组织蛋白酶G在1型糖尿病小鼠CD4^＋T淋巴细胞活化过程中的作用

Role of cathepsin G in the activation of CD4^＋ T lymphocytes in mice with type 1 diabetes

目的验证调节组织蛋白酶G（CatG）对1型糖尿病（T1DM）NOD小鼠CD4^＋T细胞活化水平的影响，并探讨特异性阻断CatG对T1DM的防治作用。方法选取6周龄雌性NOD小鼠84只，体重16-18 g，采用随机数字表法将其中36只分为3组：血糖正常组（正常组）、糖尿病前期组和糖尿病组（每组12只），检测各组CatG的基因表达与CD4^＋T细胞活化状态。另外48只小鼠进行T1DM造模，糖尿病前期组给予CatG小干扰RNA（CatG siRNA）干预（n=12），糖尿病组给予CatG抑制剂（0.1 mg腹腔注射，隔日1次，n=12）。设立相应模型对照组（各12只），干预8周后处死取材。观察CatG抑制剂治疗糖尿病小鼠对CD4^＋T细胞活化、胰岛功能、胰岛炎症的影响；观察早期应用CatG siRNA对NOD小鼠CD4^＋T细胞活化状态及糖尿病进程的影响。3组资料比较采用单因素方差分析，2组资料比较采用独立样本t检验。结果（1）血糖正常组、糖尿病前期组和糖尿病组小鼠CatG基因表达量分别为0.42±0.21、0.60±0.33、0.94±0.45，差异有统计学意义（F=12.89，P〈0.05），正常组明显低于其他2组；与正常组相比，糖尿病前期组和糖尿病组小鼠脾脏和外周血CD4^＋T细胞逐渐活化，表现为辅助性T细胞（Th）1比例明显升高，而Th2细胞和调节性T（Treg）细胞显著下降（F=11.85-21.36，均P〈0.01）。（2）与对照组相比，特异性CatG抑制剂组NOD小鼠血糖水平明显降低（t=11.26-12.45，均P〈0.01）；胰岛素分泌水平升高（t=11.89-13.78，均P〈0.01）；外周血和脾脏CD4^＋T细胞的活化水平减轻，差异具有统计学意义（均P〈0.05）。（3）与对照组相比，早期应用CatG siRNA后Th1细胞比例下降[外周血：（5.1±1.4）%比（2.4±0.3）%，脾脏：（10.6±2.0）%比（6.3±1.5）%，t=20.78、27.96，均P〈0.05]，Th2和Treg细胞比例明显升高（t=20.59-30.25，均P〈0.05），小鼠血糖降至正常或处于糖尿病前期水平，胰岛素水平显著升高（t=31.69-33.98，均P〈0.01）。结论CatG是T1DM发病中CD4^＋T细胞活化过程中的主要效应蛋白酶。特异性阻断CatG可能通过抑制CD4^＋T细胞的活化来保护胰岛β细胞功能，从而延缓或阻断T1DM的发生发展。

Objective To identify whether regulation of Cathepsin G （CatG） expression can influence CD4^＋T lymphocytes （T cells） activation in non-obese diabetic （NOD） mice, and to demonstrate the role of CatG inhibition in prevention and treatment for type 1 diabetes mellitus （T1DM）.Methods A total of 36 female NOD mice （6 weeks old） were randomly divided into 3 groups according to the duration and blood glucose： normal control group, pre-diabetes （pre-DM） group, and diabetes （DM） group. CatG gene expression and CD4^＋T cells activation were examined in these three groups of NOD mice （12 mice in each group）. After modelling, CatG small interfering RNA （siRNA） was administrated to treat the other pre-diabetic NOD mice, and CatG inhibitor was administrated to treat diabetic NOD mice （12 mice in each intervention group） . Also control groups （12 mice in each group） were respectively set. After 8 weeks, all the mice were executed and histological analysis were performed. The effect of CatG inhibitor was investigated in NOD mice on CD4^＋T cells activation, islet β cell function, and islet inflammation. Furthermore, NOD mice were injected with CatG siRNA in early stage to observe the effect of CatG knockdown on CD4^＋T cells activation status and on diabetes progression. The data of three groups were analyzed with single factor analysis of variance, and the data of two groups were compared with independent samples t test.Results （1） The gene level of CatG of the 3 groups was 0.42±0.21, 0.60±0.33, 0.94±0.45, respectively （F=12.89, P〈0.05）. The gene level of CatG in pre-DM group and DM group was significantly higher than that in normal control group. Meanwhile, compared with normal control group, the CD4^＋T cells in spleen and peripheral blood were gradually activated in pre-DM group and DM group, resulting in more T helper （Th）1 cells and fewer Th2 and regulatory T（Treg） cells （F=11.85-21.36, all P〈0.01）. （2） After administration with CatG inhibitor, the level of blood glucose was decreased （t=11.26-12.45, all P〈0.01） and insulin was increased （t=11.89-13.78, all P〈0.01）;Meanwhile, the activation of CD4^＋T cells was decreased, and the difference was statistically significant （all P〈0.05） . （3） Compared with the control group, after early application of CatG siRNA, the percentage of Th1 cells was decreased [peripheral blood： （5.1±1.4）% vs （2.4±0.3）%, spleen： （10.6±2.0） % vs （6.3±1.5） %, t=20.78, 27.96, all P〈0.05]. The percentage of Th2 and Treg cells was increased （t=20.59-30.25, all P〈0.05）. Meanwhile, the mice could maintain in normal glucose status or pre-DM status, and the insulin level could be enhanced significantly in CatG siRNA group （t=31.69-33.98, all P〈0.01）.Conclusion CatG is the major effector protease in CD4^＋T cells activation in the pathogenesis of T1DM. Targeted inhibition of CatG may delay or block T1DM progression through alleviating CD4^＋T cells activation.