EGCG受体在星形胶质细胞中的确证及其跨膜作用机制研究

Recent studies showed that the health benefits ascribed to tea consumption are believed to rely on its bioactive flavanol class-related catechins. The most aboundant catechin is (-)-epigallocatechin-3-3gallate (EGCG), which demonstrated to act as antioxidant agent and thus, may contribute to the beneficial effects attributed to tea in health benefits. Reported findings and our previous researches indicated that EGCG was found to have neuronprotective activity.However,it is enigma whether there exists the membrane recptor of EGCG, and how EGCG moves across cell membrane into astrocyte. Therefore, in this program, great attention should be given to EGCG to clarify how EGCG plays neuroprotective roles：(1)after identificaton of the EGCG receptor, which is located in membrane by EGCG affinity chromatographic column, ESI-MS and MALDI-TOF-MS, molecular biotechnology and immunocytochemistry methods will be used to analysis the EGCG receptor RNA and protein expression levels in EGCG treated and control groups in order to demenstrate the roles of EGCG receptor; (2)after extraction of lipid raft and identification of the EGCG receptor in lipid raft by density gradient centrifugation and Western Blot, respectively, the alteration of lipid raft will be observed by atomic force microscopy in EGCG treated and control groups to indicate the roles of lipid raft in EGCG internalization;(3)using lipid raft inhibitor and EGCG receptor antibody to verify EGCG trans-membrane pathway.Based on these results, we hope to illustrate the receptor mediated EGCG trans-membrane mechanisms in astrocyte, which will be helpful for our understanding the internal mechanisms of EGCG in neuroprotection.

表没食子儿茶素没食子酸酯（(-)-epigallocatechin-3-gallate， EGCG）是儿茶素中含量最高，抗氧化能力最强的单体，同时茶的多种保健功能都依赖于EGCG的生物学活性。已有研究和本课题前期研究均发现EGCG具有神经保护功能，而目前对神经细胞上的EGCG受体和其跨膜作用机制知之甚少。本课题拟采用亲和色谱层析及质谱技术纯化鉴定EGCG在星形胶质细胞膜上的受体；利用分子生物学和免疫细胞技术研究不同浓度EGCG处理星形胶质细胞后受体的表达变化，明确EGCG与其受体作用的效应关系；通过密度梯度离心及Western Blot技术分离脂筏并鉴定EGCG受体蛋白，原子力显微镜观察不同浓度EGCG处理星形胶质细胞后脂筏的变化，明确脂筏在EGCG内化中的作用；最后利用脂筏抑制剂和EGCG受体的特异性抗体，确证受体介导的EGCG跨膜作用分子机制,为深入阐明EGCG的功能提供理论基础。

茶中的主要成分EGCG可以通过血管屏障，进而在神经系统病变中表现出较好的应用前景。本研究针对EGCG进入神经细胞的机制开展了一下研究：.（1）高浓度的EGCG可抑制星形胶质细胞生长，但抑制67 LR与EGCG的结合，EGCG的活性消失，提示EGCG与67 LR结合后发挥生物学活性。对67 LR蛋白表达的检测发现其在EGCG刺激后未有变化，提示EGCG的生物学活性不依赖其膜受体67 LR的高表达。.（2）SAH后高浓度的EGCG保护神经细胞免受损伤。氧合血红蛋白刺激后67 LR的表达较对照组降低，而高浓度的EGCG预适应后可显著提高67 LR的表达。利用荧光探针检测发现EGCG处理后细胞膜上出现脂筏结构。利用免疫共沉淀结合质谱技术，发现67 LR可结合多种膜蛋白，其中包括integrin。对integrin beta 1蛋白表达的分析显示integrin beta 1表达显著降低，提示SAH后EGCG激活67 LR进而抑制integrin beta 1的表达，故阻断由此信号引起的细胞氧化应激。.（3）生理条件下EGCG诱导钙内流导致线粒体钙超载。线粒体膜电位去极化、mPTP的过度开放及ROS含量的升高，最终促进了星形胶质细胞死亡。而阻断EGCG与67 LR的结合可逆转上述过程。.（4）SAH后线粒体钙超载引起线粒体膜电位去极化、ROS升高及mPTP孔过度开放等现象。线粒体异常后导致细胞自噬的增加，但自噬流存在异常。而EGCG通过抑制SAH后p38的高表达逆转上述现象，进而维持细胞的正常功能。.（5）茶多酚通过保护线粒体结构的完整性，提高SAH后ATP含量，减少cyt c的释放，进而减轻了SAH后的损伤。.这些结果初步揭示了EGCG跨膜及干预SAH发生、发展机制，为后续深入开展EGCG跨膜调控SAH后细胞自噬信号转导机制的研究奠定了重要的理论基础。