TNFα增强肾小球系膜细胞Ⅰ型1,4,5-三磷酸肌醇受体表达受PKCα调控

The regulation roles of protein kinase C alpha in enhanced expression of type Ⅰ inositol 1,4,5-trisphophate receptors induced by tumor necrosis factor alpha

目的 通过研究蛋白激酶Cα（PKCα）参与调控肿瘤坏死因子α（TNFα）增强肾小球系膜细胞Ⅰ型1,4,5-三磷酸肌醇受体（IP3RI）表达的信号机制,揭示TNFα引起肝肾综合征肾小球滤过率（GFR）下降的分子机制.方法 选择大鼠系膜细胞株（GMCs）进行体外培养.按TNFα处理GMCs的不同时间点,分为对照组（D）、TNFα-2 h组、TNFα-4 h组、NFα-8 h组、TNFα-24 h组,另设2组分别为Sanfingol-8 h组（S）、TNFα＋ Sanfingol共培养8h组（TS）,共6组.应用免疫细胞化学染色、Western blot、Real time-PCR检测方法,观察TNFα孵育的GMCs中IP3RI表达的变化.结果 免疫细胞化学染色发现：IP3RI主要分布于GMCs的胞浆内,对照组阳性信号较弱,TNFα处理各组棕褐色阳性信号增强,以TNFα-8 h组最明显.Western blot分析发现：TNFα-4 h组、TNFα-8 h组、TNFα-24 h组与对照组差异有统计学意义（4 h：1.82±0.63,8 h：2.95±0.66,24 h：2.48±0.72,D：1±0.02,F=9.24,P＜0.05）.其中TNFα-8 h组、TNFα-24 h组IP3RI表达最高（P＜0.05）.S组、TS组与对照组差异无统计学意义（S：1.39 ±0.65,TS：1.35±0.37,P＞0.05）.Real time-PCR发现：TNFα处理各时间点组与对照组相比IP3 RI表达均明显增加,差异有统计学意义（2 h：3.35±1.97;4 h：3.16±1.35,8 h：3.70±1.76;24 h：4.49±1.70,D：1±0.01,F=6.167,P＜0.05）. TNFα各处理组组间差异无统计学意义（P＞0.05）.S组、TS组与对照组比较差异无统计学意义（S：1.53±0.79,TS：1.32±0.38,P＞0.05）.结论 IP3 RI主要分布于GMCs的胞浆内,TNFα可增加肾小球系膜细胞IP3 RI蛋白、mRNA的表达,并可被PKCα抑制剂Sanfingol所阻断,这可能是肝肾综合征时TNFα引起GFR下降的重要信号机制.

Objective To investigate the signal mechanism of protein kinase C alpha（PKC-α）participated in enhanced expression of type Ⅰ inositol 1,4,5-trisphophate receptors （IP3 RI） induced by tumor necrosis factor alpha（TNFα）,in order to delineate the mechanisms of decreased glomerular filtration rate （GFR） in hepatorenal syndrome caused by TNFα.Methods The glomerular mesangial cells （GMCs）line from rats was chosen as experimental material.GMCs were divided into control （D）,TNFα-2 h,TNFα-4 h,TNFα-8 h,and TNFα-24 h groups.Moreover,another two groups were sanflngol-8h （S）,TNFα ＋ Sanfingol-8h（TS）groups.The effect of TNFα on the expression of IP3RI was detected by immunocytochemical staining,Western blotting,teal time-polymerase chain reaction （PCR） assays.Results Immunocytochemical staining demonstrated that IP3 RI was mainly distributed in cytoplasm of GMCs.Enhanced positive staining was determined in all TNFα-treated groups,especially in TNFα-8 h group.Western blotting demonstrated that the expression of IP3RI protein was significantly higher in TNFα-4 h,TNFα-8h and TNFα-24 h groups than control group（4 h：1.82 ± 0.63 ; 8 h：2.95 ± 0.66 ; 24 h：2.48 ± 0.72 ; D：1 ±0.02 ; F =9.24,P 〈 0.05）.The expression of IP3 RI protein was the highest in TNFα-8 h and TNFα-24 h groups（P 〈0.05）.No difference was found among S,TS,and control groups（S：1.39 ±0.65; TS：1.35± 0.37 ; P 〉 0.05）.Real time-PCR found the expression of IP3 RImRNA was significantly higher in all TNFα-treated groups than control group（2 h：3.35 ± 1.97; 4 h：3.16 ± 1.35; 8 h：3.70 ± 1.76; 24 h：4.49±1.70; D：1 ±0.01; F =6.167,P 〈0.05）.No difference was found among all TNFα-treated groups（P 〉0.05）.No difference was found among S,TS,and control groups（S：1.53 ±0.79; TS：1.32 ± 0.38 ; P 〉 0.05）.Conclusions IP3 RI was mainly distributed in cytoplasm of GMCs.TNFa could enhance the expression of IP3 RI protein and IP3 RI mRNA,which could be blocked by sanfingol,a PKCα inhibitor.It might be an important signal in the mechanisms of GFR decrease caused by TNFα in hepatorenal syndrome.