骨髓间充质干细胞移植联用山胡椒叶提取物可改善脊髓损伤区域的炎症反应
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摘要
背景:研究显示,骨髓间充质干细胞积极早期治疗脊髓损伤具有很高的研究价值。由于山胡椒叶富含黄酮、皂甙等物质,具有清除自由基、抗脂质过氧化等作用。两者联用治疗脊髓损伤的研究尚属罕见。目的:观察骨髓间充质干细胞移植联合山胡椒叶提取物治疗脊髓损伤的效果,并探索其机制。方法:将52只成年SD大鼠随机分为假手术组(n=12)、脊髓损伤组(n=14)、单纯骨髓间充质干细胞组(n=13)及联合治疗组(n=13)。脊髓损伤组、单纯骨髓间充质干细胞组及联合治疗组以Allen’s打击法建立脊髓损伤模型,假手术组仅暴露脊髓,不进行Allen’s打击。造模后即刻单纯骨髓间充质干细胞组在距损伤脊髓中线0.5 mm处注射骨髓间充质干细胞1.5×10~5个,联合治疗组在注射骨髓间充质干细胞基础上,灌胃5 mg/kg山胡椒叶提取物,1次/d,连续7 d。在造模后1,5,7 d,每组各取4只大鼠进行BBB评分、脊髓细胞凋亡计数,免疫组化法检测脊髓中肿瘤坏死因子α表达。结果与结论:①与脊髓损伤组相比,单纯骨髓间充质干细胞组、联合治疗组大鼠后肢功能开始逐渐恢复,BBB评分在造模后5,7 d有所上升(P <0.05或P <0.01),联合治疗组造模后5,7 d的BBB评分明显高于单纯骨髓间充质干细胞组(P <0.05);②脊髓损伤大鼠经骨髓间充质干细胞联合山胡椒叶提取物治疗后,损伤部位的组织水肿、坏死等迹象明显减轻,凋亡细胞数量明显减少,肿瘤坏死因子α表达明显下降,上述指标均优于单纯骨髓间充质干细胞组;③结果表明,骨髓间充质干细胞移植联用胡椒叶提取物可有效治疗脊髓损伤,且其作用可能与减轻肿瘤坏死因子α介导的炎症反应有关。
BACKGROUND: Studies have shown that bone marrow mesenchymal stem cells have a high value in the early treatment of spinal cord injury.Lindera glauca leaves rich in flavonoids, saponins and other substances can scavenge free radicals and suppress lipid peroxidation. However,the combination treatment of bone marrow mesenchymal stem cells and Lindera glauca leaf extract in the spinal cord injury model is still rare.OBJECTIVE: To observe the effect and mechanism of bone marrow mesenchymal stem cell transplantation combined with Lindera glauca leaf extract on spinal cord injury.METHODS: Fifty-two adult Sprague-Dawley rats were randomly divided into sham operation(n=12), spinal cord injury(n=14), cell transplantation(n=13) and combination treatment(n=13) groups. The spinal cord injury model was established by the Allen's method in the rats of spinal cord injury, cell transplantation and combination treatment groups. In the sham operation group, the spinal cord was only exposed but not subjected to the Allen's striking method. The rats in the cell transplantation and combination treatment groups were administered bone marrow mesenchymal stem cells(1.5×105) 0.5 mm away from the midline of the injured spinal cord. Moreover, the rats in the combination treatment group were then intragastrically given 5 mg/kg Lindera glauca leaf extract, once a day for 7 continuous days. At 1, 5,7 days after model establishment, 4 rats from each group were used for Basso, Beattie and Bresnahan score and spinal cord cell apoptosis counting, and the expression of tumor necrosis factor-α in the spinal cord was detected by immunohistochemistry.RESULTS AND CONCLUSION:(1) Compared with the spinal cord injury group, the hindlimb motor function was significantly restored in the rats of cell transplantation and combination treatment groups. The Basso, Beattie and Bresnahan scores in the cell transplantation and combination treatment groups were markedly higher than those in the spinal cord injury group at 5 and 7 days after modeling(P < 0.05 or P <0.01). And the Basso, Beattie and Bresnahan scores in the combination treatment group were higher than those in the cell transplantation group(P < 0.05).(2) After treatment, the rats in the combination treatment group exhibited significantly reduced signs of tissue edema and necrosis at the injury site, in which the number of apoptotic cells and the expression of tumor necrosis factor α were significantly decreased.All the sighs in the combination treatment group were improved as compared with the cell transplantation group. These findings indicate that bone marrow mesenchymal stem cell transplantation combined with Lindera glauca leaf extract can treat the spinal cord injury effectively, and its effect may be related to the reduction of tumor necrosis factor α-mediated inflammatory response.
BACKGROUND: Studies have shown that bone marrow mesenchymal stem cells have a high value in the early treatment of spinal cord injury.Lindera glauca leaves rich in flavonoids, saponins and other substances can scavenge free radicals and suppress lipid peroxidation. However,the combination treatment of bone marrow mesenchymal stem cells and Lindera glauca leaf extract in the spinal cord injury model is still rare.OBJECTIVE: To observe the effect and mechanism of bone marrow mesenchymal stem cell transplantation combined with Lindera glauca leaf extract on spinal cord injury.METHODS: Fifty-two adult Sprague-Dawley rats were randomly divided into sham operation(n=12), spinal cord injury(n=14), cell transplantation(n=13) and combination treatment(n=13) groups. The spinal cord injury model was established by the Allen's method in the rats of spinal cord injury, cell transplantation and combination treatment groups. In the sham operation group, the spinal cord was only exposed but not subjected to the Allen's striking method. The rats in the cell transplantation and combination treatment groups were administered bone marrow mesenchymal stem cells(1.5×105) 0.5 mm away from the midline of the injured spinal cord. Moreover, the rats in the combination treatment group were then intragastrically given 5 mg/kg Lindera glauca leaf extract, once a day for 7 continuous days. At 1, 5,7 days after model establishment, 4 rats from each group were used for Basso, Beattie and Bresnahan score and spinal cord cell apoptosis counting, and the expression of tumor necrosis factor-α in the spinal cord was detected by immunohistochemistry.RESULTS AND CONCLUSION:(1) Compared with the spinal cord injury group, the hindlimb motor function was significantly restored in the rats of cell transplantation and combination treatment groups. The Basso, Beattie and Bresnahan scores in the cell transplantation and combination treatment groups were markedly higher than those in the spinal cord injury group at 5 and 7 days after modeling(P < 0.05 or P <0.01). And the Basso, Beattie and Bresnahan scores in the combination treatment group were higher than those in the cell transplantation group(P < 0.05).(2) After treatment, the rats in the combination treatment group exhibited significantly reduced signs of tissue edema and necrosis at the injury site, in which the number of apoptotic cells and the expression of tumor necrosis factor α were significantly decreased.All the sighs in the combination treatment group were improved as compared with the cell transplantation group. These findings indicate that bone marrow mesenchymal stem cell transplantation combined with Lindera glauca leaf extract can treat the spinal cord injury effectively, and its effect may be related to the reduction of tumor necrosis factor α-mediated inflammatory response.
引文
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[2]Sabapathy V,Tharion G,Kumar S.Cell Therapy Augments Functional Recovery Subsequent to Spinal Cord Injury under Experimental Conditions.Stem Cells Int.2015;2015:132172.
[3]Wang L,Wang Q,Zhang XM.Progress on bone marrow mesenchymal stem cells transplantation for spinal cord injury.Zhongguo Gu Shang.2014;27(5):437-440.
[4]Rasini V,Dominici M,Kluba T,et al.Mesenchymal stromal/stem cells markers in the human bone marrow.Cytotherapy.2013;15(3):292-306.
[5]Tang C,Jin C,Du X,et al.An autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold applied with bone marrow stimulation for cartilage repair.Tissue Eng Part A.2014;20(17-18):2455-2462.
[6]Han D,Wu C,Xiong Q,et al.Anti-inflammatory Mechanism of Bone Marrow Mesenchymal Stem Cell Transplantation in Rat Model of Spinal Cord Injury.Cell Biochem Biophys.2015;71(3):1341-1347.
[7]Jia Y,Wu D,Zhang R,et al.Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats.Neurosci Lett.2014;573:46-51.
[8]Cantinieaux D,Quertainmont R,Blacher S,et al.Conditioned medium from bone marrow-derived mesenchymal stem cells improves recovery after spinal cord injury in rats:an original strategy to avoid cell transplantation.PLoS One.2013;8(8):e69515.
[9]孙慧玲,王俊霞,顾雪竹,等.山胡椒叶及果实挥发性成分分析[J].中国实验方剂学杂志,2011,17(7):94-97.
[10]张嘉穗,孔琳,雷洁萍,等.四川山胡椒叶抗氧化作用研究[J].现代中药研究与实践,2015,29(6):29-30,33.
[11]孔琳.绒毛山胡椒和四川山胡椒叶挥发油提取及活性研究[D].成都:西南交通大学,2014.
[12]Stanko P,Kaiserova K,Altanerova V,et al.Comparison of human mesenchymal stem cells derived from dental pulp,bone marrow,adipose tissue,and umbilical cord tissue by gene expression.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub.2014;158(3):373-377.
[13]Lee WJ,Hah YS,Ock SA,et al.Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs.Exp Cell Res.2015;333(2):273-288.
[14]Song K,Huang M,Shi Q,et al.Cultivation and identification of rat bone marrow-derived mesenchymal stem cells.Mol Med Rep.2014;10(2):755-760.
[15]Ye J,Gong P,Zhou F,et al.Culture and identification of human bone marrow mesenchymal stem cells from alveolar ridge dental implant site.J Craniofac Surg.2013;24(5):1539-1543.
[16]Chen XZ,Zhang JY,Zeng JY,et al.Differentiation of rat bone marrow mesenchymal stem cells into adipocytes and blockage of the differentiation.Zhonghua Zhong Liu Za Zhi.2012;34(11):826-830.
[17]Hu JZ,Long H,Wu TD,et al.The effect of estrogen-related receptorαon the regulation of angiogenesis after spinal cord injury.Neuroscience.2015;290:570-580.
[18]Basso DM,Beattie MS,Bresnahan JC.A sensitive and reliable locomotor rating scale for open field testing in rats.J Neurotrauma.1995;12(1):1-21.
[19]Li G,Yu F,Lei T,et al.Bone marrow mesenchymal stem cell therapy in ischemic stroke:mechanisms of action and treatment optimization strategies.Neural Regen Res.2016;11(6):1015-1024.
[20]Zemel'ko VI,Kozhukharova IV,Kovaleva ZV,et al.BDNF secretion in human mesenchymal stem cells isolated from bone marrow,endometrium and adipose tissue.Tsitologiia.2014;56(3):204-211.
[21]Jeong CH,Kim SM,Lim JY,et al.Mesenchymal stem cel s expressing brain-derived neurotrophic factor enhance endogenous neurogenesis in an ischemic stroke model.Biomed Res Int.2014;2014:129145.
[22]Wei L,Fraser JL,Lu ZY,et al.Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats.Neurobiol Dis.2012;46(3):635-645.
[23]Hao L,Hao J,Fang W,et al.Dual isotope simultaneous imaging to evaluate the effects of intracoronary bone marrow-derived mesenchymal stem cells on perfusion and metabolism in canines with acute myocardial infarction.Biomed Rep.2015;3(4):447-452.
[24]Ramalho BDS,Almeida FM,Sales CM,et al.Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice.Neural Regen Res.2018;13(6):1046-1053.
[25]刘立鼎,顾静文,陈京达.山胡椒叶子化学成分及其应用[J].江西科学,1992(1):38-44.
[26]张荣玉,赵德化,盛宝恒.山胡椒等三种中药挥发油的药理观察[J].第四军医大学学报,1985(3):200-204,273-274.
[27]王玉兰,高贤明,余孝平,等.我国山胡椒资源及其开发利用的研究[J].河南科学,1994(4):331-334.
[28]Schültke E,Kendall E,Kamencic H,et al.Quercetin promotes functional recovery following acute spinal cord injury.JNeurotrauma.2003;20(6):583-591.
[29]Fu J,Sun H,Zhang Y,et al.Neuroprotective Effects of Luteolin Against Spinal Cord Ischemia-Reperfusion Injury by Attenuation of Oxidative Stress,Inflammation,and Apoptosis.J Med Food.2018;21(1):13-20.
[30]陈秉耀,游思维,刘惠玲,等.Tarlov及BBB运动功能评估体系的比较[J].中华外科杂志,2002,40(3):236.
[31]陈向荣,游思维,金大地.BBB评分评估脊髓损伤大鼠后肢运动功能的探讨[J].中国脊柱脊髓杂志,2004,14(9):547-549.
[32]于德水,吕刚,梅晰凡,等.骨髓基质细胞移植对大鼠脊髓损伤后神经细胞凋亡的影响[J].辽宁医学院学报,2009,30(2):100-101,159,
[33]吴永超,郑启新,谢宗平,等.骨髓间充质干细胞表达神经营养因子及治疗脊髓损伤的研究[J].中华实验外科杂志,2005,22(2):139-141.
[34]Cizkova D,Cubinkova V,Smolek T,et al.Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury.Int J Mol Sci.2018;19(3):E870.
[35]Pu Y,Meng K,Gu C,et al.Thrombospondin-1 modified bone marrow mesenchymal stem cells(BMSCs)promote neurite outgrowth and functional recovery in rats with spinal cord injury.Oncotarget.2017;8(56):96276-96289.
[36]Ciccarelli F,De Martinis M,Sirufo MM,et al.Psoriasis Induced by Anti-Tumor Necrosis Factor Alpha Agents:A Comprehensive Review of the Literature.Acta Dermatovenerol Croat.2016;24(3):169-174.
[37]Cordero-Coma M,Sobrin L.Anti-tumor necrosis factor-αtherapy in uveitis.Surv Ophthalmol.2015;60(6):575-589.
[38]Peng RJ,Jiang B,Ding XP,et al.Effect of TNF-αInhibition on Bone Marrow-Derived Mesenchymal Stem Cells in Neurological Function Recovery after Spinal Cord Injury via the Wnt Signaling Pathway in a Rat Model.Cell Physiol Biochem.2017;42(2):743-752.
[39]Yang XM,Cheng YY,Zhang ZL,et al.Role of Methylprednisolone in Treatment of Spinal Cord Injured with Bone Marrow Mesenchymal Stem Cells Transplantation in Rats and Its Effect on the Expressions of Tumor Necrosis Factor-αand Interleukin-1β.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2017;39(5):615-622.
[40]康聪,孟宪勇,杨新明,等.小剂量雌激素肌肉注射联合骨髓间充质干细胞移植修复脊髓损伤[J].中国组织工程研究,2017,21(25):4020-4025.
[41]Li J,Guo W,Xiong M,et al.Effect of SDF-1/CXCR4 axis on the migration of transplanted bone mesenchymal stem cells mobilized by erythropoietin toward lesion sites following spinal cord injury.Int J Mol Med.2015;36(5):1205-1214.
[2]Sabapathy V,Tharion G,Kumar S.Cell Therapy Augments Functional Recovery Subsequent to Spinal Cord Injury under Experimental Conditions.Stem Cells Int.2015;2015:132172.
[3]Wang L,Wang Q,Zhang XM.Progress on bone marrow mesenchymal stem cells transplantation for spinal cord injury.Zhongguo Gu Shang.2014;27(5):437-440.
[4]Rasini V,Dominici M,Kluba T,et al.Mesenchymal stromal/stem cells markers in the human bone marrow.Cytotherapy.2013;15(3):292-306.
[5]Tang C,Jin C,Du X,et al.An autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold applied with bone marrow stimulation for cartilage repair.Tissue Eng Part A.2014;20(17-18):2455-2462.
[6]Han D,Wu C,Xiong Q,et al.Anti-inflammatory Mechanism of Bone Marrow Mesenchymal Stem Cell Transplantation in Rat Model of Spinal Cord Injury.Cell Biochem Biophys.2015;71(3):1341-1347.
[7]Jia Y,Wu D,Zhang R,et al.Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats.Neurosci Lett.2014;573:46-51.
[8]Cantinieaux D,Quertainmont R,Blacher S,et al.Conditioned medium from bone marrow-derived mesenchymal stem cells improves recovery after spinal cord injury in rats:an original strategy to avoid cell transplantation.PLoS One.2013;8(8):e69515.
[9]孙慧玲,王俊霞,顾雪竹,等.山胡椒叶及果实挥发性成分分析[J].中国实验方剂学杂志,2011,17(7):94-97.
[10]张嘉穗,孔琳,雷洁萍,等.四川山胡椒叶抗氧化作用研究[J].现代中药研究与实践,2015,29(6):29-30,33.
[11]孔琳.绒毛山胡椒和四川山胡椒叶挥发油提取及活性研究[D].成都:西南交通大学,2014.
[12]Stanko P,Kaiserova K,Altanerova V,et al.Comparison of human mesenchymal stem cells derived from dental pulp,bone marrow,adipose tissue,and umbilical cord tissue by gene expression.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub.2014;158(3):373-377.
[13]Lee WJ,Hah YS,Ock SA,et al.Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs.Exp Cell Res.2015;333(2):273-288.
[14]Song K,Huang M,Shi Q,et al.Cultivation and identification of rat bone marrow-derived mesenchymal stem cells.Mol Med Rep.2014;10(2):755-760.
[15]Ye J,Gong P,Zhou F,et al.Culture and identification of human bone marrow mesenchymal stem cells from alveolar ridge dental implant site.J Craniofac Surg.2013;24(5):1539-1543.
[16]Chen XZ,Zhang JY,Zeng JY,et al.Differentiation of rat bone marrow mesenchymal stem cells into adipocytes and blockage of the differentiation.Zhonghua Zhong Liu Za Zhi.2012;34(11):826-830.
[17]Hu JZ,Long H,Wu TD,et al.The effect of estrogen-related receptorαon the regulation of angiogenesis after spinal cord injury.Neuroscience.2015;290:570-580.
[18]Basso DM,Beattie MS,Bresnahan JC.A sensitive and reliable locomotor rating scale for open field testing in rats.J Neurotrauma.1995;12(1):1-21.
[19]Li G,Yu F,Lei T,et al.Bone marrow mesenchymal stem cell therapy in ischemic stroke:mechanisms of action and treatment optimization strategies.Neural Regen Res.2016;11(6):1015-1024.
[20]Zemel'ko VI,Kozhukharova IV,Kovaleva ZV,et al.BDNF secretion in human mesenchymal stem cells isolated from bone marrow,endometrium and adipose tissue.Tsitologiia.2014;56(3):204-211.
[21]Jeong CH,Kim SM,Lim JY,et al.Mesenchymal stem cel s expressing brain-derived neurotrophic factor enhance endogenous neurogenesis in an ischemic stroke model.Biomed Res Int.2014;2014:129145.
[22]Wei L,Fraser JL,Lu ZY,et al.Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats.Neurobiol Dis.2012;46(3):635-645.
[23]Hao L,Hao J,Fang W,et al.Dual isotope simultaneous imaging to evaluate the effects of intracoronary bone marrow-derived mesenchymal stem cells on perfusion and metabolism in canines with acute myocardial infarction.Biomed Rep.2015;3(4):447-452.
[24]Ramalho BDS,Almeida FM,Sales CM,et al.Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice.Neural Regen Res.2018;13(6):1046-1053.
[25]刘立鼎,顾静文,陈京达.山胡椒叶子化学成分及其应用[J].江西科学,1992(1):38-44.
[26]张荣玉,赵德化,盛宝恒.山胡椒等三种中药挥发油的药理观察[J].第四军医大学学报,1985(3):200-204,273-274.
[27]王玉兰,高贤明,余孝平,等.我国山胡椒资源及其开发利用的研究[J].河南科学,1994(4):331-334.
[28]Schültke E,Kendall E,Kamencic H,et al.Quercetin promotes functional recovery following acute spinal cord injury.JNeurotrauma.2003;20(6):583-591.
[29]Fu J,Sun H,Zhang Y,et al.Neuroprotective Effects of Luteolin Against Spinal Cord Ischemia-Reperfusion Injury by Attenuation of Oxidative Stress,Inflammation,and Apoptosis.J Med Food.2018;21(1):13-20.
[30]陈秉耀,游思维,刘惠玲,等.Tarlov及BBB运动功能评估体系的比较[J].中华外科杂志,2002,40(3):236.
[31]陈向荣,游思维,金大地.BBB评分评估脊髓损伤大鼠后肢运动功能的探讨[J].中国脊柱脊髓杂志,2004,14(9):547-549.
[32]于德水,吕刚,梅晰凡,等.骨髓基质细胞移植对大鼠脊髓损伤后神经细胞凋亡的影响[J].辽宁医学院学报,2009,30(2):100-101,159,
[33]吴永超,郑启新,谢宗平,等.骨髓间充质干细胞表达神经营养因子及治疗脊髓损伤的研究[J].中华实验外科杂志,2005,22(2):139-141.
[34]Cizkova D,Cubinkova V,Smolek T,et al.Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury.Int J Mol Sci.2018;19(3):E870.
[35]Pu Y,Meng K,Gu C,et al.Thrombospondin-1 modified bone marrow mesenchymal stem cells(BMSCs)promote neurite outgrowth and functional recovery in rats with spinal cord injury.Oncotarget.2017;8(56):96276-96289.
[36]Ciccarelli F,De Martinis M,Sirufo MM,et al.Psoriasis Induced by Anti-Tumor Necrosis Factor Alpha Agents:A Comprehensive Review of the Literature.Acta Dermatovenerol Croat.2016;24(3):169-174.
[37]Cordero-Coma M,Sobrin L.Anti-tumor necrosis factor-αtherapy in uveitis.Surv Ophthalmol.2015;60(6):575-589.
[38]Peng RJ,Jiang B,Ding XP,et al.Effect of TNF-αInhibition on Bone Marrow-Derived Mesenchymal Stem Cells in Neurological Function Recovery after Spinal Cord Injury via the Wnt Signaling Pathway in a Rat Model.Cell Physiol Biochem.2017;42(2):743-752.
[39]Yang XM,Cheng YY,Zhang ZL,et al.Role of Methylprednisolone in Treatment of Spinal Cord Injured with Bone Marrow Mesenchymal Stem Cells Transplantation in Rats and Its Effect on the Expressions of Tumor Necrosis Factor-αand Interleukin-1β.Zhongguo Yi Xue Ke Xue Yuan Xue Bao.2017;39(5):615-622.
[40]康聪,孟宪勇,杨新明,等.小剂量雌激素肌肉注射联合骨髓间充质干细胞移植修复脊髓损伤[J].中国组织工程研究,2017,21(25):4020-4025.
[41]Li J,Guo W,Xiong M,et al.Effect of SDF-1/CXCR4 axis on the migration of transplanted bone mesenchymal stem cells mobilized by erythropoietin toward lesion sites following spinal cord injury.Int J Mol Med.2015;36(5):1205-1214.
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