融合蛋白LTβR-Fc对卵清蛋白诱发的皮炎小鼠模型的影响

Effects of a fusion protein LTβR-Fc on ovalbumin-induced dermatitis in a mouse model

目的：探讨单纯疱疹病毒侵入介质配体信号通路 LIGHT-HVEM 阻断剂 LTβR-Fc 融合蛋白对卵清蛋白诱发的特应性皮炎小鼠模型的影响。方法将 BALB/c 小鼠随机分为空白对照组（以100μl 生理氯化钠溶液进行刺激）、模型组（将100μg 卵清蛋白溶于100μl 生理氯化钠溶液中进行刺激）以及阻断剂组（在卵清蛋白致敏前24 h 将100μg LTβR-Fc 溶于100μl 生理氯化钠溶液作为阻断剂进行刺激），在开始致敏后第0、4、8、12、15、20、23、27、31、34天分别对各组实验小鼠进行病情严重程度评分，同时测量记录皮损面积大小，最后于造模结束时行眼眶取血并提取血清，处死各组小鼠，首先分别采用 RT-PCR 法和 ELISA 法检测小鼠皮损和脾细胞上清液内干扰素γ（IFN-γ）、白细胞介素（IL）-4和 IL-5的 mRNA 和蛋白的表达情况，随后采用 ELISA 方法检测血清内卵清蛋白特异性以及总 IgE 和 IgG1的表达水平。结果 LTβR-Fc 显著抑制了卵清蛋白诱发的皮炎小鼠模型的炎症反应。与模型组相比，阻断剂组小鼠皮损面积显著减少（P 〈0.05），湿疹面积与严重度指数（EASI）评分降低（P 〈0.05）。皮炎小鼠模型组与阻断剂组小鼠皮损处 IL-4 mRNA（1.81±0.25比0.88±0.25， P 〈0.05）、IL-5 mRNA（1.24±0.26比0.75±0.15，P 〈0.05）、IFN-γ mRNA（1.11±0.19比0.62±0.09，P 〈0.05）水平差异均有统计学意义，脾细胞上清液中 IL-4蛋白（20.12±5.39比9.58±1.44，P 〈0.05）、IL-5蛋白[（22.77±4.07）ng/L 比（11.37±2.02）ng/L，P 〈0.05）]、IFN-γ蛋白[（23930±44.20）ng/L 比（16167±950.40）ng/L，P 〈0.05）]水平差异亦具有统计学意义。经 LTβR-Fc 处理后，特应性皮炎小鼠模型血清内总 IgE 和卵清蛋白特异性IgE 水平较模型组显著下降，IgG1的表达均显著下调。结论融合蛋白 LTβR-Fc 可通过阻断 LIGHT-HVEM 信号通路显著缓解卵清蛋白诱发的皮炎小鼠的临床症状，提示 LIGHT-HVEM 信号通路可作为治疗皮炎（特应性皮炎）的潜在靶点。

Objective To evaluate effects of a fusion protein LTβR-Fc, which can block the herpesvirus entry mediator ligand （LIGHT-HVEM）signaling pathway, on ovalbumin-induced dermatitis in a mouse model. Methods Thirty BALB/c mice were randomly and equally divided into 3 groups： blank control group treated with 100 μl of sodium chloride physiological solution, model group sensitized with 100 μl of sodium chloride physiological solution containing 100 μg ovalbumin, blocker group firstly blocked with 100 μl of sodium chloride physiological solution containing 100 μg LTβR-Fc followed by sensitization with 100 μl of sodium chloride physiological solution containing 100 μg ovalbumin at 24 hours after the blocking. Disease severity was evaluated by eczema area and severity index （EASI）score, and lesional size was measured on day 0, 4, 8, 12, 15, 20, 23, 27, 31 and 34 after the first sensitization. A total of three sessions of sensitization were carried out. At the end of treatment, all the mice were sacrificed after serum was obtained from their orbital cavities. Thereafter, tissue specimens were obtained from skin lesions, and single cell suspensions of the spleen were prepared. RT-PCR was performed to detect mRNA expressions of interferon γ （IFN-γ）, interleukin 4 （IL-4）and IL-5 in murine lesions, ELISA to measure IFN-γ, IL-4 and IL-5 levels in culture supernatants of murine splenocytes, as well as ovalbumin-specific and total IgE and IgG1 levels in murine sera. Results LTβR-Fc significantly suppressed inflammatory response in the mouse model of dermatitis induced by ovalbumin. Compared with the model group, the blocker group showed significantly decreased lesion area and EASI score （both P 〈 0.05）. In addition, a significant decrease was observed in the mRNA expressions of IL-4 （0.88 ± 0.25 vs. 1.81 ± 0.25, P 〈 0.05）, IL-5 （0.75 ± 0.15 vs. 1.24 ± 0.26, P 〈 0.05）and IFN-γ （0.62 ± 0.09 vs. 1.11 ± 0.19, P 〈 0.05）in murine lesions, and in supernatant levels of IL-4 （9.58 ± 1.44 ng/L vs. 20.12 ± 5.39 ng/L, P 〈 0.05）, IL-5 （11.37 ± 2.02 ng/L vs. 22.77 ± 4.07 ng/L, P 〈 0.05）and IFN-γ （16 167 ± 950.40 ng/L vs. 23 930 ± 44.20 ng/L, P 〈 0.05）in the blocker group compared with the model group. The serum levels of both total IgE and ovalbumin-specific IgE were significantly lower in the blocker group than in the model group（total IgE： 27 466.67 ± 2 052.64 μg/L vs. 32 277 ± 407.53 μg/L, P 〈 0.05; ovalbumin-specific IgE： 1 296.33 ± 32.72 μg/L vs. 2 323.33 ± 502.43 μg/L, P 〈 0.05）, so were those of total IgG1 （0.46 ± 0.11 μg/L vs. 0.84 ± 0.11 μg/L, P 〈 0.05）and ovalbumin-specific IgG1 （0.62 ± 0.11 μg/L vs. 0.86 ± 0.07 μg/L, P 〈 0.05）. Conclusion The fusion protein LTβR-Fc can alleviate symptoms of ovalbumin-induced dermatitis in the mouse model likely by suppressing the LIGHT-HVEM signaling pathway, suggesting that this signaling pathway may serve as a target for the treatment of dermatitis（such as atopic dermatitis）.