三七皂苷R_(1)调控Nrf2介导的铁死亡途径改善ApoE^(−/−)小鼠动脉粥样硬化

Notoginsenoside R_(1) improves atherosclerosis in ApoE^(−/−)mice by regulating ferroptosis mediated by Nrf2 signaling pathway

目的探究三七皂苷R_(1)对高脂诱发的载脂蛋白E基因敲除(ApoE^(−/−))小鼠动脉粥样硬化(atherosclerosis,AS)的治疗作用及机制。方法高脂喂养雄性ApoE^(−/−)小鼠12周建立AS模型,将其随机分为模型组及三七皂苷R_(1)低、高剂量(25、35 mg/kg)组和铁死亡抑制剂Ferrostatin-1(Fer-1,1 mg/kg)组。以相同周龄的雄性C57BL/6J小鼠作为对照组,给予普通饲料饲养。给药5周后采用全自动生化分析仪检测血清总胆固醇(total cholesterol,TC)、三酰甘油(triglycerides,TG)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)和高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)水平;苏木素-伊红(HE)染色和油红O染色观察主动脉粥样斑块程度;加强普鲁士蓝染色法评估斑块铁沉积;生化试剂盒检测主动脉超氧化物歧化酶(superoxidedismutase,SOD)活力及谷胱甘肽(glutathione,GSH)、丙二醛(malondialdehyde,MDA)水平;免疫组化法检测主动脉4-羟基壬烯醛(4-hydroxynonenal,4-HNE)表达;Western blotting检测主动脉核因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)及4-HNE蛋白表达;qRT-PCR法检测主动脉Nrf2、Gpx4、前列腺素内过氧化物合酶2(prostaglandin-endoperoxide synthase 2,Ptgs2)mRNA表达。结果三七皂苷R_(1)低、高剂量组及Fer-1组不同程度地影响ApoE^(−/−)小鼠的血脂水平(P<0.05、0.01),明显减轻主动脉粥样斑块程度,抑制铁沉积(P<0.01),提高SOD、GSH抗氧化能力(P<0.05、0.01),减少脂质过氧化MDA水平和4-HNE蛋白表达(P<0.05、0.01),上调铁死亡相关核Nrf2、SLC7A11、GPX4蛋白及Nrf2、Gpx4 mRNA的表达(P<0.01),下调Ptgs2 mRNA的表达(P<0.01),以三七皂苷R_(1)高剂量组效果最为显著。结论三七皂苷R_(1)改善ApoE^(−/−)小鼠脂代谢紊乱,抑制动脉粥样斑块形成,具有抗AS的作用,其机制可能与减少斑块铁沉积、激活Nrf2/SLC7A11/GPX4通路减少脂质过氧化,抑制铁死亡有关。

Objective To explore the therapeutic effect and mechanism of notoginsenoside R_(1)(NGR_(1))on ApoE^(−/−)mice with high fat diet induced atherosclerosis(AS).Methods Male ApoE^(−/−)mice were fed high-fat diet for 12 weeks to establish atherosclerotic model,then mice were randomly divided into model group,NGR_(1) low-and high dose(25,35 mg/kg)groups,Ferrostatin-1(Fer-1,1 mg/kg)group.Male C57BL/6J mice of the same week age were used as control group and were fed on a general diet.After five weeks of feeding,the levels of total cholesterol(TC),triglycerides(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)were measured by automatic biochemical analyzer;Hematoxylin-eosin(HE)and oil red O staining were used to observe the degree of aortic atherosclerotic plaque;Enhanced Prussian blue staining was used for the assessment of iron deposits in aortic plaques;The activity of super oxide dismutase(SOD)and levels of glutathione(GSH),malondialdehyde(MDA)in aortic tissues were determined by biochemical assay;The expression of 4-hydroxynonenal(4-HNE)protein in aorta was detected by immunohistochemistry;Western blotting was performed to determine the protein expressions of nuclear factor-erythroid 2-related factor 2(Nrf2),solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4)and 4-HNE in aortic plaques;qRT-PCR was performed to measure the mRNA expressions of Nrf2,Gpx4 and prostaglandin-endoperoxide synthase 2(Ptgs2).Results The blood lipid level of ApoE^(−/−)mice was affected to different degrees(P<0.05,0.01),the degree of atherosclerosis plaque in aortic plaques was significantly reduced,iron deposition was inhibited(P<0.01),the antioxidant capacities of SOD and GSH were improved(P<0.05,0.01),the level of lipid peroxidation MDA content and 4-HNE protein expression were reduced(P<0.05,0.01),the expressions of nuclear Nrf2,SLC7A11,GPX4 protein and Nrf2,Gpx4 mRNA associated with ferroptosis were increased(P<0.01),the expression of Ptgs2 mRNA was reduced(P<0.01)in NGR_(1) low-and high-dose groups and Fer-1 group.NGR_(1) high-dose group had more significant improvement effect on the above indexes.Conclusion NGR_(1) can improve lipid metabolism disorder in ApoE^(−/−)mice,reduce atherosclerotic plaque formation in ApoE^(−/−)mice,and has antiatherogenic effect.Its mechanism may be related to reducing iron deposition,activating Nrf2/SLC7A11/GPX4 pathway to reduce lipid peroxidation and inhibit ferroptosis.