重组人促红细胞生成素对3T3-L1脂肪细胞促红细胞生成素受体表达及下游信号通路的影响

Effects of recombinant human erythropoietin on expression of erythropoietin receptor and its downstream signaling pathway in 3T3-L1 adipocytes

目的探讨重组人促红细胞生成素(rHuEPO)对正常和胰岛素抵抗3T3-L1脂肪细胞促红细胞生成素受体(EPOR)表达及其下游信号通路的调控作用。方法以1μmol/L地塞米松诱导建立3T3-L1脂肪细胞胰岛素抵抗模型(模型组),以分化成熟的正常3T3-L1脂肪细胞作为对照组。采用细胞免疫荧光法和Western blotting法检测细胞EPOR的蛋白表达,采用Real-TimePCR技术检测细胞EPOR mRNA的表达。分别以rHuEPO、磷脂酰肌醇3激酶(PI3K)抑制剂(LY294002)及信号转导和转录激活因子5(STAT5)抑制剂对模型组和对照组细胞进行干预,采用Western blotting法检测不同药物干预对EPOR蛋白的表达及其下游分子丝(苏)氨酸蛋白激酶(Akt)和STAT5磷酸化水平(p-Akt和p-STAT5蛋白的表达)的影响。结果与对照组比较,模型组EPOR蛋白和mRNA的表达均显著下调(P<0.05)。干预实验结果显示:与相应对照组或模型组比较,rHuEPO+对照组或模型组EPOR、p-Akt和p-STAT5蛋白的相对表达量均显著上调(P<0.05);与相应rHuEPO+对照组或模型组比较,rHuEPO+LY29400或STAT5阻断剂+对照组或模型组的p-Akt和p-STAT5蛋白的相对表达量显著下调(P<0.05)。结论正常3T3-L1脂肪细胞上存在EPOR,胰岛素抵抗3T3-L1脂肪细胞EPOR蛋白和mRNA的表达下调,rHuEPO可通过EPOR介导激活PI3K-Akt和JAK2-STAT5信号通路。

Objective To investigate the effects of recombinant human erythropoietin（rHuEPO） on the expression of erythropoietin receptor（EPOR） and its downstream signaling pathway in normal and insulin-resistant 3T3-L1 adipocytes.Methods The model of insulin-resistant 3T3-L1 adipocytes were induced by 1 μmol/L dexamethasone（model group）,and the well-differentiated normal 3T3-L1 adipocytes were served as control group.The expression of EPOR protein in cells was detected by immunofluorescence staining and Western blotting,and the expression of EPOR mRNA was determined by Real-Time PCR.Cells in model group and control group were intervened with rHuEPO,phosphatidylinositol-3-kinase（PI3K） inhibitor（LY294002） and signal transducer and activator of transcription 5（STAT5）,and the effects of different interventions on the expression of EPOR protein and phosphorylation of downstream molecules of serine/threonine kinase（AKT） and STAT5（p-Akt and p-STAT5 protein） were examined by Western blotting.Results The expression of EPOR protein and mRNA in model group was significantly lower than that in control group（P0.05）.The intervention experiment revealed that the relative expression of EPOR,p-Akt and p-STAT5 protein in rHuEPO＋control group or model group was significantly higher than that in corresponding control group or model group（P0.05）,and the relative expression of p-Akt and p-STAT5 protein in rHuEPO＋LY29400 or STAT5 blocker＋control group or model group was significantly lower than that in corresponding rHuEPO＋control group or model group（P0.05）.Conclusion EPOR exists in normal 3T3-L1 adipocytes,the expression of EPOR protein and mRNA in insulin-resistant 3T3-L1 adipocytes decreases,and rHuEPO can activate PI3K-Akt and JAK2-STAT5 signaling pathway through EPOR mediation.