凉血解毒透邪法调节皮肤常驻记忆性T 细胞缓解寻常型银屑病复发的作用及机制研究
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R285.5;R758.63

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国家自然科学基金项目(82274527);广东省自然科学基金项目(2021A1515011245)


Study on Effect of Expelling Pathogen Method of Cooling the Blood and Resolving Toxins on Mitigation of Psoriasis Vulgaris Relapse by Adjusting Tissue-Resident Memory T Cells and Its Mechanism
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    摘要:

    目的:观察凉血解毒透邪法(银屑灵方) 对寻常型银屑病复发症状的影响及其对皮肤常驻记忆性 T细胞(TRM) 的影响。方法:选择72只SPF级Balb/C小鼠,体质量18~22 g,雄性,随机分为正常对照组、 单发模型组,复发模型组、阳性药组(甲氨蝶呤,0.3 mg·kg-1·d-1)、银屑灵方低、高剂量组(生药23.5 g·kg-1·d-1, 47.0 g·kg-1·d-1),共6组,每组12只。除正常对照组外 ,其它小鼠裸背处涂抹5%咪喹莫特乳膏(IMQ) 50 mg, 每天1次,连续7 d。放置不作处理,为银屑病缓解期模型。4周后进行2次造模(复发造模),除了正常对照 组和单发模型组以外,其余小鼠背部涂抹5% IMQ 50 mg,每天1次,连续5 d。给药:给药时间为首次造模期 间和造模后2周。在2次造模的第3天,对各组小鼠皮损进行了第1次银屑病面积与严重性指数(PASI) 评分; 在2次造模的第5天,再次对各组小鼠皮损进行了PASI评分;并处死动物,对小鼠体质量丢失量、脾脏指数进 行分析。采用HE染色,在显微镜下评价黏膜病理损伤指数;采用免疫组织化学检测小鼠皮损的PCNA蛋白的 表达;采用荧光定量qPCR检测银屑病相关炎症因子mRNA的表达;采用流式细胞术检测皮损区TRM细胞数量 的变化,并用荧光定量qPCR检测TRM细胞产生与存活相关因子的表达水平,采用免疫组织化学和蛋白免疫印 迹法检测TRM细胞表型和存活相关蛋白的表达。结果:与复发模型组比较,银屑灵方能够缓解银屑病复发模 型小鼠皮损外观症状,抑制体质量丢失和表皮增厚;减低皮损处白细胞介素(IL) 17、IL23、肿瘤坏死因子 α (TNFα) mRNA 的表达和皮肤PCNA 蛋白的表达。银屑灵方能够减少银屑病小鼠缓解期皮损区表型为 CD3+TCRγδ+CD8+CD103+的TRM细胞数量;并能抑制皮损区IL7、IL15、BCL6和PRDM1 mRNA的表达水平及 STAT5磷酸化水平。结论:银屑灵方有效减缓银屑病复发小鼠的皮损,其机制可能是通过抑制TRM形成的转 录因子水平、调控TRM存活与增殖的信号STAT5通路及下游信号分子IL7、IL15的表达,减少缓解期原皮损区 TRM细胞数量,达到改善银屑病复发的效果。

    Abstract:

    Abstract: Objective: To observe the effect of expelling pathogen method of cooling the blood and resolving toxins (Yinxieling Prescription) on the recurrent symptoms of psoriasis vulgaris and the tissue-resident memory T cells( TRM). Methods:A total of 72 SPF male Balb/C mice,weighing about 18~22 g,were randomly divided into six groups, including the normal control group, the single-onset model group, the relapse model group, the positive medicine group (Methotrexate, 0.3 m·kg-1·d-1), the low-dose Yinxieling Prescription group and the high-dose Yinxieling Prescription group (crude herbs,23.5 g·kg-1·d-1 and 47.0 g·kg-1·d-1),with 12 mice in each group. Except for the normal control group, the other mice were smeared with 50 mg of 5% Imiquimod Cream (IMQ) on the nude back,once a day for seven consecutive days. The mice given no any treatment were used as the models for psoriasis in remission stage. After four weeks,the second modeling was done (modeling for relapse). Except for the normal control group and the single-onset mode group,the other mice were smeared with 50 mg of 5% IMQ on the back,once a day for five consecutive days. Administration:the administration was conducted respectively during the first modeling period and two weeks after modeling. The first psoriasis area and severity index( PASI) scoring of skin lesions was performed in each group on the third day of the second modeling. The PASI scoring of skin lesions in each group was evaluated again on the fifth day of the second modeling; after the mice were sacrificed, their weight loss and spleen index were analyzed. HE staining was used to evaluate the pathological injury index of mucous membrane under the microscope; the expression of PCNA protein in skin lesions of mice was detected by immunohistochemistry;quantitative real-time PCR (qPCR) was used to detect the mRNA expression of psoriasis-related inflammatory factors; flow cytometry was used to detect the changes in the number of TRM cells in the lesion area,and qPCR was used to detect the expression levels of survival-related factors produced by TRM cells;immunohistochemistry and western blot were used to detect the phenotype of TRM cells and the expression of survival-related proteins. Results:Compared with the relapse model group,Yinxieling Prescription can alleviate the appearance symptoms of skin lesions in mice model of psoriasis relapse, inhibit the weight loss and epidermal thickening,and reduce the mRNA expression of interleukin (IL) 17,IL23,and tumor necrosis factor α( TNF-α) and the expression of PCNA protein in the skin. Yinxieling Prescription can reduce the number of TRM cells with phenotype of CD3+TCRγδ+CD8+CD103+ in the skin lesions of psoriasis mice in the remission stage;it can also inhibit the mRNA expression levels of IL7,IL15,BCL6 and PRDM1 and the phosphorylation level of STAT5 in the skin lesions. Conclusion: Yinxieling Prescription can effectively alleviate the skin lesions of mice model of psoriasis relapse, whose mechanism may lie in the inhibition of the level of transcription factors formed by TRM, regulation of the STAT5 signal pathway of TRM survival and proliferation and the expression of downstream signal molecules (IL7 and IL15),reduction of the number of TRM cells in the primary skin lesion area in the remission stage so as to improve the recurrent symptoms of psoriasis.

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张文娟,胡冯菊,黎莉,卢月,吴晶晶,韩凌,危建安.凉血解毒透邪法调节皮肤常驻记忆性T 细胞缓解寻常型银屑病复发的作用及机制研究[J].新中医,2025,57(2):166-175

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  • 在线发布日期: 2025-02-05
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