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
穆清爽 1李沛珊 2李艳霞 3李瑞晟 1杨新玲4
作者信息
- 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
- 2. 新疆医科大学第二附属医院神经内科
- 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
- 折叠
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-OH-DA 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.
Abstract
Objective To investigate the effect of vitamin D(VD)on PC12 cell damage induced by 6-hy-droxy-dopamine(6-OHDA),iron death and activation of Nrf2/HO-1 pathway.Methods The Parkinson's disease(PD)model of PC12 cells was constructed using 6-OHDA.After the intervention of different con-centrations of VD combined with iron death activator(Erastin)or Nrf2 inhibitor(ML385),CCK-8 detec-ted 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+vita-min D group(ML385+VD)was Annexin V-FITC/PI double stained to detect apoptosis level after the in-tervention.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.Results Compared with control group,6-OHDA,Erastin and ML385 significantly inhibited PC12 cell via-bility(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 in-hibited 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 on the above indexes.Conclusion Vitamin D can activate Nrf2/HO-1 pathway and alleviate iron death in PC12 cells in-duced by 6-OHDA.
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1Key words
Parkinson's disease(PD)/iron death/vitamin D/6-hydroxydopamine/nuclear factor E2 related factor 2/heme oxygenase 1引用本文复制引用
基金项目
国家自然科学基金地区科学基金(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(SKL-HIDCA-2022-NKX8)
出版年
2023
NETL
NSTL
维普
万方数据