JAK/STAT信号介导高糖诱导系膜细胞转化生长因子β_1及其基质蛋白的分泌

JAK/STAT SIGNALING IN HIGH GLUCOSE-INDUCED SECRETION OF TRANSFORMING GROWTH FACTOR-β_1 AND EXTRACELLULAR MATRIX PROTEINS IN MESANGIAL CELLS

目的观察高糖和AG490对大鼠肾小球系膜细胞Janus激酶/信号转导和转录活化因子(Januskinase/signal transducer and activators of transcription,JAK/STAT)信号激活以及对系膜细胞转化生长因子β1(transforming growth factor-β1,TGF-β1)和细胞外基质蛋白表达的影响。方法体外培养大鼠肾小球系膜细胞,分别给予高糖和AG490干预,采用免疫沉淀和Western印迹检测JAK2磷酸化表达,Western印迹检测信号蛋白STAT1、STAT3、磷酸化-STAT1(phospho-STAT1,p-STAT1)和p-STAT3表达;酶联免疫吸附实验(enzyme-linkedimmunoadsorbent assay,ELISA)和放射免疫法测定细胞上清液中TGF-β1、纤维连接蛋白(fibronectin,FN)和IV型胶原的分泌,逆转录-聚合酶链反应(reverse transcription and polymerase chain reaction,RT-PCR)检测TGF-β1mRNA表达。结果与低糖对照组比较,高糖培养的系膜细胞JAK2、p-STAT1和p-STAT3表达明显上调,TGF-β1、FN和IV型胶原分泌增加,TGF-β1mRNA表达增加;AG490能够明显抑制高糖培养系膜细胞JAK2的磷酸化,下调p-STAT1和p-STAT3表达,明显抑制TGF-β1、FN和Ⅳ型胶原的分泌,同时降低TGF-β1mRNA表达。结论JAK/STAT信号途径参与高糖诱导的肾小球系膜细胞TGF-β1和细胞外基质的分泌。

Objective To investigate the effect of blockade of JAK/STAT signaling pathway on the secretion of transforming growth factor-β1 （TGF-β1） and extracellular matrix proteins in glomerular mesangial cells（GMCs） under high concentration of glucose. Methods By using the high concentration of glucose and AG490 to stimulate the cultured rat GMCs in vitro. Immunoprecipitation and Western blot analysis were used to determine tryosine phosphorylated Janus kinase 2（JAK2）. The protein expression of STAT1 and STAT3,p-STAT1 and p-STAT3 were observed by Western blot. The protein synthesis of TGF-β1, fibronectin and type Ⅳ collagen in the supernatants of the GMCs were detected by enzyme-linked immunoadsorbent assay （ELISA） and radioimmunoassay. TGF-β1 mRNA were measured by reverse transcription and polymerase chain reaction （RT-PCR）. Results Compared with low glucose control group, the expression of JAK2, p-STAT1, p-STAT3 and TGF-β1 mRNA was significantly increased in GMCs under high glucose medium and the high levels of TGF-β1, fibronectin and collagen Ⅳ were observed in the supernatants. The expression of JAK2, p-STAT1, p-STAT3 and TGF-β1 mRNA and the levels of TGF-β1, extracellular matrix（ECM） protein in the supernatants in AG490 group were obviously lower than those of the high glucose control group. Conclusion Blockade of JAK/STAT signaling pathway may be involved in the high glucoseinduced overproduction of TGF-β1 and ECM proteins in GMCs.