The Pharmacodynamic Test Of Arotinoid Ethylester On Psoriasiform Mice Model

ObjectiveArotinoid Ethylester (AE) has various pharmacological effects such as immunomodulation, inducing cell differentiation, anti-tumor, anti-cuticularization, anti-rheumatism, anti-inflammation, supress sebum production, dissolving horny cells, depigmentation and inhibiting some bacterial activity, etc.To evaluate the therapeutic efficacy for psoriasis,We detected the pharmacodynamic effect on psoriasiform mice, in regard to epidermic cell proliferation, apoptosis of keratinocytes,augmentation of granular cells, inflammation inhibition, supression of chemotaxis of PMN, etc. Retinoid acitretion was used as a positive control.Materials and MethodsⅠ. Grouping of mice1.Mice: SPF glass adult BALB/c mice, half female and male (weight 20±5g) were obtained from the Institute of Laboratory Animal Science of Chinese Academy of Medical Science.2.Grouping: The mice were allocated into several groups randomly as followes:2.1 Group: Effects on the proliferative psoriasiform pathological changes: 230 mice were separated into 7 groups randomly, 30～40 mice in each group, each group was separated again into 3～4 time- point groups (2-week, 4-week, 6-week). The normal control group and the model group, each has an extra group(0-week-group), 10 mice in each. After setting up the model, different doses of drugs were given to mice by way of intragastrie administration. Mice of different groups were sacrificed, the skin sample was excised from the back of mice with psoriasiform pathological changes. The samples was divided into triplicate, deposited respectively at-20℃refrigerator after embedded with OCT, -80℃refrigerator and fixed with 4％formalin.2.2 Group: Test against inflammation in the ear of mice: 70 mice were allocated into 7 groups randomly, 10 mice each group, with female and male half. We droped 50 ul croton oil on the right ear in mice respectively (except the normal control group), and gave the drug to mice by way of intragastric administration. The left ear of mice acted as normal blank control. After 2 hours, the mice were sacrificed and the left and right ear along the ear baseline were excised, a 6 millimeter diameter punch was obtained from the left and the right ear. Punched samples were weighted with a electronic scale.2.3 Group: Test on the ability of inducing the proliferation of the epidermical granular layer: 72 mice were divided into 2 groups which were subgrouped to 6 mice in each subgroup, with half female and male mice. All the mice were painted with different drugs with cotton bud every day. At day 10 and 14, tail skin was obtained, respectively, and fixed in 70％alcohol, embedded with paraffin, and ready for histological examination.Ⅱ. Establishing the psoriasiform model in mice1. Establishment of the psoriasiform proliferative model.All the mice to be tested were ablated their back hair with the depilator before the test day. According to the literature, DMBA and croton oil were applied to the back of the mice, respectively. On the first day and the eighth day, 75％ DMBA(150ug/200ul acetone) was applied to the 2.5*2.0 cm~2 back skin of the depilated area.On the fifteenth day, the 0.25％croton oil(50ug/200ul acetone) was applied to the same area, 2 times per week, totally four weeks.2. Evaluating the therapeutical effect on the psoriasiform hyperplastic model in mice.①The HP (histopathology) examination on the pathological changes in the skin (H.E. technique).②Detection of the number of the epidermal corneum layers. (H.E. technique).③Detection of the expression of PCNA in the skin (I.H.technique).④Detection of the expression of TNFR-p75 in the skin (I.H.technique).⑤Detection of the expression of IL-8RⅡin the skin (I. H.technique).⑥Detection of the expression of Bax in the skin (I.H.technique).Ⅲ. Normalization of the granular cell in the epidermis of the tail scales in the miceWe examined the tail skin by light microscope, to count the number of granular cell layers.Ⅳ.Detection of anti-inflammation effect on the ears of the mice1. Detection of weight incrementHaving established the inflammation model of ears in mice by croton oil, we applied different drug to the mice by way of intragastric administration. 2 hours later, we used a puncher of 6 millimeter diameter to punch an ear patch from the illed ear (right side) and the normal control one (left side), and the ear weights of mice were weighed by an electronic balance. Counting the difference between the two ears and the result was considered as Swelling-Degree according to the literature. ①Swelling-Degree:the difference of the ear weight between two ears.②Inhibition radio of inflammation: 1—Swelling- Degree of testing-group/Swelling-Degree of control group×100％.Ⅴ. Statistical analysisSPSS software was used, the measurement data was analyzed by One Way ANOVA, the numeration data was analyzed by rank sum test. A P value that is less than 0.05 was considered to be significant statistically; A P value less than 0.01 very significant.ResultⅠ. Evaluation of the therapeutic effect on the psoriasiform proliferative model.1.Detection of the expression of IL-8RⅡin epidermis(1) The expression of IL-8RⅡin model and control- groups were obvious.(2) The three AE groups shows not only dose-dependancy, but also time-dependancy, in regard to IL-8RⅡexpression. The expression of IL-8RⅡin High dose AE group was the lowest, which decreased progressively with the time course.(3) The expression of IL-8RⅡinn Acitretin-Group was obviously less than the three AE groups at the early time of administration(2W,4W),P＜0.05(2W) and P＜0.01, respectively(4W). But the expression of which became very high at later stage, and the difference between Acitretin and the three AE groups was statistically significant(P＜0.05).2. Detection of the expression of TNFR-p75.(1) The expression of the model group was obviously highter than the normal control, and it increased progressively with the continous stimulation by the croton oil.(2) The expression of the three AE-groups were less than the Acitretin-Group at each time point statistically. At the later time it showed a very high expression of TNFR-p75, even higher than that of the model group(P＜0.01).(3) The expression in high-dose group was lower significantly than in low-dose groups(P=0.03), and showed higher tendancy with the time course.3. Effect on the number of epidermal cells of mice:(1) The three AE-groups showed that AE promoted the hyperplastic epidermis to proliferate at early time and then inhibite progressively with time.(2) The Acitretin group showed a good therapeutic effect of inhibiting the hyperplastic epidermis.4. Detection of the expression of PCNA in the hyperplastic epidermis.(1)The AE-Group showed dose-dependant effect: The low dose group showed a better effect of inhibiting the expression of PCNA and the effect of which was time dependant.(2) The Acitretin showed a therapeutic effect of inhibiting the expression of PCNA at each time point, but the effect was obviously weaker than the low dose group of AE.5. Detection of the expression of Bax in the hyperplastic epidermis.(1) Three AE group all showed the effect of enhancing the expression of Bax and the effect was either dose-dependent: the higher the dose, the better the effect; or time dependant in the mid-dose group and the high-dose one: the effect was better gradually with the time course. (2) The Acitretin-Goup showed a weak effect on enhancing the expression of Bax in epidermis at the three time point, The difference between the high-dose group of AE and the Acitretin group was statistically significant(P＜0.01).ⅡEvaluation on the therapeutic effect against inflammation in the ear of mice1. Gross observation of the ear of miceAfter applied croton oil in the ears of mice 2 hours later, only the high-dose group of AE showed no obvious inflammatory change, the other groups all showed distinct inflammatory changes: hyperemia, swelling, etc. The Acitretin group showed light inflammatory change.2.Detection of the Swelling-Degree of the ear of mice.The Swelling-Degree were all higher at the Acitretin group, the mid-dose and low-dose groups of AE, compared with the normal control group(P＜0.05). Among the administration groups, only the high dose group of AE had no obvious difference with the normal control group(P＞0.05)and was statistically different compared with the model control group(P＜0.05). The Acitretin group showed no statistical difference with the model control group(P＞0.05); The high-dose group of AE had significant difference with the mid-dose and low-dose group of AE(P＜0.05).ⅢDetection on the effect of cellular proliferation in granular layer of epidermis of tail scales in mice.1. 10 days after application, only the high-dose group of AE and the Acitretin group showed obvious effect in inducing cellular proliferation.2. 14 days after applichation, Acitretin and the mid-dose AE groups showed obvious inducing effect, and the high-dose group of AE was the strongest, the difference was statistically significant, compared with Acitretin and the mid or low dose group of AE.Conclusion1. DMBA/croton can successfully induce the psoriasiform changes in mice, such as hyperplasia of KC, the inflammatory change in skin and some biochemical and immunological changes were similar to that in psoriasis.2. AE either at high or low dose has the effect of anti-chemotaxis effect on leukocyte. The effect was most obvious with higher dose of AE at early time point, but the long-term medication of AE can cause the toxic reaction which don't appear in the low-dose of AE; so the low-dose of AE is more safe for the long-term medication. Acitretin shows a good effect at earlier time but it has severe toxic reaction on long-term use.3. AE can inhibit the proliferation of epidermal cells, the effect shows either time-dependancy:the longer term use, the better effect, or dose-dependency:the lower dose, the better effect. Acitretin has a good effect at earlier time point, but the long-term effect is weaker than that of AE.4. AE and Acitretin are all effective on inducing the apoptosis of the hyperplastic epidermic cells. The effect of AE is either time or dose-dependant. Acitretin is less potent than that of AE, especially the high dose of AE.5. AE and Acitretin both have the therapeutic effect on inducing the proliferation of granular layer of epidermic cells in the mice tail. The effect of AE shows either time or dose-dependency.