鬼箭羽醇提取物对大鼠肾移植免疫排斥和免疫耐受信号通路的调节机制

At present,the major measures to treat renal failure (RF) are hemodialysis and kidney transplant allograft.One of which the most effective measure is to use allogeneic transplantation. But the most dangerous factor for the donor kidney is a graft rejection that may lead to loss of its function. It has been found that effective ways to prevent and treat the rejection are to suppress immune rejection or induce immune tolerance. At present, the study of drugs for suppressing immune rejection has made certain great progress, while the study of inducing immune tolerance still remains in its animal or vitro experiments stage which has only received a number of strategies in some areas.Yet the study of drugs with the dual roles of the suppressing immune rejection and inducing immune tolerance has not started.We have found in recent experiments that the ethanolic extracts of Euonymus alatus (EEEA) can meet this requirement, that is, it has dual roles of the suppress immune rejection and induce immune tolerance, and there is no relevant reports for those dual effects..In the study, some of molecular biology methodes will be used for exploring its acting mechanisms from molecular level.The RT-PCR, Western blot, 3H-TdR incorporation and Northen hybridization will be used in the study for detecting of effects, such as on the expressions of nucleoprotein that are encoded by the HLA-Cw3/Cw4, IL, CD40/CD154, CD28—B7T and CTLA4-IgT; on the phosphorylation and ubiquitination of IκB; and on the adjustment of IKKα/β/γ, gene modification of IDO/Foxp3 and the related signaling pathways (NF-κB,TCR-CD3), etc..The study will offer certain new evidence for further clarification of the mechanism of allograft rejection in organ transplant. The dual effects of the EEEA that suppress immune rejection and induce immune tolerance will make great breakthrough for treatment of allograft rejection in organ transplan.

目前肾功能衰竭的主要治疗措施是透析和肾移植，其中最有效的措施是肾移植，而排斥反应又是移植肾失去功能的最危险因素，防治排斥反应的有效途径是免疫排斥抑制或免疫耐受诱导，目前免疫排斥抑制药物研究已取得了一定进展，但免疫耐受诱导药物研究大多还停留在动物实验或体外实验阶段，只是在某些领域中取得了一些策略，对于同时具备免疫排斥抑制和免疫耐受诱导双重作用药物研究尚未起步。我们发现鬼箭羽醇提取物（EEEA）具有对大鼠肾移植免疫排斥抑制和免疫耐受诱导双重作用。本项目就是用PCR、Western blot、Northen杂交等技术探究其机制，如对HLA-Cw3与CTLA4-IgT表达、IκB磷酸化与泛素化、Foxp3基因修饰及相关信号通路（NF-κB）的影响等。这一双重作用机制研究，将为深入阐明器官移植排斥反应机制提供新证据，并为解决目前器官移植排斥反应治疗的两大难题（免疫排斥抑制、免疫耐受诱导）带来重大突破

目前解决肾移植排斥反应的有效方法是免疫排斥抑制和免疫耐受诱导。本项目是在目前已用于临床的仅为免疫抑制药物（不良反应多而重，价昂），且发现鬼箭羽醇提物（EEEA）具备免疫排斥抑制和免疫耐受诱导双重药理作用基础上，提出用PCR、Western blot等技术，从分子信号通路解释其机制（如NK 细胞活性和包括 5 个家族成员的 NF-κB 信号通路三个环节、IL-2、TGF、CD4+CD25+FOXP3+Tregs等蛋白与mRNA表达等）。.结果证实：.（1）急性毒性试验表明EEEA无毒性；.（2）EEEA有利于阻止家兔肾缺血再灌注所导致的肾损伤，表现为血清中IL-2、IL-6、IL-17、TGF-β1、INF-γ减少，其机制与抑制肾组织和脾脏组织中TGF-β1 、NF-κB、INF-γ和p65蛋白与mRNA表达，增强FOXP3 蛋白与mRNA表达有关，且这种效应联合地塞米松更佳。.（3）EEEA对大鼠异体肾移植和家兔异体肾移植均有明显免疫排斥抑制作用和免疫耐受诱导作用，且这种效应联合CsA更佳。其免疫排斥抑制机制与抑制血、肾和脾中TGF-β1、NF-κB、INF-γ、IL-17蛋白与mRNA表达有关，也与与抑制CXCL10快速生成或加速CXCL10消除有关，且这种抑制机制主要在脾脏。免疫耐受诱导机制与促进大鼠血、肾、脾中IL-35、IL-10、IL-2、IL-6、IL-23、p65表达而增强FOXP3 蛋白与mRNA表达，进一步提高CD4+、CD8+、CD4+CD25+、CD4+CD25+FXOP3+Tregs的百分比而增强免疫耐受有关，且这种诱导机制主要在脾脏，免疫排斥抑制和免疫耐受诱导效应联合CsA更佳。.（4）由于肾缺血再灌注炎症反应过程和肾移植免疫排斥与免疫耐受机制复杂，加上EEEA成分复杂，表明EEEA对家兔肾缺血再灌注所导致肾损伤的阻止作用，对大鼠异体肾移植和家兔异体肾移植均有明显的抑制作用和免疫耐受诱导作用，是基于EEEA多成分的多靶向干预效应和机体复杂病理过程中多分子信号通路、细胞因子等机制的多靶路互补效应。.鉴于EEEA的这些效应与机制及与糖皮质激素和环孢素A合用效应更佳的事实，加上初步验证其无毒性，在对器官缺血再灌注损伤、异体器官移植免疫排斥抑制和免疫耐受诱导的长期预防、治疗方面，EEEA有良好的科学研究价值、临床应用前景及农村药业产业链发展意义。