Exendin-4通过调节MAPK—NF—κB相关的炎症抑制高糖诱导肾小球系膜细胞的细胞外基质分泌

Secretion of extracellular matrix were inhibited by Exendin-4 treatment through MAPK-NF-κB related inflammation in high glucose-induced glomerular mesangial cells

目的研究Exendin-4对高糖诱导大鼠肾小球系膜细胞（GMCs）的细胞外基质分泌作用及相关机制。方法体外培养GMCs，分为4组：正常葡萄糖组（NG组）、正常葡萄糖＋Exendin-4组（NGE组）、高糖组（HG组）、高糖＋Exendin-4组（HGE组）。HGE组细胞分别予3、5、10、15、30 nmol/L的Exendin-4处理12、24、48 h；采用CCK8法测定细胞增殖；酶联免疫吸附法（ELISA）测定细胞上清中细胞外基质蛋白纤维连接蛋白（FN）和Ⅳ型胶原蛋白（Col-Ⅳ）的分泌水平；逆转录PCR（RT-PCR）检测FN、Col-Ⅳ及相关炎症介质肿瘤坏死因子-α（TNF-α）、单核细胞趋化蛋白1（MCP-1）、细胞间黏附因-1（ICAM-1）、转化生长因子-β1（TGF-β1）等mRNA的表达；Western印迹测定丝裂原活化蛋白激酶（MAPK）信号通路磷酸化及核转录因子-κB（NF-κB）、胰升糖素样肽1受体（GLP-1R）的表达。结果（1）高糖培养的GMCs给予10 nmol/L的Exendin-4处理24 h后，其细胞增殖率较3 nmol/L、5 nmol/L的Exendin-4处理组明显减低（均P〈0.05）；与15 nmol/L、30 nmol/L组相比，10 nmol/L的Exendin-4处理对GMCs增殖率无明显变化（均P〉0.05）。（2）与NG组比较，HG组GMCs中FN、Col-Ⅳ的分泌及基因表达量明显增多（均P〈0.05），相关炎症介质MCP-1、TNF-α、ICAM-1、TGF-β1 mRNA的表达显著升高（均P〈0.05）；Exendin-4处理后GMCs的FN、Col-Ⅳ的分泌明显减少，TNF-α、MCP-1、ICAM-1、TGF-β1 mRNA被明显抑制，表达量明显降低（均P〈0.05）。（3）与NG组比较，HG组GMCs中NF-κB水平、细胞外调节蛋白激酶（Erk1/2）、Jun氨基末端激酶（JNK）、p38MAPK的磷酸化水平明显增多（均P〈0.05），同时GLP-1R的表达明显下降（P〈0.05）；Exendin-4处理后NF-κB水平及Erk1/2、JNK磷酸化水平被抑制（均P〈0.05），GLP-1R的表达明显升高（P〈0.05），而p38MAPK磷酸化水平无明显变化（P〉0.05）。高糖培养的GMCs给予Erk1/2、JNK、NF-κB的特异性抑制剂PD98059、SP600125、Bay11-7082预孵育后，Exendin-4对FN、Col-Ⅳ的分泌抑制作用被解除。结论Exendin-4可以抑制高糖诱导GMCs的细胞外基质的分泌，其机制可能与抑制Erk1/2、JNK磷酸化及NF-κB介导的炎症反应有关。

ObjectiveTo explore the effects and related mechanism of Exendin-4 on secretion of extracellular matrix in high glucose-induced glomerular mesangial cells（GMCs）.MethodsGMCs were incubated in medium with glucose or Exendin-4 for the four groups： normal glucose group（NG group）： cells were treated in medium with 5.6 mmol/L glucose; NG with Exendin-4 treatment group（NGE group）： cells were treated with 5.6 mmol/L glucose and Exendin-4; high glucose group（HG group）： cells were cultured with 30 mmol/L glucose; HG with Exendin-4 treatment group（HGE group）： cells were treated with 30 mmol/L glucose and Exendin-4 at concentration of 3, 5, 10, 15, 30 nmol/L separately, which were cultured for 12, 24, 48 hours. GMCs treated with Exendin-4 were determined by assessing proliferation using a CCK8 method. The levels of fibronectin（FN）, collagen type Ⅳ（Col-Ⅳ）in the cell supernatant were measured using enzyme-linked immunosorbent assay（ELISA）. The gene levels of Col-Ⅳ, FN, and expression of inflammatory mediators including monocyte chemotactic protein 1（MCP-1）, tumor necrosis factor-α（TNF-α）, intercellular cell adhesion molecule-1（ICAM-1）, transforming growth factor-β1（TGF-β1）were evaluated using reverse transcription PCR（RT-PCR）; The expression of nuclear transcription factor-κB（NF-κB）, glucagon-like peptide-l receptor（GLP-1R）, and phosphorylation levels of mitogen-activated protein kinases（MAPKs）were evaluated by Western blot.Results（1）After treatment with 10 nmol/L Exendin-4 for 24 hour, the proliferation rate of GMCs was significantly decreased compared with 3 nmol/L, 5 nmol/L Exendin-4 treatment（P〈0.05）, while there was no difference compared with 15 nmol/L, 30 nmol/L Exendin-4 treatment（both P〉0.05）. （2）The gene expression of FN, Col-Ⅳ and the inflammatory mediators, MCP-1, TNF-α, ICAM-1, TGF-β1 in HG group were significantly increased compared with the NG group, （all P〈0.05）. After treatment with Exendin-4, the levels of FN, Col-Ⅳ and the gene expression of TNF-α, MCP-1, ICAM-1, TGF-β1 were decreased（all P〈0.05）. （3）Compared with NG group, the expression of NF-κB and the phosphorylation of extracellular regulated protein kinases（p-Erk1/2）, Jun N-terminal kinase（p-JNK）and, p38MAPK（p-p38MAPK）in the group of HG group were increased significantly, accompanied by the decrease of GLP-1R protein level（all P〈0.05）. Importantly, Exendin-4 treatment significantly reduced protein expression of p-Erk1/2, p-JNK, and NF-κB（all P〈0.05）, and the level of GLP-1R protein increased（P〈0.05）. Furthermore, specific Erk1/2, JNK or NF-κB inhibitors markedly blocked Exendin-4-mediated decrease in the levels of FN, Col-Ⅳ.Conclusion Exendin-4 treatment inhibits the secretion of extracellular matrix potentially through Erk1/2, JNK/NF-κB signaling in higher glucose induced GMCs.