山东医药

目的 观察刺山柑果提取物（CSE）乳膏外用对系统性硬皮病（SSc）小鼠病变皮肤组织Toll样受体3(TLR3)/β干扰素TIR结构域衔接蛋白（TRIF）信号通路的影响，探讨CSE乳膏外用治疗SSc的机制。方法 BALB/c小鼠90只，随机分成空白对照组、模型组、低剂量组、中剂量组、高剂量组、青霉胺组，每组15只。模型组、低剂量组、中剂量组、高剂量组、青霉胺组小鼠采用皮下注射博来霉素法构建SSc模型，空白对照组每天皮下注射等体积生理盐水，持续28 d。造模成功后，低剂量组、中剂量组、高剂量组小鼠分别外用225、450、900 mg/kg CSE乳膏，青霉胺组小鼠125 mg/kg青霉胺药液灌胃，空白对照组和模型组小鼠外用无药物乳膏基质，每天1次，持续60 d。末次给药4 h后，采用颈椎脱臼法处死小鼠。采用HE染色法观察各组小鼠病变皮肤组织病理形态学变化，采用Western blotting法检测各组小鼠病变皮肤组织中TLR3、TNF受体相关因子6(TRAF6)蛋白，采用免疫组化法检测TRIF蛋白，采用qPCR法检测各组小鼠病变皮肤组织中白细胞介素6(IL-6)、IL-13 mRNA。结果 模型组小鼠皮肤出现明显炎症和纤维化现象。中、高剂量组和青霉胺组小鼠真皮层变薄，胶原纤维显著减少并呈现排列松散和均质化的变化，同时皮脂腺数量增加，炎症细胞浸润明显减少。模型组小鼠病变皮肤组织中TLR3、TRAF6、TRIF蛋白和IL-6、IL-13mRNA相对表达量均高于空白对照组（P均<0.05）。低、中、高剂量组及青霉胺组小鼠病变皮肤组织中TLR3、TRAF6、TRIF蛋白和IL-6、IL-13 mRNA相对表达量均低于模型组，且高剂量组小鼠病变皮肤组织中TRAF6蛋白相对表达量高于低剂量组和青霉胺组，中剂量组小鼠病变皮肤组织中IL-6 mRNA相对表达量高于低剂量组和高剂量组（P均<0.05）。结论 CSE乳膏外用可有效改善SSc小鼠的真皮纤维化，其作用机制可能与抑制TLR3/TRIF信号通路的异常激活有关。

Objective To investigate the effect of topical application of capparis spinosa fruit extract(CSE) cream on the Toll-like receptor 3(TLR3)/TIR-domain-containing adaptor inducing interferon-β(TRIF) signaling pathway in lesional skin tissues of systemic scleroderma(SSc) mice and to explore the mechanism of CSE cream in treating SSc.Methods A total of 90 BALB/c mice were randomly divided into six groups: the blank control group, model group, lowdose, medium-dose and high-dose CSE cream groups, and the penicillamine group, with 15 mice in each group.Mice in the model group, low-dose group, medium-dose group, high-dose group, and penicillamine group were subjected to bleomycin-induced SSc model construction via subcutaneous injection, while mice in the blank control group received subcutaneous injections of an equal volume of saline daily for 28 days.After successful model induction, mice in the low-dose, medium-dose, and high-dose groups were treated topically with CSE cream at doses of 225, 450, and 900 mg/kg, respectively, while mice in the penicillamine group received intragastric administration of penicillamine at 125 mg/kg.Mice in the blank control group and model group were treated with a drug-free cream base topically, once daily for 60 days.Four hours after the final treatment, the mice were euthanized by cervical dislocation.Histopathological changes in the skin lesions of each group were observed via HE staining.Western blotting was used to detect TLR3 and TNF receptor-associated factor 6(TRAF6) proteins in the skin lesions of each group.Immunohistochemistry was employed to detect TRIF protein, and qPCR was performed to measure interleukin-6(IL-6) and IL-13 mRNA levels in the skin lesions of each group.Results The skin of mice in the model group showed significant inflammation and fibrosis.The dermis of mice in the medium-dose and high-dose groups, as well as the penicillamine group, became thinner, collagen fibers significantly decreased, and showed loose and homogeneous arrangement; at the same time, the number of sebaceous glands increased, and the infiltration of inflammatory cells significantly decreased.The relative expression levels of TLR3, TRAF6, TRIF proteins, IL-6, IL-13 mRNA in the pathological skin tissues of the model group were higher than those in the blank control group(all P<0.05).The relative expression levels of TLR3, TRAF6, TRIF proteins, IL-6, and IL-13 mRNA in the pathological skin tissues of mice in the low-, medium-, and high-dose groups, as well as the penicillamine group, were all lower than those in the model group.In addition, the relative expression level of TRAF6 protein in the pathological skin tissues of mice was higher in the high-dose group than in the low-dose group and the penicillamine group.The relative expression level of IL-6 mRNA in the pathological skin tissues of mice was higher in the medium-dose group than in the low-dose group and the high-dose group(both P<0.05).Conclusion Topical application of CSE cream can effectively improve dermal fibrosis in SSc mice, and its mechanism of action may be related to the inhibition of abnormal activation of the TLR3/TRIF signaling pathway.