补阳还五汤联合骨髓间充质干细胞移植脑缺血再灌注大鼠脑组织紧密连接蛋白的表达
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摘要
背景:急性脑缺血时内皮细胞间的紧密连接受到破坏,紧密连接蛋白转录或表达发生改变,引起血脑屏障通透性增加而形成脑水肿。目的:旨在从紧密连接蛋白的调控方面,探讨补阳还五汤联合骨髓间充质干细胞移植对脑缺血再灌注大鼠血脑屏障修复的作用机制。方法:80只大鼠(山东省动物实验中心提供)随机分为假手术组(n=16),模型组(n=16),骨髓间充质干细胞组(n=16),补阳还五汤组(n=16),补阳还五汤联合骨髓间充质干细胞组(联合组,n=16)。利用改良线栓法制备大脑中动脉栓塞缺血再灌注大鼠模型,造模前3 d开始,补阳还五汤组、联合组给予补阳还五汤15 mL/kg灌胃,假手术组、模型组、骨髓间充质干细胞组给予等量生理盐水灌胃;造模后1d时,骨髓间充质干细胞组、联合组尾静脉注射含1×10~6个骨髓间充质干细胞悬液1m L,其他组给予等量生理盐水。在造模后3,5d取缺血侧脑组织,采用Western Blot法检测脑组织中Occludin、Claudin-5、JAM-A、ZO-1等紧密连接相关蛋白的表达。结果与结论:(1)与假手术组相比,模型组大鼠缺血脑组织中Occludin、Claudin-5、JAM-A、ZO-1的蛋白表达显著下降(P<0.01);(2)与模型组相比,补阳还五汤组、骨髓间充质干细胞组和联合组缺血脑组织中Occludin、Claudin-5、JAM-A、ZO-1的蛋白表达量上升(P <0.05),以联合组上调最为明显(P <0.01);(3)结果表明,补阳还五汤联合骨髓间充质干细胞对血脑屏障的修复具有协同作用,可能是通过上调缺血脑组织中Occludin、Claudin、JAM-A、ZO-1的蛋白表达来维持血脑屏障紧密连接的完整性,以实现脑保护的作用。
BACKGROUND: In acute cerebral ischemia, the tight junction between endothelial cells is damaged, and variation in the transcription or expression of tight junction proteins increases the blood-brain barrier permeability eventually triggering the formation of brain edema. OBJECTIVE: To explore the effect mechanism by which Buyang Huanwu Decoction combined with bone marrow mesenchymal stem cell(BMSCs) transplantation treats a cerebral ischemia-reperfusion rat model via the blood brain barrier. METHODS: Eighty rats provided by the Animal Experimental Central of Shandong Province, China, were randomized into sham operation group(n=16), model group(n=16), transplantation group(n=16), Buyang Huanwu Decoction group(n=16), and Buyang Huanwu Decoction+transplantation group(combination group, n=16). Ischemia-reperfusion rat model was established via middle cerebral artery occlusion using improved Zea Longa's suture-occlusion method. In the Buyang Huanwu Decoction group and combination group, the rats were gavaged with 15 ml/kg Buyang Huanwu Decoction 3 days before modeling, while in the sham operation group, model group and transplantation group, the rats were gavaged with the equal volume of normal saline. The transplantation group and combination group were injected with bone marrow mesenchymal stem cell suspension(1 x106 mL, 1 mL) 1 day after modeling, and the other groups were gavaged with the equal volume of normal saline. The expression of Occludin, Claudin-5, Jam-A, and ZO-1 in ischemic brain tissues was detected by western blot assay at 3 and 5 days after modeling. RESULTS AND CONCLUSION: Compared with the sham operation group, the protein expression of Occludin, Claudin-5, Jam-A, and ZO-1 in the ischemic brain tissue of rats in the model group decreased significantly(P < 0.01). Compared with the model group, the protein expression levels of Occludin, Claudin-5, Jam-A, and ZO-1 in the rat ischemic brain tissue in the Buyang Huanwu Decoction group, transplantation group and combination group were up-regulated significantly(P < 0.05), with the most significant increase in the combination group(P < 0.01). Buyang Huanwu Decoction combined with bone marrow mesenchymal stem cell transplantation exerts a synergistic effect in blood-brain barrier repair, and keeps the tight junction with the blood-brain barrier through the upregulation of ischemic brain tissue Occludin, Claudin-5, Jam-A and ZO-1 protein expression, to achieve the brain protection.
BACKGROUND: In acute cerebral ischemia, the tight junction between endothelial cells is damaged, and variation in the transcription or expression of tight junction proteins increases the blood-brain barrier permeability eventually triggering the formation of brain edema. OBJECTIVE: To explore the effect mechanism by which Buyang Huanwu Decoction combined with bone marrow mesenchymal stem cell(BMSCs) transplantation treats a cerebral ischemia-reperfusion rat model via the blood brain barrier. METHODS: Eighty rats provided by the Animal Experimental Central of Shandong Province, China, were randomized into sham operation group(n=16), model group(n=16), transplantation group(n=16), Buyang Huanwu Decoction group(n=16), and Buyang Huanwu Decoction+transplantation group(combination group, n=16). Ischemia-reperfusion rat model was established via middle cerebral artery occlusion using improved Zea Longa's suture-occlusion method. In the Buyang Huanwu Decoction group and combination group, the rats were gavaged with 15 ml/kg Buyang Huanwu Decoction 3 days before modeling, while in the sham operation group, model group and transplantation group, the rats were gavaged with the equal volume of normal saline. The transplantation group and combination group were injected with bone marrow mesenchymal stem cell suspension(1 x106 mL, 1 mL) 1 day after modeling, and the other groups were gavaged with the equal volume of normal saline. The expression of Occludin, Claudin-5, Jam-A, and ZO-1 in ischemic brain tissues was detected by western blot assay at 3 and 5 days after modeling. RESULTS AND CONCLUSION: Compared with the sham operation group, the protein expression of Occludin, Claudin-5, Jam-A, and ZO-1 in the ischemic brain tissue of rats in the model group decreased significantly(P < 0.01). Compared with the model group, the protein expression levels of Occludin, Claudin-5, Jam-A, and ZO-1 in the rat ischemic brain tissue in the Buyang Huanwu Decoction group, transplantation group and combination group were up-regulated significantly(P < 0.05), with the most significant increase in the combination group(P < 0.01). Buyang Huanwu Decoction combined with bone marrow mesenchymal stem cell transplantation exerts a synergistic effect in blood-brain barrier repair, and keeps the tight junction with the blood-brain barrier through the upregulation of ischemic brain tissue Occludin, Claudin-5, Jam-A and ZO-1 protein expression, to achieve the brain protection.
引文
[1]Mac Gregor DG, Avshalumov MV, Rice ME. Brain edema induced by in vitro ischemia:causal factors and neuroprotection. J Neurochem.2003;85(6):1402-1411.
[2]Steeg PS, Camphausen KA, Smith QR. Brain metastases as preventive and therapeutic targets. Nat Rev Cancer. 2011;11(5):352-363.
[3]Day RE, Kitchen P, Owen DS, et al. Human aquaporins:regulators of transcellular water flow. Biochim Biophys Acta. 2014;1840(5):1492-1506.
[4]张运克,佟帅.补阳还五汤抗脑缺血及再灌注损伤作用机理研究进展[J].河南中医,2002,22(4):75-76,80.
[5]Haas S, Weidner N, Winkler J. Adult stem cell therapy in stroke. Curr Opin Neurol. 2005;18(1):59-64.
[6]Wang C, Fei Y, Xu C, et al. Bone marrow mesenchymal stem cells ameliorate neurological deficits and blood-brain barrier dysfunction after intracerebral hemorrhage in spontaneously hypertensive rats. Int J Clin Exp Pathol. 2015;8(5):4715-4724.
[7]Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A. 1999;96(19):10711-10716.
[8]Liu H, Honmou O, Harada K, et al. Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia. Brain. 2006;129(Pt 10):2734-2745.
[9]Ito M, Kuroda S, Sugiyama T, et al. Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats. Neuropathology. 2012;32(5):522-533.
[10]Zhang YK, Han XY, Che ZY. Effects of buyang huanwu tang combined with bone marrow mesenchymal stem cell transplantation on the expression of VEGF and Ki-67 in the brain tissue of the cerebral ischemia-reperfusion model rat. J Tradit Chin Med. 2010;30(4):278-282.
[11]张运克,高峰,张丹,等.补阳还五汤联合骨髓间充质干细胞移植对脑缺血再灌注大鼠脑组织NSE和GFAP表达的影响[J].中医杂志, 2013,54(23):2043-2045.
[12]张运克,车志英,梅雪蕊.补阳还五汤联合骨髓间充质干细胞移植对大鼠脑缺血再灌注模型神经细胞凋亡的影响[J].中华中医药学刊, 2009,27(8):1660-1662.
[13]张运克,车志英,梅雪蕊.补阳还五汤联合骨髓间充质干细胞移植对脑缺血再灌注大鼠行为学及脑梗死体积的影响[J].辽宁中医杂志, 2010,37(2):366-368.
[14]张运克,张振强,宋军喜.补阳还五汤联合MSCs移植对脑缺血再灌注模型大鼠VEGF及信号通路的影响[J].时珍国医国药, 2016,27(4):806-809.
[15]张运克,宋军营,张振强.补阳还五汤联合骨髓间充质干细胞对大脑中动脉闭塞再灌注治疗的作用机制[J].中国组织工程研究, 2015,19(50):8120-8125.
[16]Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20(1):84-91.
[17]Kiley MA, Oxbury JM, Coley SC. Intracranial hypertension in Sturge-Weber/Klippel-Trenaunay-Weber overlap syndrome due to impairment of cerebral venous outflow. J Clin Neurosci. 2002;9(3):330-333.
[18]邹伟,孙晓伟,于学平,等.血脑屏障与脑缺血再灌注损伤研究进展[J].中华中医药学刊,2009,27(3):466-478.
[19]徐姝燕,富建华.紧密连接相关蛋白Occludin的研究进展[J].国际儿科学杂志,2012,39(5):51-454.
[20]Jia W, Lu R, Martin TA, et al. The role of claudin-5 in blood-brain barrier(BBB)and brain metastases(review). Mol Med Rep. 2014;9(3):779-785.
[21]Garrido-Urbani S, Bradfield PF, Imhof BA. Tight junction dynamics:the role of junctional adhesion molecules(JAMs). Cell Tissue Res. 2014;355(3):701-715.
[22]Liu W, Wang P, Shang C, et al. Endophilin-1 regulates blood-brain barrier permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway. Brain Res. 2014;1573:17-26.
[23]孙伟,杨鲲鹏,盛利,等.大鼠局灶性脑缺血再灌注血脑屏障超微结构及紧密连接蛋白Occludin变化的研究[J].中风与神经疾病杂志, 2007,24(4):425-427.
[24]王静娥,田国萍,周进,等.大鼠脑缺血时紧密连接相关蛋白occludin和claudin-5表达的变化[J].解剖科学进展, 2010,16(2):149-152.
[25]Yeung D, Manias JL, Stewart DJ, et al. Decreased junctional adhesion molecule-A expression during blood-brain barrier breakdown. Acta Neuropathol. 2008;115(6):635-642.
[26]Musch MW, Walsh-Reitz MM, Chang EB. Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol. 2006;290(2):G222-231.
[27]陈易简.血脑屏障完整性与中枢神经系统疾病的发展和转归的相关性研究进展[J].国际神经病学神经外科学杂志, 2017,44(3):319-321.
[28]逯锐锐,龚圆渊,张力华.中医药对血脑屏障通透性影响的研究进展[J].湖南中医杂志,2016,32(7):187-188.
[29]范文涛,王利胜,范有明,等.补阳还五汤对大鼠脑微血管内皮细胞氧糖剥夺再灌注损伤的保护作用[J].中国实验方剂学杂志, 2016,22(14):111-115.
[30]刘玉晖,易文凤,游宇.补阳还五汤抗动脉粥样硬化作用与紧密连接蛋白的关系[J].中国实验方剂学杂志,2014,20(24):155-160.
[31]张楠,王思迪,涂盼春,等.紧密连接相关蛋白对血脑屏障通透性影响的研究进展[J].沈阳医学院学报,2017,19(1):72-75.
[2]Steeg PS, Camphausen KA, Smith QR. Brain metastases as preventive and therapeutic targets. Nat Rev Cancer. 2011;11(5):352-363.
[3]Day RE, Kitchen P, Owen DS, et al. Human aquaporins:regulators of transcellular water flow. Biochim Biophys Acta. 2014;1840(5):1492-1506.
[4]张运克,佟帅.补阳还五汤抗脑缺血及再灌注损伤作用机理研究进展[J].河南中医,2002,22(4):75-76,80.
[5]Haas S, Weidner N, Winkler J. Adult stem cell therapy in stroke. Curr Opin Neurol. 2005;18(1):59-64.
[6]Wang C, Fei Y, Xu C, et al. Bone marrow mesenchymal stem cells ameliorate neurological deficits and blood-brain barrier dysfunction after intracerebral hemorrhage in spontaneously hypertensive rats. Int J Clin Exp Pathol. 2015;8(5):4715-4724.
[7]Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A. 1999;96(19):10711-10716.
[8]Liu H, Honmou O, Harada K, et al. Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia. Brain. 2006;129(Pt 10):2734-2745.
[9]Ito M, Kuroda S, Sugiyama T, et al. Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats. Neuropathology. 2012;32(5):522-533.
[10]Zhang YK, Han XY, Che ZY. Effects of buyang huanwu tang combined with bone marrow mesenchymal stem cell transplantation on the expression of VEGF and Ki-67 in the brain tissue of the cerebral ischemia-reperfusion model rat. J Tradit Chin Med. 2010;30(4):278-282.
[11]张运克,高峰,张丹,等.补阳还五汤联合骨髓间充质干细胞移植对脑缺血再灌注大鼠脑组织NSE和GFAP表达的影响[J].中医杂志, 2013,54(23):2043-2045.
[12]张运克,车志英,梅雪蕊.补阳还五汤联合骨髓间充质干细胞移植对大鼠脑缺血再灌注模型神经细胞凋亡的影响[J].中华中医药学刊, 2009,27(8):1660-1662.
[13]张运克,车志英,梅雪蕊.补阳还五汤联合骨髓间充质干细胞移植对脑缺血再灌注大鼠行为学及脑梗死体积的影响[J].辽宁中医杂志, 2010,37(2):366-368.
[14]张运克,张振强,宋军喜.补阳还五汤联合MSCs移植对脑缺血再灌注模型大鼠VEGF及信号通路的影响[J].时珍国医国药, 2016,27(4):806-809.
[15]张运克,宋军营,张振强.补阳还五汤联合骨髓间充质干细胞对大脑中动脉闭塞再灌注治疗的作用机制[J].中国组织工程研究, 2015,19(50):8120-8125.
[16]Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989;20(1):84-91.
[17]Kiley MA, Oxbury JM, Coley SC. Intracranial hypertension in Sturge-Weber/Klippel-Trenaunay-Weber overlap syndrome due to impairment of cerebral venous outflow. J Clin Neurosci. 2002;9(3):330-333.
[18]邹伟,孙晓伟,于学平,等.血脑屏障与脑缺血再灌注损伤研究进展[J].中华中医药学刊,2009,27(3):466-478.
[19]徐姝燕,富建华.紧密连接相关蛋白Occludin的研究进展[J].国际儿科学杂志,2012,39(5):51-454.
[20]Jia W, Lu R, Martin TA, et al. The role of claudin-5 in blood-brain barrier(BBB)and brain metastases(review). Mol Med Rep. 2014;9(3):779-785.
[21]Garrido-Urbani S, Bradfield PF, Imhof BA. Tight junction dynamics:the role of junctional adhesion molecules(JAMs). Cell Tissue Res. 2014;355(3):701-715.
[22]Liu W, Wang P, Shang C, et al. Endophilin-1 regulates blood-brain barrier permeability by controlling ZO-1 and occludin expression via the EGFR-ERK1/2 pathway. Brain Res. 2014;1573:17-26.
[23]孙伟,杨鲲鹏,盛利,等.大鼠局灶性脑缺血再灌注血脑屏障超微结构及紧密连接蛋白Occludin变化的研究[J].中风与神经疾病杂志, 2007,24(4):425-427.
[24]王静娥,田国萍,周进,等.大鼠脑缺血时紧密连接相关蛋白occludin和claudin-5表达的变化[J].解剖科学进展, 2010,16(2):149-152.
[25]Yeung D, Manias JL, Stewart DJ, et al. Decreased junctional adhesion molecule-A expression during blood-brain barrier breakdown. Acta Neuropathol. 2008;115(6):635-642.
[26]Musch MW, Walsh-Reitz MM, Chang EB. Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol. 2006;290(2):G222-231.
[27]陈易简.血脑屏障完整性与中枢神经系统疾病的发展和转归的相关性研究进展[J].国际神经病学神经外科学杂志, 2017,44(3):319-321.
[28]逯锐锐,龚圆渊,张力华.中医药对血脑屏障通透性影响的研究进展[J].湖南中医杂志,2016,32(7):187-188.
[29]范文涛,王利胜,范有明,等.补阳还五汤对大鼠脑微血管内皮细胞氧糖剥夺再灌注损伤的保护作用[J].中国实验方剂学杂志, 2016,22(14):111-115.
[30]刘玉晖,易文凤,游宇.补阳还五汤抗动脉粥样硬化作用与紧密连接蛋白的关系[J].中国实验方剂学杂志,2014,20(24):155-160.
[31]张楠,王思迪,涂盼春,等.紧密连接相关蛋白对血脑屏障通透性影响的研究进展[J].沈阳医学院学报,2017,19(1):72-75.