Wedelolactone alleviates lipopolysaccharide-induced pyroptosis of alveolar epithelial cells by inhibiting AMPK/NLRP3/Caspase-1 signaling pathway
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目的 探讨蟛蜞菊内酯(wedelactone,WEL)对脂多糖(lipolyaccharide,LPS)诱导的肺泡上皮细胞焦亡及腺苷酸活化蛋白激酶(adenylate activates protein kinase,AMPK)/核苷酸结合寡聚化结构域样受体3(nucleotide-bound oligomerized domain-like receptor 3,NLRP3)/半胱氨酸天冬氨酸蛋白酶 1(cysteinyl aspartate specific proteinase-1,Caspase-1)信号通路的影响.方法 使用 5～200 μmol/L 的 WEL 处理 BEAS-2B 细胞,MTT法检测细胞活性.将BEAS-2B细胞分为对照组、脂多糖组(LPS组)、10 μmol/L蟛蜞菊内酯组(WEL-L组)、20 μmol/L蟛蜞菊内酯组(WEL-M组)、40 μmol/L蟛蜞菊内酯组(WEL-H组)、40 μmol/L蟛蜞菊内酯+10 μmol/L AMPK 抑制剂 Compound C 组(WEL-H+Compound C 组)、20 μmol/L Caspase-1 抑制剂 Z-YVAD-FMK 组(Z-YVAD-FMK组).透射电镜观察细胞超微结构,酶联免疫吸附试验检测炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-8 水平.免疫荧光检测 Caspase-1、Gasdermin 家族蛋白(gasdermin family proteins,DGSDMD);Western blot 检测 AMPK、NLRP3、Caspase-1 表达水平.结果 选择浓度为10、20、40 μmol/L的WEL进行后续实验.与对照组相比,LPS组细胞活性下降,损伤严重,细胞皱缩,线粒体嵴断裂且数量减少,TNF-α、IL-1β、IL-8水平及GSDMD、NLRP3、Caspase-1表达升高,p-AMPK/AMPK表达下降(P＜0.05).WEL处理可明显改善LPS诱导的肺泡上皮细胞焦亡(P＜0.05)o Compound C可部分逆转WEL对LPS诱导的肺泡上皮细胞焦亡改善作用(P＜0.05).Z-YVAD-FMK处理同样可明显改善LPS诱导的肺泡上皮细胞焦亡(P＜0.05).结论 WEL可以通过抑制AMPK/NLRP3/Caspase-1信号通路抑制LPS诱导的肺泡上皮细胞焦亡.
Objective To investigate the effects of wedelolactone(WEL)on lipopolysaccharide(LPS)-induced pyroptosis of alveolar epithelial cells and AMP-activated protein kinase/nucleotide binding oligomeric domain like receptor 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)signaling pathway.Methods Human lung epithelial cells BEAS-2B were treated with 5-200 μmoI/L wedelolactone,and cell activity was detected using MTT assay.The alveolar epithelial cells were divided into control group,lipopolysaccharide group(LPS group),10 μmol/L wedelolactone group(WEL-L group),20 μmol/L wedelolactone group(WEL-M group),40 μmol/L wedelolactone group(WEL-H group),40 μmol/L wedelolactone+10 μmol/L AMPK inhibitor Compound C group(WEL-H+Compound C group),and 20 μmol/L Caspase-1 inhibitor Z-YVAD-FMK group(Z-YVAD-FMK group).Transmission electron microscopy was applied to observe the microstructure of cells.ELISA was applied to detect levels of inflammatory factors such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-8(IL-8).Immunofluorescence was applied to detect Caspase-1 and gasdermin family proteins(DGSDMD).Western blot was applied to detect protein expression levels of AMPK,NLRP3,and Caspase-1.Results Wedelolactone concentrations of 10,20 and 40 µmol/L were selected for follow-up experiments.Compared with Control group,LPS group showed decreased cell activity,severe damage,cell contraction,mitochondrial ridge breakage and decreased number,increased levels of TNF-α,IL-1β,IL-8 and GSDMD,NLRP3,Caspase-1 expression,and decreased p-AMPK/AMPK expression(P＜0.05).Wedelolactone treatment could significantly improve LPS-induced pyrosis of alveolar epithelial cells(P＜0.05).Compound C could partially reverse the effect of wedelactone on LPS-induced pyrodeath of alveolar epithelial cells(P＜0.05).Z-YVAD-FMK treatment also significantly improved LPS-induced pyroptosis of alveolar epithelial cells(P＜0.05).Conclusion Wedclolactone can inhibit LPS-induced pyroptosis of pulmonary alveolar epithelial cells by inhibiting AMPK/NLRP3/Caspase-1 signaling pathway.
Wedelolactoneacute lung injuryAMP-activated protein kinasenucleotide binding oligomeric domain like receptor 3cysteinyl aspartate specific proteinase-1lipopolysaccharidepulmonary alveolar epithelial cellspyroptosis
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