摘要
目的:观察槲皮素对类风湿性关节炎的大鼠铜死亡的影响及治疗作用,并基于溶质载体家族31成员1(SLC31A1)/铁氧还蛋白1(FDX1)通路探究其可能机制.方法:60只雄性SD大鼠分为6组:正常组、模型组、槲皮素高、低剂量(150、50 mg·kg-1)组、铜死亡抑制剂(TTM)组(10 mg·kg-1)、甲氨蝶呤(2 mg·kg-1)组,每组10只.除正常组外,采用Ⅱ型胶原诱导法制备类风湿性关节炎模型(CIA)大鼠.造模成功后,各给药组按相应剂量药物进行干预,正常组、模型组使用等体积生理盐水灌胃,每天1次,干预周期为4周.观察大鼠足肿胀度并测定关节炎临床积分;酶联免疫吸附测定法(ELISA)检测大鼠血清类风湿因子(RF)、基质金属蛋白酶-3(MMP-3)、肿瘤坏死因子-α(TNF-α)及白细胞介素(IL)-1β、IL-10、铜蓝蛋白(Cp)水平;检测关节组织铜(Cu)、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)染色法观察关节组织病理形态变化;免疫荧光法(IF)检测关节组织活性氧(ROS)及二氢硫辛酸转乙酰酶(DLAT)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SLC31A1、FDX1、硫辛酸合成酶(LIAS)、热休克蛋白70(HSP70)、丙酮酸脱氢酶E1亚基β(PDHB)、铜转运P型ATP酶β(ATP7B)蛋白及mRNA表达.结果:与正常组比较,模型组大鼠关节红肿畸形,关节炎临床积分明显升高,关节组织骨破坏、滑膜增生及炎性细胞浸润,病理改变明显,血清中RF、MMP-3、TNF-α、IL-1β、Cp水平明显上升,IL-10水平降低;关节组织Cu、MDA、ROS,DLAT水平明显升高,SOD、GSH含量明显降低;SLC31 A1、HSP70蛋白及mRNA表达均明显上调,FDX1、LIAS、PDHB、ATP7B蛋白及mRNA表达下调(P＜0.01).与模型组比较,各给药组均不同程度改善大鼠关节肿胀变形程度,减低关节炎临床积分,降低大鼠关节骨破坏程度及炎细胞浸润及滑膜增生,血清RF、MMP-3、TNF-α、IL-1β、Cp水平明显降低,IL-10水平上升,关节组织Cu、MDA、ROS、DLAT水平明显降低,SOD、GSH水平升高,SLC31A1、HSP70蛋白及mRNA表达下调,FDX1、LIAS、PDHB、ATP7B蛋白及mRNA表达上调(P＜0.05).结论:槲皮素可有效减轻类风湿性关节炎大鼠滑膜增生及炎性浸润,缓解关节组织病理损伤,其机制或与阻断SLC31A1/FDX1信号通路激活,抑制过度的铜死亡有关.
Abstract
Objective:To observe the influence and therapeutic effect of quercetin on cuproptosis in rheumatoid arthritis rats and to explore its possible mechanism based on the solute carrier family 31 member 1(SLC31A1)/ferredoxin 1(FDX1)pathway.Methods:Sixty male SD rats were divided into six groups:A control group,a model group,high-and low-dose quercetin groups(150 and 50 mg·kg-1),a cuproptosis inhibitor(tetrathiomolybdate,TTM)group(10 mg·kg-1),and a methotrexate group(2 mg·kg-1),10 rats in each group.Except for the control group,the model of rheumatoid arthritis(CIA)rats was established by type Ⅱ collagen induction method.After successful modeling,each drug group was intervened according to the corresponding dose of drugs,and the control group and the model group were given the same amount of normal saline by gavage,once a day,which lasted for 4 weeks.The swelling degree of rats'feet was observed,and the clinical arthritis scores were determined.The levels of serum rheumatoid factor(RF),matrix metalloproteinase-3(MMP-3),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-10(IL-10),and ceruloplasmin(Cp)were detected by enzyme-linked immunosorbent assay(ELISA).The content of copper ion(Cu),malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)in joint tissue was detected.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of joint tissue.The levels of reactive oxygen species(ROS)and dihydrolipoic acid transacetylase(DLAT)were detected by immunofluorescence(IF).The protein and mRNA expression of SLC31A1,FDX1,lipoic acid synthase(LIAS),heat shock protein 70(HSP70),pyruvate dehydrogenase El subunit β(PDHB),and copper transporting P-type ATPase β(ATP7B)was detected by immunohistochemistry(IHC)and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Results:Compared to the control group,the model group exhibited joint swelling and deformity,significantly increased clinical arthritis scores,obvious bone destruction,synovial hyperplasia,and inflammatory cell infiltration in joint tissue.In addition,the serum levels of RF,MMP-3,TNF-α,IL-1β,and Cp showed significant elevation,while the level of IL-10 was significantly reduced.The levels of Cu,MDA,ROS,and DLAT in joint tissue were markedly increased,whereas SOD and GSH content was significantly decreased.The protein and mRNA expression of SLC31A1 and HSP70 was significantly up-regulated,while the protein and mRNA expression of FDX1,LIAS,PDHB,and ATP7B was significantly down-regulated(P＜0.01).Compared to the model group,each treatment group exhibited varying degrees of improvement in joint swelling and deformation as well as clinical arthritis scores in rats.Additionally,there was a reduction in joint bone destruction,inflammatory cell infiltration,and synovial hyperplasia in rats.Furthermore,the serum levels of RF,MMP-3,TNF-α,IL-1β,and Cp significantly decreased,while the level of IL-10 increased significantly.In joint tissue,the levels of Cu,MDA,ROS,and DLAT showed significant decreases,while SOD and GSH content exhibited significant increases.The protein and mRNA expression of SLC31A1 and HSP70 was down-regulated,while the protein and mRNA expression of FDX1,LIAS,PDHB,and ATP7B was up-regulated(P＜0.05).Conclusion:Quercetin effectively reduces synovial hyperplasia and inflammatory infiltration in rats with rheumatoid arthritis,thereby alleviating pathological damage to joint tissue.This effect may be attributed to the blockade of the SLC31A1/FDX1 signaling pathway activation and inhibition of excessive cuproptosis.
国家科技期刊平台
NSTL
维普
万方数据