate specific proteinase1(caspase1)and gasdermin family member(GSDMD-N terminal)in lung tissue and MH-S cells were detected with Western blot.RESULTS ① Compared with the normal control group,the histopathological changes,Injury score,W/D ratio,pulmonary vascular permeability,MPO content,leukocyte count,pro-inflammatory factor IL-1β,TNF-α,IL-6 levels,iNOS,phosphorylated NF-κB p65,NLRP3,caspase1 and GSDMD-N-terminal protein expression in lung tissues were signifi-cantly increased in the model group(P<0.05,P<0.01)while the expression levels of anti-inflammatory factors IL-10 and ARG1 were decreased(P<0.05,P<0.01).Compared with the model group,the Injury score,W/D ratio,pulmonary vascular permeability,leukocyte number,MPO content,proinflammatory factor content and iNOS,phosphorylated NF-κB p65,NLRP3,caspase1 and GSDMD-N-terminal protein levels were significantly decreased in the model+farrerol group(P<0.05,P<0.01)while the levels of anti-inflammatory factor IL-10 and ARG1 protein were increased(P<0.05,P<0.01).② The results of in vitro experiments showed that compared with the cell control group,the contents of IL-1β,TNF-α and IL-6 and the expression levels of iNOS,phosphorylated NF-κB p65,NLRP3,caspase1 and GSD-MD-N-terminal protein were increased(P<0.05,P<0.01),and that the content of anti-inflammatory factor IL-10 and expression level of ARG1 protein were decreased(P<0.01).Compared with the model group,the content of proinflammatory factor and the expressions of iNOS,phosphorylated NF-κB p65,NLRP3,caspase1 and GSDMD-N protein in the model+farrerol group were significantly decreased(P<0.05,P<0.01)while the expression levels of IL-10 and ARG1 protein were increased(P<0.05,P<0.01).CONCLUSION Farrerol can alleviate acute lung injury induced by LPS in mice,possibly by inhibiting the phosphorylation of NF-κB p65 and activation of NLRP3 inflammatome,alleviating pyroptosis of cells and regulating macrophage polarization.
维普
万方数据