炎症因子C5a和肿瘤坏死因子α刺激对小鼠主动脉内皮细胞衰老与代谢的差异化网络调控作用

Mechanism of Differential Protein Network in Senescence,Angiogenesis and Related Metabolisms of Mouse Aortic Endothelial cells Stimulated by C5a and Tumor Necrosis Factorα

目的探讨炎症因子C5a和肿瘤坏死因子α(TNF-α)对内皮细胞代谢、衰老和功能行为的调控网络。方法体外培养小鼠胸主动脉内皮细胞,分别给予C5a(100 ng/ml)和TNF-α(30 ng/ml)刺激12 h,收集细胞和培养基上清,进行蛋白质组学质谱检测,提取细胞mRNA进行实时定量逆转录PCR(RT-qPCR)检测特异基因的表达情况,利用生物信息学方法对蛋白质组学检测结果进行细胞组分(GO)和京都基因和基因组百科全书(KEGG)分析。结果GO分析结果显示,体外培养的小鼠胸主动脉内皮细胞在TNF-α刺激下,以血管紧张素4(ANGPT4)、Ⅳ型胶原α2链(COL4A2)为主的血管生成功能相关蛋白的表达水平降低,以血管细胞黏附分子-1(VCAM1)、趋化因子配体2(CCL2)为主的衰老相关蛋白表达水平升高,且血管生成与衰老作用通过CCL2和ENG相互作用,展现出一定的相关性;C5a刺激引起体外培养的小鼠胸主动脉内皮细胞中与血管生成功能相关的ANGPT4、COL4A2等蛋白表达水平下降,然并未影响衰老相关蛋白的表达。经过KEGG分析,两种炎症刺激展现出不同的代谢特征,TNF-α调控丙氨酸、天冬氨酸和谷氨酸等参与的糖代谢和脂肪代谢过程,并通过PI3K-Akt、缺氧诱导因子1(HIF-1)等信号通路分别调控细胞凋亡和细胞增殖;而C5a调节钙重吸收的代谢过程,通过PI3K-Akt信号通路、蛋白质消化吸收等调控细胞增殖。体外实验验证在TNF-α刺激下,小鼠胸主动脉内皮细胞Vcam1和Ccl2的mRNA水平增加,Angpt4和Col4a2 mRNA水平降低;C5a刺激也下调Angpt4和Col4a2 mRNA水平,与蛋白基因芯片结果一致。结论TNF-α通过糖酵解等代谢途径和HIF-1等信号通路参与血管生成和细胞衰老,C5a参与血管生成过程,但参与的细胞代谢相关的途径较少。本研究提示不同的炎症因子在内皮细胞衰老与代谢方面存在明显的功能差异。

Objective To investigat the regulatory network of inflammatory factors tumor necrosis factorα(TNF-α)and C5 a on metabolisms,aging and functions of endothelial cells.Methods Mouse thoracic aortic endothelial cells were cultured in vitro and stimulated with C5 a(100 ng/ml)and TNF-α(30 ng/ml)for 12 hours,respectively.The cells and supernatant were collected and detected by protein omics mass spectrometry.The results were analyzed by gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG).The mRNA was extracted and the expression of specific genes was detected by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).Results Through GO analysis,we found that the expressions of angiogenesis-related proteins(mainly angiopoietin-4[ANGPT4]and collagen typeⅣα2[COL4 A2])were decreased while the expressions of aging-related proteins,(mainly vascular cell adhesion molecule-1[VCAM1]and chemokines ligand 2[CCL2])were increased in endothelial cells stimulated by TNF-α.The relationship between angiogenesis and aging was shown by interaction between CCL2 and ENG.C5 a stimulation down-regulated angiogenesis related proteins expression levels in endothelial cells(mainly ANGPT4 and COL4 A2),but did not affect the expressions of aging related proteins.According to KEGG analysis,the two inflammatory factors showed different metabolic characteristics.TNF-αregulated glucose metabolism and fat metabolism involving alanine,aspartic acid and glutamic acid,and regulated apoptosis and cell proliferation through PI3 K-Akt signal pathway and hypoxia-inducible factor-1(HIF-1)signal pathway respectively.C5 a regulated the metabolic process of calcium reabsorption,and regulated cell proliferation through PI3 K-Akt signal pathway and protein digestion and absorption.In vitro experiments verified that under the stimulation of TNF-α,the mRNA levels of Vcam1 and Ccl2 in mouse thoracic aortic endothelial cells increased,and the levels of Angpt4 and Col4 a2 mRNA decreased.C5 a stimulation also down-regulated the levels of Angpt4 and Col4 a2 mRNA,which was consistent with the results of protein gene chip.Conclusion In cultured aortic endothelial cells,TNF-αregulated angiogenesis and cell aging through glycolysis and HIF-1 signal pathways.C5 a was involved in angiogenesis.This study suggested that different inflammatory factors function differently in endothelial cell aging and metabolisms.