桑色素抑制NLRP3/Caspase-1/IL-1β信号通路对慢性阻塞性肺疾病大鼠肺泡上皮细胞焦亡的影响

Influences of Morin hydrate on pyroptosis of alveolar epithelial cells in rats with chronic obstructive pulmonary disease by inhibiting NLRP 3/Caspase-1/IL-1βsignaling pathway

目的:探讨桑色素(MH)抑制核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸酶-1(Caspase-1)/IL-1β信号通路对慢性阻塞性肺疾病(COPD)大鼠肺泡上皮细胞焦亡的作用机制。方法:采用烟熏法建立COPD模型大鼠,将其随机分模型组(COPD组)、MH低、中、高剂量组(MH-L、MH-M、MH-H组)(50、100、200 mg/kg)、阳性对照组(地塞米松组,DXMS,0.09 mg/kg),另取10只健康大鼠作为正常对照组(NC组)。检测大鼠肺功能指标[潮气量(TV)、肺活量(FVC)和呼气峰流速(PEF)];HE染色观察肺组织病理学变化;免疫荧光双染观察肺泡上皮细胞焦亡情况;ELISA检测肺泡灌洗液(BALF)中炎症因子水平;RT-qPCR检测NLRP3、Caspase-1、消皮素D(GSDMD)、IL-1βmRNA水平;Western blot检测肺组织SP-C、NLRP3、C-Caspase-1、GSDMD、GSDMD-N、IL-1β蛋白表达。结果:与COPD组比较,DXMS组、MH各组肺组织管腔狭窄、黏液分泌和支气管黏膜上皮细胞坏死等病理学变化明显改善,肺功能指标(TV、FVC、PEF)显著上升(P<0.05),且MH-L、MH-M、MH-H组依次上升(P<0.05);TNF-α、IL-6、IL-8水平、NLRP3及C-Caspase-1免疫荧光表达、NLRP3、Caspase-1、GSDMD、IL-1βmRNA水平、NLRP3、C-Caspase-1、GSDMD、GSDMD-N、IL-1β蛋白水平显著下降,SP-C蛋白表达逐渐上升(P<0.05)。结论:MH可抑制NLRP3/Caspase-1/IL-1β信号通路及其介导的炎症因子释放和细胞焦亡,减轻COPD大鼠的肺组织病变。

Objective:To investigate the mechanism of Morin hydrate(MH)on pyroptosis of alveolar epithelial cells in rats with chronic obstructive pulmonary disease(COPD)by inhibiting nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)/IL-1βsignaling pathway.Methods:COPD model rats were estab⁃lished by smoking method,which were randomly divided into model group(COPD group),low,medium and high doses MH groups(MH-L,MH-M,MH-H groups)(50,100,200 mg/kg)and positive control group(dexamethasone group,DXMS,0.09 mg/kg),another 10 healthy rats were taken as normal control group(NC group).Pulmonary function indexes[tidal volume(TV),forced vital capacity(FVC)and peak expiratory flow rate(PEF)]were measured;HE staining was used to observe pathological changes of lung tissue;pyroptosis of alveolar epithelial cells was observed by immunofluorescence double staining;levels of inflammatory factors in bronchoalveolar lavage fluid(BALF)were detected by ELISA;levels of NLRP3,Caspase-1,gasdermin D(GSDMD)and IL-1βmRNA were detected by RT-qPCR;protein expressions of SP-C,NLRP3,C-Caspase-1,GSDMD,GSDMD-N and IL-1βin lung tissue were detected by Western blot.Results:Compared with COPD group,pathological changes of lung tissue including lumen stenosis,mucus secretion and bronchial epithelial cell necrosis in DXMS group and MH groups were significantly improved,the pulmonary function indexes(TV,FVC,PEF)were increased obviously(P<0.05),and increased in turn in MH-L,MH-M,MH-H groups(P<0.05);levels of TNF-α,IL-6,IL-8,immunofluorescence expressions of NLRP3 and C-Caspase-1,and mRNA levels of NLRP3,Cas⁃pase-1,GSDMD,IL-1β,protein levels of NLRP 3,C-Caspase-1,GSDMD,GSDMD-N and IL-1βwere decreased obviously,while SP-C protein expression was gradually increased(P<0.05).Conclusion:MH can inhibit the NLRP 3/Caspase-1/IL-1βsignaling path⁃way,the release of inflammatory factors and pyroptosis by it,and alleviate lung pathological changes in COPD rats.