基于核因子红细胞系 2 相关因子 2(nuclear factor erythroid-2-related factor 2，Nrf2)/血红素加氧酶-1(heme oxygenase-1，HO-1)/谷胱甘肽过氧化酶4(glutathione peroxidase 4，GPX4)信号通路观察黄芪甲苷(astragaloside Ⅳ，AS-Ⅳ)对ApoE-/-小鼠动脉粥样硬化的干预作用，探究AS-Ⅳ改善动脉粥样硬化小鼠铁死亡的潜在机制。该研究通过采用高脂饲料喂养建立动脉粥样硬化小鼠模型。造模8周后将ApoE-/-小鼠随机分为模型(model)组、AS-Ⅳ组、AS-Ⅳ+Nrf2抑制剂(ML385)组和ferrostatin-1(Fer-1)组，同时另设对照(control)组，分别给予相应药物腹腔注射治疗，control组与model组进行等量生理盐水腹腔注射。实验结束后采用全自动血脂分析仪检测血清生化水平，HE染色观察主动脉窦组织形态学变化，比色法检测各组小鼠血清中Fe2+、丙二醛(malondialdehyde，MDA)、谷胱甘肽(glutathione，GSH)、超氧化物歧化酶(superoxide dismutase，SOD)水平，免疫荧光观察各组小鼠主动脉窦铁蛋白重链1(ferritin heavy chain 1，FTH1)、铁蛋白轻链(ferritin light chain，FTL)蛋白表达，Western blot法检测小鼠主动脉脏组织Nrf2、HO-1、GPX4蛋白水平，透射电镜观察主动脉组织超微结构改变。结果显示，与control组比较，model组小鼠主动脉窦钙化面积及斑块沉积面积明显，线粒体膜密度增加、线粒体嵴减少或消失，血清总胆固醇(total cholesterol，TC)、甘油三酯(triglyceride，TG)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol，LDL-C)、Fe2+、MDA 水平升高，高密度脂蛋白胆固醇(high density lipoprotein cholesterol，HDL-C)、SOD、GSH 水平下降，主动脉 Nrf2、HO-1、GPX4 蛋白及铁储存蛋白FTH1、FTL表达明显抑制;与model组相比，AS-Ⅳ治疗后血清TC、TG、LDL-C、Fe2+、MDA水平下降，HDL-C、SOD、GSH水平上升，主动脉Nrf2、HO-1、GPX4蛋白及铁储存蛋白FTH1、FTL蛋白表达增加，主动脉组织形态学明显改善;与AS-Ⅳ组相比，Nrf2抑制剂ML385能逆转AS-Ⅳ对AS小鼠的治疗作用。以上表明，AS-Ⅳ可抑制铁死亡改善ApoE-/-小鼠动脉粥样硬化，其作用机制可能与调控Nrf2/HO-1/GPX4信号通路有关。
Mechanism of astragaloside Ⅳ modulation of Nrf2/HO-1/GPX4 pathway to inhibit ferroptosis and ameliorate atherosclerosis in ApoE-/-mice
The intervention effect of astragaloside Ⅳ(AS-Ⅳ)on atherosclerosis in apolipoprotein E gene knockout(ApoE)-/-mice was observed based on the nuclear factor erythroid-2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4)signaling pathway to explore the potential mechanism of AS-Ⅳ in improving ferroptosis in atherosclerotic mice.This study established an atherosclerosis mouse model by feeding them a high-fat diet.After modeling for 8 weeks,ApoE-/-mice were randomly divided into the model group,AS-Ⅳ group,AS-Ⅳ+Nrf2 inhibitor(ML385)group,and ferrostatin-1(Fer-1)group.Additionally,a blank control group was also established.Corresponding drugs were administered via intraperitoneal injection,with the control group receiving an equivalent amount of normal saline injection as the model group.After the experiment,serum biochemical levels were measured using an automatic blood lipid analyzer,hematoxylin-eosin(HE)staining was used to observe morphological changes in aortic sinus tissues,colorimetric methods were used to detect levels of ferrous ion(Fe2+),malondialdehyde(MDA),glutathione(GSH),and superoxide dismutase(SOD)in mouse serum,immunofluorescence was used to observe the expressions of ferritin heavy chain 1(FTH1)and ferritin light chain(FTL)proteins in the aortic sinus of mice,Western blot was used to detect the protein levels of Nrf2,HO-1,and GPX4 in mouse aortic tissues,and transmission electron microscopy was used to observe ultrastructural changes in aortic tissues.Results showed that compared to the control group,the model group of mice had significantly increased calcification and plaque deposition areas in the aortic sinus,increased mitochondrial membrane density,decreased or disappeared mitochondrial cristae,elevated levels of total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),Fe2+,and MDA,decreased levels of high-density lipoprotein cholesterol(HDL-C),SOD,and GSH,and significant inhibition of Nrf2,HO-1,GPX4 proteins,as well as iron storage proteins FTH1 and FTL expressions in the aorta.Compared to the model group,AS-Ⅳ treatment resulted in decreased serum TC,TG,LDL-C,Fe2+,and MDA levels,increased HDL-C,SOD,and GSH levels,increased expressions of Nrf2,HO-1,and GPX4 proteins,and iron storage proteins FTH1 and FTL,and significant improvement in aortic tissue morphology.Compared to the AS-Ⅳ group,the Nrf2 inhibitor ML385 could reverse the therapeutic effect of AS-Ⅳ on atherosclerosis mice.These findings suggest that AS-Ⅳ can inhibit ferroptosis and improve atherosclerosis in ApoE-/-mice,and its mechanism of action may be related to the regulation of the Nrf2/HO-1/GPX4 signaling pathway.
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