Influence Of Caffeic Acid Phenethyl Ester On Immunological Function Of Mouse And Cerebral Ischemia Reperfusion Injury

Aim:In order to study the effect of Caffeic acid phenethyl ester(CAPE) on cellular immunological system of mouse by investigating the activation,proliferation,cell cycle distribution,apotosis and Intracellular Ca²⁺ concentrations of T lymphocytes;In order to elucidate the effect of CAPE on native immunological system by investigating its effect on proliferation,phagocytosis and NO production of mouse peritoneal and RAW264.7 macrophage in vitro.Through building cerebral ischemia reperfusion injury(CIR/I) mouse model to study the effect of CAPE on the experimental mouse.Methods:Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69,CD25 on T lymphocytes activated by Con A; Intracellular Ca²⁺ concentrations were analyzed by Fluo-4/AM staining together with flow cytometry analysis.CFDA-SE staining together with flow cytometry was used to analyse the proliferation of T lymphocytes stimulated by Con A.The distribution of the cell-cycle was analyzed by PI staining together with flow cytometry analysis. Annexin-V/PI double staining together with flow cytometry was used to detect the effect of CAPE on the apoptosis of thymocytes induced by Dex.The proliferation of mouse peritoneal and RAW264.7 macrophage was analyzed by MTT;Fluorescence conjugated microbeads and flow cytometry were used to detect phagocytosis of the macrophages in vitro;NO production from the macrophages activated by LPS was detected by Griess kit.Experimental cerebral ischemia reperfusion injury model was established by surgical procedures with ligation of the common carotid arterys(CCA).CAPE was administeried introvenously 30 mins before CCA ligation.Water content of cerebral tissue was determined by dry-wet method;NO concentration in the serum was detected by Griess kit;H2DCFDA method and FCM were used to detected the intracellular ROS level of mouse blood neutrophils.Results:1.CAPE can down regulate the expression rate of CD69,CD25 and Intracellular Ca²⁺ concentrations of T cells in response to Con A,Also,CAPE can inhibit proliferation of activated T cells in response to Con A.FCM analysis plus PI staining implied that CAPE decreased the ratio of S and G2/M phase cells and increased the ratio of apoptosis and G0/G1 phase cells plus Con A group.FCM analysis of Annexin-V/PI staining implied that CAPE promote apoptosis of thymocytes induced by Dex.2.CAPE can inhibit proliferation of mouse peritoneal macrophage and of RAW264.7 macrophage stimulated by LPS;CAPE can promote phagocytosis of resting macrophages for microbeads,However,It inhibit phagocytosis of LPS-activated macrophage for microbeads;CAPE can inhibit NO production of macrophage stimulated by LPS.3.Experimental result displayed that,Comparing with the sham group,The water content of cerebral tissue rose obviously in CIR/I group,Illustrating the development of cerebral edema.Meanwhile,The NO concentrantions in serum and intracellular ROS level of neutrophil rose obviously in CIR/I group.But in the CAPE pretreated group,CAPE can degrade the water content of cerebral tissue and improve the cerebral edema caused by CIR/I significantly.Also CAPE can down-regulate the NO concentrantions in serum and intracellular ROS level of neutrophil notably comparing with the CIR/I groups.Conclusion:1.CAPE can inhibit activation and proliferation of mouse T lymphocytes and block the progression of T lymphocytes into cell division phase.CAPE also promote the apoptosis of thymocytes induced by Dex.All of these demonstrates that CAPE is a potential effective immunosuppressive agent for mouse T lymphocyte.2.CAPE can inhibit the proliferation of macrophage and dual-directionaly regulate the phagocytosis function of macrophage.Meanwhile,CAPE can depress the NO production of macrophage.All of these demonstrates that CAPE exerts definite regulation effect on innate immunity.3.As a effective antioxidant agent,CAPE exert protection on the CIR/I mouse.