西维来司钠对COPD大鼠GRP78/PERK/CHOP信号通路及气道高分泌的影响

Effects of sivelestat sodium on the GRP78/PERK/CHOP signaling pathway and airway hypersecretion in COPD rats

目的探究西维来司钠对慢性阻塞性肺疾病(COPD)大鼠葡萄糖调节蛋白78/蛋白激酶R样内质网激酶/CCAAT增强子结合蛋白同源蛋白(GRP78/PERK/CHOP)信号通路及气道高分泌的影响。方法采用香烟烟熏联合脂多糖(LPS)气管滴注制备COPD大鼠模型。成模大鼠随机分为COPD组、西维来司钠干预组(低、中、高剂量),另取正常饲养大鼠为对照组(NC组),每组12只,西维来司钠低、中、高剂量组分别尾静脉注射西维来司钠2.5 mg/kg、5.0 mg/kg、10.0 mg/kg,NC组、COPD组注射等量生理盐水,每天2次,连续干预21 d。检测大鼠肺功能指标0.1 s用力呼气量(FEV0.1)、用力肺活量(FVC)水平,采用原位末端标记法(TUNEL)检测大鼠肺泡上皮细胞凋亡情况,显微镜观察大鼠肺组织病理学改变,实时荧光定量PCR(RT-qPCR)检测大鼠肺组织黏蛋白5AC(MUC5AC)、葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)、CCAAT/增强子结合蛋白同源蛋白(CHOP)mRNA表达水平,蛋白免疫印迹(Western blot)法检测大鼠肺组织MUC5AC、GRP78、PERK、CHOP蛋白表达量。结果NC组大鼠肺泡组织结构完整,无明显病理改变;COPD组大鼠肺组织支气管壁增高,肺泡组织壁变薄,纤毛脱落、倒伏明显,有大量炎性细胞浸润;西维来司钠各干预组大鼠肺组织病理改变均减轻。与NC组比较,COPD组大鼠FEV0.1、FVC水平显著降低,肺泡上皮细胞凋亡率、肺组织MUC5AC、GRP78、PERK、CHOP mRNA及蛋白水平显著增加(P<0.05);与COPD组比较,西维来司钠低、中、高剂量组大鼠FEV0.1、FVC水平依次升高,肺泡上皮细胞凋亡率、肺组织MUC5AC、GRP78、PERK、CHOP mRNA及蛋白水平依次降低(P<0.05)。结论西维来司钠可减轻COPD大鼠气道黏液高分泌,可能与抑制GRP78/PERK/CHOP信号通路激活,抑制COPD肺组织上皮细胞凋亡有关。

Objective To investigate the effect of sivelestat sodium on the glucose-regulated protein 78/protein kinase R-like endoplasmic reticulum kinase/CCAAT enhancer-binding protein homologous protein(GRP78/PERK/CHOP)signaling pathway and airway hypersecretion in rats with chronic obstructive pulmonary disease(COPD).Methods The COPD rat model was established by exposure to cigarette smoke combined with endotracheal instillation of lipopolysaccharide(LPS).The model rats were randomly divided into a COPD group,sivelestat sodium intervention groups(low,medium,and high dose),and a control group(NC group)of normal rats,with 12 rats in each group.The rats in the low,medium,and high dose sivelestat sodium groups were injected via their tail vein with 2.5 mg/kg,5.0 mg/kg,and10.0 mg/kg sivelestat sodium,respectively,and the NC group and COPD group were injected with the same amount of normal saline twice a day for 21 days.The levels of lung function indexes,including the forced expiratory volume in 0.1 s(FEV0.1)and forced vital capacity(FVC),were measured.The apoptosis of alveolar epithelial cells was detected by Td T-mediated d UTP nick and labeling(TUNEL),and microscope histopathological changes of the rat lungs were assessed.The mRNA expression levels of mucin 5 AC(MUC5 AC),glucose-regulated protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),and CCAAT/enhancer binding protein homologous protein(CHOP)were detected by real-time fluorescence quantitative PCR(RT-qPCR),and the protein expression of MUC5 AC,GRP78,PERK,and CHOP in the lung tissue of rats was detected by Western blot.Results In the NC group,the alveolar structure was intact without obvious pathological changes.In the COPD group rats,the wall thickness of lung tissues was increased,the wall of alveoli was thin,the cilia were collapsed and detached,and there was extensive inflammatory cell infiltration.However,the pathological changes in the lung tissue of rats in each sivelestat sodium intervention group were alleviated.Compared with those in the NC group,the FEV0.1 and FVC in the COPD group were significantly lower,the apoptosis rate of alveolar epithelial cells was higher,and the mRNA and protein levels of MUC5 AC,GRP78,PERK,and CHOP in lung tissues were significantly increased(P<0.05).Compared with those in the COPD group,the FEV0.1 and FVC in the rats in the low,medium,and high dose sivelestat sodium groups were increased,the apoptosis rate of alveolar epithelial cells was reduced,and the mRNA and protein levels of MUC5 AC,GRP78,PERK,and CHOP in lung tissues were decreased(P<0.05).Conclusions Sivelestat sodium can reduce airway mucus hypersecretion in COPD rats,which may be related to the inhibition of GRP78/PERK/CHOP signaling pathway activation and reduction in lung epithelial cell apoptosis in COPD.