Evaluation of The Number of CD4(+) CD25(+) FoxP3(+) Treg Cells in Normal Mice Exposed to AFB(1) and Treated with Aged Garlic Extract

OBJECTIVE: Aflatoxin B1 (AFB(1)) suppresses the immune system. To decrease such suppressive effects on the immune system, a wide range of herbal medicines like garlic are utilized. Biological activities of garlic in vitro and in vivo have also been verified. Our previous studies demonstrated that aged garlic (dry garlic bulbs preserved in the freezer for six months at -20˚C) have increased immunostimulator fractions and reduced immunosuppressor fractions. This study focuses on the immunosuppressor activity of AFB(1) and immunostimulator activity of aged garlic extract (AGE) through the evaluation of CD4(+) CD25(+) FoxP(+) regulator cell (Treg) counts and the pattern of cytokine production in Balb/c normal mice. MATERIALS AND METHODS: In this experimental research, AFB(1) was separated from Aspergillus flavus (PTCC 5004) by HPLC and AGE prepared using the Mantis method. The Delayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectiveness of different doses of AGE and AFB(1), which can both have an effect on the immune system. Subsequent experiments were carried out on 20 Balb/c mice to estimate the effects of AGE and AFB(1) on the number of Treg cell in 4 groups: 10 µl/kg/day of AFB(1) and AGE diluents were administered for 4 consecutive days to group 1. AFB(1), 2. control, 3. AGE + AFB(1) and 4. AGE via intraperitoneal (IP) route, respectively. Mice were sacrificed and splenocytes harvested and the percentage of splenic Treg cells was measured by flow cytometry analysis. The ELISA method was utilized to measure Cytokine production. RESULTS: The findings reveal that AGE increased the level of IFN-λ and IL-4 cytokines produced by splenocytes stimulated by specific tumor antigen and decreased the number of Treg cells in the spleen (p<0.05). AFB(1) increased the number Treg cells in the spleen and decreased cytokine production (p<0.05). In groups 2 (control) and 4 (AGE) the number of Treg cells decreased (p value<0.05) whereas in groups 1 and 3 the number of Treg cells increased (p<0.05). CONCLUSION: This study indicated that AGE is able to alter the cytokine production in normal mice into a Th(1) protective pattern which is beneficial to the immune system in general and anti-tumor immunity in particular. AFB(1) is able to alter the cytokine production into a Th(2) protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB(1).