细胞因子信号传导抑制蛋白1抑制高糖诱导的肾小球系膜细胞单核细胞趋化蛋白1的表达

Suppressor of cytokine signaling-1 inhibits high glucose-induced expression of monocyte chemoattractant protein-1 in glomerular mesangial cells

目的 探讨细胞因子信号传导抑制蛋白1（SOCS-1）对高糖状态下肾小球系膜细胞单核细胞趋化蛋白1（MCP-1）表达的影响.方法 体外培养人肾小球系膜细胞,应用脂质体2000分别转染pCR3.1-SOCS-1表达质粒和pCR3.1空质粒载体,G418筛选阳性克隆.分别采用低糖（5.5 mmol/L）、高糖（30 mmol/L）、低糖＋甘露醇（24.5 mmol/L甘露醇）和JAK-STAT信号通路抑制剂AG490（10 μmol/L）进行刺激.Western印迹检测系膜细胞SOCS-1、信号转导和转录活化因子1、3（STAT1、STAT3）及其磷酸化蛋白（p-STATI、p-STAT3）的表达.ELISA法和放免法测定细胞上清液中MCP-1、FN和Ⅳ型胶原的含量.RT-PCR法检测SOCS-1和MCP-1mRNA的表达.结果 高糖刺激系膜细胞SOCS-1蛋白和mRNA表达呈时间依赖性变化,4 h表达达到峰值,然后逐渐减低,24 h达基线水平.与低糖组相比,高糖组系膜细胞STAT1和STAT3磷酸化水平显著上调（P＜0.01）;MCP-1 mRNA水平表达显著上调（0.39±0.05）比（0.16±0.02）,P＜0.01];上清液中MCP-1[（459±67）比（241±19）ng/L]、FN[（5.84±0.61）比（3.41±0.31）mg/L]和Ⅳ型胶原[（16.45±2.30）比（9.56±1.52）μg/L]含量均显著增加（均P＜0.01）.与空载体对照组相比,SOCS-1过表达组系膜细胞STAT1和STAT3的磷酸化水平显著下降（P＜0.05）;MCP-1 mRNA表达下调[（0.34±0.04）比（0.42±0.05）,P＜0.05];上清液中MCP-1[（387±47）比（463±56）ng/L]、FN[（4.61±0.57）比（5.76±0.74）mg/L]和Ⅳ型胶原[（13.4±2.32）比（17.1±2.57）μg/L]含量显著减少（均P＜0.05）.与高糖组相比,AG490组系膜细胞MCP-1 mRNA（0.31±0.04）表达显著下调;上清液中MCP-1[（361±53）ng/L]、FN[（5.46±0.71）mg/L]和Ⅳ型胶原[（15.2±1.97）μg/L]含量均减少.结论 SOCS-1过表达抑制高糖状态下肾小球系膜细胞MCP-1及细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现.

Objective To investigate the effect of suppressor of cytokine signaling-1 （SOCS-1）on expression of monocyte chemoattractant protein-1 （MCP-1）in human glomerular mesangial cells （HMCs） under high concentration of glucose. Methods Stable transfections of HMC with pCR3.1 vector and pCR3. 1-SOCS-1 were performed with hpofectamine 2000, and cells were selected with geneticin. Cells were stimulated with low glucose （LG, 5.5 mmol/L）, high glucose （HG, 30 mmol/L）, LG plus mannitol （24.5 mmol/L） and AG490 （10 μmol/L）. The protein expression levels of SOCS-1, signal transducer and activators of transcription 1,3 （STAT1, STAT3）,p-STAT1 and p-STAT3 were observed by Western blotting. The protein synthesis of MCP-1, FN and type Ⅳ collagen in the supernatants of the HMCs were detected by ELISA and radioimmunoassay. The expression level of SOCS-1 and MCP-1 mRNA was measured by BT-PCR.Results HG induced the expression of SOCS-1 protein and mRNA in HMCs in time-dependant manner, peaked at 4 h, and gradually decreased to baseline at 24 h. Compared with low glucose control group, the phosphorylation levels of STAT1 and STAT3 and the expression of MCP-1 mRNA [（0.39±0.05） vs （0.16±0.02）]were significantly increased in HMCs under high glucose medium （P 〈0.01 ）. Exposure of HMCs to high glucose conditions showed high concentration of MCP-1 [（459±67） ng/L vs （241±19） ng/L], fibronectin [（5.84±0.61） mg/L vs （3.41±0.31） mg/L]and type Ⅳ collagen [（16.45±2.30） μg/L vs （9.56±1.52） μg/L]in the supernatants （all P〈0.01）.Overexpression of SOCS-1 inhibited the phosphorylation levels of STAT1 and STAT3 and the expression of MCP-1 mRNA [（0.34±0.04） vs （0.42±0.05）]in HMCs under high glucose condition （all P〈0.05）. Compared with vector control group, the concentration of MCP-1 [（387±47） ng/L vs （463±56） ng/L], fibronectin [（4.61±0.57） mg/L vs （5.76±0.74） mg/L]and type Ⅳ collagen [（13.4±2.32） μg/L vs （17.1±2.57） μg/L]was decreased in supernatants of HMCs with SOCS-1 overexpression （all P〈0.05）. Compared with HG group, the expression of MCP-1 mRNA （0.31±0.04） and the concentration of MCP-1 [（361±53） ng/L], FN [（5.46±0.71） mg/L]and type Ⅳ collagen [（15.2±1.97） μg/L]in supernatants were decreased in HMCs treated with AG490.Conclusion Overexpression of SOCS-1 inhibits overproduction of MCP-1 and ECM proteins in HMCs under high glucose conditions, which may be partly by regulating the phosphoryhtion of STAT1 and STAT3.