The Effects Of Bererine On The Damage And Immune System Of Middle Cerebral Artery Occlusion-reperfusion Injury Mouse

ObjectiveTo investigate the variances of peripheral and central immune system of focal middle cerebral artery MCAO/R mouse and the protective effect and the mechanism of Bererine in peripheral and central immune system of focal middle cerebral artery occlusion-reperfusion(MCAO/R) mouse.Methods1.The mouse model of focal cerebral ischemia reperfusion injury was induced by middle cerebral artery occlusion(MCAO) and was given 1.0 h occlusion of the MCAO and 24 h reperfusion.The model's success is detemined by the neurologic deficit,TTC and HE staining. The neurologic deficit score was investigated according to Zea-Longa's Standard.The infarct volume was assessed with software(Adobe Photo Shop 5.0) after TTC(2,3,5-Triphenyl Tetrazolium Chloride) staining.Ber was administered intraperitoneal immediately after 0.5 h occlusion and 12 h reperfusion.And the concentration of Ber in vivo was determined by the decrease of neurologic deficit score and infarct volume.Four groups were randomly divided: Normal group,sham-operation group,MCAO/R group and(M+Ber) group.Then,lymphocytes, splenocytes,thymic cells and bone marrow were incubated with different stimulator in vitro to test the variances.2.The protective effect of Ber on the peripheral immune system was focus on lymphocytes prepared from lymph nodes and spleen and change of peripheral blood.Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69,CD25 and CD71 by T cells both naive and stimulated by Con A from 4 groups.CFDA-SE staining and MTT test were used to detect the effect of Ber on the proliferation of lymphocytes stimulated by Con A.The distribution of the cell-cycle was analyzed by PI staining together with flow cytometry analysis.Calcein and DIOC₆(3) staining was used to analyze the apoptosis of lymphocytes induced by Cycloheximide from microbial(CHX).Changes of reactive oxygen species(ROS) in peripheral blood were analyzed by H₂DCFDA staining together with flow cytometry.Concentrations of NO and H₂O₂ in serum were measured by NO assay kit and H₂O₂ assay kit.3.The protective effect of Ber on the central immune system was focus on the atrophy of thymus and the apoptosis of thymic cells detected by Calcein and DIOC₆(3) staining.NO concentration secreted by bone marrow induced by(LPS+IFN-γ) or(LPS+IFN-γ+ATP) with or without cAMP were measured by NO assay kit.NO fluorescence probe(DAF-FM DA) was used to detect the secretion of NO by bone marrow stimulated by(LPS+IFN-γ).Results1.Ber(5 mg/kg) was administered to the MCAO/R mouse to improve the neurologic deficit score and the infarct volume in vivo.2.Our findings in vitro suggest that Ber exerts immunosuppressive effect on the peripheral and central immune system,which significantly inhibits the activation and proliferation of the mouse lymphocytes,blocks the progression of mouse lymphocytes into S and G2/M phases, prevents the apoptosis of lymphocytes and thymus induced by CHX or DEX,decrease the production of NO from bone marrow induced by(LPS+IFN-γ)/(LPS+IFN-γ+ATP) in a dose-dependent manner.3.Compared to Normal group,the activation and proliferation of lymphocytes from MCAO/R was weaker,and the distribution of cell cycle of lymphocytes was more blocked to the G0/G1. The apoptosis,the level of ROS in the peripheral blood,the content of NO and H₂O₂ from the serum,NO from bone marrow were smiliar between the sham-operation group and Ber-group. But there were large differences in the model group and normal groups.ConclusionsIt was weaker that the cell response of immune cells(lymphocytes,splenocytes and thymic cells) in the model group comparing to the normal group while the Ber-group was improved obviously.The rate of apoptosis of the lymphocytes and thymic cells,the output of free radical(eg:ROS,NO and H₂O₂) which do more damage to the brain was larely increased than the Normal group.The rate of apoptosis,the production of free radicals,the atophy of immune organs and the reaction of immune cells were all improved in the(M+Ber) groups.It suggested that Ber can protect the peripheral and central immune system of MCAO/R mouse.