土茯苓、乌梅及其配伍对银屑病样模型小鼠皮损组织炎症因子及Notch信号通路的影响

Effect of Tu Fu Ling(Rhizoma Smilacis Glabrae),Wu Mei(Fructus Mume)and Their Compatibility on Inflammatory Factors and Notch Signaling Pathway in Skin Lesions of Psoriasis-like Model Mice

目的探讨土茯苓、乌梅及其配伍对咪喹莫特诱导的银屑病样小鼠模型的干预作用及其可能的作用机制。方法将60只BALB/c小鼠随机分为正常组、模型组、甲氨蝶呤组、土茯苓+乌梅组、土茯苓组、乌梅组,每组10只。各给药组小鼠提前预防性给药3天,土茯苓组、乌梅组、土茯苓+乌梅组小鼠分别给予相应药物1.95、0.78、2.73 g/(kg·d)灌胃,甲氨蝶呤组给予甲氨蝶呤片1 mg/(kg·d)灌胃,正常组及模型组给予10 ml/(kg·d)纯净水灌胃,每日1次。除正常组外,其余各组小鼠第4天开始每日上午涂抹5%咪喹莫特乳膏62.5 mg制备银屑病样小鼠模型,每日下午各组继续按预防性给药剂量灌胃,连续6天。观察记录各组小鼠的一般情况,对皮损组织进行银屑病面积和严重程度指数(PASI)评分,并用金(正均)氏公式定量分析土茯苓配伍乌梅的增效作用;HE染色观察小鼠皮损组织的病理学变化并进行Baker评分;ELISA法检测皮损组织白细胞介素6(IL-6)、白细胞介素12(IL-12)、白细胞介素17(IL-17)、白细胞介素23(IL-23)含量;Western Blot法检测皮损组织神经源性位点缺口同源蛋白1抗体(Notch1)、Hes家族BHLH转录因子1抗体(Hes1)、锯齿典型Notch配体1抗体(Jagged1)蛋白表达。结果与正常组比较,模型组小鼠毛发、精神状态等一般情况较差,皮损区可见明显的红斑、鳞屑及皮损浸润增厚,PASI评分、Baker评分升高,皮损组织中IL-6、IL-12、IL-23含量及Notch1、Hes1蛋白表达升高(P<0.05或P<0.01)。与模型组比较,土茯苓组、乌梅组、土茯苓+乌梅组、甲氨蝶呤组小鼠一般情况改善,皮损区红斑和鳞屑有不同程度的减少,PASI评分降低,皮损组织中IL-6、IL-12、IL-23含量降低(P<0.05或P<0.01);土茯苓组、土茯苓+乌梅组、甲氨蝶呤组小鼠Baker评分降低,土茯苓+乌梅组、甲氨蝶呤组小鼠Notch1、Hes1蛋白表达降低(P<0.05或P<0.0l)。与土茯苓组、乌梅组比较,土茯苓+乌梅组小鼠PASI评分、Baker评分降低(P<0.05);与土茯苓组比较,土茯苓+乌梅组小鼠皮损组织中Notch1蛋白表达降低(P<0.05);与甲氨蝶呤组比较,土茯苓+乌梅组小鼠各指标差异均无统计学意义(P>0.05)。金(正均)氏公式计算得出土茯苓+乌梅联用指数Q值达1.29(>1.15),表明土茯苓、乌梅配伍表现为协同增效作用。结论土茯苓、乌梅配伍可协同改善银屑病小鼠的皮损症状,其疗效较单用药物更好,其作用机制可能与调节Nocth信号通路、抑制炎症反应有关。

Objective To investigate the intervention effect of Tu Fu Ling(Rhizoma Smilacis Glabrae),Wu Mei(Fructus Mume)and their compatibility on imiquimod-induced psoriasis-like model mice and the underlying mechanism.Methods A total of 60 BALB/c mice were randomly divided into normal group,model group,methotrexate group,combination(Tu Fu Ling plus Wu Mei)group,Tu Fu Ling group and Wu Mei group with 10 mice in each group.As prophylaxis,interventions were provided to the mice for three days.The corresponding herbal medicine at a dose of 1.95,0.78 and 2.73 g/(kg·d)was given to the mice by gavage in the Tu Fu Ling group,Wu Mei group and the combination group,respectively;1 mg/(kg·d)of methotrexate tablets was given to mice in the methotrexate group by gavage;and 10 ml/(kg·d)of purified water was given by gavage to the mice in the normal group and model group;all interventions were provided once daily.Since the fourth day,mice in all groups but the model group were smeared by 62.5 mg of 5%imiquimod cream every morning to establish the psoriasis-like mice model;the prophylaxis interventions were given continuously every afternoon for six consecutive days.The general conditions of mice in each group were observed,and psoriasis area severity index(PASI)score in each group was compared.Quantitative analysis in Jin’s(Zheng jun)formula was performed to analyze the synergistic effect by the combination of Tu Fu Ling and Wu Mei.Changes in the skin lesions of histopathology were observed with HE staining and evaluated by Baker score.The levels of interleukin 6(IL-6),interleukin 12(IL-12),interleukin 17(IL-17)and interleukin 23(IL-23)in the skin lesions was determined by ELISA.The protein expressions of Neurogenic locus Notch homologous protein 1 antibody(Notch1),Hes family BHLH transcription factor 1 antibody(Hes1)and Jagged canonical Notch ligand 1 antibody(Jagged1)in the skin lesions were detected by western blotting.Results Compared to those in the normal group,poor general condition of hair and mental state,obvious erythema,scales,and thickened skin lesions were observed,and the PASI score,the Baker score,the levels of IL-6,IL-12 and IL-23,and the protein expressions of Notch1 and Hes1 in the skin significantly increased in the model group(P<0.05 or P<0.01).Compared to those in the model group,the general condition of the mice was improved,and the erythema and scale of skin lesions of mice,the PASI score,the levels of IL-6,IL-12 and IL-23 in the skin were significantly reduced in the Tu Fu Ling group,Wu Mei group,combination group and methotrexate group(P<0.05 or P<0.01);the Baker score in the Tu Fu Ling group,combination group and methotrexate group,and the protein expressions of Notch1 and Hes1 in the combined group and methotrexate group significantly decreased(P<0.05 or P<0.01).The combination group had lower PASI score and Baker score than the Tu Fu Ling group and Wu Mei group(P<0.05),as well as decreased Notch1 expression than the Tu Fu Ling group(P<0.05).There was no difference between the combined group and methotrexate group in all the indicators(P>0.05).A synergistic effect with Q value up to 1.29(>1.15)was shown in the combination of Tu Fu Ling and Wu Mei with Jin’s(Zheng jun)formula.Conclusion Tu Fu Ling in combination with Wu Mei could improve the symptoms of skin lesions in psoriasis model mice with better effect than used alone,and its mechanism might be related to regulation of Notch1 signaling pathway and inhibition of inflammatory response.