尿酸刺激对前列腺上皮细胞的影响及其作用机制  

The stimulation effect of uric acid on prostate epithelial cells and its mechanism

作　　者：张坤 胡建鹏[1] 徐致鹏 谈建 汤华明[1] 崔飞伦[1] Zhang Kun;Hu Jianpeng;Xu Zhipeng;Tan Jian;Tang Huaming;Cui Feilun(Department of Urology, the Affiliated People’s Hospital of Jiangsu University, Zhenjiang 212002, China)

机构地区：[1]江苏大学附属人民医院,镇江212002

出　　处：《中华实验外科杂志》2019年第4期698-701,共4页Chinese Journal of Experimental Surgery

基　　金：江苏省医学重点人才项目(QNRC2016457);江苏省社会发展重点疾病研发计划项目(EB2016715);江苏省卫计委基金项目(H2017089).

摘　　要：目的探讨尿酸在诱导前列腺上皮RWPE-1细胞发生炎性反应中的作用及其作用机制。方法浓度分别为0、8、12、16 mg/dl的尿酸处理前列腺上皮细胞RWPE-1细胞,倒置显微镜观察RWPE-1细胞的生长及形态学变化;Western blot方法检测RWPE-1细胞内Toll样受体4(TLR- 4)、核转录因子-κBp65(NF-κBp65)、κB抑制蛋白激酶α(IKKα)、κB抑制蛋白激酶β(IKKβ)、Nod样受体蛋白3(NLRP3)炎性蛋白的表达变化;实时定量反转录聚合酶链反应(RT-qPCR)技术检测IKKα、IKKβ、NLRP3、半胱氨酰天冬氨酸特异性蛋白酶1(Caspase-1)的mRNA表达变化;酶联免疫吸附试验(ELISA)法检测细胞培养上清液中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达变化。结果倒置显微镜观察显示,RWPE-1细胞形态学变化与尿酸作用具有浓度依赖关系;RWPE-1细胞中TLR4、NF-κBp65、IKKα、IKKβ、NLRP3炎性蛋白的表达水平随尿酸浓度的增高而增高,呈正相关(P<0.05);随着尿酸的浓度升高,IKKα、IKKβ、NLRP3、Caspase-1基因的表达呈上调趋势,呈正相关(P<0.05);尿酸刺激RWPE-1细胞12 h后,各组(0、8、12、16 mg/dl)TNF-α含量分别为:(9.69±0.22)、(9.56±0.47)、(9.63±0.51)、(10.08±0.11) ng/L,IL-1β分别为:(7.75±0.52)、(7.85±0.87)、(9.27±1.11)、(9.62±0.44) ng/L,差异无统计学意义(P>0.05);24 h后各组TNF-α分别为:(24.86±0.61)、(25.14±0.83)、(30.34±0.61)、(30.77±1.84) ng/L,IL-1β分别为:(14.81±0.23)、(15.53±0.30)、(22.87±0.65)、(25.60±0.70) ng/L;48 h后各组TNF-α分别为:(26.72±0.37)、(26.80±1.14)、(31.60±0.76)、(42.98±1.49) ng/L,IL-1β分别为:(25.36±0.70)、(30.29±0.62)、(38.91±1.52)、(40.97±1.17) ng/L,尿酸处理24、48 h后,高浓度尿酸促进了RWPE-1细胞中IL-1β和TNF-α的分泌(P<0.05)。结论尿酸可以诱导前列腺上皮RWPE-1细胞发生炎性反应,而激活TLR4/NF-κB和NLRP3/Caspase- 1信号通路是其机制之一。Objective To investigate the stimulation effect and mechanism of uric acid induce the response of inflammation in prostatic epithelial cells (RWPE-1 cells). Methods Prostatic epithelial cells (RWPE-1 cells) were cultured with uric acid at concentrations of 0, 8, 12 and 16 mg/dl. The proliferation and morphological changes of RWPE-1 cells were observed by an inverted microscope. Western blotting method was used to detect protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor-κB p65(NF-κBp65), inhibitor of nuclear factor-κB kinase subunit alpha (IKKα), inhibitor of nuclear factor-κB kinase subunit beta (IKKβ) and nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) in RWPE-1 cells. Gene expression of IKKα, IKKβ, NLRP3 and Caspase-1 was quantified by real-time polymerase chain reaction. Cell culture supernatants were measured for inflammatory interleukin (IL)-1β and tumor necrosis factor-α(TNF-α) by enzyme linked immunosorbent assay (ELISA). Results Morphological changes in RWPE-1 cells were concentration-dependent with uric acid from inverted microscopy. Compared with untreated RWPE-1 cells, the levels of TLR4, NF-κBp65, IKKα, IKKβ and NLRP3 in the uric acid-stimulated group were elevated, and the levels of TLR4, NF-κBp65, IKKα, IKKβ and NLRP3 were signaficantly correlated with uric acid concentration(P<0.05). Stimulation of soluble uric acid increased the expression of the IKKα, IKKβ, NLRP3 and Caspase-1 genes, which were positively correlated with the increase in uric acid concentration (P<0.05). After uric acid stimulated RWPE-1 cells for 12 h, the TNF-α levels of each group (0, 8, 12, 16 mg/dl) were (9.69±0.22),(9.56±0.47),(9.63±0.51),(10.08±0.11) ng/L, and IL-1β were (7.75±0.52),(7.85±0.87),(9.27±1.11),(9.62±0.44) ng/L, the difference was not statistically significant (P>0.05). After 24 h, the TNF-α levels of each group were (24.86±0.61),(25.14±0.83),(30.34±0.61),(30.77±1.84) ng/L, IL-1β were (14.81±0.23),(15.53±0.30),(

关 键 词：尿酸 RWPE-1细胞 炎性反应 Toll样受体4/核转录因子-κB信号通路 Nod样受体蛋白3/半胱氨酰天冬氨酸特异性蛋白酶-1信号通路 

分 类 号：R589.7[医药卫生—内分泌]