基于Keap1/Nrf2/ARE通路探讨槲皮素对脂多糖诱导的急性肾损伤大鼠铁死亡影响  

Quercetin Attenuates Ferroptosis Against LPS-induced Acute Kidney Injury Rats via Modulating Keap1/Nrf2/ARE Pathway

作　　者：杨昊若 于大君[2] 张昱[2] 杨斌[2] YANG Haoruo;YU Dajun;ZHANG Yu;YANG Bin(Tianjin University of Chinese Medicine,Tianjin 301617,China;Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)

机构地区：[1]天津中医药大学,天津301617 [2]中国中医科学院西苑医院,北京100091

出　　处：《中国实验方剂学杂志》2025年第5期65-75,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81873300);中国中医科学院科技创新工程项目(CI2021A01207)。

摘　　要：目的:基于Kelch样环氧氯丙烷相关蛋白1(Keap1)/核因子E_(2)相关因子2(Nrf2)/抗氧化反应元件(ARE)通路,探讨槲皮素对脂多糖(LPS)诱导急性肾损伤(AKI)大鼠铁死亡的影响及治疗作用。方法:60只雄性SD大鼠随机分为正常组、模型组、槲皮素高、低(100、10 mg·kg^(-1))剂量组、铁死亡抑制剂(FER1)组(Ferrostatin 1,5 mg·kg^(-1))、槲皮素高剂量+Nrf2抑制剂(ML385)组(ML385,30 mg·kg^(-1))。除正常组外各组通过腹腔注射脂多糖(LPS,10 mg·kg^(-1))制备AKI大鼠模型。造模成功后,各给药组相应剂量药物干预,正常组、模型组给予等量生理盐水,干预周期为3周。生化检测血清肌酐(SCr)、尿素氮(BUN)水平,考察大鼠肾功能;酶联免疫吸附测定法(ELISA)检测血清肿瘤坏死因子-α(TNF-α)含量及白细胞介素(IL)-1β、IL-6;检测肾组织Fe^(2+)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平;苏木素-伊红(HE)染色、马松染色(Masson)及高碘酸希夫染色(PAS)法观察肾组织病理形态变化;透射电子显微镜观察线粒体形态变化;免疫荧光法(IF)检测肾组织活性氧(ROS)水平;免疫组化法(IHC)及实时荧光定量聚合酶链式反应(Real-time PCR)检测Keap1、Nrf2、血红素加氧酶-1(HO-1)、谷胱甘肽过氧化物酶4(GPX4)、转铁蛋白受体(TFR1)、肾损伤分子-1(KIM-1)蛋白及mRNA表达。结果:与正常组比较,模型组大鼠血清SCr、BUN、TNF-α、IL-1β、IL-6,肾组织Fe^(2+)、MDA含量均显著升高,SOD、GSH含量显著降低(P<0.01);大鼠肾组织病理损伤严重;线粒体铁死亡特征性损伤明显且数量减少;肾组织ROS水平明显上升;肾组织中Keap1、TFR1、KIM-1蛋白及mRNA表达显著升高,Nrf2、HO-1、GPX4表达显著降低(P<0.01)。与模型组比较,槲皮素各剂量组及FER1组血清SCr、BUN、TNF-α、IL-1β、IL-6,肾组织Fe^(2+)、MDA水平呈现不同程度的降低,SOD、GSH含量明显增强(P<0.05);大鼠肾组织病理损伤得到明显缓解;线粒�Objective:To investigate the effect and therapeutic role of quercetin on ferroptosis in lipopolysaccharide(LPS)-induced acute kidney injury(AKI)rats based on the Kelch-like epichlorohydrin-related protein-1(Keap1)/nuclear factor erythroid-2-related factor 2(Nrf2)/antioxidant response element(ARE)pathway.Methods:Sixty male SD rats were randomly divided into normal group,model group,quercetin high-dose(100 mg·kg^(-1))and low-dose(10 mg·kg^(-1))groups,ferroptosis inhibitor Ferrostatin 1(FER1)group(5 mg·kg^(-1)),and quercetin high-dose+Nrf2 inhibitor group(ML385,30 mg·kg^(-1)).Except for the normal group,the AKI rat model was established in each group by intraperitoneal injection of LPS(10 mg·kg^(-1)).Following successful modeling,each treatment group received the corresponding dose of drug intervention,while the normal and model groups were administered an equal volume of normal saline.The intervention lasted for 3 weeks.Serum creatinine(SCr)and blood urea nitrogen(BUN)levels were measured biochemically to assess renal function.Serum tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βand IL-6 levels were detected by enzyme-linked immunosorbent assay(ELISA).The levels of Fe^(2+),malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)in renal tissue were detected.Hematoxylin-eosin(HE),Masson,and periodic acid-Schiff(PAS)staining were employed to observe pathological morphological changes in renal tissue.Mitochondrial morphological changes were observed using transmission electron microscopy.Reactive oxygen species(ROS)levels in renal tissue were detected by immunofluorescence(IF).The protein and mRNA expression levels of Keap1,Nrf2,heme oxygenase-1(HO-1),glutathione peroxidase 4(GPX4),transferrin receptor(TFR1),and kidney injury molecule-1(KIM-1)were assessed by immunohistochemistry(IHC)and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Results:Compared with the normal group,the model group exhibited significantly elevated serum levels of SCr,BUN,TNF-α,IL-1β,IL-6,Fe

关 键 词：急性肾损伤 槲皮素 铁死亡 Kelch样环氧氯丙烷相关蛋白1(Keap1)/核因子E_(2)相关因子2(Nrf2)/抗氧化反应元件(ARE)通路 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R287