Protective effect and mechanism of farrerol on acute lung injury induced by lipopolysaccharide in mice
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.
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