恩格列净通过抑制EGFR信号通路减轻ox-LDL诱导的人脐静脉内皮细胞损伤

Empagliflozin alleviates ox-LDL-induced injury of human umbilical vein endothelial cells by inhibiting the EGFR signaling pathway

摘要:

目的]研究恩格列净修复氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)损伤的作用机制。 [方法]体外培养原代HUVEC；用ox-LDL诱导HUVEC损伤,CCK-8法测定细胞存活率；EDU法检测细胞增殖；Western blot法检测HUVEC中Ki-67、Bcl-2、cleaved Caspase-3、Bax、内皮型一氧化氮合酶(eNOS)、细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)和表皮生长因子受体(EGFR)的蛋白表达水平；RT-qPCR检测HUVEC中白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的mRNA表达水平；利用Swiss targets、Gene Cards databases、GO和KEGG、蛋白质网络互作分析(PPI)、分子对接(Click Docking,https://mcule.com/apps/1-click-docking/)进行恩格列净作用靶标预测。 [结果]CCK-8结果表明,0.025 μmol/L恩格列净可明显减轻ox-LDL诱导的HUVEC损伤(P＜0.01)。EDU结果表明,ox-LDL处理HUVEC 24 h后,细胞增殖受到明显抑制(P＜0.01),而加入恩格列净后可明显缓解细胞增殖抑制情况(P＜0.01)。Western blot结果显示,HUVEC经ox-LDL处理后,Ki-67、Bcl-2和eNOS的蛋白表达水平显著减少、cleaved Caspase-3、Bax、ICAM-1和VCAM-1的蛋白表达水平明显增加(P＜0.05)；加入恩格列净后,上述蛋白表达趋势被逆转(P＜0.05)；RT-qPCR结果表明,HUVEC经ox-LDL处理后,IL-6和TNF-α的表达水平上升(P＜0.01),加入恩格列净后,IL-6和TNF-α的表达水平有所下降(P＜0.05)；而加入EGFR激动剂NSC 228155后,恩格列净拮抗ox-LDL介导的内皮细胞损伤作用被明显逆转(P＜0.05)。 [结论]恩格列净可显著减轻ox-LDL所致的HUVEC损伤,该作用机制可能涉及EGFR信号通路。

Abstract:

Aim To investigate the protective and reparative effect of empagliflozin on oxidized-low density lipoprotein (ox-LDL) -induced injury in human umbilical vein endothelial cells (HUVEC) and its mechanism of action. Methods Primary HUVEC were cultured in vitro. ox-LDL was used to induce HUVEC injury model, and the cell survival rate was measured by CCK-8 assay. EDU method was used to detect cell proliferation. Western blot was used to detect the protein levels of Ki-67, Bcl-2, cleaved Caspase-3, Bax, endothelial nitric oxide synthase (eNOS), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and epidermal growth factor receptor (EGFR) in HUVEC. RT-qPCR was used to detect the mRNA expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in HUVEC. Using Swiss targets, GeneCards databases, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG), protein-protein interaction (PPI) network analysis,and ClickDocking (https://mcule.com/apps/1-click-docking/) to predict the target of empagliflozin. Results CCK-8 results showed that 0.025 μmol/L empagliflozin significantly alleviated ox-LDL-induced HUVEC injury (P＜0.01). EDU results showed that ox-LDL treatment for 24 h significantly inhibited the proliferation of HUVEC (P＜0.01), while empagliflozin treatment significantly alleviated the inhibition of cell proliferation (P＜0.01). The results of Western blot showed ox-LDL treatment significantly decreased the protein expression levels of Ki-67, Bcl-2, and eNOS, and increased the protein expression levels of cleaved Caspase-3, Bax, ICAM-1, and VCAM-1 in HUVEC (all P＜0.05). However, empagliflozin treatment reversed these changes (all P＜0.05). RT-qPCR results showed that ox-LDL treatment increased the mRNA expression levels of IL-6 and TNF-α in HUVEC (P＜0.01), while empagliflozin treatment decreased their expression levels (P＜0.05). However, after adding EGFR agonist NSC 228155, the protective effect of empagliflozin against ox-LDL-mediated HUVEC injury was significantly reversed(P＜0.05). Conclusion Empagliflozin can significantly reduce ox-LDL-induced HUVEC injury, which may be related to EGFR signaling pathway.