刘谊蓉,黄振兴
LIU Yirong,HUANG Zhenxing

• 1:西宁市第一人民医院肾内科

摘要(Abstract)：

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%PDS1.5 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NLRP3、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NLRP3、TGF-β1及IL-1βmRNA表达增加（P<0.05）,2.50%PDS组大鼠PM组织NLRP3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NCsiRNA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 siRNANLRP3) and siRNA-NLRP3 + 4. 25% PDS group( transfected with siRNA-NLRP3 + 4. 25% PDS1. 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 factor β1(TGF-β1) in HPMC,1. 50% PDS and 2. 50% PDS groups. The levels of NLRP3, apoptosisassociated 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 ofNLRP3,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 the1. 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 ofNLRP3,TGF-β1 andIL-1βwere increased in PM tissues of 4. 25% PDS group(P< 0. 05), and mRNA expression levels of NLRP3 andTGF-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-1β were 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-β1in 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 PDrelated PF.

关键词(KeyWords)： 腹膜纤维化;腹膜透析;RNA干扰;大鼠细胞;NLRP3炎症小体
peritoneal fibrosis(PF);peritoneal dialysis(PD);RNA interference;rats cells;NLRP3 inflammasome

Abstract:

Keywords:

基金项目(Foundation): 西宁市科技计划项目（2020-M-26）

作者(Author): 刘谊蓉,黄振兴
LIU Yirong,HUANG Zhenxing

DOI: 10.19367/j.cnki.2096-8388.2022.04.006

参考文献(References)：

• [1]董晶莹，李婧波，韩春艳，等．血液透析和腹膜透析治疗老年终末期肾病的临床疗效[J]．中国老年学杂志，2021,41(11):2353-2356.
• [2]刘璐璐．腹膜透析作为紧急开始肾脏替代治疗选择的进展[J]．临床与病理杂志，2021,41(9):2207-2210.
• [3]卓建钦．腹膜透析对终末期肾病患者心肌损伤、钙磷代谢及免疫功能的影响[J]．医学理论与实践，2021,34(8):1325-1327.
• [4] KOBAYASHI T, NOGUCHI T, SAITO K, et al. Eosinophilic inflammation in peritoneal fibrosis patients undergoing peritoneal dialysis[J]. Contributions to Nephrology,2018, 196:1-4.
• [5] ALATAB S, NAJAFI I, ATLASI R, et al. A systematic review of preclinical studies on therapeutic potential of stem cells or stem cells products in peritoneal fibrosis[J]. Minerva Urologica E Nefrologica, 2018, 70(2):162-178.
• [6]曹曼，吕勇，张磊，等．基于“脾主腹”理论观察加味参苓白术散对腹膜透析患者腹膜功能及纤维化指标的影响[J]．安徽中医药大学学报，2021,40(3):17-21.
• [7]陈瑞丹，张彦，杨培丽，等．迷迭香酸通过抑制NADPH氧化酶/ROS/NLRP3炎症小体通路改善高血压肾病[J]．第三军医大学学报，2021,43(12):1153-1161.
• [8] LIU W, SUN J, GUO Y, et al. Calhex231 ameliorates myocardial fibrosis post myocardial infarction in rats through the autophagy-NLRP3 inflammasome pathway in macrophages[J]. Journal of Cellular and Molecular Medicine, 2020, 24(22):13440-13453.
• [9] HAUTEM N, MORELLE J, SOW A, et al. The NLRP3inflammasome has a critical role in peritoneal dialysis-related peritonitis[J]. Journal of American Society of Nephrology, 2017, 28(7):2038-2052.
• [10]WU J, XING C, ZHANG L, et al. Autophagy promotes fibrosis and apoptosis in the peritoneum during long-term peritoneal dialysis[J]. Journal of Cellular and Molecular Medicine, 2018, 22(2):1190-1201.
• [11] STRIPPOLI R, MORENOVICENTE R, BATTISTELLI C, et al. Molecular mechanisms underlying peritoneal EMT and fibrosis[J]. Stem Cells International, 2016,2016:3543678.
• [12]韩艳奇，高志丹，黄翠萍．NLRP3炎症小体及其在支气管哮喘中的研究进展[J]．湖北科技学院学报（医学版），2021,35(3):273-276.
• [13]LIAO X, JIANG Y, DAI Q, et al. Fluorofenidone attenuates renal fibrosis by inhibiting the mtROS-NLRP3 pathway in a murine model of folic acid nephropathy[J]. Biochemical Biophysical Research Communications, 2021,534:694-701.
• [14]赵静，张丽英，康红霞．基于自噬途径探讨黄芪甲苷抑制糖尿病肾病系膜细胞NLRP3炎症小体活化通路及机制[J]．中医药导报，2021,27(9):41-46.
• [15]NAM S A, KIM W Y, KIM J W, et al. Autophagy in FOXD1 stroma-derived cells regulates renal fibrosis through TGF-β and NLRP3 inflammasome pathway[J].Biochemical Biophysical Research Communications,2019, 508(3):965-972.
• [16]孙珍珠，黄兴晓，林娜，等．二氢杨梅素通过抑制NLRP3炎症小体减轻多柔比星引起的大鼠心肌损伤[J]．中华病理学杂志，2020,49(10):1046-1051.
• [17]WANG Y J, CHEN Y Y, HSIAO C M, et al. Induction of autophagy by pterostilbene contributes to the prevention of renal fibrosisviaattenuating NLRP3 inflammasome activation and epithelial-mesenchymal transition[J]. Frontiers in Cell and Developmental Biology, 2020, 8:436.
• [18]刘晶，朱微，蒋春明，等．低密度脂蛋白受体表达失调在高糖腹膜透析液诱导腹膜纤维化中的作用[J]．中华肾脏病杂志，2018, 34(6):432-438.
• [19]范为民．通痹七物方对痛风性关节炎NLRP3炎症小体、线粒体自噬蛋白的影响[J]．中药新药与临床药理，2020,31(7):769-774.
• [20]刘孟楠，罗钢，杨思进．中药提取物通过抑制NLRP3途径治疗动脉粥样硬化性疾病研究进展[J]．甘肃中医药大学学报，2020,37(5):91-96.
• [21]葛凡，王文恺，朱景天，等．黄芪甲苷通过NLRP3炎性小体调节糖尿病动脉粥样硬化早期大鼠血脂及炎症因子的研究[J]．南京中医药大学学报，2021,37(3):383-387.
• [22] MRIDHA A R, WREE A, ROBERTSON A B, et al.NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice[J]. J Hepatol, 2017, 66(5):1037-1046.
• [23]李淑华，张旭帆，何訸，等．腹膜透析患者腹膜纤维化与腹膜透析液中MMP-2、TIMP-1及IL-6水平的相关性研究[J]．解放军医药杂志，2021,33(5):48-50,55.
• [24]李福记，霍冬梅，梁靖梅，等．苦参碱调控Snail2表达水平抑制转化生长因子β1诱导的人腹膜间皮细胞上皮间充质转分化[J]．中华肾脏病杂志，2020(3):221-226.
• [25]HE W T, WAN H, HU L, et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion[J]. Cell Res, 2015, 25(12):1285-1298.
• [26] ZENG C Y, LI C G, SHU J X, et al. ATP induces caspase-3/gasdermin E-mediated pyroptosis in NLRP3pathway-blocked murine macrophages[J]. Apoptosis,2019, 24(9-10):703-717.
• [27]LEE B L, MIRRASHIDI K M, STOWE I B, et al. ASCand caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages[J]. Scientific Reports, 2018, 8(1):3788.

文章评论(Comment)：