维生素D通过Nrf2/HO-1抑制铁死亡缓解6-羟基多巴胺所致PC12细胞损伤的作用机制研究  

Study on mechanism of vitamin D inhibiting iron death through Nrf2/HO-1 to alleviate 6-hydroxy-dopamine induced PC12 cell damage

作　　者：穆清爽[1] 李沛珊 李艳霞[3] 李瑞晟[1] 杨新玲[4] MU Qingshuang;LI Peishan;LI Yanxia;LI Ruisheng;YANG Xinling(The First Department of Cadre,the Second Affiliated Hospital of Xinjiang Medical University,Xinjiang Key Laboratory of Nervous System Diseases Research,Urumqi 830063,China;Department of Neurology,Urumqi 830063,China;Department of Rehabilitation,the Second Affiliated Hospital of Xinjiang Medical University,Urumqi 830063,China;Xinjiang Medical University,Urumqi 830017,China)

机构地区：[1]新疆医科大学第二附属医院干部一科,新疆神经系统疾病研究重点实验室,乌鲁木齐830063 [2]新疆医科大学第二附属医院神经内科,乌鲁木齐830063 [3]新疆医科大学第二附属医院康复科,乌鲁木齐830063 [4]新疆医科大学,乌鲁木齐830017

出　　处：《新疆医科大学学报》2023年第12期1580-1588,共9页Journal of Xinjiang Medical University

基　　金：国家自然科学基金地区科学基金项目(82160232);中央引导地方科技发展专项资金项目(ZYYD2022C17);省部共建中亚高发病成因与防治国家重点实验室开放课题项目(SKL-HIDCA-2022-NKX8)。

摘　　要：目的 探讨维生素D(VD)缓解6-羟基多巴胺(6-OHDA)所致PC12细胞损伤的作用与铁死亡及Nrf2/HO-1通路激活的关系。方法 利用6-OHDA构建PC12细胞帕金森病(PD)模型,不同浓度的VD分别联合铁死亡激活剂(Erastin)或Nrf2抑制剂(ML385)干预后,CCK-8法检测PC12细胞活力变化。将PC12细胞分成空白对照组(Control)、6-羟基多巴胺组(6-OHDA)、维生素D组(VD)、铁死亡激活剂组(Erastin)、铁死亡激活剂+维生素D组(Erastin+VD)、Nrf2抑制剂组(ML385)、Nrf2抑制剂+维生素D组(ML385+VD),对应干预结束后,Annexin V-FITC/PI双染检测细胞凋亡水平,检测丙二醛(MDA)、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、谷胱甘肽(GSH)含量,铁离子试剂盒检测铁离子含量,qRT-PCR检测谷胱甘肽过氧化酶4(GPX4)、铁蛋白重链多肽1(FTH-1)、胱氨酸/谷氨酸反向转运蛋白(XCT,也称SLC7A11)、转铁蛋白受体(TFR-1)表达,Western blot检测Nrf2/HO-1通路激活情况。结果 与Control组比较,6-OHDA、Erastin、ML385均显著抑制PC12细胞活力(P<0.01),促进细胞凋亡(P<0.01);LDH、MDA水平升高(P<0.01), GSH、SOD水平降低(P<0.01),铁离子含量升高(P均<0.01),GPX4、FTH-1、XCT等的表达降低(P均<0.01),TFR-1的表达显著升高(P<0.01),Nrf2、HO-1的表达被显著抑制(P<0.01);与6-OHDA组比较,VD可显著促进PC12细胞活力(P<0.01)、抑制细胞凋亡(P<0.01),降低LDH、MDA水平(P均<0.01),升高GSH、SOD水平(P均<0.01),抑制铁离子沉积(P均<0.01),GPX4、FTH-1、XCT等的表达均显著升高(P均<0.01),TFR-1的表达显著降低(P<0.01),Nrf2、HO-1表达升高(P<0.01);ML385可显著抑制VD对上述指标的调控作用。结论 维生素D可激活Nrf2/HO-1通路,缓解6-OHDA所致PC12细胞铁死亡。Objective To investigate the effect of vitamin D(VD)on PC12 cell damage induced by 6-hydroxy-dopamine(6-OHDA),iron death and activation of Nrf2/HO-1 pathway.MethodsThe Parkinson′s disease(PD)model of PC12 cells was constructed using 6-OHDA.After the intervention of different concentrations of VD combined with iron death activator(Erastin)or Nrf2 inhibitor(ML385),CCK-8 detected the changes of PC12 cell viability.PC12 cells were divided into blank control group(Control),6-hydroxydopamine group(6-OHDA),vitamin D group(VD),iron death activator group(Erastin),iron death activator+vitamin D group(Erastin+VD),Nrf2 inhibitor group(ML385),Nrf2 inhibitor+vitamin D group(ML385+VD)was Annexin V-FITC/PI double stained to detect apoptosis level after the intervention.The biochemical kit was used to detect the expression of malondialdehyde(MDA),super oxide dismutase(SOD),lactic dehydrogenase(LDH),glutathione(GSH)and the iron ion kit was used to detect the iron ion content.qRT-PCR was used to detect the expression of Glutathione peroxidase 4(GPX4),FTH-1,SLC7A11 and TFR-1.Western blot was used to detect the activation of Nrf2/HO-1 pathway.ResultsCompared with control group,6-OHDA,Erastin and ML385 significantly inhibited PC12 cell viability(P<0.01)and promoted apoptosis(P<0.01).LDH and MDA levels were increased(P<0.01).GSH and SOD levels were decreased(P<0.01).Iron ion content was increased(P<0.01).GPX4,FTH-1 and XCT expressions were decreased(P<0.01).TFR-1 expression was significantly increased(P<0.01).The expressions of Nrf2 and HO-1 were significantly inhibited(P<0.01).Compared with 6-OHDA group,VD significantly promoted PC12 cell viability(P<0.01),inhibited apoptosis(P<0.01),decreased LDH and MDA levels(P<0.01),increased GSH and SOD levels(P<0.01),and inhibited iron ion deposition(P<0.01).GPX4,FTH-1 and XCT were significantly increased(P<0.01),while the expression of TFR-1 was significantly decreased(P<0.01),and the expression of Nrf2 and HO-1 was increased(P<0.01).ML385 can significantly inhibit the regulatory effect of VD

关 键 词：帕金森病 铁死亡 维生素D 6-羟基多巴胺 核因子E2相关因子2/血红素加氧酶1 

分 类 号：R742.5[医药卫生—神经病学与精神病学]