高磷诱导内皮细胞凋亡的代谢组学以及MAPK通路研究

Metabolomics and MAPK pathway in endothelial cell apoptosis induced by high inorganic phosphate

摘要:

目的研究高磷对血管内皮细胞代谢影响并观察MAPK通路变化情况。方法体外培养人脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVEC）24 h,分为正常浓度组（1.0 mmol/L）、高磷浓度组（3.0 mmol/L）。通过气相色谱/质谱（gas chromatography/mass spectrometry,GC/MS）方法分析代谢物图谱,并采用正交信号校正和偏最小二乘判别分析方法（OSC-PLS-DA）。根据OSC-PLS-DA模型的变量权重（variable importance for projection,VIP）〉1及显著性差异检验结果筛选出差异性代谢物。同时用AnnexinV-FITC/PI双染法检测细胞凋亡率;Western印记法检测MAPK通路ERK1/2、p-ERK1/2、p38 M APK、p-p38 M APK、JNK、p-JNK蛋白表达。结果鉴别出16个显著差异物质,涉及碳水化合物、氨基酸以及脂质代谢通路紊乱;与1.0 mmol/L Pi组相比,3.0 mmol/L Pi组细胞凋亡率明显升高（P〈0.05）,p-ERK1/2蛋白表达明显升高（P〈0.05）,p-p38 M APK蛋白表达明显下降（P〈0.05）,ERK1/2、p38 M APK、JNK和p-JNK蛋白表达无明显改变。结论高磷诱导内皮细胞凋亡是通过上调p-ERK信号通路及下调p-p38MAPK信号通路的活性来实现的。

Abstract:

Objective To investigate the metabolomics and MAPK pathway in apoptosis of endothelial cells induced by high inorganic phosphate. Methods Human umbilical vein endothelial cells （HUVECs） were treated with 1.0 or 3.0 nmol/L phosphate for 24 h, respectively. Gas- chromatography/mass-chromatography （GC/MS） was applied to analyze metabolic spectrum of HUVECs. Orthogonal signal correction-partial least square discriminate analysis （OSC-PLS-DA） was used to process the metabolic data. Metabolites with variable importance for projection （VIP） values exceeding 1 and P〈0.05 were generally selected as potential identified metabolites. Endothelial cellapoptosis rate was measured by flowcytometry with AnnexinV-HTC/PI staining. The expression of ERK1/2, p-ERK1/2, p38MAPK, p-p38MAPK, JNK, p-JNK proteins was detected by Western blotting. Results Sixteen distincted metabolites were identified involving carbohydrate, amino acid and lipid metabolic pathway. Compared with 1.0 mmol/L phosphate group, the apoptosis rate in HUVECs cultured with 3.0 mmol/L phophate was significantly increased （P〈0.05 ） meanwhile the expression of p-ERK1/2 protein was increased and the expression of p-p38MAPK was decreased （both P〈0.05 ）. The expression of ERK, p38MAPK, JNK and p-JNK proteins were not changed after treatment of phosphate. Conclusion High concentration of inorganic phosphate can induce apoptosis of human umbilical vein endothelial cells with distinct metabolomic changes, which is associated with up-regulation of ERK signaling pathway and down-regulation of p38MAPK signaling pathway.