广谱半胱氨酸天冬氨酸蛋白酶抑制剂抗小鼠变应性接触性皮炎的研究

Attenuation of allergic contact dermatitis by Z-VAD-FMK, a broad caspase inhibitor： experiment with mice

目的研究广谱半胱氨酸天冬氨酸蛋白酶（caspase）抑制剂Z-VAD—FMK对小鼠变应性接触性皮炎（ACD）的抑制作用。方法以Franz垂直扩散池进行Z-VAD-FMK的体外透皮实验，高效液相色谱方法检测其透皮量。建立ACD小鼠模型；实验组分别以0．1、0．5、2．5及5mmol／L4种浓度的Z—VAD-FMK溶液外涂于ACD模型小鼠一侧耳背部；阴性对照组以磷酸盐缓冲液（PBS）或含二甲基亚砜（DMSO）的PBS外涂，每组6只小鼠。以耳肿度、双侧耳重量之差及组织切片中双侧耳双面距离之差为指标，观察Z-VAD-FMK对小鼠ACD的抑制作用；以酶联免疫吸附试验（ELISA）法检测各组小鼠耳皮损中自细胞介素2（IL-2）及1-干扰素（INF-1）的含量。结果在供给池为5mmol／LZ—VAD—FMK 200μl、接收池容积为4ml、有效扩散面积为1cm^2的条件下，Z—VAD—FMK6、12及24h的透皮率分别为2．15％、9．62％及12．85％。2．5mmol／LZ—VAD—FMK组小鼠耳肿度为（12．5±1．4）×10^-2mm，重量差为（3．2±0．3）mg，镜下距离差为（11．8±1．3）×10^-2mm，均低于阴性对照组[（19．1±2．0）×10^-2mm，（4．3±0．4）mg，（16．8±1．7）×10^-2mm，q=4．76，4．34，4．87，均P〈0．05]。2．5mmol／LZ—VAD-FMK组小鼠耳皮损中IL-2及INF-γ的表达均低于阴性对照组[IL-2：（148±10）pg／ml vs（205±18）pg／ml，t=2．205，P〈0．05；INF-γ（650±45）pg／ml vs（1030±58）pg／ml，t=2．375，P〈0．05]。结论广谱easpase抑制剂Z-VAD-FMK能够透过皮肤，并抑制T细胞的活化与增殖，抑制ACD。

Objective To investigate the effects of N-benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone （Z-VAD-FMK）, a broad caspase inhibitor, on allergic contact dermatitis （ACD）. Methods A Balb/c mouse was killed and its back skin was peeled, put into a Franz diffusion cell, and smeared with Z-VAD-FMK. High performance liquid chromatography was used to examine the permeation rate. Dinitro-fluorobenzene （DNFB） was applied on the depilated back of mice as inducer and then on the back-side of right ear as provocative agent 6 clays later so as to establish mouse ACD models. Two hours after the provocation 24 mouse models were divided into 4 equal groups to undergo the application of Z-VAD-FMK at the concentration of 0. 1, 0.5, 2. 5, and 5 mmol/L on the back-side of right ear twice. PBS was applied in the negative control group. Then the mice were killed with their 2 ears cut off. Microscopy was used to measure the right ear swelling degree, and differences in weight and thickness between the 2 ears. ELISA was used to detect the levels of IL-2 and INF-γ in the ear tissues. Results The transdermal permeation rates of Z-VAD-FMK at the time points 6, 12, and 24 h were 2. 15%, 9. 62%, and 12. 85% respectively. The right ear swelling degree, and differences in weight and thickness between the 2 ears in the 2. 5 mmol/L Z- VAD-FMK group were （12.5 ±1.4） ×10^-2 mm, （3.2±0.3） mg, and （11.8±1.3） ×10^-2 mm respectively, all significantly lower than those of the negative control group [ （ 19. 1 ± 2.0） × 10^-2 mm, （4. 3 ±0. 4） mg, and （ 16. 8 ± 1.7） × 10^-2 mm, all P 〈0. 05]. The IL-2 and INF-γ levels in the ear skin lesion of the 2. 5 mmol/L Z-VAD-FMK group were （ 148 ± 10） and （ 650 ± 45 ） pg/ml respectively, both significantly lower than those of the negative control group [ （205 ± 18） and （ 1030 ± 58） pg/ml, both P 〈 0. 05 ]. Conclusion Z-VAD-FMK can permeate through mouse skin and inhibit the activation and proliferation of T lymphocytes, leading to the inhibitory effect of contact allergic reaction.