NLRP3炎症小体对腹膜透析相关性腹膜纤维化的作用

Role of NLRP3 inflammasome in the pathogenesis of peritoneal dialysis-related peritoneal fibrosis

目的探讨核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体对腹膜透析(PD)相关性腹膜(PM)纤维化(PF)的作用。方法取对数生长期的人PM间皮细胞(HPMC)分为HPMC组(不予以任何处理)、1.50%PD液(PDS)组(1.50%PDS1.5 mL)、2.50%PDS组(2.50%PDS1.5 mL)、4.25%PDS组(4.25%PDS 1.5 mL)、NC-siRNA组(转染NC-siRNA)、siRNA-NLRP3组(转染siRNA-NLRP3)及siRNA-NLRP3+4.25%PDS组(转染siRNA-NLRP3+4.25%PDS 1.5 mL),培养24 h,制作切片,采用透射电镜观察各组HPMC细胞形态学特征,采用免疫印迹法(Western blot)检测HPMC组、1.50%PDS组及2.50%PDS组HPMC细胞NLRP3、转化生长因子β1(TGF-β1)蛋白表达,检测NC-siRNA组、4.25%PDS组及siRNA-NLRP3组、siRNA-NLRP3+4.25%PDS组HPMC细胞中NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)、TGF-β1及白细胞介素-1β(IL-1β)蛋白质的相对表达;取24只健康SD大鼠随机均分为生理盐水组、1.50%PDS组、2.50%PDS组及4.25%PDS组,100 mL/(kg·d)处理,连续6周,末次干预后取PM组织,采用苏木精-伊红(HE)染色观察各组大鼠PM组织学特征,分别采用实时荧光定量聚合酶链式反应(qRT-PCR)和Western blot检测各组大鼠PM组织NLRP 3、TGF-β1、IL-1βmRNA表达和NLRP3、TGF-β1蛋白质的相对表达量。结果生理盐水组大鼠PM组织结构较为完整、清晰,1.50%PDS组、2.50%PDS组及4.25%PDS组大鼠PM组织增厚,增生区域炎细胞浸润,局部区域见少量血管增生;与生理盐水组比较,4.25%PDS组大鼠PM组织NLRP 3、TGF-β1及IL-1βmRNA表达增加(P<0.05),2.50%PDS组大鼠PM组织NLRP 3和TGF-β1 mRNA表达增加(P<0.05);与生理盐水组比较,4.25%PDS组和2.50%PDS组大鼠PM组织NLRP3、TGF-β1蛋白表达增加(P<0.05);Vimentin(红色荧光)和PCK(绿色荧光)双阳性率>90%,HPMC组HPMC细胞形态结构较正常、细胞核呈不规则多边形,1.50%PDS组和2.50%PDS组HPMC细胞核呈不规则多边形、可见少量自噬,4.25%PDS组HPMC细胞中积累了与自噬相关的结构和凋亡空泡;与HPMC组比较,2.50%PDS组和4.25%PDS组HPMC细胞NLRP3、TGF-β1蛋白表达明显升高(P<0.01);NC-siRNA组、4.25%PDS组、siRNA-NLRP3组及siRNA-NLRP3+4.25%PDS组HPMC细胞的细胞器形态结构都较完整,4.25%PDS组HPMC细胞胞浆中含大量空泡,且除NC-siRNA组外、其余3组HPMC细胞均存在不同程度的自噬;4.25%PDS组HPMC细胞NLRP3、ASC、Caspase-1、TGF-β1及IL-1β蛋白表达较NC-siRNA组升高(P<0.05),siRNA-NLRP3+4.25%PDS组HPMC细胞中NLRP3、ASC、Caspase-1及TGF-β1蛋白表达较4.25%PDS组降低(P<0.05)。结论NLRP3炎症小体可能是预防和治疗PD相关PF的潜在靶点。

Objective To investigate the role of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome in the pathogenesis of peritoneal dialysis(PD)-associated peritoneal fibrosis(PF).Methods Human PM mesothelial cells(HPMC)at logarithmic growth phase were divided into HPMC group(no treatment),1.50%PD solution(PDS)group(1.50%PDS1.5 mL),2.50%PDS group(2.50%PDS 1.5 mL),4.25%PDS group(4.25%PDS1.5 mL),NC-siRNA group(transfected with NC-siRNA),siRNA-NLRP3 group(transfected with siRNA-NLRP3)and siRNA-NLRP3+4.25%PDS group(transfected with siRNA-NLRP3+4.25%PDS 1.5 mL).Above cells were cultured for 24 h.Sections were made and the morphological characteristics of HPMC cells in each group were observed by transmission electron microscopy(TME).Western blot was used to detect the expression levels of NLRP3 and transforming growth factorβ1(TGF-β1)in HPMC,1.50%PDS and 2.50%PDS groups.The levels of NLRP3,apoptosis-associated spot-like protein(ASC),cysteine protease-1(Caspase-1),TGF-β1,and interleukin-1β(IL-1β)proteins in NC-siRNA,4.25%PDS,siRNA-NLRP3,siRNA-NLRP3+4.25%groups were detected by Western blot.Twenty-four healthy SD rats were randomly divided into saline,1.50%PDS,2.50%PDS and 4.25%PDS groups and treated with 100 mL/(kg·d)for 6 continous weeks.After the last intervention,PM tissues were taken.Hematoxylin-eosin(HE)staining was applied to observe the pathological characteristics of PM tissues.Quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was used to measure the relative mRNA expressions of NLRP 3,TGF-β1,IL-1β.The protein levels of NLRP3 and TGF-β1 were detected by and Western blot.Results The PM tissues in physiological saline group were relatively intact and clear,while the PM tissues in the 1.50%PDS,2.50%PDS and 4.25%PDS groups were gradually thickened and infiltrated by inflammatory cells in the hyperplastic areas,with a small amount of vascular hyperplasia seen in local areas.Compared with physiological saline group,mRNA expression levels of NLRP 3,TGF-β1 and IL-1βwere increased in PM tissues of 4.25%PDS group(P<0.05),and mRNA expression levels of NLRP 3 and TGF-β1 were increased in PM tissues of 2.50%PDS group(P<0.05).Compared with physiological saline group,protein expression levels of NLRP3 and TGF-β1 were increased in PM tissues of 4.25%PDS and 2.50%PDS groups(P<0.05),and cells with Vimentin+(red fluorescence)and PCK+(green fluorescence)were>90%.The morphological structure of HPMC cells in the HPMC group was normal,and the nuclei showed irregular polygonal shape and a small amount of autophagy was visible in 1.50%PDS and 2.50%PDS groups.Structures associated with autophagy and apoptotic vacuoles were accumulated in HPMC cells of 4.25%PDS group.Compared with HPMC group,the protein expression levels of NLRP3 and TGF-β1 in 2.50%PDS and 4.25%PDS groups were significantly increased(P<0.01).The morphological structures of cell organelles in the NC-siRNA,4.25%PDS,siRNA-NLRP3,and siRNA-NLRP3+4.25%PDS groups were intact,but the cytoplasm of 4.25%PDS group contained a large number of vacuoles.Except for NC-siRNA group,the other 3 groups of HPMC cells had different degrees of autophagy.The protein expression levels of NLRP3,ASC,Caspase-1,TGF-β1,and IL-1βwere higher in 4.25%PDS group than those in NC-siRNA group(P<0.05).The protein expression levels of NLRP3,ASC,Caspase-1,and TGF-β1 in was lower in siRNA-NLRP3+4.25%PDS group than those in 4.25%PDS group(P<0.05).Conclusion NLRP3 inflammasome may be a potential target for preventing and treating PD-related PF.