基于NLRP3信号通路探讨辛伐他汀抑制COPD大鼠肺组织细胞焦亡的作用研究

Study on the effect of simvastatin on the pyroptosis of lung tissue cells in COPD rats based on NLRP3 signaling pathway

目的基于核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路探讨辛伐他汀抑制慢性阻塞性肺疾病(COPD)大鼠肺组织细胞焦亡的作用。方法取40只雄性SD大鼠,采用香烟烟熏方式构建COPD模型。辛伐他汀药物组(8只)分别于建模第1~14天、第16~29天和第31~59天在烟熏前使用辛伐他汀以5 mg/kg剂量灌胃,NLPR3下调组(8只)分别于上述时间点经尾静脉注射NLPR3-siRNA转染重组载体,空载组(8只)同时经尾静脉注射NC-siRNA转染重组载体,对照组(8只)和COPD模型组(8只)同时进行等体积生理盐水灌胃。采用酶联免疫吸附试验检测肺泡灌洗液(BALF)上清液和血清中白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)水平,采用苏木精-伊红染色观察大鼠肺组织病理改变,采用末端标记法染色观察肺细胞凋亡情况,采用实时荧光定量PCR检测肺组织中NLRP3、凋亡相关点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)、IL-1β、IL-18、TNF-αmRNA水平,采用免疫组化法检测肺组织中NLRP3、ASC、Caspase-1、IL-1β、IL-18、TNF-α蛋白水平。结果与COPD模型组和空载组比较,辛伐他汀药物组、NLPR3下调组肺功能指标均改善,BALF上清液和血清中IL-1β、IL-18、TNF-α水平均降低(P<0.05),肺组织病理改变减轻,肺组织中细胞凋亡率、NLRP3、Caspase-1、IL-1β、ASC、IL-18、TNF-αmRNA和蛋白水平降低(P<0.05)。结论辛伐他汀可抑制COPD大鼠肺组织细胞焦亡,其机制可能与抑制NLRP3/Caspase-1/IL-1β信号通路,进而抑制ASC、IL-18、TNF-α表达相关。

Objective To investigate the effect of simvastatin on pyroptosis in lung tissue of rats with chronic obstructive pulmonary disease(COPD)based on the nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway.Methods Totally 40 male SD rats were enrolled,and COPD model was established by smoking.Simvastatin drug group(8 rats)was given 5 mg/kg simvastatin by gavage before smoking from day 1 to day 14,day 16 to day 29 and day 31 to day 59,respectively.NLPR3 siRNA transfection recombinant vector was injected into the tail vein of the NLPR3 down-regulation group(8 rats)respectively at the above time points,and NC-siRNA transfection recombinant vector was injected into the tail vein of the empty vector group(8 rats)at the same time.The control group(8 rats)and COPD model group(8 rats)were given same volume of normal saline by gavage at the same time.The levels of interleukin(IL)-1β,IL-18 and tumor necrosis factor-α(TNF-α)in the supernatant of bronchoalveolar lavage fluid(BALF)and serum were detected by enzyme-linked immunosorbent assay.Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue.The apoptosis of lung cells was observed by terminal labeling staining.The mRNA levels of NLRP3,apoptosis-associated dot-like protein(ASC),cysteinyl aspartate specific proteinase-1(Caspase-1),IL-1β,IL-18,and TNF-αin lung tissue were detected by real-time fluorescence quantitative PCR.The protein levels of NLRP3,ASC,Caspase-1,IL-1β,IL-18 and TNF-αin lung tissue were detected by immunohistochemistry.Results Compared with the COPD model group and the empty vector group,the simvastatin drug group and NLPR3 down-regulation group had significant improvements in the lung function indexes and significant reductions in the levels of IL-1β,IL-18 and TNF-αin BALF and serum(P<0.05).The pathological changes of lung tissue were alleviated,and the apoptosis rate and the mRNA and protein levels of NLRP3,Caspase-1,IL-1β,ASC,IL-18 and TNF-αin lung tissue were decreased(P<0.05).Conclusion Simvastatin can inhibit pyroptosis in the lung tissue of COPD rats,which may be related to the inhibition of NLRP3/Caspase-1/IL-1βsignaling pathway and the expression of ASC,IL-18 and TNF-α.