外用广谱半胱氨酸天冬氨酸蛋白酶抑制剂抑制小鼠变应性接触性皮炎的激发

Inhibition of elicitation of allergic contact dermatitis by topical use of Z-VAD-FMK, a broad caspaseinhibitor： experiment in mice

目的研究外用广谱半胱氨酸天冬氨酸蛋白酶（caspase）抑制剂苄氧羰基．缬氨酰一丙氨酰．门冬氨酰氟甲基酮（Z-VAD-FMK）对小鼠变应性接触性皮炎（ACD）激发阶段的抑制作用，探讨其对T细胞的作用。方法以2，4二硝基氟苯（DNFB）制作小鼠经典ACD模型，于激发前外用不同浓度的Z．VAD．FMK，以耳肿度、双耳重量之差及组织切片中同一位置双耳双面距离之差为指标，观察Z—VAD．FMK对小鼠ACD激发阶段的抑制作用；以酶联免疫吸附法（ELISA）检测小鼠耳皮损中T细胞因子干扰素1（INF．1）及白细胞介素2（IL-2）的含量，以实时反转录PCR（real—timeRT．PCR）方法检测上述因子mRNA水平（实验结果以“相对于每百万管家基因的拷贝数”表示）；进行局部淋巴结分析（LLNA），以5-溴脱氧尿核苷（BrdU）一流式细胞术检测耳引流淋巴结中淋巴细胞增殖活性，以流式细胞术检测淋巴细胞表面活化标记。结果1．25mmol／LZ-VAD—FMK组小鼠耳肿度为（12．6±1．2）×10^-2mm，双耳重量差为（3．1±0．2）mg，镜下双耳双面距离差为（12．1±1．1）×10^-2mm，均低于阴性对照组[（17．4±1．6）×10^-2mm，（4．2±0．3）mg，（16．7±1．5）×10^-2mm；q=3．25、2．98、3．12，均P〈0．05]。1．25mmol／LZ-VAD-FMK组小鼠耳皮损中INF-1与IL-2mRNA及蛋白的表达均低于阴性对照组[INF一1mRNA：152±12比220±15，IL-2mRNA：96±8比156±11，q=3．15、3．42；INF-y蛋白：（856±45）pg／mt比（1180±58）pg／ml，IL-2蛋白：（167±12）pg／ml比（225±16）pg／ml，q=3．11、3．14；均P〈0．05]。1．25mmol／LZ-VAD-FMK组小鼠耳引流淋巴结中T细胞内掺入的BrdU的平均荧光强度明显低于阴性对照组（185±15比298±21，q=3．02，P〈0．05），T细胞3种表面活化标记CD59、CD25与Ia阳性细胞的百分率亦均明显低于阴性对照组（7．8％±0．7％比10．5％±1．O％，9．8％±0．8％比14．5％±1.1％，31.2％±2．8％比46．5％±3．2％，q：3．16、3．52、3．11，均P〈0．05）。结论在小鼠ACD激发前外用广谱caspase抑制剂Z—VAD—FMK可以抑制小鼠皮损局部及耳引流淋巴结中T细胞的增殖与活化，从而抑制小鼠ACD的发生。

Objective To explore the effects of N-benzyloxycarbonyl-Val-Ala-Asp- fluoromethylketone （Z-VAD-FMK）, a broad caspase inhibitor, on the elieitation of murine allergic contact dermatitis （ACD） and examine the effects on T lymphocytes. Methods 1-fluoro-2,4-dinitrobenzene （DNFB） was used to establish the classical murine model of ACD. Different concentrations of Z-VAD-FMK were applied before ear provocation. Several parameters were detected, including ear swelling degree, weight differences and thickness of ear tissue under microscope between 2 ears. Enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of Thl cytokines （INF-/and IL-2） in ear tissues. Real-time reverse transcription-polymerase chain reaction （RT-PCR） was used to detect their levels of mRNA and theresults were shown as ＂copies relative to one million housekeeping genes＂. Local lymph node assay （LLNA） was conducted. Bromodeoxyuridine-flow cytometry was used to detect the proliferation of T lymphocytes in local lymph node and flow cytometry to detect the activation of T lymphocytes. Results The right ear swelling degree, weight differences and thickness between two ears in the 1.25 mmol/L Z-VAD-FMK group were （12.6±1.2） x1O^-2mm, （3.1±0.2） mg, and （12.1±1.1） x10^-2 mmrespectively. And they were all significantly lower than those of the negative control group（ （17. 4±1.6） x 10^-2 mm, （4. 2±0. 3） rag, （16.7 ＋1.5）x10^-2 mm;q =3.25,2.98,3.12, all P 〈0.05）. The levels of INF-y/ and IL-2 in the ear skin lesions of 1.25 mmol/L Z-VAD-FMK group were （ 856±45 ） and （ 167±12 ） pg/ml respectively and they were both significantly lower than those of the negative control group （ （ 1180±58） and （225 ＋ 16） pg/ml;q =3.11,3.14,both P 〈0. 05）. The mRNA levels of the above two cytokines were 152±12 and 96±8 respectively and they were both significantly lower than those of the negative control group （220 15 and 156±11 ;q = 3.15,3.42, both P 〈 0. 05 ）. In LLNA, the mean intensity of BrdU in T lymphocytes of 1.25 mmol/L Z-VAD-FMK-treated group was significantly weaker than that of the negative control group （ 185±15 vs 298±21, q = 3.02, P 〈 0. 05 ）. The percents of activation markers-positive T lymphocytes of the Z-VAD-FMK group were 7.8% ±0. 7%, 9. 8% ±0. 8% and 31.2% ±2. 8% respectively and they were all significantly lower than those of the negative control group （ 10. 5% ±1.0%, 14. 5%±1.1% ,46. 5% ±3.2%, q = 3.16,3.52,3.11, all P 〈 0.05 ）. Conclusion Topical use of Z-VAD-FMK prior to ear provocation can suppress the proliferation and activation of T lymphocytes in both skin tissues and local lymph nodes and thus result in the inhibitory effect of allergic contact dermatitis.