中国药理学与毒理学杂志2025，Vol.39Issue(12)：891-902,12.DOI:10.3867/j.issn.1000-3002.2025.08649
牛大力总黄酮通过抑制焦亡改善脂多糖诱导的脓毒症急性肺损伤
Total flavonoids of Nanhaia speciosa ameliorate lipopolysaccha-ride-induced acute lung injury in sepsis via inhibition of pyroptosis
摘要
Abstract
OBJECTIVE To investigate the ameliorative effect of total flavonoids of Nanhaia speciosas(TFN)on lipopolysaccharide(LPS)-induced acute lung injury(ALI)and the mechanisms related to pyroptosis.METHODS ①Thirty-six SD rats were randomly divided into normal control,model,model+dexamethasone(DXM),and model+TFN 100,200 and 300 mg·kg-1 groups.Rats in the model+TFN 100,200 and 300 mg·kg-1 group were given the corresponding dose of TFN(ig),and pretreated for 14 consecutive days once daily.Rats in model,molde+TFN 100,200 and 300 mg·kg-1 groups received intraperitoneal(ip)LPS at 1 mg·kg-1 6 h after the final drug administration.The normal control group received an equal volume of saline ip.Rats in the model+DXM group were given DXM 5 mg·kg-1 ip 30 min prior to LPS 1 mg·kg-1 ip administration.After 16 h,the model,model+DXM,and model+TFN groups were subjected to tracheal nebulization of LPS 5 mg·kg-1 to establish a rat sepsis-ALI model.The normal control group underwent tracheal nebulization of an equivalent volume of saline.All rats were euthanized 6 h later.HE was used to observe the pathological changes in lungs.Lung edema was assessed via the wet to dry weight ratio(W/D)of lung tissues.BCA protein quantification was used to detect the concentration of total proteins in bronchoalveolar lavage fluid(BALF).ELISA was adopted to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-18 in lung tissues.The BALF lactate dehydrogenase(LDH)kit was employed to detect LDH activity in BALF.Western blot-ting was used to detect the phosphorylation level of AMP-activated protein kinase(AMPK),and the expression levels of NOD-like receptor family pyrin domain containing 3(NLRP3),gasdermin D(GSD-MD),and GSDMD N-terminal domain(GSDMD-N)in lung tissues.RT-qPCR was used to detect the levels of cysteine aspartic acid specific protease-1(Caspase-1)and NLRP3 mRNA in lung tissues.②A549 cells were divided into the cell control group and TFN 50,100,200,300 and 400 mg·L-1 groups.CCK-8 assay was used to measure cell viability to determine the safe concentration of TFN.A549 cells were divided into the following groups:cell control group,model group,model+pyroptosis inhibitor(MCC950)group,model+TFN 50,100 and 200 mg·L-1 group,model+AMPK inhibitor Compound C group,and model+TFN 200 mg·L-1+Compound C group.The Model+MCC950 group,Model+TFN 50,100 and 200 mg·L-1 group,Model+Compound C group,and Model+TFN 200 mg·L-1+Compound C group were pretreated for 1 h with MCC95010 μmol·L-1,Compound C 5 μmol·L-1,and the corresponding doses of TFN,respectively.Subsequently,except the cell control group,all the groups were incubated with LPS 40 mg·L-1 for 24 h to establish an in vitro ALI cell model.Cell morphology was observed under an inverted microscope,and Western blotting analysis was performed to detect the phosphoryla-tion levels of AMPK and the expression levels of IL-1β,IL-18,NLRP3,GSDMD,and GSDMD-N pro-teins in A549 cells.RESULTS ① Compared with the normal control group,alveolar hemorrhage,inflammatory cell infiltration and widening of alveolar septa were observed in lung tissues of the model group.The W/D ratio,BALF total protein concentration,LDH activity and TNF-α,IL-1β,IL-18 content in lung tissues as well as the protein expression levels of NLRP3 and GSDMD-N and the Caspase-1 and NLRP3 mRNA levels were significantly elevated.The AMPK phosphorylation level in lung tissues was not significantly elevated.Compared with the model group,alveolar hemorrhage,inflammatory cell infil-tration and alveolar septal widening were significantly mitigated in the model+DXM group and model+TFN 100,200 and 300 mg·kg-1 group.The W/D ratio,total BALF protein concentration,LDH activity and TNF-α,IL-1β,IL-18 content,and the expression levels of NLRP3 and GSDMD-N protein in lung tissues were significantly increased.Caspase-1 and NLRP3 mRNA levels were significantly reduced,but AMPK phosphorylation levels in lung tissues were significantly increased.② Compared with the cell control group,TFN 50,100 and 200 mg·kg-1 showed no significant effect on A549 cell viability.Cells in the model group exhibited significant swelling and ballooning changes,and the expression levels of IL-1β,IL-18,NLRP3,and GSDMD-N proteins were significantly increased.Compared with the model group,the morphology of cells in the model+MCC950 group and the model+TFN 50,100,200 mg·L-1 groups was significantly improved,the expression levels of IL-1β,IL-18,NLRP3 and GSDMD-N proteins of cells in the model+TFN 100 and 200 mg·L-1 groups were significantly reduced while the phosphorylation levels of AMPK were significantly increased.③ Compared with the model group,the protein expression level of GSDMD-N was significantly increased in the model+Compound C group.Compared with the model+TFN 200 mg·L-1 group,the AMPK phosphorylation level was significantly reduced in the model+TFN 200 mg·L-1+Compound C group,while the protein expression levels of NLRP3 and GSDMD-N were significantly increased.CONCLUSION TFN has a protective effect against LPS-induced sepsis-associated ALI,and the mechanism may involve the activation of AMPK phosphorylation and inhibition of pyroptosis.关键词
牛大力/总黄酮/急性肺损伤/脂多糖/脓毒症/细胞焦亡Key words
Nanhaia speciosa/total flavonoids/acute lung injury/lipopolysaccharide/sepsis/pyroptosis分类
医药卫生引用本文复制引用
李莉兰,邓芳,刘丽花,周诗尧,麦希田,张毅,梁秋云,黄慧学..牛大力总黄酮通过抑制焦亡改善脂多糖诱导的脓毒症急性肺损伤[J].中国药理学与毒理学杂志,2025,39(12):891-902,12.基金项目
国家自然科学基金(82160778) （82160778）
国家级大学生创新训练项目(202110601023) National Natural Science Foundation of China(82160778) （202110601023）
and National Innovation Training Program for College Students(202110601023) （202110601023）
