复健片保护缺血性卒中血脑屏障的作用研究
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摘要
目的:探讨滋补肝肾中药复健片对缺血性卒中后血脑屏障的保护机制。方法:基于“肾生髓”的中医理论,以大脑中动脉闭塞(MCAO)大鼠为实验对象,通过观察复健片调控血脑屏障通透性和结构的研究,初步探索滋补肝肾中药保护血脑屏障的机制。利用渗出血管外的依文氏蓝(EB)染色法,以依文氏蓝含量为指标,观察复健片对血脑屏障通透性的影响;以脑组织含水量为指标,观察复健片对脑水肿的影响;利用酶联免疫法,以组织型纤溶酶原激活剂(t-PA)为指标,观察复健片对血脑屏障基膜损伤的纤溶酶系统的影响;利用酶联免疫法,以基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)为指标,观察复健片对血脑屏障基膜损伤的明胶酶系统的影响;利用原位杂交法,以内皮细胞内ET-1 mRNA、ecNOS mRNA和P-gp mRNA为指标,观察复健片对内皮细胞功能活性的影响;利用放免法,以环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)为指标,观察复健片对星形胶质细胞活性的影响;利用酶联免疫法,以肿瘤坏死因子-α(TNF-α)和白介素10(IL-10)为指标,观察复健片对炎性细胞因子的影响。结果:复健片可显著降低血脑屏障的通透性,减轻脑水肿;抑制t-PA、MMP-2和MMP-9的表达,防止血脑屏障的基膜损伤;抑制血管内皮细胞内病理性上调的ET-1 mRNA表达,增强ecNOS mRNA、P-gp mRNA表达以保护内皮细胞;降低cGMP表达、提高cAMP,有促使星形胶质细胞协同诱导维持血-脑屏障特性的作用;降低TNF-α含量,提高IL-10含量,降低炎症反应对脑毛细血管内皮、基底膜和星形胶质细胞的损害,使血脑屏障的通透性降低。结论:以滋补肝肾立法组方的复健片可在体调控MCAO大鼠血脑屏障的基膜-星形胶质细胞-内皮细胞功能,从而有利于保护血脑屏障的功能,防止脑水肿的发生。
Objective: To investigate the effect of the traditional Chinese herbal medicine called Fujian Tablet on the BBB after cerebral ischemic stroke. Fujian Tablet is ascribed the therapeutic method called“the nourishing of liver and kidney”by Traditional Chinese Medicine (TCM). Methods: Based on the TCM theory that“nourishing the liver and kidney can produce sui”(with sui indicating bone marrow, spinal cord and brain, all related structures in Traditional Chinese Medicine), we set up MCAO rat model to explore the mechanisms of protection that may occur from applying the Traditional Chinese Medicine therapeutic method of Nourishing Liver and Kidney by the use of the Fujian Tablet and observing it’s effects of on regulating permeability and structure of BBB. EB was measured by AZO-blue dyeing method to evaluate the effects of Fujian Tablet on the permeability of BBB. Moisture capacity of brain tissue was measured to evaluate the effects of Fujian Tablet on encephaledema. t-PA was measured by euzymelinked immunosorbent assay to evaluate the effects of Fujian Tablet on the fibrinolysin system in order to help prevent destruction of the basilemma. MMP-2 and MMP-9 were measured by euzymelinked immunosorbent assay to evaluate the effects of Fujian Tablet on the gelatinase system in helping prevent destruction of the basilemma. The expression of ET-1 mRNA, ecNOS mRNA and P-gpmRNA in the endothelium were observed through in situ hybridization in order to reflect the effects of Fujian Tablet on the function of the endothelium. cAMP and cGMP were measured by radio-immunity method to reflect the effects of Fujian Tablet on the function of astrocyte activity. TNF-αand IL-10 were measured by euzymelinked immunosorbent assay to evaluate the effects of Fujian Tablet on inflammatory factors. Results: Fujian Tablet can decrease the permeability of BBB and lessen hydrocephalus. Fujian Tablet can restrain the expression of t-PA, MMP-2 and MMP-9 thus protecting the basilemma. Fujian Tablet can decrease the expression of ET-1mRNA and increase the expression of ecNOS mRNA and P-gp mRNA thus protecting endotheliocytes. Fujian Tablet can decrease the expression of cGMP and increase the expression of cAMP to help the astrocytes maintain BBB in co-induction with the endotheliocytes. Fujian Tablet can decrease the expression of TNF-αand increase the expression of IL-10 lowering the inflammatory reaction and helping prevent the destruction of endotheliocytes, astrocytes and basilemma and the resultant further decrease in the permeability of BBB. Conclusions: Fujian tablet with its action of“nourishing the liver and kidney”can regulate the function of basilemma, astrocytes and endothelium in vivo, protecting BBB in order to prevent hydrocephalus.
Objective: To investigate the effect of the traditional Chinese herbal medicine called Fujian Tablet on the BBB after cerebral ischemic stroke. Fujian Tablet is ascribed the therapeutic method called“the nourishing of liver and kidney”by Traditional Chinese Medicine (TCM). Methods: Based on the TCM theory that“nourishing the liver and kidney can produce sui”(with sui indicating bone marrow, spinal cord and brain, all related structures in Traditional Chinese Medicine), we set up MCAO rat model to explore the mechanisms of protection that may occur from applying the Traditional Chinese Medicine therapeutic method of Nourishing Liver and Kidney by the use of the Fujian Tablet and observing it’s effects of on regulating permeability and structure of BBB. EB was measured by AZO-blue dyeing method to evaluate the effects of Fujian Tablet on the permeability of BBB. Moisture capacity of brain tissue was measured to evaluate the effects of Fujian Tablet on encephaledema. t-PA was measured by euzymelinked immunosorbent assay to evaluate the effects of Fujian Tablet on the fibrinolysin system in order to help prevent destruction of the basilemma. MMP-2 and MMP-9 were measured by euzymelinked immunosorbent assay to evaluate the effects of Fujian Tablet on the gelatinase system in helping prevent destruction of the basilemma. The expression of ET-1 mRNA, ecNOS mRNA and P-gpmRNA in the endothelium were observed through in situ hybridization in order to reflect the effects of Fujian Tablet on the function of the endothelium. cAMP and cGMP were measured by radio-immunity method to reflect the effects of Fujian Tablet on the function of astrocyte activity. TNF-αand IL-10 were measured by euzymelinked immunosorbent assay to evaluate the effects of Fujian Tablet on inflammatory factors. Results: Fujian Tablet can decrease the permeability of BBB and lessen hydrocephalus. Fujian Tablet can restrain the expression of t-PA, MMP-2 and MMP-9 thus protecting the basilemma. Fujian Tablet can decrease the expression of ET-1mRNA and increase the expression of ecNOS mRNA and P-gp mRNA thus protecting endotheliocytes. Fujian Tablet can decrease the expression of cGMP and increase the expression of cAMP to help the astrocytes maintain BBB in co-induction with the endotheliocytes. Fujian Tablet can decrease the expression of TNF-αand increase the expression of IL-10 lowering the inflammatory reaction and helping prevent the destruction of endotheliocytes, astrocytes and basilemma and the resultant further decrease in the permeability of BBB. Conclusions: Fujian tablet with its action of“nourishing the liver and kidney”can regulate the function of basilemma, astrocytes and endothelium in vivo, protecting BBB in order to prevent hydrocephalus.
引文
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