The Effect Of Trichosanthin On The Prevention Of Type 1 Diabetes In NOD Mice

Objective:To investigate the role of trichosanthin (TCS) on the prevention of type 1 diabetes and the severity of insulitis in NOD mice. Further study the possible mechanism through investigate the change of T lymphocyte subsets in thymus and spleen, investigate the production of Th1 cytokines such as IFN-γ and Th2 cytokines such as IL-4 in serum and supernatant of incubated splenocytes.Methods:Seventy-two female (>18g) 8-wk-old NOD mice were obtained and randomized into three experiments for different treatment.Experiment 1: Forty mice were randomized into 2 groups, The first 20 mice were treated with 5μg/0.1ml TCS by intraperitoneally every 3 weeks, another 20 mice with 0.1ml PBS as control group. At the time of developed diabetes or at the end of the fifth month, mice were killed to observe the incidence of diabetes and the severity of insulitis.Experiment 2: Twenty-two mice were randomized into 3 groups and treated with 5μg/0.1ml TCS, 10μg/0.1ml TCS or 0.1ml PBS byintraperitoneally every 3 weeks separately. At the end of the forth week, mice were killed to investigate the change of T lymphocyte subsets in thymus and spleen and the production of EL-4 and IFN-r in serum.Experiment 2: Eight mice in this group were killed and their splenocytes were cultured in culture medium containing the TCS (at the final concentration of 0.1μg/ml TCS or 1.0μg/ml TCS) or the same PBS. Production of IL-4 and IFN-y in supernatant was determined by ELISA.Result:1. The incidence of diabetes in TCS group (20%) was obviously less than PBS group (60%)(P<0.05);2. The severity of insulitis in TCS group was lower than PBS group (P<0.001);3. In thymus CD4+T cells in 5μgTCS (44.62±1.20) and 10μgTCS (33.10±3.18) group increased markedly than in PBS group (21.88±7.31) (P<0.01) and CD8+T cells in 5μgTCS (13.57±1.25) and 10μgTCS (9.89±1.18) groups also increased markedly than in PBS group (5.63±1.65)(P<0.01);4. In spleen CD4+T cells in 5μgTCS (41.96±1.48) and 10μgTCS group (32.54±1.94) increased markedly than in PBS group (30.34±2.33)(P<0.01 and P<0.05) but CD8+T cells only in 5μgTCS group (7.56±1.06) increased markedly than in PBS group (6.03±1.08)(P<0.01), 10μgTCS group (6.78±1.00) have no significant increased;5. In serums the production of IL-4 in 5μg TCS group (53.52±2.74) washigher than in PBS group (34.13±3.13)(P<0.01) while in 10μg TCS group (35.75±1.82) was no significant increase. The production of IFN-r shows no significant difference in three groups (167.95±1.98, 176.18±5.24 vs169.74±4.28);6. In supernatant, the level of IL-4 in two TCS groups (16.68±1.22 and 10.70±0.51) was obviously higher than in control group (7.05±0.06)(P<0.01 and P<0.05) and IFN-r show no difference in three groups (208.44±50.08, 226.68±70.71 vs225.89±28.04).Conclusion:1, TCS could interfere with the development of diabetes and severity of insulitis in NOD mice.2. The possible mechanism seems to be related to:1) TCS could help to the development of T lymphocytes in thymus and spleen and also help to the transform from CD4+Th0 cells to CD4+Th2 cells.2) TCS could increase the systematic production of Th2 cytokines IL-4 toward the shift of Th1/Th2 balance.3) TCS could stimulate the spleen to secret Th2 cytokines IL-4, which have active effect in regulating Th1/Th2 balance.3. The role of TCS in NOD mice maybe related to the dose of TCS and the mechanism.4. This study provides further support for the importance of the balance of Thl/Th2 and the protective effect of IL-4 on the development of type 1dibetes in NOD mice.