| IntroductionGarlic is a kind of plant containing many organic sulfur compounds, and one of the most effective components is allicin. People used garlic to treat infection, wound, snake bite, diarrhea, rheumatism, heart diseases, diabetes, and other physiologic function disorders. Researches on the garlic reveal lowering the blood fat, blood pressure and blood sugar, anti-cancer, antibiosis and immunomodulator. The organic sulfur compounds derived from garlic can inhibit some cancers, such as prostate cancer, breast cancer, colon cancer, lung cancer, and bladder cancer. The chemical constituents of garlic are very complex containing over 100 different compounds which are regarded as contributing to beneficial biological effects. The most important and unique feature is the high content of organo-sulfur substance, which are at least four times more than other high sulfur-containing vegetables such as onion, broccoli, and cauliflower. Recent years, some Japanese researchers have developed a new type of black garlic by processing ordinary garlic under control of the temperature and humidity. The irritating odor is removal and Black garlic with fruity taste is readily edible without further treatments. There has been report on the black garlic containing more amino acid, organic sulfur and S-allyl-L-cysteine than ordinary garlic. However, there are no reports home and abroad on the black garlic affecting the immune functions. Therefore, we intraperitoneally injected the mice with black garlic extracts solution and observe the effects of black garlic extracts as a type of immunomodulator, and provide the immunological and experiment evidences for the practical application.To further approach the black garlic on the immune function in mcie, we give the normal BALB/c mice intraperitoneal injection with BGE solution, and detect the effects on the immune function. Materials1. RegentsBGE (black garlic extracts, dry powder, granted by Prof. Sasaki from Hirosaki University), RPMI 1640 Medium, fetal bovine serum,0.01 M phosphate buffer solution, Red blood cells lysis solution, LDH substrate solution, Griess reagent, ELISA Kit for IL-2, IFN-γ, TNF-a, and IL-4.2. AnimalFemale BALB/c mice,6-8 weeks old (Academia Sinica Shanghai experimental animal center).Methods1. Animal grouping and treatments40 female BALB/c mice were randomly divided into eight groups, and Group 1-7 were test groups and Group 8 was control group.The black garlic extract solution was prepared with normal saline and the concentration was 1 mg/ml. Mice in test groups were intraperitoneally injected 0.1 ml black garlic extract solution and that in control group with normal saline for successive five days. From the first day to the seventh day after the last injection, one test group of mice were sacrificed each day and mice in the control group were executed on the seventh day.2. Samples collection and analysis(1) Separate spleen cells and prepare splenic suspensionSacrifice the mice by cervical location, prepare the spleen cell suspension by conventional method, and adjust the cell concentration into 1×107/ml.(2) Th1 cytokines IL-2, TNF-a,IFN-γand Th2 cytokine IL-4 in spleen cell culture supernatant were detected by Elisa.(3) The cytotoxicity of NK cells was detected by LDH release assay(4) The NO secreting level of cultured spleen cells by Griess method(5) FasL mRNA level of spleen cells was detected by RT-PCR semi-quantity assay(6) Statistical analysisT-test (SPSS 16.0 software) was applied to compare the experimental data between test group and control group. Results1. The result of cytotoxicity of NK cellsFollowing by giving the black garlic solution by intraperitoneally injection for five successive days, the data of the cytotoxicity of NK cells in test group suggests that the toxicity of NK cells was significantly elevated from the fourth to sixth days post treatment compared with control group (5.82±1.38%) and reached the peak value (11.21±1.17%), then decreased.2. The detection results of cytokines level in cultured spleen cells supernatantWe detected the secreting level of Thl cytokines IL-2, IFN-y, TNF-a and Th2 cytokine IL-4 after treat the mice with the BGE solution and the results indicate that the IL-2 secreting level was significantly elevated on the sixth day post treatment, there existing statistical significance (P< 0.05) compared with control group (20.14±3.88 pg/ml). TNF-a secreting level was significantly increased (P＜0.01) from the first day after treatment, and reached the peak value (68.73±0.64 pg/ml)on the third day, then decreased. However, it's still higher than that in control group (27.36±8.30 pg/ml) on the seventh day. IFN-y secreting level was also increased after treatment, and reached the peak value (105.05±55.73 pg/ml), then decreased to the level slose to the control group (17.38±1.19 pg/ml) on the seventh day. Th2 cytokine IL-4 secreting level has been lower than control, and the expressing level was significantly lower (P< 0.01) than that in control group (19.98±2.13 pg/ml), but the variation was also in the base line.3. The detection results of NO level in cultured spleen cell supernatantNO2- level was always higher than control group (11.58±0.86μM) from the third to seventh day after treatment with BGE, there existing statistical significance (P< 0.01).4. The detection results of spleen cells'FasL mRNA levelWe detected the spleen cells FasL mRNA expressing level by Semi-quantity RT-PCR. The results suggested there was no difference of FasL mRNA expressing level between test and control group. Conclusion1. BGE can increase the cytotoxicity of NK cells.2. FasL mRNA expressing level is not elevated which suggests that BGE doesn't induce killer cells'cytotoxicity by Fas/FasL pathway.3. Black garlic can highly increase the NO secreting level of cultured spleen cell supernatant, which suggests that BGE can enhance macrophage's activity.4. BGE can enhance the production of IL-2, IFN-γ,TNF-αin the supernatant of the cultured spleen cells, which suggests that BGE can enhance Thl immune response. |
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