人脐带间充质干细胞来源外泌体减轻大鼠脊髓星形胶质细胞氧糖剥夺/复氧损伤所致的水肿
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摘要
背景:外泌体对神经细胞具有调控修复作用,从而促进神经细胞增生,减轻脊髓损伤,但外泌体对脊髓损伤水肿作用的相关研究较少。目的:探讨人脐带间充质干细胞来源外泌体减轻氧糖剥夺/复氧损伤大鼠脊髓星形胶质细胞水肿的作用及机制。方法:超高速离心法分离提取人脐带间充质干细胞外泌体,采用胰酶消化法提取新生SD大鼠脊髓星形胶质细胞。第一部分实验分组:①对照组,细胞正常培养,未进行氧糖剥夺/复氧处理;②模型组:氧糖剥夺6 h/复氧24 h;③人脐带间充质干细胞外泌体组:氧糖剥夺6 h/复氧24 h,在复氧过程的培养基中分别加入30,60,90μg人脐带间充质干细胞外泌体,作用24 h。Western blot检测细胞AQP4蛋白表达,活细胞工作站检测细胞体积,透射电镜观察细胞水肿超微结构变化。第二部分实验分组:①对照组:细胞正常培养,未进行氧糖剥夺/复氧处理;②模型组:氧糖剥夺6 h/复氧24 h;③人脐带间充质干细胞外泌体组:氧糖剥夺6 h/复氧24h,在复氧过程的培养基中加入90μg人脐带间充质干细胞外泌体,作用24h;④JNK受体抑制剂SP600125组:氧糖剥夺6 h/复氧24 h,在复氧过程的培养基中加入5μmol/L JNK受体抑制剂SP600125,作用24 h。Western blot检测细胞AQP4及p-JNK蛋白表达,活细胞工作站检测细胞体积。结果与结论:①与模型组比较,人脐带间充质干细胞外泌体干预24 h后,透射电镜可见线粒体、内质网水肿减轻,溶酶体数量减少,细胞水肿体积显著降低(P <0.05),AQP4蛋白的表达量减少(P <0.05);②与模型组比较,人脐带间充质干细胞外泌体与JNK抑制剂SP600125干预后,细胞中AQP4、p-JNK表达明显降低(P <0.05),细胞水肿体积显著降低(P <0.05);③结果表明,人脐带间充质干细胞外泌体可通过抑制JNK信号通路降低氧糖剥夺/复氧损伤后脊髓星形胶质细胞AQP4蛋白的表达,减轻细胞水肿。
BACKGROUND: Exosomes can regulate and repair nerve cells, thus promoting the proliferation of nerve cells to alleviate spinal cord injury.Exosomes have been less reported to alleviate edema after spinal cord injury.OBJECTIVE: To study the effect and mechanism by which human umbilical cord mesenchymal stem cell-derived exosomes(hucMSCs-exo)alleviate the edema of spinal astrocytes induced by oxygen-glucose deprivation/reoxygenation.METHODS: Ultrahigh speed centrifugation method was used to isolate and extract hucMSCs-exo. Spinal astrocytes were extracted from newborn Sprague-Dawley rats by trypsin digestion. Part One: The cells were(1) cultured normally in normal control group,(2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group, or(3) cultured in medium containing 30, 60, and 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group. Western blot assay was performed to detect the expression of AQP4. Living cell workstation was used to detect cell volume. Transmission electron microscope was used to observe the ultrastructure of intracellular edema. Part Two: The cells were(1) cultured normally in normal control group,(2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group,(3) cultured in medium containing 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group, or(4) cultured in medium containing 5 μmol/L JNK inhibitor sp600125 for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in JNK inhibitor sp600125 group. AQP4 expression and p-JNK protein expression were measured by western blot assay. Living cell workstation was used to detect cell volume RESULTS AND CONCLUSION:(1) After 24-hour hucMSCs-exo intervention, mitochondrial and endoplasmic reticular edemas were alleviated, the number of lysosomes decreased, the volume of intracellular cell edema was significantly reduced, and the expression of AQP4 in the cells was significantly lowered as compared with the model group(P < 0. 05).(2) Intervention with hucMSCs-exo and JNK inhibitor SP600125 significantly reduced the expression of AQP4 and p-JNK(P < 0.05) and the volume of intracellular edema(P < 0.05) as compared with the model group. Therefore, hucMSCs-exo can reduce the expression of AQP4 protein in spinal astrocytes after oxygen-glucose deprivation/reoxygenation by inhibiting JNK signaling pathway, thereby alleviating cell edema.
BACKGROUND: Exosomes can regulate and repair nerve cells, thus promoting the proliferation of nerve cells to alleviate spinal cord injury.Exosomes have been less reported to alleviate edema after spinal cord injury.OBJECTIVE: To study the effect and mechanism by which human umbilical cord mesenchymal stem cell-derived exosomes(hucMSCs-exo)alleviate the edema of spinal astrocytes induced by oxygen-glucose deprivation/reoxygenation.METHODS: Ultrahigh speed centrifugation method was used to isolate and extract hucMSCs-exo. Spinal astrocytes were extracted from newborn Sprague-Dawley rats by trypsin digestion. Part One: The cells were(1) cultured normally in normal control group,(2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group, or(3) cultured in medium containing 30, 60, and 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group. Western blot assay was performed to detect the expression of AQP4. Living cell workstation was used to detect cell volume. Transmission electron microscope was used to observe the ultrastructure of intracellular edema. Part Two: The cells were(1) cultured normally in normal control group,(2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group,(3) cultured in medium containing 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group, or(4) cultured in medium containing 5 μmol/L JNK inhibitor sp600125 for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in JNK inhibitor sp600125 group. AQP4 expression and p-JNK protein expression were measured by western blot assay. Living cell workstation was used to detect cell volume RESULTS AND CONCLUSION:(1) After 24-hour hucMSCs-exo intervention, mitochondrial and endoplasmic reticular edemas were alleviated, the number of lysosomes decreased, the volume of intracellular cell edema was significantly reduced, and the expression of AQP4 in the cells was significantly lowered as compared with the model group(P < 0. 05).(2) Intervention with hucMSCs-exo and JNK inhibitor SP600125 significantly reduced the expression of AQP4 and p-JNK(P < 0.05) and the volume of intracellular edema(P < 0.05) as compared with the model group. Therefore, hucMSCs-exo can reduce the expression of AQP4 protein in spinal astrocytes after oxygen-glucose deprivation/reoxygenation by inhibiting JNK signaling pathway, thereby alleviating cell edema.
引文
[1]Anwar MA,Al Shehabi TS,Eid AH.Inflammogenesis of Secondary Spinal Cord Injury.Front Cell Neurosci.2016;10:98.
[2]Kasuya Y,Umezawa H,Hatano M.Stress-Activated Protein Kinases in Spinal Cord Injury:Focus on Roles of p38.Int J Mol Sci.2018;19(3):E867.
[3]He FY,Feng WZ,Zhong J,et al.Effects of propofol and dexmedetomidine anesthesia on Th1/Th2 of rat spinal cord injury.Eur Rev Med Pharmacol Sci.2017;21(6):1355-1361.
[4]Yang J,Zhang X,Chen X,et al.Exosome Mediated Delivery of miR-124 Promotes Neurogenesis after Ischemia.Mol Ther Nucleic Acids.2017;7:278-287.
[5]Qi J,Zhou Y,Jiao Z,et al.Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway.Cell Physiol Biochem.2017;42(6):2242-2254.
[6]Ma J,Zhao Y,Sun L,et al.Exosomes Derived from Akt-Modified Human Umbilical Cord Mesenchymal Stem Cells Improve Cardiac Regeneration and Promote Angiogenesis via Activating Platelet-Derived Growth Factor D.Stem Cells Transl Med.2017;6(1):51-59.
[7]Guo ZY,Sun X,Xu XL,et al.Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms.Neural Regen Res.2015;10(4):651-658.
[8]Li Z,Qin H,Feng Z,et al.Human umbilical cord mesenchymal stem cell-loaded amniotic membrane for the repair of radial nerve injury.Neural Regen Res.2013;8(36):3441-3448.
[9]Xin H,Li Y,Buller B,et al.Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.Stem Cells.2012;30(7):1556-1564.
[10]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-κB signaling in an IL-6-dependent manner.JNeuroinflammation.2017;14(1):231.
[11]Wang YF,Fan ZK,Cao Y,et al.2-Methoxyestradiol inhibits the up-regulation of AQP4 and AQP1 expression after spinal cord injury.Brain Res.2011;1370:220-226.
[12]Zheng Y,Wang L,Chen M,et al.Upregulation of miR-130b protects against cerebral ischemic injury by targeting water channel protein aquaporin 4(AQP4).Am J Transl Res.2017;9(7):3452-3461.
[13]Zeng Z,Leng T,Feng X,et al.Silencing TRPM7 in mouse cortical astrocytes impairs cell proliferation and migration via ERK and JNK signaling pathways.PLoS One.2015;10(3):e0119912.
[14]李浩阳,陈旭,李强,等.人脐带华通胶间充质干细胞对白细胞介素1β诱导髓核细胞退变的保护作用[J].中国组织工程研究,2017,21(25):3943-3948.
[15]Caradec J,Kharmate G,Hosseini-Beheshti E,et al.Reproducibility and efficiency of serum-derived exosome extraction methods.Clin Biochem.2014;47(13-14):1286-1292.
[16]Alvarez ML,Khosroheidari M,Kanchi Ravi R,et al.Comparison of protein,microRNA,and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.Kidney Int.2012;82(9):1024-1032.
[17]宋君来,李满,孙麟,等.抑制高迁移率族蛋白1减轻大鼠脊髓星形胶质细胞氧糖剥夺/复氧后的损伤[J].中国组织工程研究,2018,22(28):4537-4543.
[18]Jian Z,Ding S,Deng H,et al.Probenecid protects against oxygen-glucose deprivation injury in primary astrocytes by regulating inflammasome activity.Brain Res.2016;1643:123-129.
[19]Ou-Yang L,Liu Y,Wang BY,et al.Carnosine suppresses oxygen-glucose deprivation/recovery-induced proliferation and migration of reactive astrocytes of rats in vitro.Acta Pharmacol Sin.2018;39(1):24-34.
[20]Takebayashi H,Nabeshima Y,Yoshida S,et al.The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages.Curr Biol.2002;12(13):1157-1163.
[21]Jian Z,Ding S,Deng H,et al.Probenecid protects against oxygen-glucose deprivation injury in primary astrocytes by regulating inflammasome activity.Brain Res.2016;1643:123-129.
[22]Ti D,Hao H,Tong C,et al.LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b.J Transl Med.2015;13:308.
[23]Rosselli DD,Mumaw JL,Dickerson V,et al.Efficacy of allogeneic mesenchymal stem cell administration in a model of acute ischemic kidney injury in cats.Res Vet Sci.2016;108:18-24.
[24]Wang D,Wang S,Ji B,et al.Spatiotemporal expression of FOXA1correlates with reactive gliosis after spinal cord injury.Neuropeptides.2017;66:36-44.
[25]Nathan FM,Li S.Environmental cues determine the fate of astrocytes after spinal cord injury.Neural Regen Res.2017;12(12):1964-1970.
[26]Abdullah M,Takase H,Nunome M,et al.Amyloid-βReduces Exosome Release from Astrocytes by Enhancing JNKPhosphorylation.J Alzheimers Dis.2016;53(4):1433-1441.
[27]王天仪,刘勇,原文琦,等.微小RNA-199a-5p下调后通过c-jun氨基端激酶通路促进脊髓后索损伤修复的研究[J].中华实验外科杂志,2015,32(7):1498-1502.
[28]Tang Z,Sun X,Huo G,et al.Protective effects of erythropoietin on astrocytic swelling after oxygen-glucose deprivation and reoxygenation:mediation through AQP4 expression and MAPKpathway.Neuropharmacology.2013;67:8-15.
[29]Buffo A,Rolando C,Ceruti S.Astrocytes in the damaged brain:molecular and cellular insights into their reactive response and healing potential.Biochem Pharmacol.2010;79(2):77-89.
[30]Cho YE,Seo W,Kim DK,et al.Exogenous exosomes from mice with acetaminophen-induced liver injury promote toxicity in the recipient hepatocytes and mice.Sci Rep.2018;8(1):16070.
[31]Jia C,Keasey MP,Lovins C,et al.Inhibition of astrocyte FAK-JNKsignaling promotes subventricular zone neurogenesis through CNTF.Glia.2018;66(11):2456-2469.
[2]Kasuya Y,Umezawa H,Hatano M.Stress-Activated Protein Kinases in Spinal Cord Injury:Focus on Roles of p38.Int J Mol Sci.2018;19(3):E867.
[3]He FY,Feng WZ,Zhong J,et al.Effects of propofol and dexmedetomidine anesthesia on Th1/Th2 of rat spinal cord injury.Eur Rev Med Pharmacol Sci.2017;21(6):1355-1361.
[4]Yang J,Zhang X,Chen X,et al.Exosome Mediated Delivery of miR-124 Promotes Neurogenesis after Ischemia.Mol Ther Nucleic Acids.2017;7:278-287.
[5]Qi J,Zhou Y,Jiao Z,et al.Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway.Cell Physiol Biochem.2017;42(6):2242-2254.
[6]Ma J,Zhao Y,Sun L,et al.Exosomes Derived from Akt-Modified Human Umbilical Cord Mesenchymal Stem Cells Improve Cardiac Regeneration and Promote Angiogenesis via Activating Platelet-Derived Growth Factor D.Stem Cells Transl Med.2017;6(1):51-59.
[7]Guo ZY,Sun X,Xu XL,et al.Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms.Neural Regen Res.2015;10(4):651-658.
[8]Li Z,Qin H,Feng Z,et al.Human umbilical cord mesenchymal stem cell-loaded amniotic membrane for the repair of radial nerve injury.Neural Regen Res.2013;8(36):3441-3448.
[9]Xin H,Li Y,Buller B,et al.Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.Stem Cells.2012;30(7):1556-1564.
[10]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-κB signaling in an IL-6-dependent manner.JNeuroinflammation.2017;14(1):231.
[11]Wang YF,Fan ZK,Cao Y,et al.2-Methoxyestradiol inhibits the up-regulation of AQP4 and AQP1 expression after spinal cord injury.Brain Res.2011;1370:220-226.
[12]Zheng Y,Wang L,Chen M,et al.Upregulation of miR-130b protects against cerebral ischemic injury by targeting water channel protein aquaporin 4(AQP4).Am J Transl Res.2017;9(7):3452-3461.
[13]Zeng Z,Leng T,Feng X,et al.Silencing TRPM7 in mouse cortical astrocytes impairs cell proliferation and migration via ERK and JNK signaling pathways.PLoS One.2015;10(3):e0119912.
[14]李浩阳,陈旭,李强,等.人脐带华通胶间充质干细胞对白细胞介素1β诱导髓核细胞退变的保护作用[J].中国组织工程研究,2017,21(25):3943-3948.
[15]Caradec J,Kharmate G,Hosseini-Beheshti E,et al.Reproducibility and efficiency of serum-derived exosome extraction methods.Clin Biochem.2014;47(13-14):1286-1292.
[16]Alvarez ML,Khosroheidari M,Kanchi Ravi R,et al.Comparison of protein,microRNA,and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.Kidney Int.2012;82(9):1024-1032.
[17]宋君来,李满,孙麟,等.抑制高迁移率族蛋白1减轻大鼠脊髓星形胶质细胞氧糖剥夺/复氧后的损伤[J].中国组织工程研究,2018,22(28):4537-4543.
[18]Jian Z,Ding S,Deng H,et al.Probenecid protects against oxygen-glucose deprivation injury in primary astrocytes by regulating inflammasome activity.Brain Res.2016;1643:123-129.
[19]Ou-Yang L,Liu Y,Wang BY,et al.Carnosine suppresses oxygen-glucose deprivation/recovery-induced proliferation and migration of reactive astrocytes of rats in vitro.Acta Pharmacol Sin.2018;39(1):24-34.
[20]Takebayashi H,Nabeshima Y,Yoshida S,et al.The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages.Curr Biol.2002;12(13):1157-1163.
[21]Jian Z,Ding S,Deng H,et al.Probenecid protects against oxygen-glucose deprivation injury in primary astrocytes by regulating inflammasome activity.Brain Res.2016;1643:123-129.
[22]Ti D,Hao H,Tong C,et al.LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b.J Transl Med.2015;13:308.
[23]Rosselli DD,Mumaw JL,Dickerson V,et al.Efficacy of allogeneic mesenchymal stem cell administration in a model of acute ischemic kidney injury in cats.Res Vet Sci.2016;108:18-24.
[24]Wang D,Wang S,Ji B,et al.Spatiotemporal expression of FOXA1correlates with reactive gliosis after spinal cord injury.Neuropeptides.2017;66:36-44.
[25]Nathan FM,Li S.Environmental cues determine the fate of astrocytes after spinal cord injury.Neural Regen Res.2017;12(12):1964-1970.
[26]Abdullah M,Takase H,Nunome M,et al.Amyloid-βReduces Exosome Release from Astrocytes by Enhancing JNKPhosphorylation.J Alzheimers Dis.2016;53(4):1433-1441.
[27]王天仪,刘勇,原文琦,等.微小RNA-199a-5p下调后通过c-jun氨基端激酶通路促进脊髓后索损伤修复的研究[J].中华实验外科杂志,2015,32(7):1498-1502.
[28]Tang Z,Sun X,Huo G,et al.Protective effects of erythropoietin on astrocytic swelling after oxygen-glucose deprivation and reoxygenation:mediation through AQP4 expression and MAPKpathway.Neuropharmacology.2013;67:8-15.
[29]Buffo A,Rolando C,Ceruti S.Astrocytes in the damaged brain:molecular and cellular insights into their reactive response and healing potential.Biochem Pharmacol.2010;79(2):77-89.
[30]Cho YE,Seo W,Kim DK,et al.Exogenous exosomes from mice with acetaminophen-induced liver injury promote toxicity in the recipient hepatocytes and mice.Sci Rep.2018;8(1):16070.
[31]Jia C,Keasey MP,Lovins C,et al.Inhibition of astrocyte FAK-JNKsignaling promotes subventricular zone neurogenesis through CNTF.Glia.2018;66(11):2456-2469.