基于IL-4/STAT6通路研究吴茱萸次碱对特应性皮炎模型小鼠的改善作用

Study on Effect of Evodiamine on Atopic Dermatitis Mice Based on IL-4/STAT6 Pathway

目的:研究吴茱萸次碱对特应性皮炎小鼠的作用及其机制。方法:50只4周龄SPF级雄性C57BL/6小鼠随机分为正常组、模型组、阳性对照组、吴茱萸次碱低剂量组和吴茱萸次碱高剂量组,每组10只,除正常组外,其余组小鼠建立特应性皮炎模型。造模成功4 h后,阳性对照组小鼠灌胃给予10 mg·kg^(-1)泼尼松龙溶液,吴茱萸次碱低、高剂量组小鼠灌胃给予10 mg·kg^(-1)、20 mg·kg^(-1)吴茱萸次碱溶液,正常组和模型组小鼠灌胃等量生理盐水,每天1次,连续14 d。末次给药结束后,测定小鼠右耳炎症评分,并测量耳部厚度;ELISA检测小鼠血清中细胞因子干扰素γ(interferon gamma,IFN-γ)、白细胞介素-4(interleukin-4,IL-4)、IL-5和免疫球蛋白E(immunoglobulin E,IgE)的水平;HE染色检测小鼠耳部组织结构的变化;Western Blot检测耳组织中IL-4/信号转导及转录激活因子6(signal transducer and activator of transcription 6,STAT6)信号通路相关蛋白表达水平。结果:与正常组比较,模型组小鼠耳部出现充血水肿、糜烂、干燥脱屑、结痂等炎性症状,皮炎评分、右耳厚度及血清中IL-4、IL-5、IgE的水平显著升高(P<0.05),耳组织IL-4、STAT6和嗜酸粒细胞趋化因子(eotaxin)蛋白表达水平显著升高(P<0.05),血清中IFN-γ的水平及耳组织细胞因子信号抑制因子1(suppressor of cytokine signaling 1,SOCS1)蛋白表达水平显著降低(P<0.05);与模型组比较,吴茱萸次碱低、高剂量组小鼠耳部炎性症状减轻,皮炎评分、右耳厚度及血清中IL-4、IL-5、IgE的水平明显降低(P<0.05),耳组织IL-4、STAT6和eotaxin蛋白表达水平显著降低(P<0.05);血清中IFN-γ的水平及耳组织SOCS1蛋白表达水平显著升高(P<0.05),且呈剂量依赖性。HE染色显示:正常组小鼠耳部组织皮肤结构正常完整,无水肿和炎性细胞浸润;模型组小鼠皮肤结构缺失,明显水肿,可见大量炎性细胞浸润;各给药组小鼠耳部组织病理损伤得到显著改善,其中吴茱萸次碱高剂量组小鼠皮肤结构较完整,细胞轻度水肿,少量炎性细胞浸润。结论:吴茱萸次碱可明显改善特应性皮炎小鼠的炎症反应,其作用可能与调节IL-4/STAT6信号通路有关。

Objective:To investigate the effect and mechanism of evodiamine on atopic dermatitis mice.Methods:Fifty 4-week-old SPF male C57BL/6 mice were randomly divided into a normal group,a model group,a positive control group,a low-dose evodiamine group,and a high-dose evodiamine group,with 10 mice in each group.Except for the normal group,the other groups of mice established atopic dermatitis models.4 hours after the successful modeling,the mice in the positive control group were given 10 mg·kg^(-1) prednisolone solution by gavage,and the mice in the low-dose and high-dose evodiamine groups were given 10 mg·kg^(-1) and 20 mg·kg^(-1) evodiamine solution by gavage,and the mice in the normal group and model group were given the same amount of normal saline,once a day,for 14 consecutive days.After the last administration,the inflammation score of the right ear of the mouse was determined,and the thickness of the ear was measured.ELISA was used to detect the cytokines interferon-gamma(IFN-γ),interleukin-4(IL-4),IL-5,and immunoglobulin E(IgE)levels in mouse serum.HE staining was used to detect changes in the structure of mouse ear tissue.Western Blot was used to detect the expression levels of IL-4/signal transducer and activator of transcription 6(STAT6)signaling pathway-related proteins in ear tissue.RESULTS:Compared with the normal group,the mice in the model group had inflammatory symptoms such as congestion,edema,erosion,dry desquamation,and crusting in the ears.The dermatitis score,the thickness of the right ear,and the levels of IL-4,IL-5,and IgE in serum significantly increased(P<0.05).The expression levels of IL-4,STAT6,and eotaxin protein in ear tissue were significantly increased(P<0.05).The level of IFN-γin serum and the expression level of suppressor of cytokine signaling 1(SOCS1)protein in ear tissue were significantly decreased(P<0.05).Compared with the model group,the ear inflammatory symptoms of the mice in the low-dose and high-dose evodiamine groups were alleviated,and the dermatitis score,the thickness of the right ear,and the levels of IL-4,IL-5,and IgE in serum were significantly decreased(P<0.05).The expression levels of IL-4,STAT6,and eotaxin protein in ear tissue were significantly decreased(P<0.05).The level of IFN-γin serum and the expression of SOCS1 protein in ear tissue were significantly increased(P<0.05)in a dose-dependent manner.HE staining showed that:the skin structure of the ear tissue of the mice in the normal group was normal and intact,without edema and inflammatory cell infiltration.However,the skin structure of the mice in the model group was missing,with obvious edema and a large number of inflammatory cell infiltration.The pathological damage of the ear tissue of the mice in each administration group was significantly improved,and the skin structure of the mice in the high-dose evodiamine group was relatively intact,with slight cell edema,and a small amount of inflammatory cell infiltration.Conclusion:Evodiamine can significantly alleviate the inflammatory response in mice with atopic dermatitis,and its effect may be related to the regulation of the IL-4/STAT6 signaling pathway.