实验动物：C57BL/6，7周龄，雌性

造模试剂：OXA（恶唑酮）

造模方法：致敏—Day0涂抹右耳+背部

激发—Day7~Day25涂抹右耳+背部

模型评价内容

• 小鼠体重
• 背部及耳部表皮厚度测量
• 血清中总IgE检测
• 病理分析：皮肤炎症等病变，嗜酸性粒细胞等

The AD model was induced by OXA in wild-type C57BL/6 mice. (A) Mouse body Weight. (B) Ear thickness. (C) Serum total IgE concentration. Ear thickness and the serum IgE were significantly increased in the OXA -treated group. Values are expressed as mean ± SEM. **p<0.01.

Analysis of ear skin pathology and lymphocyte infiltration in a wild-type C57BL/6 AD model. (A) Hematoxylin and eosin (H&E) staining of mouse ear tissue sections; (B) Eosinophil infiltration scores in the ear epidermis. Values are expressed as mean ± SEM. ****p<0.0001.

治疗策略

可以选择在免疫接种时开始治疗-预防性策略，也可以在小鼠发病后开始治疗-治疗性策略。

AD小鼠疾病模型（C57BL/6）用于评价Dexamethasone药效

• 从B图可以看出，Oxa造模组的耳部厚度有明显的增加，同时血清中总的IgE（图C）有明显的升高，证明造模成功。
• 从耳部厚度和IgE变化情况可以看出，不同浓度的Dexamethasone对AD疾病具有不同程度的缓解作用。
• 高剂量组（0.09%DPD）对耳部皮肤水肿的缓解作用达到了与对照组持平的效果。但与对照组相比，也会导致明显的体重减轻。

The efficacy of dexamethasone (Dex) was verified in a wild-type C57BL/6 AD model.

病理分析

组别设置

从病理统计结果发现，除对照组外，其余四组动物背部和耳朵皮肤表皮均可见不同程度的基质细胞增生、表皮增厚、角化过度伴角化不全、表皮痂皮，真皮及皮下组织可见混合炎细胞浸润等特异性皮炎相关的病理性改变，提示G2造模组和G3-G5给药组的模型动物均造模成功。对各组动物的背部皮肤和耳朵皮肤嗜酸性粒细胞浸润改变进行了病理学评分和统计。受试品组G3-G5背部和耳朵皮肤嗜酸粒细胞浸润均低于G2造模组。其中，背部皮肤中，受试品G3-G5组嗜酸粒细胞浸润程度未见显著差异。耳朵皮肤中，受试品G4、G5组嗜酸粒细胞浸润程度要低于G2和G3组。上述结果提示三组浓度的受试品均对特异性皮炎相关症状有一定改善作用，其中浓度为0.06%和0.09%的DPD药效较好。

Efficacy Validation on AD model (B-hIL4/hIL4RA mice) for dupilumab (in house)

Fig 1. Dupilumab alleviated OXA induced atopic dermatitis in B-hIL4/IL4RA  mice. Mice received 0.8% OXA on ear and skin on day 0, then received 0.4% OXA application three times a week from day 7 to day 25. Body weight and ear thickness were recorded after OXA application. Mice received PBS or dupilumab (10， 25 mg/kg) twice a week from day 6 for totally 6 times. Ear, back skin and blood were collected at the end (A). Dupilumab produced no effects on body weight (C). Ear thickness (B) and serum IgE (D) were decreased in dupilumab treated mice.

Fig 2. Efficacy of anti-human IL4R antibody in B-hIL4/hIL4R mice. Dose dependent effects of anti-human IL-4R antibody (in house) in oxazolone induced skin lesions in B-hIL4R mice.

Dupilumab reduced OXA induced human IL-4 increase in B-hIL4/hIL4RA mice (Elisa)

Fig 3. Protain expression analysis in OXA-induced AD model of B-hIL4/hIL4RA mice by ELISA. Ear and skin samples of modeling area were collected from at day 9, 16, 23 and 26 and analyzed ELISA. Tissue sample homogenate supernatants were loaded for ELISA detection, and the results were standardized by total protein concentration of the corresponding sample.

Protein expression analysis in OXA-induced AD model of B-hIL4/hIL4RA mice by Luminex

Fig 4. Protain expression analysis in OXA-induced AD model of B-hIL4/hIL4RA mice by Luminex. Ear and skin samples of modeling area were collected from at day 9, 16, 23 and 26 and analyzed by Luminex. Tissue sample homogenate supernatants were loaded for detection.

Fig 5. Protain expression analysis in OXA-induced AD model of B-hIL4/hIL4RA mice by MSD. Ear and skin samples of modeling area were collected from at day 9, 16, 23 and 26 and analyzed by Luminex. Tissue sample homogenate supernatants were loaded for detection.

Fig 6. Mouse IL-5 and IL-13 expression analysis in OXA-induced AD model of B-hIL4/hIL4RA mice by qRT-PCR. Ear were collected at day 9 and 26 and total RNA of these samples were extracted. Then RNA samples were reverse transcribed and mouse IL-5, mouse IL-13 levels were measured by qRT-PCR, while mouse GAPDH was used as house keeping gene.