减少氧糖剥夺/复氧后脊髓星形胶质细胞凋亡:抑制高迁移率族蛋白B1/核转录因子κB通路的作用
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摘要
背景:脊髓损伤后,星形胶质细胞核内高迁移率族蛋白B1被释放到细胞外,通过与细胞膜表面受体结合激活核转录因子κB引起脊髓水肿或者炎症等一系列病理反应,但有关高迁移率族蛋白B1/核转录因子κB通路调节氧糖剥夺/复氧损伤后脊髓星形胶质细胞凋亡的研究尚少。目的:分析高迁移率族蛋白B1/核转录因子κB通路对氧糖剥夺/复氧后脊髓星形胶质细胞凋亡的调节作用。方法:体外培养新生一二天SD大鼠(山西医科大学动物中心提供)脊髓星形胶质细胞,建立氧糖剥夺细胞损伤模型,并分别复氧培养6,12,24 h,ELISA法检测培养基内高迁移率族蛋白B1质量浓度,Western blot法检测细胞内高迁移率族蛋B1、Bcl-2、BAX蛋白表达量,MTT法检测细胞存活率,以此筛选最佳复氧时间进行以下实验。取第4代脊髓星形胶质细胞,分4组培养:正常组、氧糖剥夺/复氧组、氧糖剥夺/复氧+丙酮酸乙酯组、氧糖剥夺/复氧+核转录因子κB抑制剂组,复氧培养24h后,ELISA法检测培养基内高迁移率族蛋白B1质量浓度,Westernblot法检测细胞内高迁移率族蛋B1、核转录因子κB、Bcl-2、BAX蛋白表达量,MTT法检测细胞存活率,流式细胞仪检测细胞凋亡。结果与结论:(1)各检测结果显示复氧培养24 h为最佳复氧时间,用于后续实验;(2)与正常组比较,氧糖剥夺6 h/复氧24 h组高迁移率族蛋B1、核转录因子κB蛋白表达及细胞凋亡率升高(P <0.05),细胞存活率、Bcl-2/BAX比值降低(P <0.05);与氧糖剥夺6 h/复氧24 h组比较,氧糖剥夺6 h/复氧24 h+丙酮酸乙酯组、氧糖剥夺6 h/复氧24 h+核转录因子κB抑制剂组细胞存活率、Bcl-2/BAX比值升高(P <0.05),核转录因子κB蛋白表达、细胞凋亡率降低(P <0.05);(3)结果表明,高迁移率族蛋白B1/核转录因子κB通路参与调节氧糖剥夺/复氧后脊髓星形胶质细胞的凋亡。
BACKGROUND: Spinal cord injuries triggers the release of high mobility group box 1(HMGB1) from nerve cells to activate the nuclear factor-kappa B(NF-κB) by binding to cell membrane surface receptor, thereby inducing a series of pathological reactions, such as spinal cord edema or inflammation. However, little is reported on whether inhibition of HMGB1/NF-κB pathway could attenuate spinal cord astrocytes apoptosis after oxygen-glucose deprivation/reoxygenation(OGD/R). OBJECTIVE: To investigate the effect of HMGB1/NF-κB pathway on spinal cord astrocytes apoptosis after OGD/R. METHODS: Spinal cord astrocytes of newborn Sprague-Dawley rats(provided by the Laboratory Animal Center of Shanxi Medical University, China) were cultured in vitro and an OGD/R model was established. Spinal cord astrocytes were subjected to reoxygenation 6, 12, and 24 hours. Release of HMGB1 in the culture medium was detected by ELISA. The expression of HMGB1, Bcl-2 and Bax were detected by western blot and the cell survival rate was determined by MTT. The optimal reoxygenation time was then selected for the following experiments. Spinal cord astrocytes at passage 4 were divided into normal group, OGD6 h/R24 h group, OGD6 h/R24 h+ethyl pyruvate group, OGD6 h/R24 h+Bay 11-7082 group. After 24 hours of reoxygenation, the release of HMGB1 was detected by ELISA, the expression of HMGB1, NF-κB, Bcl-2 and BAX was analyzed by western blot, the survival rate of astrocytes was determined by MTT, and the apoptosis of astrocytes was measured by flow cytometry. RESULTS AND CONCLUSION:(1) The results showed that the best reoxygenation time was 24 hours, which was used for subsequent experiments.(2) Compared with the normal group, the release and expression of HMGB1 and NF-κB as well as the apoptotic rate of astrocytes were obviously increased in the OGD6 h/R24 h group(P < 0.05), while the survival rate of astrocytes and the ratio of Bcl-2/Bax were obviously declined in the OGD6 h/R24 h group(P < 0.05). Compared with the OGD6 h/R24 h group, the astrocyte survival rate and the ratio of Bcl-2/Bax were significantly raised(P < 0.05), while the expression of NF-κB and the apoptosis rate of astrocytes were remarkably decreased(P < 0.05) in the OGD6 h/R24 h+ethyl pyruvate and OGD6 h/R24 h+Bay 11-7082 groups. To conclude, the HMGB1/NF-κB signal pathway is involved in the regulation of apoptosis in spinal cord astrocytes after OGD/R.
BACKGROUND: Spinal cord injuries triggers the release of high mobility group box 1(HMGB1) from nerve cells to activate the nuclear factor-kappa B(NF-κB) by binding to cell membrane surface receptor, thereby inducing a series of pathological reactions, such as spinal cord edema or inflammation. However, little is reported on whether inhibition of HMGB1/NF-κB pathway could attenuate spinal cord astrocytes apoptosis after oxygen-glucose deprivation/reoxygenation(OGD/R). OBJECTIVE: To investigate the effect of HMGB1/NF-κB pathway on spinal cord astrocytes apoptosis after OGD/R. METHODS: Spinal cord astrocytes of newborn Sprague-Dawley rats(provided by the Laboratory Animal Center of Shanxi Medical University, China) were cultured in vitro and an OGD/R model was established. Spinal cord astrocytes were subjected to reoxygenation 6, 12, and 24 hours. Release of HMGB1 in the culture medium was detected by ELISA. The expression of HMGB1, Bcl-2 and Bax were detected by western blot and the cell survival rate was determined by MTT. The optimal reoxygenation time was then selected for the following experiments. Spinal cord astrocytes at passage 4 were divided into normal group, OGD6 h/R24 h group, OGD6 h/R24 h+ethyl pyruvate group, OGD6 h/R24 h+Bay 11-7082 group. After 24 hours of reoxygenation, the release of HMGB1 was detected by ELISA, the expression of HMGB1, NF-κB, Bcl-2 and BAX was analyzed by western blot, the survival rate of astrocytes was determined by MTT, and the apoptosis of astrocytes was measured by flow cytometry. RESULTS AND CONCLUSION:(1) The results showed that the best reoxygenation time was 24 hours, which was used for subsequent experiments.(2) Compared with the normal group, the release and expression of HMGB1 and NF-κB as well as the apoptotic rate of astrocytes were obviously increased in the OGD6 h/R24 h group(P < 0.05), while the survival rate of astrocytes and the ratio of Bcl-2/Bax were obviously declined in the OGD6 h/R24 h group(P < 0.05). Compared with the OGD6 h/R24 h group, the astrocyte survival rate and the ratio of Bcl-2/Bax were significantly raised(P < 0.05), while the expression of NF-κB and the apoptosis rate of astrocytes were remarkably decreased(P < 0.05) in the OGD6 h/R24 h+ethyl pyruvate and OGD6 h/R24 h+Bay 11-7082 groups. To conclude, the HMGB1/NF-κB signal pathway is involved in the regulation of apoptosis in spinal cord astrocytes after OGD/R.
引文
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[31]Veneruso V,Rossi F,Villella A,et al.Stem cell paracrine effect and delivery strategies for spinal cord injury regeneration.J Controll Release.2019;300:141-153.
[32]Obara K,Tohgi N,Shirai K,et al.Hair-Follicle-Associated Pluripotent(HAP)Stem Cells Encapsulated on Polyvinylidene Fluoride Membranes(PFM)Promote Functional Recovery from Spinal Cord Injury.Stem Cell Rev.2019;15(1):59-66.
[33]Takuma K,Baba A,Matsuda T.Astrocyte apoptosis:implications for neuroprotection.Prog Neurobiol.2004;72(2):111-127.
[34]Zeug A,Muller FE,Anders S,et al.Control of astrocyte morphology by Rho GTPases.Brain Res Bull.2018;136:44-53.
[35]Kwan T,Floyd CL,Kim S,et al.RNA Binding Protein Human Antigen R Is Translocated in Astrocytes following Spinal Cord Injury and Promotes the Inflammatory Response.J Neurotrauma.2017;34(6):1249-1259.
[36]Anwar MA,Al Shehabi TS,Eid AH.Inflammogenesis of Secondary Spinal Cord Injury.Front Cell Neurosci.2016;10:98.
[37]Coulson-Thomas VJ,Lauer ME,Soleman S,et al.Tumor Necrosis Factor-stimulated Gene-6(TSG-6)Is Constitutively Expressed in Adult Central Nervous System(CNS)and Associated with Astrocytemediated Glial Scar Formation following Spinal Cord Injury.J Biol Chem.2016;291(38):19939-19952.
[38]Shi Y,Kim S,Huff TB,et al.Effective repair of traumatically injured spinal cord by nanoscale block copolymer micelles.Nat Nanotechnol.2010;5(1):80-87.
[39]Pekny M,Wilhelmsson U,Pekna M.The dual role of astrocyte activation and reactive gliosis.Neurosci Lett.2014;565:30-38.
[40]Meneses CS,Muller HY,Herzberg DE,et al.Microglia and astrocyte activation in the spinal cord of lame horses.Vet Anaesth Analg.2018;45(1):92-102.
[41]Cory S,Adams JM.The Bcl2 family:regulators of the cellular life-or-death switch.Nat Rev Cancer.2002;2(9):647-656.
[42]Liu D,Zhang M,Rong Xet al.Potassium 2-(1-hydroxypentyl)-benzoate attenuates neuronal apoptosis in neuron-astrocyte co-culture system through neurotrophy and neuroinflammation pathway.Acta Pharm Sin B.2017;7(5):554-563.
[43]Sun L,Li M,Ma X,et al.Inhibiting high mobility group box-1 reduces early spinal cord edema and attenuates astrocyte activation and aquaporin-4 expression after spinal cord injury in rats.J Neurotrauma.2019;36(3):421-435.
[44]Kim SW,Lim CM,Kim JB,et al.Extracellular HMGB1 released by NMDAtreatment confers neuronal apoptosis via RAGE-p38 MAPK/ERKsignaling pathway.Neurotox Res.2011;20(2):159-169.
[45]Anton M,Alen F,Gomez de Heras R,et al.Oleoylethanolamide prevents neuroimmune HMGB1/TLR4/NF-kB danger signaling in rat frontal cortex and depressive-like behavior induced by ethanol binge administration.Addict Biol.2017;22(3):724-741.
[46]Bhat SM,Massey N,Karriker LA,et al.Ethyl pyruvate reduces organic dust-induced airway inflammation by targeting HMGB1-RAGEsignaling.Respir Res.2019;20(1):27.
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[2]Scintu F,Scorciapino L,Carta M,et al.Human astrocytes can be induced to differentiate into cells with neuronal phenotype.Exp Cell Res.2006;312(12):2336-2346.
[3]Zhao X,Zhou KS,Li ZH,et al.Knockdown of Ski decreased the reactive astrocytes proliferation in vitro induced by oxygen-glucose deprivation/reoxygenation.J Cell Biochem.2018;119(6):4548-4558.
[4]Chen J,Wang Q,Zhou W,et al.GPCR kinase 2-interacting protein-1protects against ischemia-reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling.FASEB J.2018:fj201800548.
[5]Li H,Zhang X,Qi X,et al.Icariin Inhibits Endoplasmic Reticulum Stress-induced Neuronal Apoptosis after Spinal Cord Injury through Modulating the PI3K/AKT Signaling Pathway.Int J Biol Sci.2019;15(2):277-286.
[6]Jiang H,Fang J,Xing J,et al.Tilianin mediates neuroprotection against ischemic injury by attenuating CaMKII-dependent mitochondrionmediated apoptosis and MAPK/NF-kappaB signaling.Life Sci.2019;216:233-245.
[7]Brunelle JK,Letai A.Control of mitochondrial apoptosis by the Bcl-2family.J Cell Sci.2009;122(Pt 4):437-441.
[8]Lu Z,Miao Y,Muhammad I,et al.Colistin-induced autophagy and apoptosis involves the JNK-Bcl2-Bax signaling pathway and JNK-p53-ROS positive feedback loop in PC-12 cells.Chem Biol Interact.2017;277:62-73.
[9]Haas C,Fischer I.Human astrocytes derived from glial restricted progenitors support regeneration of the injured spinal cord.JNeurotrauma.2013;30(12):1035-1052.
[10]Lepore AC,O'Donnell J,Kim AS,et al.Reduction in expression of the astrocyte glutamate transporter,GLT1,worsens functional and histological outcomes following traumatic spinal cord injury.Glia.2011;59(12):1996-2005.
[11]Xu Z,Zhang K,Wang Q,et al.MicroRNA-124 improves functional recovery and suppresses Bax-dependent apoptosis in rats following spinal cord injury.Mol Med Rep.2019;19(4):2551-2560.
[12]Pillai R,Scintu F,Scorciapino L,et al.Human astrocytes can be induced to differentiate into cells with neuronal phenotype.Exp Cell Res.2006;312(12):2336-2346.
[13]Xu W,Gao L,Li T,et al.Mesencephalic Astrocyte-Derived Neurotrophic Factor(MANF)Protects Against Neuronal Apoptosis via Activation of Akt/MDM2/p53 Signaling Pathway in a Rat Model of Intracerebral Hemorrhage.Front Molr Neurosci.2018;11:176.
[14]Tran AP,Warren PM,Silver J.The Biology of Regeneration Failure and Success After Spinal Cord Injury.Physiol Rev.2018;98(2):881-917.
[15]Qiu J,Nishimura M,Wang Y,et al.Early release of HMGB-1 from neurons after the onset of brain ischemia.J Cereb Blood Flow Metab.2008;28(5):927-938.
[16]Bi Y,Zhu Y,Zhang M,et al.Effect of Shikonin on Spinal Cord Injury in Rats Via Regulation of HMGB1/TLR4/NF-kB Signaling Pathway.Cell Physiol Biochem.2017;43(2):481-491.
[17]Kang N,Hai Y,Yang J,et al.Hyperbaric oxygen intervention reduces secondary spinal cord injury in rats via regulation of HMGB1/TLR4/NF-kappa B signaling pathway.Int J Clin Exp Pathol.2015;8(2):1141-1153.
[18]Kigerl KA,Lai W,Wallace LM,et al.High mobility group box-1(HMGB1)is increased in injured mouse spinal cord and can elicit neurotoxic inflammation.Brain Behav Immun.2018;72:22-33.
[19]Papatheodorou A,Stein A,Bank M,et al.High-Mobility Group Box 1(HMGB1)Is Elevated Systemically in Persons with Acute or Chronic Traumatic Spinal Cord Injury.J Neurotrauma.2017;34(3):746-754.
[20]Gwak GY,Moon TG,Lee DH,et al.Glycyrrhizin attenuates HMGB1-induced hepatocyte apoptosis by inhibiting the p38-dependent mitochondrial pathway.World J Gastroenterol.2012;18(7):679-684.
[21]Gong G,Xiang L,Yuan L,et al.Protective effect of glycyrrhizin,a direct HMGB1 inhibitor,on focal cerebral ischemia/reperfusion-induced inflammation,oxidative stress,and apoptosis in rats.PLoS One.2014;9(3):e89450.
[22]Yang L,Wang F,Yang L,et al.HMGB1 a-Box Reverses Brain Edema and Deterioration of Neurological Function in a Traumatic Brain Injury Mouse Model.Cell Physiol Biochem.2018;46(6):2532-2542.
[23]Zhang M,Ma Y,Chai L,et al.Storax Protected Oxygen-Glucose Deprivation/Reoxygenation Induced Primary Astrocyte Injury by Inhibiting NF-kappaB Activation in vitro.Front Pharmacol.2018;9:1527.
[24]Bao G,Li C,Qi L,et al.Tetrandrine protects against oxygen-glucose-serum deprivation/reoxygenation-induced injury via PI3K/AKT/NF-kappa B signaling pathway in rat spinal cord astrocytes.Biomed Pharmacother.2016;84:925-930.
[25]Shin JH,Kim ID,Kim SW,et al.Ethyl pyruvate inhibits HMGB1phosphorylation and release by chelating calcium.Mol Med.2015;20:649-657.
[26]Sun L,Li M,Ma X,et al.Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-kappaB signaling in an IL-6-dependent manner.J Neuroinflam.2017;14(1):231.
[27]Kerstetter A,Miller R.Isolation and culture of spinal cord astrocytes.Methods Mol Biol.2012;814:93-104.
[28]Wei W,Shurui C,Zipeng Z,et al.Aspirin suppresses neuronal apoptosis,reduces tissue inflammation,and restrains astrocyte activation by activating the Nrf2/HO-1 signaling pathway.Neuroreport.2018;29(7):524-531.
[29]Lu X,Xue P,Fu L,et al.HAX1 is associated with neuronal apoptosis and astrocyte proliferation after spinal cord injury.Tissue Cell.2018;54:1-9.
[30]Xu P,Yang X.The Efficacy and Safety of Mesenchymal Stem Cell Transplantation for Spinal Cord Injury Patients:A Meta-Analysis and Systematic Review.Cell Transplant.2019;28(1):36-46.
[31]Veneruso V,Rossi F,Villella A,et al.Stem cell paracrine effect and delivery strategies for spinal cord injury regeneration.J Controll Release.2019;300:141-153.
[32]Obara K,Tohgi N,Shirai K,et al.Hair-Follicle-Associated Pluripotent(HAP)Stem Cells Encapsulated on Polyvinylidene Fluoride Membranes(PFM)Promote Functional Recovery from Spinal Cord Injury.Stem Cell Rev.2019;15(1):59-66.
[33]Takuma K,Baba A,Matsuda T.Astrocyte apoptosis:implications for neuroprotection.Prog Neurobiol.2004;72(2):111-127.
[34]Zeug A,Muller FE,Anders S,et al.Control of astrocyte morphology by Rho GTPases.Brain Res Bull.2018;136:44-53.
[35]Kwan T,Floyd CL,Kim S,et al.RNA Binding Protein Human Antigen R Is Translocated in Astrocytes following Spinal Cord Injury and Promotes the Inflammatory Response.J Neurotrauma.2017;34(6):1249-1259.
[36]Anwar MA,Al Shehabi TS,Eid AH.Inflammogenesis of Secondary Spinal Cord Injury.Front Cell Neurosci.2016;10:98.
[37]Coulson-Thomas VJ,Lauer ME,Soleman S,et al.Tumor Necrosis Factor-stimulated Gene-6(TSG-6)Is Constitutively Expressed in Adult Central Nervous System(CNS)and Associated with Astrocytemediated Glial Scar Formation following Spinal Cord Injury.J Biol Chem.2016;291(38):19939-19952.
[38]Shi Y,Kim S,Huff TB,et al.Effective repair of traumatically injured spinal cord by nanoscale block copolymer micelles.Nat Nanotechnol.2010;5(1):80-87.
[39]Pekny M,Wilhelmsson U,Pekna M.The dual role of astrocyte activation and reactive gliosis.Neurosci Lett.2014;565:30-38.
[40]Meneses CS,Muller HY,Herzberg DE,et al.Microglia and astrocyte activation in the spinal cord of lame horses.Vet Anaesth Analg.2018;45(1):92-102.
[41]Cory S,Adams JM.The Bcl2 family:regulators of the cellular life-or-death switch.Nat Rev Cancer.2002;2(9):647-656.
[42]Liu D,Zhang M,Rong Xet al.Potassium 2-(1-hydroxypentyl)-benzoate attenuates neuronal apoptosis in neuron-astrocyte co-culture system through neurotrophy and neuroinflammation pathway.Acta Pharm Sin B.2017;7(5):554-563.
[43]Sun L,Li M,Ma X,et al.Inhibiting high mobility group box-1 reduces early spinal cord edema and attenuates astrocyte activation and aquaporin-4 expression after spinal cord injury in rats.J Neurotrauma.2019;36(3):421-435.
[44]Kim SW,Lim CM,Kim JB,et al.Extracellular HMGB1 released by NMDAtreatment confers neuronal apoptosis via RAGE-p38 MAPK/ERKsignaling pathway.Neurotox Res.2011;20(2):159-169.
[45]Anton M,Alen F,Gomez de Heras R,et al.Oleoylethanolamide prevents neuroimmune HMGB1/TLR4/NF-kB danger signaling in rat frontal cortex and depressive-like behavior induced by ethanol binge administration.Addict Biol.2017;22(3):724-741.
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