Effects Of GAs And GBEP On The Immune Function Of Mice

ObjectivesTo evaluate the effect of Ginkgolic Acids (GAs) and Ginkgo Biloba Exocarp Polysaccharide (GBEP) on the immune function of mice. To study the immunomodulatory effect of GBEP in the immuosuppressive mice induced by cyclophosphamide. To study the sensitivity of immune toxicity indicators and explore more comprehensive and reasonable combination of immune assays. Moreover, to provide references for establishing immunotoxicology evaluation and screen system in China.Methods1 Evaluation of the immune toxicity of Ginkgolic AcidsFemale BALB/c mice were divided into 5 immunization groups.Each immunization group was composed of six experimental groups with 15 mice in each group—a water control group, a oil control group, a positive control group and three GAs groups. High dose of GAs was equal to 25 percent of LD50 in oral acute toxicity test. High, medium and low doses of GAs were 350,117 and 39 mg/kg bw respectively and were given to mice by ig. CTX (60mg/kg bw) was daily given to positive control group by ig. The expetiment period was 28 days. Then, the following indicators were detected:Immunization groupⅠ:Sheep red blood cell (SRBC) primary immune response test, hemolysis test; Immunization groupⅡ:NK cell activity, splenocyte proliferation reaction induced by mitogen LPS and ConA, cytotoxic T cell activity assay; Immunization groupⅢ:hematology, biochemistry, immunoglobulins, histopathology, lymphocyte phenotyping, the weight of major immune organs and other important organs; Immunization groupⅣ:phagocytic activity of macrophages. Immunization groupⅤ:delayed-type hypersensitivity reaction (DTH).2 Evaluation of the immune function of Ginkgo Biloba Exocarp Polysaccharide2.1 Evaluation of immune function of Ginkgo Biloba Exocarp Polysaccharide in normal miceFemale BALB/c mice were divided into five immunization groups. Each immunization group were composed of 4 experiment groups with 10 mice in each group—a control group and three doses of GBEP group. According to "The tolerant maximum amount given to animals", high dose of GBEP was 10.00g/kg bw. Medium and low doses of GBEP were 3.33g /kg bw and 1.11g/kg bw, GBEP was given to mice by ig. Distilled water was given to control mice by ig. The expetiment period was 28 days. Then, the following indicators were detect Immunization group I:Sheep red blood cell (SRBC) primary immune response t hemolysis test; Immunization groupⅡ:NK cell activity, splenocyte proliferation reaction induced by mitogen LPS and ConA, cytotoxic T cell activity assay; Immunization groupⅢ: hematology, biochemistry, immunoglobulins, histopathology, lymphocyte phenotyping, the weight of major immune organs and other important organs; Immunization groupⅣ: phagocytic activity of macrophages. Immunization groupⅤ:delayed-type hypersensitivity reaction (DTH).2.2 Evaluation of the immune function of Ginkgo Biloba Exocarp Polysaccharide in immunosuppressive miceFemale BALB/c mice were divided into six groups. Each group comprised of ten animals.Negative control group received stilled water; Positive control group, CY 60mg/kg body weight; group A, CY(60mg/kg body weight) and GBEP (10.00g/kg body weight) group B, CY(60mg/kg body weight) and GBEP (3.33g/kg body weight); group C, CY(60mg/kg body weight) and GBEP (1.11g/kg body weight), group D, CY(60mg/kg body weight) and GBEP (0.10g/kg body weight). The amount of GBEP and other chemicals given to mice was adjusted according to body weight weekly. The expetiment period was 28 days. Then, the following indicators were detected:Immunization groupⅠ:Sheep red blood cell (SRBC) primary immune response test, hemolysis test; Immunization groupⅡ:NK cell activity, splenocyte proliferation reaction induced by mitogen LPS and ConA, cytokine production by splenocytes; Immunization groupⅢ:immunoglobulins, lymphocyte phenotyping, the weight of major immune organs; Immunization groupⅣ:phagocytic activity of macrophages. Immunization groupⅤ:delayed-type hypersensitivity reaction (DTH).Results1 The effect of GAs on the immune function of mice1.1 Immunotoxicity evaluation procedure and methods was established with CTXIn our study, the STS results showed that significant reductions were found in body weight, thymus weight, liver and urine weight in CTX group animals when compared with control group. Histology results showed that analosis was found in splenic corpuscle. The normal structure of lymphaden in CTX group animals disappeared. Hematology test results showed that significant reductions were found in the number of WBC and the percentage of LYM. Moreover, significant reductions were found in the concentration of IgA and IgG, the percentage of T, B, Th and Ts lymphocytes, the ratio of CD4+/CD8+, PFC/106 cells and HC Moreover, significant reductions were found in ConA- and LPS-induced lymphoc proliferation, NK cell activity, CTL cell activity and DTH reaction in CTX group animals.1.2 The effect of GAs on the immune function of miceIn our study, significant differences were not found between the water group and oil group animals. The STS results had shown that at high dose of GAs, a significant reduction was found in body weight and spleen weight. At all doses of GAs, significant reductions were found in liver weight and urine weight. Blood biochemistry test results had shown that at at all dose of GAs, a significant reduction was found at the BUN level. At low and high dose of GAs, a significant reduction was found in the ratio of A/G. Moreover, significant increases were found at percentage of T and Th lymphocytes at low dose of GAs group. But, a significant reduction was found in the percentage of B lymphocytes. At middle dose of GAs, a significant increase of the percentage of T lymphocytes was found. At high dose of GAs, a significant increase was found in the percentage of T lymphocytes and a significant reduction was found at the percentage of B lymphocytes. At all doses of GAs, no significant difference was found in PFC/106, HC50 and CTL cell activity compared with control group. A significant reduction was found in NK cell activity at all groups of GAs.2 The effect of GBEP on the immune function of mice2.1 The effect of GBEP on the immune function of normal miceIn our study, at high dose of GBEP, significant reductions were found at lymphaden weight. At low and middle dose of GBEP, significant reduction was found in the relative lymphaden weight compared with control animals. Blood biochemistry results showed that no significant difference was found between GBEP group and control group animals. As regard the concentration of immunoglobulins, at middle dose of GBEP, significant increases were found in the concentration of IgA and IgG.. At high dose of GBEP, a significant increase was found in the concentration of IgA. With regard to the other results, at all doses of GBEP, significant increases were found in the percentage of T lymphocytes and Th lymphocytes and the ratio of Th/Ts. No significant differences were found at DTH reaction, NK cell activity, CTL cell activity and macrophage cell activity between GBEP groups and control group animals.2.2 The immunomodulatory effect of GBEP on the immune function of immunosuppressive miceOur study had shown that a single dose of CTX (60 mg/kg) induced a significant reduction in body weight, spleen weight and thymus weight. Our study had shown that CTX had immunosuppressive effect on the subpopulation of CD3+CD19-, CD3-CD19+, CD3+CD4+ a CD3+CD8+. CY induced a significant reduction in PFC/106 cells and HC50, when compai with the control animals. As regard the concentration of three kinds of immunoglobulin in peripheral blood, a significant reduction in concentration of IgG and IgM was observed in CTX group animals, when compared with the control group. CY only elicited suppressive effect in ConA-and LPS-induced lymphocyte proliferation, when compared with the control group. CY showed a suppressive effect in the cytokine production by ConA-induced splenocytes, when compared with control animals. No significant suppressive effect in DTH assay was observed at single dose of CY. CY induced a significant suppressive effect in NK cell activity, when compared with control group.As regard the effect of GBEP, at doses of 1.11g/kg, a significant increase in relative spleen weight was observed when compared with CY group. As regard to the organ weight of thymus, a significant increase was observed at dose of 3.33 g/kg,1.11 g/kg and 0.10g/kg. At all doses of GBEP, a significant increase in PFC/106 cells and HC50 was observed when compared with CY group. At dose of 10.00g/kg, GBEP induced a significant increase in concentration of IgM. At dose of 3.33g/kg, GBEP induced a significant increase in three kinds of immunoglobulin. At dose of 1.11g/kg and 0.10g/kg, GBEP induced a significant increase in concentration of IgG and IgA. As regard ConA-induced lymphocyte proliferation in GBEP groups, significant increases were observed at all GBEP groups. At dose of 3.33g/kg, 1.11g/kg and 0.10g/kg, GBEP induced a significant increase in LPS-induced lymphocyte proliferation. At dose of 10.00g/kg, GBEP induced a significant increase in the concentration of TNF,IFN-γ,IL-5,IL-4 and IL-2. At dose of 3.33g/kg, GBEP induced a significant increase in the concentration of TNF and IFN-γ. At dose of 1.11g/kg, GBEP induced a significant increase in the concentration of TNF. At dose of 0.10g/kg, GBEP induced a significant increase in the concentration of TNF,IFN-γand IL-2. At all doses of GBEP, significant stimulatory effect in DTH assay was observed. At dose of 1.11 g/kg and 0.10g/kg, GBEP induced a significant increase in NK cell activity in immunosuppressive animals. At all doses of GBEP, significant increases were found in the percentage of phagocytic cells and phagocytic index in immunosuppressive animals.Conclusions1 The immunosuppession model was established successfully, and immunotoxicity evaluation procedure was recommended. The immunotoxicity evaluation procedure was composed of standard toxicity study and additional immune tests, in which the former v composed of hemotology, the immune organ weight, histopathology and immunoglobu and the latter was composed of lymphocyte phenotyping, the reaction to SRBC,50% serum hemolysis, phagocytic activity of macrophages, CTL activity, NK cell activity, delayed-type hypersensitivity reaction2 In our study, we found that there was pathological damage on some organs of mice received Ginkgolic acids. Moreover, immunosuppressive function of GAs was found on the humoral immune function of mice. But, the cellular immune function and non-specific immune function of mice were not affected significantly by GAs.3 The immunotoxicity of GBEP was not found on the normal mice. 4 In our study,3.33g/kg,1.11 g/kg and 0.10g/kg dose of GBEP had significant immodulatory function on the immunosuppressive mice induced by CTX. At high dose of 10.00g/kg, the immune regulatory function of GEBP was not obvious and may need further study.