Effect Of A Broad-Spectrum Caspase Inhibitor On Mouse Contact Dermtitis

ObjectiveTo investigate the effect of Z-VAD-FMK,a broad-spectrum caspase inhibitor,on mouse allergic contact dermatitis(ACD) and mouse irritant contact dermatitis(ICD) and study its effect on T lymphocytes.To interpret its anti-inflammatory mechanism and provide important data for developing new anti-dermatitis drugs.Methods1.The in vitro permeation was evaluated using Franz diffusion cells fitted with normal mouse skin and high pressure liquid chromatography(HPLC) was used to detect drug concentration.As to in vivo permeation,20%ethylene alcohol was used to remove epidermis of mouse ears on which Z-VAD-FMK was painted and then ears were grinded in liquid nitrogen.The drug concentration was determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS).2.1-Fluoro-2,4-dinitrobenzene(DNFB) was used to produce classical ACD mouse model.At different time after elicitation,the scoring system was evaluated. Several parameters were detected,including ear swelling,difference of ear weight between different side,and thickness of ear tissue under microscopic observation.3.DNFB was used to produce primary ACD mouse model.Two models were compared with each other in three aspects.Firstly,ear swelling was detected by a digital vernier caliper and used as clinical index.Secondly,levels of T lymphocyte cytokines and their mRNA in ear tissues were detected using ELISA and real time RT-PCR respectively.In the last place,local lymph node assay(LLNA) was carried out and flow cytometry was used to detect proliferation and activation of T lymphocytes in local lymph node.4.Classical ACD mouse model and primary ACD mouse model were used to investigate the effect of Z-VAD-FMK on ACD in three aspects:therapeutical effect,preventive effect on elicitation,and preventive effect on induction.5.ELISA was used to detect levels of Th1 cytokines(INF-γand IL-2) in ear tissues in investigating the effect of Z-VAD-FMK on mouse ACD.Real time RT-PCR was used to detect levels of their mRNA.6.Local lymph node assay(LLNA) was carried out.BrdU-flow cytometry was used to detect proliferation of T lymphocytes in local lymph node and flow cytometry to detect activation of T lymphocytes after topical use of Z-VAD-FMK.7.Croton oil was used to produce ICD mouse model.To investigate its effect on ICD,Z-VAD-FMK was externally applied on the surface of the ear.Results1.In vitro permeation rate of Z-VAD-FMK in 6h,12h and 24h were 5.35%,15.52, and 22.76%respectively,with 200μl of 20mmol/1 Z-VAD-FMK in donor compartment,4ml of 0.9%saline in receptor,and a diffusion area of lcm~2.As to in vivo permeation,the concentration of Z-VAD-FMK in ear tissue achieved 100μmol/1 after twice topical use of 15μl of 2.5mmol/1 Z-VAD-FMK with the interval of 12h,which is similar to the effective concentration in vitro.2.In classical ACD mouse model,similar to ear swelling,difference of ear weight between different side and thickness of ear tissue under microscopic observation also indicate that the degree of inflammation went up to the peak(1-2)d after elicitation.Then the degree of inflammation declined gradually,and reached about half of the peak at day 4. 3.In primary ACD mouse model,ear swelling response was observed 6 days after ear painting of DNFB,which was the same as the time interval from back elicitation to occurrence of ear inflammation in classical ACD.Kinetics of the inflammatory response to DNFB during primary ACD was just like that during classical ACD.Infiltrating cells in ear tissues were mostly mononuclear cells in the two models.Levels of IL-2 and INF-γwere all significantly higher in both models,compared with that in normal control,but there was no significant difference in the level of IL-4.Proliferation and activation of T lymphocytes in local lymph node was observed in both models,but not in normal control.The difference between them was significant.4.Z-VAD-FMK was externally used on mouse ACD at three time points:2h after elicitation,before elicitation,and before induction.Results showed that in these three conditions,three evaluation indexes of Z-VAD-FMK-treated mice were all significantly lower than that of vehicle-treated mice.5.In the above three conditions,levels of both INF-γand IL-2 in ear tissues of Z-VAD-FMK-treated mice were significantly lower than that of vehicle-treated mice.6.In LLNA,mean intensity of BrdU in T lymphocytes of Z-VAD-FMK-treated mice was significantly weaker than that of vehicle-treated mice,and percents of activation markers-positive T lymphocytes were all significant lower than that of vehicle-treated mice too.7.In investigation of the therapeutic effect and preventive effect of Z-VAD-FMK on ICD,there was no significant difference between Z-VAD-FMK-treated mice and vehicle-treated mice at all.Conclusions1.Z-VAD-FMK can permeate through mouse skin,which provides possibility to its clinical use as topical medication.2.Just as classical ACD,primary ACD is also a type of Th1 reaction.They can take ??the place of each other in application,providing new choice for investigation of new anti-inflammatory and anti-immune drugs.3.Topical application of Z-VAD-FMK can effectively inhibit allergic contact dermatitis in three aspects:treating,preventing induction,and preventing elicitation.4.Z-VAD-FMK failed to inhibit ICD,which suggests that Z-VAD-FMK produces a marked effect mainly by anti-immune response;its anti-irritant inflammation effect is weaker however.5.Topical use of Z-VAD-FMK can inhibit the activation and proliferation of T lymphocytes in both skin tissue and local lymph node,leading to the inhibitory effect of contact allergic reaction.6.In order to bring the anti-inflammatory effect of Z-VAD-FMK into full play,we should pay more efforts to develop its dosage form and molecular structure.