【摘要】 背景:龟板散由龟板、黄连和冰片组成,是治疗皮肤疮疡的有效方剂。中药单体机制明确,龟板的有效单体十四酸甾醇酯可以激活并调控毛囊干细胞中的Wnt/β-catenin信号通路。目的:探讨龟板软膏对大鼠急性皮肤创面的修复效果,以及其作用是否通过调控毛囊干细胞的Wnt/β-catenin信号通路实现。方法:取龟板散各组分中的有效单体十四酸甾醇酯(S8)、盐酸小檗碱及右旋龙脑制备龟板软膏。构建SD大鼠急性皮肤创面模型并随机分为3个药物组:龟板软膏组、S8组和空白对照组,连续给药10d。造模后连续3 d注射BrdU(50 mg/kg)标记增殖细胞。分别于修复第3,5,7及10天计算创面愈合率,苏木精-伊红染色和Masson染色观察创面病理学变化,Western blot法和免疫荧光检测Wnt/β-catenin信号通路蛋白β-catenin、LEF1、C-myc及毛囊干细胞的表面标志物CD34和Integrinβ1的表达情况。实验方案经广州中医药大学动物实验伦理委员会批准,批准号为20190218001。结果与结论:①修复第5,7,10天,龟板软膏组的创面愈合率均高于其他组(P <0.05);②龟板软膏组新生表皮与真皮连接紧密,新生皮肤附属器较S8组和空白对照组多,胶原纤维致密,修复效果优于其他组;③龟板软膏组和S8组Wnt/β-catenin信号通路蛋白β-catenin、LEF1、C-myc及毛囊干细胞的表面标志物CD34、Integrinβ1的阳性表达量均高于空白对照组,β-catenin、CD34和Integrinβ1的阳性表达均上调,LEF1和C-myc的表达呈先上调后下降变化,差异有统计学意义(P <0.05);④龟板软膏组中BrdU标记的增殖细胞数量较其他组多,且同时表达CD34阳性和LEF1阳性;⑤结果提示:龟板软膏可能通过调控毛囊干细胞中的Wnt/β-catenin信号通路,加快大鼠急性皮肤创面的修复,龟板软膏由S8、盐酸小檗碱及右旋龙脑3种有效单体组成,具有稳定性和安全性,其对急性皮肤创面的促愈合效果优于单一有效单体。更多还原

【Abstract】 BACKGROUND: Traditional prescription Guiban San composed of tortoise shell, coptis chinensis and borneol is used for treating skin injury.As per the clear mechanism of traditional Chinese medicine monomer, the effective monomer cholesterol myristate of tortoise shell can activate and regulate the Wnt/β-catenin signal pathway in hair follicle stem cells.OBJECTIVE: To explore the effect of tortoise shell ointment on acute skin wound in rats and the mechanism whether is by regulating Wnt/β-catenin signal pathway in hair follicle stem cells.METHODS: Tortoise shell ointment was prepared with cholesterol myristate, berberine and borneol--the effective monomers from components of Guiban San. Acute skin wound model was made in Sprague-Dawley rats and rat models were then randomly divided into three groups: tortoise shell ointment group, cholesterol myristate(S8) group and control group. Medication in each group was administered continuously for 10 days. BrdU(50 mg/kg) for proliferative cell labeling was administered for 3 consecutive days. The healing rate of the wound was analyzed in different groups at 3, 5, 7, and 10 days after repair. Hematoxylin-eosin staining and Masson staining were conducted for histopathological observations. The expression of β-catenin, LEF1, C-myc in Wnt/β-catenin signal pathway and CD34, Integrin β1 in hair follicle stem cells were detected by western blot and immunofluorescence. The study protocol was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine with an approval No. 20190218001.RESULTS AND CONCLUSION:(1) At days 5, 7, and 10 after repair, the wound healing rate in the tortoise shell ointment group was significantly higher than that in the other groups(P < 0.05).(2) Compared with the S8 and control groups, tortoise shell ointment group showed better repair effect with more neoformative hair follicles and denser collagen fibers.(3) The expressions of β-catenin, LEF1, C-myc,CD34, Integrin β1 in the tortoise shell ointment and S8 group were higher than those in the control group. The positive expression of β-catenin,CD34 and Integrin β1 were increased during wound healing. LEF1 and C-myc were increased during the prophase and metaphase of wound healing but decreased during the later stage. The difference was statistically significant among three groups(P < 0.05). The number of BrdU-labeled proliferative cells in the tortoise shell ointment group was higher than that in the S8 and control groups, with concurrent positive expressions of CD34 and LEF1. To conclude, tortoise shell ointment may stably and safely promote the repair of acute skin wound in rats via regulating Wnt/β-catenin signal pathway in hair follicle stem cells, and the healing effect is superior to any single effective monomer.更多还原