RIP1/RIP3通路介导氯化血红素诱导的HT-22海马神经细胞损伤的研究

doi:10.3877/cma.j.issn.2095-9141.2018.05.008

中华神经创伤外科电子杂志 ›› 2018, Vol. 04 ›› Issue (05) : 283 -290. doi: 10.3877/cma.j.issn.2095-9141.2018.05.008

所属专题：文献；

基础研究

RIP1/RIP3通路介导氯化血红素诱导的HT-22海马神经细胞损伤的研究

苏兴奋¹^,^†(

), 王汉东², 林元相¹, 陈伏祥¹

^1. 350005　福州，福建医科大学附属第一医院神经外科
^2. 210002　南京，南京大学医学院附属金陵医院神经外科

收稿日期:2018-07-23 出版日期:2018-10-15
通信作者: 苏兴奋
基金资助:
福建省自然科学基金(2015J05149); 国家自然科学基金青年课题(81501012)

Receptor interacting protein kinases 1 and 3 mediate hemin induced cell death in HT-22 hippocampal neuronal cells

Xingfen Su¹^,^†(), Handong Wang², Yuanxiang Lin¹, Fuxiang Chen¹

^1. Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China
^2. Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China

Received:2018-07-23 Published:2018-10-15
Corresponding author: Xingfen Su
About author:
Corresponding author: Su Xingfen, Email: flying828@163.com

全文 ( ) 下载PDF ( ) 导出引用

目的

探索受体相互作用蛋白激酶1（RIP1）/RIP3通路是否参与氯化血红素（hemin）诱导的HT-22海马神经细胞损伤以及研究Necrostatin-1（Nec-1）在hemin诱导的HT-22细胞死亡中潜在的神经保护作用。

方法

给予不同浓度的hemin（0、25、50、100 μmol/L）作用HT-22细胞24 h后测定碘化丙啶（PI）阳性细胞数和细胞存活率。用Necrostatin-1、zVAD和活性氧（ROS）清除剂叔丁基茴香醚（BHA）分别处理hemin诱导的HT-22细胞，24 h后分别测定各组PI⁺细胞数、细胞存活率，使用MitoSox Red指示ROS水平。最后研究使用siRNA敲低RIP3水平对hemin诱导HT-22细胞死亡的影响，测定各组PI⁺细胞数、细胞存活率及ROS水平。

结果

Hemin可剂量依赖性的诱导HT-22神经细胞死亡；RIP1抑制剂Necrostatin-1可显著抑制hemin诱导的HT-22细胞死亡，PI⁺细胞数明显减少，细胞存活率提高，并且减少ROS积聚；BHA可显著减少hemin诱导的HT-22细胞的PI⁺细胞数。更进一步的使用siRNA敲低RIP3表达水平可以显著减少hemin诱导的HT-22细胞死亡，PI⁺细胞数明显减少，细胞存活率明显提高，ROS积聚水平显著减低。

结论

RIP1/RIP3通路及ROS可能介导hemin诱导的HT-22海马神经元细胞死亡，Necrostatin-1在其中起神经保护作用。

关键词: 氯化血红素, 程序性坏死, 受体相互作用蛋白激酶1, 受体相互作用蛋白激酶3, Necrostatin-1

Objective

To explore whether receptor interacting protein kinase 1 (RIP1)/RIP3 pathways participate in hemin induced cell death in HT-22 hippocampal neuronal cells and investigate the potential neuroprotection of necrostatin-1 in hemin induced cell death in HT-22.

Methods

First, different concentrations of hemin (0, 25, 50, 100 μmol/L) were added to HT-22 cells. Propidium iodide (PI) positive cells and cell viability were measured at 24 h after hemin treatment. Then, necrostatin-1, zVAD and reactive oxygen species (ROS) scavenger butylated hydroxyanisole (BHA) were applied to hemin-treated HT-22 cells. PI⁺ cells and cell viability were measured at 24 h after hemin treatment. MitoSox Red was used to indicate ROS level. Last, the effect of RIP3 in hemin induced HT-22 cell death was explored through RIP3 knockdown using siRNA. PI⁺ cells, cell viability and ROS lever were measured at 24 h after hemin treatment.

Results

Hemin could induce a dose dependent cell death in HT22 neural cells. RIP1 specific inhibitor necrostatin-1 significantly inhibited cell death induced by hemin in HT-22 cells, greatly reducing PI⁺ cells, dramatically improving cell viability and decreasing ROS accumulation. BHA could significantly inhibit PI⁺ cells induced by hemin in HT-22 cells. Furthermore, silencing of RIP3 using siRNA attenuated hemin induced cell death in HT-22 cells, greatly reducing PI⁺ cells, dramatically improving cell viability and decreasing ROS accumulation.

Conclusion

These data revealed that RIP1/ RIP3 pathway and ROS might mediate hemin induced cell death in HT-22 hippocampus neural cells, and necrostatin-1 played a neuroprotection role in hemin induced cell death in HT-22.

Key words: Hemin, Necroptosis, Receptor interacting protein kinase 1, Receptor interacting protein kinase 3, Necrostatin-1

图1 氯化血红素剂量依赖性的诱导HT-22细胞死亡

图2 Nec-1和zVAD对氯化血红素诱导的HT-22细胞死亡的影响

图3 Nec-1对氯化血红素诱导HT-22细胞产生的活性氧的影响

图4 活性氧清除剂叔丁基羟基茴香醚对氯化血红素诱导HT-22细胞死亡的影响

图5 敲低受体相互作用蛋白激酶3水平抑制氯化血红素诱导HT-22细胞死亡

[1]	Keep RF, Hua Y, Xi G. Intracerebral haemorrhage: mechanisms of injury and therapeutic targets[J]. Lancet Neurol, 2012, 11(8): 720-731.
[2]	Robinson SR, Dang TN, Dringen R, et al. Hemin toxicity: a preventable source of brain damage following hemorrhagic stroke[J]. Redox Rep, 2009, 14(6): 228-235.
[3]	Regan RF, Chen J, Benvenisti-Zarom L. Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin[J]. BMC Neurosci, 2004, 5(1): 34.
[4]	Laird MD, Wakade C, Alleyne CH Jr., et al. Hemin-induced necroptosis involves glutathione depletion in mouse astrocytes[J]. Free Radic Biol Med, 2008, 45(8): 1103-1114.
[5]	Vandenabeele P, Galluzzi L, Vanden Berghe T, et al. Molecular mechanisms of necroptosis: an ordered cellular explosion[J]. Nat Rev Mol Cell Biol, 2010, 11(10): 700-714.
[6]	Xu X, Chua CC, Kong J, et al. Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells[J]. J Neurochem, 2007, 103(5): 2004-2014.
[7]	Li Y, Yang X, Ma C, et al. Necroptosis contributes to the NMDA- induced excitotoxicity in rat’s cultured cortical neurons[J]. Neurosci Lett, 2008, 447(2-3): 120-123.
[8]	Zhang DW, Shao J, Lin J, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis[J]. Science, 2009, 325(5938): 332-336.
[9]	Galluzzi L, Vanden Berghe T, Vanlangenakker N, et al. Programmed necrosis from molecules to health and disease[J]. Int Rev Cell Mol Biol, 2011, 289: 1-35.
[10]	He S, Wang L, Miao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha[J]. Cell, 2009, 137(6): 1100-1111.
[11]	Vanlangenakker N, Vanden Berghe T, Vandenabeele P. Many stimuli pull the necrotic trigger, an overview[J]. Cell Death Differ, 2012, 19(1): 75-86.
[12]	Aronowski J, Zhao X. Molecular pathophysiology of cerebral hemorrhage: secondary brain injury[J]. Stroke, 2011, 42(6): 1781-1786.
[13]	Vandenabeele P, Declercq W, Van Herreweghe F, et al. The role of the kinases RIP1 and RIP3 in TNF-induced necrosis[J]. Sci Signal, 2010, 3(115): re4.
[14]	Degterev A, Huang Z, Boyce M, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury[J]. Nat Chem Biol, 2005, 1(2): 112-119.
[15]	You Z, Savitz SI, Yang J, et al. Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice[J]. J Cereb Blood Flow Metab, 2008, 28(9): 1564-1573.
[16]	Su X, Wang H, Kang D, et al. Necrostatin-1 ameliorates intracerebral hemorrhage-induced brain injury in mice through inhibiting RIP1/RIP3 pathway[J]. Neurochem Res, 2015, 40(4): 643-650.
[17]	Degterev A, Hitomi J, Germscheid M, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins[J]. Nat Chem Biol, 2008, 4(5): 313-321.
[18]	Zhu X, Tao L, Tejima-Mandeville E, et al. Plasmalemma permeability and necrotic cell death phenotypes after intracerebral hemorrhage in mice[J]. Stroke, 2012, 43(2): 524-531.
[19]	Zille M, Karuppagounder SS, Chen Y, et al. Neuronal death after hemorrhagic stroke in vitro and in vivo shares features of ferroptosis and necroptosis[J]. Stroke, 2017, 48(4): 1033-1043.
[20]	Lule S, Wu L, McAllister LM, et al. Genetic inhibition of receptor interacting protein kinase-1 reduces cell death and improves functional outcome after intracerebral hemorrhage in mice[J]. Stroke, 2017, 48(9): 2549-2556.

[1]	柳成林, 荀文兴, 杨海珍, 范素萌, 刘宇博, 张红梅. 程序性坏死特异性抑制剂-1对高糖环境下牙周膜干细胞增殖和成骨分化的影响[J]. 中华口腔医学研究杂志(电子版), 2022, 16(03): 160-167.
[2]	詹雅清, 赖汉津, 沈建通, 温仕宏, 黄文起. 程序性坏死的发生机制及其在缺血损伤性疾病中的研究进展[J]. 中华普通外科学文献(电子版), 2021, 15(03): 224-228.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Receptor interacting protein kinases 1 and 3 mediate hemin induced cell death in HT-22 hippocampal neuronal cells

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Receptor interacting protein kinases 1 and 3 mediate hemin induced cell death in HT-22 hippocampal neuronal cells