养血解毒方对银屑病角质形成细胞紧密连接的调节作用

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(P<0.01),增加皮损区表皮水分含量(P<0.01),减少表皮ki67,loricrin的异常表达和真皮CD3+T细胞浸润(P<0.01),并增加紧密连接蛋白claudin-1,claudin-7,occludin的表达(P<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(P<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。

Regulatory Effect of Yangxue Jiedu Decoction on Tight Junction of Keratinocytes in Psoriasis

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 (P<0.01).Compared with the model group, the skin moisture content in the lesion was increased (P<0.01).Abnormal expressions of ki67 and loricrin in epidermis and infiltration of CD3⁺T cells were reduced (P<0.01).In addition, the expressions of claudin-1, claudin-7, occludin proteins (P<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 (P<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.