补阳还五汤对脊髓损伤后Ⅰ型胶原蛋白和Ⅳ型胶原蛋白表达的影响
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摘要
背景:研究表明,Ⅰ型胶原蛋白和Ⅳ型胶原蛋白有利于脊髓损伤后肢体运动功能的恢复。目的:通过观察脊髓损伤大鼠肢体运动功能变化及Ⅰ型胶原蛋白和Ⅳ型胶原蛋白的表达,探讨补阳还五汤对脊髓损伤后大鼠肢体运动功能改善的可能机制。方法:实验方案经福建中医药大学动物实验伦理委员会批准[批准号为SYXK(闽)2013-005]。将54只大鼠随机分为3组:假手组(n=18)、模型组(n=18)和补阳还五汤组(n=18)。假手术组切除椎板后立即缝合伤口,其余2组切除椎板后应用NYU脊髓打击器建立脊髓损伤模型。造模后第1天开始补阳还五汤组给予补阳还五汤1 mL灌胃,模型组和假手术组术后蒸馏水1 mL灌胃。干预后1,3,5,7 d对各组大鼠进行BBB肢体运动功能评分;干预7 d后取材,尼式染色观察脊髓神经元形态和免疫组织化学检测Ⅰ型胶原蛋白和Ⅳ型胶原蛋白表达;电镜观察脊髓髓鞘超微结构,Western blot检测Ⅰ型胶原蛋白和Ⅳ型胶原蛋白的蛋白表达量。结果与结论:(1)干预第5天后,补阳还五汤组的BBB肢体运动功能评分显著高于同期的模型组(P <0.05);(2)尼式染色显示补阳还五汤组的细胞形态较为完整,细胞水肿较轻,瘀血面积较小,神经元恢复情况好于模型组;(3)电镜实验发现补阳还五汤组的髓鞘形态较模型组完整,水肿情况较轻,与尼式染色结果一致;(4)免疫组织化学显示补阳还五汤组的Ⅰ型胶原蛋白和Ⅳ型胶原蛋白的表达量均较模型组显著增多(P <0.05);(5)Western blot显示补阳还五汤组的Ⅰ型胶原蛋白和Ⅳ型胶原蛋白的表达量均较模型组显著增多(P <0.05);(6)结果说明,补阳还五汤能够改善脊髓损伤后大鼠的肢体运动功能障碍,其机制可能与增加Ⅰ型胶原蛋白和Ⅳ型胶原蛋白表达有关。
BACKGROUND: Collagen type I and IV are shown to be beneficial for limb locomotion recovery after spinal cord injury.OBJECTIVE: To explore the mechanism of Buyang Huanwu decoction on the rehabilitation of limb locomotion of rats after spinal cord injury by observing the changes of limb locomotion and expression levels of collagen type I and IV.METHODS: The study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine,approval number: SYXK2013-005. Fifty-four Sprague-Dawley rats were divided into three groups, sham, model and Buyang Huanwu decoction groups(n=18/group). Rats in the sham group underwent the laminectomy and rats in the other groups were used for establishing the spinal cord injury model by NYU impactor. The rats in the Buyang Huanwu decoction group, and sham and model groups were administrated 1 mL of Buyang Huanwu decoction and normal saline via gavage, respectively, at 1 day post-injury. Basso, Beattie, and Bresnahan limb locomotion scores were evaluated at 1, 3, 5 and 7 days after injury. At 7 days after injury, samples were removed for Nissl staining to observe the morphology of spinal cord neurons and the expression levels of collagen type I and IV were detected by immunohistochemistry. The ultrastructures of medullary sheath were observed under transmission electron microscope. The expression levels of collagen type I and IV proteins were detected by western blot assay.RESULTS AND CONCLUSION:(1) The Basso, Beattie, and Bresnahan limb locomotion scores from the fifth day after modeling in the Buyang Huanwu decoction group were significantly higher than those in the model group(P < 0.05).(2) Nissl staining showed that in the Buyang Huanwu decoction group, the structure of neurons was complete, cell edema was slight, hemostasis area was small, and the recovery of neurons was better than that in the model group.(3) Transmission electron microscope showed that the structure of myelin sheath in the Buyang Huanwu decoction group was better than that in the model group, and with slight edema, which were similar with the Nissl staining results.(4) Immunohistochemistry results showed that the expression levels of collagen type I and IV in the Buyang Huanwu decoction group were significantly higher than those in the model group(P < 0.05).(5) Western blot assay results showed that expressions of collagen type I and IV proteins in the Buyang Huanwu decoction group were significantly higher than those in the model group(P < 0.05).(6) In summary,Buyang Huanwu decoction can promote the rehabilitation of limb locomotion of rats after spinal cord injury, possibly by up-regulating the expressions of collagen type I and IV.
BACKGROUND: Collagen type I and IV are shown to be beneficial for limb locomotion recovery after spinal cord injury.OBJECTIVE: To explore the mechanism of Buyang Huanwu decoction on the rehabilitation of limb locomotion of rats after spinal cord injury by observing the changes of limb locomotion and expression levels of collagen type I and IV.METHODS: The study was approved by the Experimental Animal Ethics Committee of Fujian University of Traditional Chinese Medicine,approval number: SYXK2013-005. Fifty-four Sprague-Dawley rats were divided into three groups, sham, model and Buyang Huanwu decoction groups(n=18/group). Rats in the sham group underwent the laminectomy and rats in the other groups were used for establishing the spinal cord injury model by NYU impactor. The rats in the Buyang Huanwu decoction group, and sham and model groups were administrated 1 mL of Buyang Huanwu decoction and normal saline via gavage, respectively, at 1 day post-injury. Basso, Beattie, and Bresnahan limb locomotion scores were evaluated at 1, 3, 5 and 7 days after injury. At 7 days after injury, samples were removed for Nissl staining to observe the morphology of spinal cord neurons and the expression levels of collagen type I and IV were detected by immunohistochemistry. The ultrastructures of medullary sheath were observed under transmission electron microscope. The expression levels of collagen type I and IV proteins were detected by western blot assay.RESULTS AND CONCLUSION:(1) The Basso, Beattie, and Bresnahan limb locomotion scores from the fifth day after modeling in the Buyang Huanwu decoction group were significantly higher than those in the model group(P < 0.05).(2) Nissl staining showed that in the Buyang Huanwu decoction group, the structure of neurons was complete, cell edema was slight, hemostasis area was small, and the recovery of neurons was better than that in the model group.(3) Transmission electron microscope showed that the structure of myelin sheath in the Buyang Huanwu decoction group was better than that in the model group, and with slight edema, which were similar with the Nissl staining results.(4) Immunohistochemistry results showed that the expression levels of collagen type I and IV in the Buyang Huanwu decoction group were significantly higher than those in the model group(P < 0.05).(5) Western blot assay results showed that expressions of collagen type I and IV proteins in the Buyang Huanwu decoction group were significantly higher than those in the model group(P < 0.05).(6) In summary,Buyang Huanwu decoction can promote the rehabilitation of limb locomotion of rats after spinal cord injury, possibly by up-regulating the expressions of collagen type I and IV.
引文
[1]Sharifalhoseini M,Rahimimovaghar V.Hospital-based incidence of traumatic spinal cord injury in tehran,iran.Iran J Public Health.2014;43(3):331-341.
[2]李念龙,于占革.骨髓间充质干细胞移植治疗脊髓损伤的研究进展[J].中国矫形外科杂志,2012,20(4):343-345.
[3]袁慧萍.中西医结合治疗急性脊椎损伤45例疗效观察[J].中国中医急症,2013,22(8):1386-1387.
[4]祁健,张俊江,孟庆溪,等.脊髓损伤的病理变化及治疗进展[J].现代生物医学进展,2017,17(21):4179-4183.
[5]Boulenguez P,Vinay L.Strategies to restore motor functions after spinal cord injury.Curr Opin Neurobiol.2009;19(6):587-600..
[6]成军.细胞外基质的分子生物学与临床疾病[M].北京医科大学出版社,1999.
[7]郭文荣,李兴.糖尿病肾病与ADPN?TGF-β1?Collagen IV?ICAM-1关系的研究进展[J].医学研究杂志,2015,44(6):170-173.
[8]Kurt G,Yildirim Z,Cemil B,et al.Effects of curcumin on acute spinal cord ischemia-reperfusion injury in rabbits.Laboratory investigation.JNeurosurg Spine.2014;20(4):464-470.
[9]石杜鹃,凌丽,薛金伟.补阳还五汤药浴对大鼠周围神经损伤再生影响的实验研究[J].时珍国医国药,2008,19(5):1066-1067.
[10]Chen WF,Chen CH,Chen NF,et al.Neuroprotective Effects of Direct Intrathecal Administration of Granulocyte Colony-Stimulating Factor in Rats with Spinal Cord Injury.CNS Neurosci Ther.2015;21(9):698-707.
[11]范筱,吴杨鹏,汪今朝,张俐.活血通督汤对脊髓损伤后BDNF/TrkB信号表达的影响[J].中华中医药杂志,2017,32(05):2115-2120.
[12]汪今朝,范筱,张俐.活血通督汤对脊髓损伤后胶质瘢痕形成的影响[J].中国中医骨伤科杂志,2017,25(8):1-5.
[13]侯兆阳,陈哲,魏家森.补阳还五汤对急性脊髓损伤模型大鼠神经细胞凋亡的影响[J].浙江中西医结合杂志,2015,25(01):8-11+106.
[14]Bareyre FM.Neuronal repair and replacement in spinal cord injury.JNeurol Sci.2008;265(1-2):63-72.
[15]吴杨鹏,范筱,张俐.急性脊髓损伤动物模型的建立与评估[J].中国组织工程研究,2016,20(49):7341-7348.
[16]Volarevic V,Erceg S,Bhattacharya SS,et al.Stem cell-based therapy for spinal cord injury.Cell Transplantation.2013;22(8):1309-1323.
[17]Young W.Spinal cord contusion models.Prog Brain Res.2002;137:231-255.
[18]Doble A.The role of excitotoxicity in neurodegenerative disease:implications for therapy.Pharmacol Ther.1999;81(3):163-221..
[19]Xiong Y,Rabchevsky AG,Hall ED.Role of peroxynitrite in secondary oxidative damage after spinal cord injury.J Neurochem.2007;100(3):639-649.
[20]Engelhardt B,Sorokin L.The blood-brain and the blood-cerebrospinal fluid barriers:function and dysfunction.Semin Immunopathol.2009;31(4):497-511.
[21]Erbayraktar Z,G?kmen N,Y?lmaz O,et al.Experimental traumatic spinal cord injury.Methods Mol Biol.2013;982:103-112.
[22]Gelse K,P?schl E,Aigner T.Collagens-structure,function,and biosynthesis.Advanced Drug Delivery Reviews.2003;55(12):1531-1546.
[23]Giannelli G,Bergamini C,Marinosci F,et al.Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma.Int J Cancer.2002;97(4):425-431.
[24]Sweeney SM,DiLullo G,Slater SJ,et al.Angiogenesis in Collagen IRequiresα2β1 Ligation of a GFP*GER Sequence and Possibly p38MAPK Activation and Focal Adhesion Disassembly.J Biol Chem.2003;278(33):30516-30524
[25]Verrecchia F,Mauviel A.TGF-beta and TNF-alpha:antagonistic cytokines controlling type I collagen gene expression.Cell Signal.2004;16(8):873-880.
[26]张峻,季欣然,唐佩福.应用生物材料促进脊髓损伤修复的研究进展[J].中国骨与关节杂志,2017,6(4):309-312.
[27]Cardoso FL,Brites D,Brito MA.Looking at the blood-brain barrier:molecular anatomy and possible investigation approaches.Brain Res Rev.2010;64(2):328-363.
[28]Takigawa T,Yonezawa T,Yoshitaka T,et al.Separation of the perivascular basement membrane provides a conduit for inflammatory cells in a mouse spinal cord injury model.J Neurotrauma.2010;27(4):739-751.
[29]Xian-Hui D,Xiao-Ping H,Wei-Juan G.Neuroprotective effects of the BuyangHuanwu decoction on functional recovery in rats following spinal cord injury.J Spinal Cord Med.2016;39(1):85-92.
[30]张继平,王志彬,林爱华,等.补阳还五汤对脊髓损伤大鼠脊髓组织血小板活化因子含量的影响[J].中国中医药信息杂志,2011,15(11):47-49.
[31]张继平,王志彬,林爱华,等.补阳还五汤对脊髓损伤大鼠脊髓组织病理学的影响[J].中国中西医结合外科杂志,2014,20(3):274-277.
[32]陈安,廖君,熊艾君,等.补阳还五汤对脊髓损伤后胶质瘢痕及GFAP表达的影响[J].世界中西医结合杂志,2007,2(10):570-572.
[33]陈安,张建伟,伍校琼,等.补阳还五汤对红核脊髓束横断后神经元的保护作用[J].中国临床解剖学杂志,2007,25(6):665-668.
[34]王伟,谢杰,方坚,等.补阳还五汤对实验性脊髓损伤大鼠水通道蛋白-4表达的影响[J].中国康复,2005,20(1):3-5.
[2]李念龙,于占革.骨髓间充质干细胞移植治疗脊髓损伤的研究进展[J].中国矫形外科杂志,2012,20(4):343-345.
[3]袁慧萍.中西医结合治疗急性脊椎损伤45例疗效观察[J].中国中医急症,2013,22(8):1386-1387.
[4]祁健,张俊江,孟庆溪,等.脊髓损伤的病理变化及治疗进展[J].现代生物医学进展,2017,17(21):4179-4183.
[5]Boulenguez P,Vinay L.Strategies to restore motor functions after spinal cord injury.Curr Opin Neurobiol.2009;19(6):587-600..
[6]成军.细胞外基质的分子生物学与临床疾病[M].北京医科大学出版社,1999.
[7]郭文荣,李兴.糖尿病肾病与ADPN?TGF-β1?Collagen IV?ICAM-1关系的研究进展[J].医学研究杂志,2015,44(6):170-173.
[8]Kurt G,Yildirim Z,Cemil B,et al.Effects of curcumin on acute spinal cord ischemia-reperfusion injury in rabbits.Laboratory investigation.JNeurosurg Spine.2014;20(4):464-470.
[9]石杜鹃,凌丽,薛金伟.补阳还五汤药浴对大鼠周围神经损伤再生影响的实验研究[J].时珍国医国药,2008,19(5):1066-1067.
[10]Chen WF,Chen CH,Chen NF,et al.Neuroprotective Effects of Direct Intrathecal Administration of Granulocyte Colony-Stimulating Factor in Rats with Spinal Cord Injury.CNS Neurosci Ther.2015;21(9):698-707.
[11]范筱,吴杨鹏,汪今朝,张俐.活血通督汤对脊髓损伤后BDNF/TrkB信号表达的影响[J].中华中医药杂志,2017,32(05):2115-2120.
[12]汪今朝,范筱,张俐.活血通督汤对脊髓损伤后胶质瘢痕形成的影响[J].中国中医骨伤科杂志,2017,25(8):1-5.
[13]侯兆阳,陈哲,魏家森.补阳还五汤对急性脊髓损伤模型大鼠神经细胞凋亡的影响[J].浙江中西医结合杂志,2015,25(01):8-11+106.
[14]Bareyre FM.Neuronal repair and replacement in spinal cord injury.JNeurol Sci.2008;265(1-2):63-72.
[15]吴杨鹏,范筱,张俐.急性脊髓损伤动物模型的建立与评估[J].中国组织工程研究,2016,20(49):7341-7348.
[16]Volarevic V,Erceg S,Bhattacharya SS,et al.Stem cell-based therapy for spinal cord injury.Cell Transplantation.2013;22(8):1309-1323.
[17]Young W.Spinal cord contusion models.Prog Brain Res.2002;137:231-255.
[18]Doble A.The role of excitotoxicity in neurodegenerative disease:implications for therapy.Pharmacol Ther.1999;81(3):163-221..
[19]Xiong Y,Rabchevsky AG,Hall ED.Role of peroxynitrite in secondary oxidative damage after spinal cord injury.J Neurochem.2007;100(3):639-649.
[20]Engelhardt B,Sorokin L.The blood-brain and the blood-cerebrospinal fluid barriers:function and dysfunction.Semin Immunopathol.2009;31(4):497-511.
[21]Erbayraktar Z,G?kmen N,Y?lmaz O,et al.Experimental traumatic spinal cord injury.Methods Mol Biol.2013;982:103-112.
[22]Gelse K,P?schl E,Aigner T.Collagens-structure,function,and biosynthesis.Advanced Drug Delivery Reviews.2003;55(12):1531-1546.
[23]Giannelli G,Bergamini C,Marinosci F,et al.Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma.Int J Cancer.2002;97(4):425-431.
[24]Sweeney SM,DiLullo G,Slater SJ,et al.Angiogenesis in Collagen IRequiresα2β1 Ligation of a GFP*GER Sequence and Possibly p38MAPK Activation and Focal Adhesion Disassembly.J Biol Chem.2003;278(33):30516-30524
[25]Verrecchia F,Mauviel A.TGF-beta and TNF-alpha:antagonistic cytokines controlling type I collagen gene expression.Cell Signal.2004;16(8):873-880.
[26]张峻,季欣然,唐佩福.应用生物材料促进脊髓损伤修复的研究进展[J].中国骨与关节杂志,2017,6(4):309-312.
[27]Cardoso FL,Brites D,Brito MA.Looking at the blood-brain barrier:molecular anatomy and possible investigation approaches.Brain Res Rev.2010;64(2):328-363.
[28]Takigawa T,Yonezawa T,Yoshitaka T,et al.Separation of the perivascular basement membrane provides a conduit for inflammatory cells in a mouse spinal cord injury model.J Neurotrauma.2010;27(4):739-751.
[29]Xian-Hui D,Xiao-Ping H,Wei-Juan G.Neuroprotective effects of the BuyangHuanwu decoction on functional recovery in rats following spinal cord injury.J Spinal Cord Med.2016;39(1):85-92.
[30]张继平,王志彬,林爱华,等.补阳还五汤对脊髓损伤大鼠脊髓组织血小板活化因子含量的影响[J].中国中医药信息杂志,2011,15(11):47-49.
[31]张继平,王志彬,林爱华,等.补阳还五汤对脊髓损伤大鼠脊髓组织病理学的影响[J].中国中西医结合外科杂志,2014,20(3):274-277.
[32]陈安,廖君,熊艾君,等.补阳还五汤对脊髓损伤后胶质瘢痕及GFAP表达的影响[J].世界中西医结合杂志,2007,2(10):570-572.
[33]陈安,张建伟,伍校琼,等.补阳还五汤对红核脊髓束横断后神经元的保护作用[J].中国临床解剖学杂志,2007,25(6):665-668.
[34]王伟,谢杰,方坚,等.补阳还五汤对实验性脊髓损伤大鼠水通道蛋白-4表达的影响[J].中国康复,2005,20(1):3-5.
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