冠心康通过激活ERK5/Nrf2通路对ox-LDL和LPS诱导的巨噬细胞铁死亡的影响

Effect of Guanxinkang on ox-LDL and LPS induced ferroptosis in macrophages by acti⁃vating ERK5/Nrf2 pathway

目的 研究冠心康对脂多糖(LPS)和氧化低密度脂蛋白(ox-LDL)干预的巨噬细胞铁死亡的影响及其抗动脉粥样硬化的作用机制。方法 采用ox-LDL联合LPS诱导RAW264.7巨噬细胞铁死亡并给予冠心康和ERK5抑制剂XMD8-92进行干预,检测铁死亡相关指标脂质过氧化物(LPO)含量、胞内二价铁离子(Fe^(2+))含量、活性氧(ROS)及谷胱甘肽(GSH)水平,IL-6、TNF-α、MMP含量及ERK5/Nrf2信号通路mRNA及蛋白的表达。结果 LPS联合ox-LDL诱导的RAW264.7巨噬细胞LPO含量、胞内Fe^(2+)含量及ROS水平升高,GSH含量减少,IL-6、TNF-α、MMP-2、MMP-9含量显著升高,ERK5、Nrf2、HO-1、GPX-4的mRNA及P-ERK5、P-Nrf2、HO-1的蛋白表达水平降低(P<0.05)。冠心康组LPO、Fe^(2+)、ROS降低,GSH含量增加,IL-6、TNF-α、MMP-2、MMP-9水平下调,ERK5、Nrf2、HO-1、GPX-4的mRNA及P-ERK5、P-Nrf2、HO-1的蛋白表达水平升高,而XMD8-92可以抑制冠心康这一作用(P<0.05)。结论 冠心康可以抑制LPS联合ox-LDL诱导的RAW264.7巨噬细胞的铁死亡,减轻炎症反应,其机制与活化ERK5及下游Nrf2通路有关。

Objective To investigate the effect of Guanxinkang on lipopolysaccharide(LPS)and oxidized low-density lipopro⁃tein(ox-LDL)induced ferroptosis in macrophages and its anti-atherosclerosis mechanism.Methods ox-LDL combined with LPS was used to induce iron death in RAW264.7 macrophages,and Guanxinkang and ERK5 inhibitor XMD8-92 were adminis⁃tered to intervene,and the levels of lipid peroxides(LPO),intracellular ferric ion(Fe^(2+)),reactive oxygen species(ROS)and glutathione(GSH)related indexes of iron death were detected.IL-6,TNF-α,MMP and ERK5/Nrf2 signaling pathway mRNA and protein expression.Results In RAW264.7 macrophages,LPO content,intracellular Fe^(2+)content and ROS level increased,GSH content decreased,IL-6,TNF-α,MMP-2 and MMP-9 contents increased significantly after LPS combined with ox-LDL induction.mRNA levels of ERK5,Nrf2,HO-1 and GPX-4 and protein expression levels of P-erk5,p-nrF2 and HO-1 were decreased(P<0.05).In Guanxinkang group,LPO,Fe^(2+)and ROS were decreased,GSH content was increased,IL-6,TNF-α,MMP-2 and MMP-9 levels were decreased,ERK5,Nrf2,HO-1 and GPX-4 mRNA and p-ERK5,p-Nrf2 and HO-1 protein expression levels were increased.XMD8-92 could inhibit the effect of Guanxinkang(P<0.05).Conclusion Guanxinkang can inhibit the ferroptosis of RAW264.7 macrophages induced by LPS and ox-LDL,and reduce the inflammatory response.The mechanism is related to the activation of ERK5 and downstream Nrf2 pathway.