The Effect Of Gossypol Acetic Acid On Immune Function In Mice And Its Apoptotic Mechamism On Peritoneal Macrophages

Objective:The aim of the study was to investigate the effect of gossypol acetic acid(GA) on immune function in BALB/c mice and its toxicity and apoptoic mechanism on peritoneal macrophages.To explain the impact of GA on the immune system in several aspects.Moreover, to provide a theoretical and experimental basis for the development of its new biological activity and clinical use.Methods:GA was administered orally four times at24h intervals at three doses of5.0,25.0and125.0mg/kg to non-immunized mice, determining the influence on the total number of lymphocytes, weight ratio and subsets of lymphocytes in the thymus, spleen and mesenteric lymph nodes at three different times(24h,72h,120h after the last administration). The same protocol was administered to SRBC-immunized mice (prior to or after priming). And the humoral immune response to sheep erythrocytes (SRBC) which includes plaque forming cells (PFC) and anti-SRBC haemagglutinin (IgM,IgG) titer in the serum was determined only once(24h after last administration).In the experiment, RAW264.7cells were mouse peritoneal macrophages in vitro model. The cells were treated with GA at0,25,30,35μmol/L. Inhibition of cell proliferation was detected by MTT assay. The oxidative damage was evaluated by measuring activities of SOD、GSH-Px, content of MDA.The apoptosis was showed by AO/EB dyeing,TUNEL assay and Annexin-FITC/PI staining assay.Measurement the impact of GA on ROS level and mitochondrial transmenbrance potential and the protective effect of NAC(a free radical scavenging agent) by DCFA and Rh123staining. Western blotting analysis of caspase-3and-9expression, and the inhibition of Z-VAD-FMK and Ac-LEHD-FMK, NAC on cell apoptosis.Results:GA decreased the total number of lymphocytes in thymus and mesenteric lymph nodes in a dose-dependent manner. GA dependent on the dosage decreased the percentage of CD4+thymocytes but increased the percentage of CD8+lymphocytes in spleen and lymph nodes. In SRBC-immunized mice, GA at the doses of25.0and125.0mg/kg administered prior to SRBC reduced the number of PFC and the production of IgG. However, GA administered after priming decreased the production of IgM and IgG.GA inhibited the proliferation of RAW264.7significantly and the IC50was32.9μmol/L.GA decreased activities of SOD and GSH-Px. However, GA increased the content of MDA.With the increasing doses of GA, the apoptotic phenomenon and rate were more obvious.Study reveals GA might induced apoptosis in RAW264.7by caspase-dependent mitochondrial apoptotic pathway, mainly in:a significant increase of intracellular ROS levels,significantly decrease of mitochondrial membrane potential, activation of Caspase-3,9protein, the caspase inhibitor Z-VAD-FMK and caspase-9inhibitor Ac-LEHD-FMK significantly inhibited GA induced apoptosis. The protective effect of NAC was reflected in reducing the apoptosis induced by GA in RAW264.7and improving cell survival rate. It also blocked ROS generation and mitochondrial membrane potential collapse.Conclusion:GA suppressed the humoral immune function effectively, and reduced the total number of lymphocyte from thymus and lymph node. However, no obvious effect was found on the immune organ index. GA may have a complex and ambiguous effect on different lymphocyte subpopulations.GA inhibited proliferation of RAW26.47, caused oxidative damage and might induce apoptosis through caspase-dependent mitochondrial pathway.