TNF—α通过PKCα信号通路诱导人系膜细胞IP3R1表达  

PC-PLC/PKC-α pathway involves in TNF-α-induced IP3R1 expression in human mesangial cells

作　　者：王育蓉[1] 章欢[1] 孙辉[1] 刘沛[2] 

机构地区：[1]九江市第一人民医院消化内科,江西省九江332000 [2]中国医科大学附属院一院传染科

出　　处：《中华急诊医学杂志》2013年第2期153-157,共5页Chinese Journal of Emergency Medicine

基　　金：江西省卫生厅科研基金资助项目（20123192）

摘　　要：目的探讨肿瘤坏死因子（TNF—α）对人。肾小球系膜细胞（HMCs）I型1，4，5-三磷酸肌醇受体（IP，R1）蛋白和mRNA表达的影响及其分子机制，以期为肝肾综合征（HRS）肾小球滤过率下降的发生机制和防治思路提供理论依据。方法用实时定量PCR和免疫印迹法检测TNF-α刺激HMCs0、2、4、8、24h的IP3R1mRNA和蛋白表达的情况。以TNF-α刺激HMCs8h为对照，应用D609，U73122，PP1，Safingol和Rottlerin预处理HMCs后，实时定量PCR法检测JJTNF-α对IP3R1mRNA表达的影响；以TNF-α刺激HMCs24h组为对照，免疫印迹法检测上述抑制剂预处理后，TNF-α对IP3R1蛋白表达的影响。此外，应用非放射性测活法检测0、4、8、24hTNF-α对PKC-α的活化影响，并予D609、Safingo干预后，检测TNF-α对PKC-α的活化的影响。结果TNF-α刺激HMCs2h到8hIP3R1mRNA表达明显增加，于8h达高峰（P〈0．01），而于24h有所下降（P〈0．01）；而IP3R1蛋白表达于TNF-α刺激4h开始升高，至24hIP3R1蛋白升高达高峰（P〈0．01）。与8h组比较，抑制剂＋TNF-α各组中，Safingol＋TNF-α组和D609＋TNF-α组IP3R1mRNA的表达明显下降，差异具有统计学意义（3．30±0．81）vs．（1．95±0．13），P〈0．05；（2．10±0．49），P〈0．01。与24h相比，Safingol＋TNF-α组和D609＋TNF-α组，IP3R1蛋白的表达亦明显下降（3．09±0．13）vs．（1．86＋0．39），P〈0．01；（1．98±0．02），P〈0．01。非放射性测活检测显示TNF-α处理8h使PKC—α自动磷酸化而活化，而D609或Safingol预处理后，可抑制TNF-α对PKC-α的活化。结论TNF-α能上调IP3R1mRNA及蛋白的表达，PC-PLC／PKC—α信号途径可能在TNF-α上调1只R1的表达中起重要作用。Objective To explore the effects of TNF-α on the expression of IPzR1 mRNA and protein in human mesangial cells （ HMCs ） and elucidate the mechnism of TNF-ot indnces the IP3R1 expression in the occurrence of hepatorenal syndrome （HRS）. Methods HMCs was stimulated by tumor （TNF-α） with 100 ng/mL for different hours （2, 4, 8, 24 hours ）. The expression change of IP3 R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblot assay. Several inhibitors including D609, U73122, PP1, Safingol, Rottlerin and non-radioactive PKC assay to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs. Results The levels of IP3R1 mRNA at 2 h post-TNF-a： exposure were significantly enhanced and reached peak at 8 h in HMCs （ P 〈0. 01 ）, then descened at 24 h （P 〈0. 01） . The levels of IP3R1 protein at 4 h post-TNF-α exposure were obviously increased and reached peak at 24 h post-TNF-α exposure （P 〈 0. 01 ）. Compared with the control group, safingol （PKC-α inhibitor） and D609 （PC-PLC inhibitor） each significantly suppressed TNF-α- induced expression of IP3R1 mRNA （3.30 ± 0. 81 ） vs. （ 1.95 ± 0. 130, P 〈 0. 05 ; （2. 10 ± 0. 49）, P 〈 0.01 andlP3R1 protein （3.09±0.13）vs. （1.86±0.39）, P〈0.01; （1.98±0.02）, P〈0.01. TNF- α promoted autophosphorylation, and hence the activation, of PKC-α with maximal phosphorylation that occurred 8 h post-stimulation measured by non-radioactive PKC assay, and the effect was marked attenuated by pretreated with D609 or safingol. Conclusions TNF-α increased the expression of IP3R1 and this was mediated, at least in part, through the PC-PLC/PKC-α signaling oathwavs in HMCs.

关 键 词：肿瘤坏死因子-α 肝肾综合征 人肾小球系膜细胞 I型1 4 5-三磷酸肌醇受体 蛋白 激酶C-α 磷脂酰胆碱特异性磷脂酶C 

分 类 号：R285.5[医药卫生—中药学]