养血解毒方对银屑病角质形成细胞紧密连接的调节作用

刘宇; 赵京霞; 王燕; 底婷婷; 蒙玉娇; 郭昕炜; 郭简宁; 郭肖瑶; 齐聪; 张璐; 张蕾; 李萍

doi:10.13422/j.cnki.syfjx.20200301

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养血解毒方对银屑病角质形成细胞紧密连接的调节作用

药理 | 更新时间：2021-02-09

- 养血解毒方对银屑病角质形成细胞紧密连接的调节作用
- Regulatory Effect of Yangxue Jiedu Decoction on Tight Junction of Keratinocytes in Psoriasis
- 中国实验方剂学杂志 2020年26卷第3期页码：62-70
- 作者机构：
  
  1.北京中医药大学，北京　 100029
  2.首都医科大学　附属北京中医医院，北京市中医研究所，银屑病中医临床基础研究北京市重点实验室，北京　 100010
- 作者简介：
  
  [第一作者]　刘宇，在读硕士，从事中医药防治银屑病研究，E-mail：1010022635@qq.com
  *李萍，博士生导师，研究员，教授，从事中医药防治银屑病研究，E-mail：liping411@126.com
- 基金信息：
  
  国家自然科学基金项目(81673989);北京市卫生系统高层次卫生技术人才培养计划项目(2015-3-116)
- DOI：10.13422/j.cnki.syfjx.20200301
  中图分类号： R2-0; R22; R285.5; R289
- 收稿日期：2019-06-05，
  
  网络出版日期：2019-10-18，
  
  纸质出版日期：2020-02-05
- 稿件说明：
移动端阅览
刘宇, 赵京霞, 王燕, 等. 养血解毒方对银屑病角质形成细胞紧密连接的调节作用[J]. 中国实验方剂学杂志, 2020,26(3):62-70.

Yu LIU, Jing-xia ZHAO, Yan WANG, et al. Regulatory Effect of Yangxue Jiedu Decoction on Tight Junction of Keratinocytes in Psoriasis[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(3): 62-70.
刘宇, 赵京霞, 王燕, 等. 养血解毒方对银屑病角质形成细胞紧密连接的调节作用[J]. 中国实验方剂学杂志, 2020,26(3):62-70. DOI： 10.13422/j.cnki.syfjx.20200301.

Yu LIU, Jing-xia ZHAO, Yan WANG, et al. Regulatory Effect of Yangxue Jiedu Decoction on Tight Junction of Keratinocytes in Psoriasis[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(3): 62-70. DOI： 10.13422/j.cnki.syfjx.20200301.

摘要

目的：

观察银屑病样小鼠皮损紧密连接蛋白中水闸蛋白(claduin-1，claudin-7)，闭锁蛋白(occludin)的表达，明确养血解毒方对银屑病表皮通透屏障的修复作用，为养血解毒方治疗银屑病提供科学依据。

方法：

将C57BL/6J小鼠随机分为空白组、模型组、甲氨喋呤组、养血解毒方组，制备甲氨喋呤溶液、养血解毒方水煎剂对应灌胃干预，同时小鼠背部剃毛后给予咪喹莫特涂抹诱导银屑病样皮损模型。每日拍照记录皮损形态并对严重程度指数(PASI)评分；水油测试笔检测皮损表皮含水量；苏木素-伊红(HE)染色观察其病理改变、测量表皮厚度；免疫荧光法检测增殖相关的核抗原(Ki67)；免疫组化法检测表皮兜甲蛋白(loricrin)，真皮中CD3

＋

T淋巴细胞浸润和紧密连接蛋白claduin-1，claudin-7，occludin的表达；蛋白免疫印迹法(Western blot)检测皮损中claudin-7，occludin的表达。模拟银屑病皮损微环境，建立白细胞介素-17(IL-17，1 mg·L

-1

)刺激的角质形成细胞(Hacat)模型，制作养血组分、解毒组分、养血解毒方喷干粉进行干预。采用细胞增殖毒性检测试剂盒-8(CCK-8)法检测药物对Hacat细胞的毒性；细胞免疫荧光法检测药物对角质形成细胞claudin-1，claudin-7，occludin表达的干预作用。

结果：

与模型组比较，养血解毒方可显著减轻小鼠银屑病样皮损表现，降低PASI评分及皮损表皮厚度(

0.01)，增加皮损区表皮水分含量(

0.01)，减少表皮ki67，loricrin的异常表达和真皮CD3

＋

T细胞浸润(

0.01)，并增加紧密连接蛋白claudin-1，claudin-7，occludin的表达(

0.05)，增加紧密连接结构的完整性；体外研究发现，与模型组比较，养血解毒方组和养血组分组明显升高紧密连接蛋白claudin-1，claudin-7，occludin的表达(

0.05)；与模型组比较，解毒组分组蛋白表达水平无统计学差异。

结论：

养血解毒方通过调节角质形成细胞间紧密连接的表达抑制其异常的增殖分化过程，进一步恢复破坏的表皮通透屏障，可能是其治疗银屑病的作用机制之一。其中养血解毒方的养血组分对调节紧密连接的修复起主要作用。

Abstract

Objective:

To observe the expressions of tight junction proteins (claduin-1

claudin-7 occludin)of psoriasis-like lesions in mice

and clarify the effect of Yangxue Jiedu decoction on the epidermal barrier of psoriasis

so as to provide scientific basis for the treatment of psoriasis with Yangxue Jiedu decoction.

Method:

C57BL/6J mice were randomly divided into control group

model group

methotrexate group and Yangxue Jiedu decoction.Methotrexate solution and water decoction of Yangxue Jiedu decoction were prepared

and the mice were given imiquimot to induce psoriasis-skin lesions after the hair was shaved.Daily photos were taken to record the forms of skin lesions and psoriasis area and severity index(PASI) scores.Water and oil test pens were used to detect skin moisture content. The pathological changes were observed by htoxylin eosin (HE) staining

and the epidermal thickness was measured.Ki67 was detected by immunofluorescence. Immunohistochemistry was used to detect the expressions of loricrin

CD3

T lymphocyte infiltration and claduin-1

claudin-7

occludin.In addition

the expressions of claudin-7 and occludin in skin lesions were detected by Western blot.Meanwhile

interleukin-17(IL-17) (1 mg·L

-1

) was used to simulate the microenvironment of psoriasis skin lesions

and the intervention was conducted by making Yangxue component

Jiedu component and Yangxue Jiedu dry powder.The toxicity of the drug on Hacat cells was detected by cell counting kit-8(CCK-8)method.The effect of the drugs on the expressions of claudin-1

claudin-7

occludin in Hacat was detected by immunofluorescence assay.

Result:

Yangxue Jiedu decoction could significantly reduce the psoriasis skin lesions in mice

and reduce the PASI score and skin thickness (

0.01). Compared with the model group

the skin moisture content in the lesion was increased (

0.01). Abnormal expressions of ki67 and loricrin in epidermis and infiltration of CD3

T cells were reduced (

0.01). In addition

the expressions of claudin-1

claudin-7

occludin proteins (

0.05)

and the integrity of the tight junction structure were increased.

In vitro

studies

compared with the model group

the expressions of claudin-1

claudin-7

and occludin in the Yangxue Jiedu group and Yangxue group were increased (

0.05). Compared with the model group

there was no statistically significant difference in protein expression in the Jiedu group.

Conclusion:

By regulating the expressions of tight junction proteins between keratinocytes

Yangxue Jiedu decoction can inhibit the abnormal proliferation and differentiation

and further restore the broken epidermal barrier.Yangxue Jiedu decoction plays a role in regulating tight junction mainly through Yangxue component.

关键词

Keywords

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