Tortoise shell ointment for acute skin wound in rats via Wnt/beta-catenin signal pathway in hair follicle stem cells Tortoise shell ointment for acute skin wound in rats via Wnt/beta-catenin signal pathway in hair follicle stem cells

doi:10.3969/j.issn.2095-4344.1772

Abstract

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.

Key words: tortoise shell ointment, cholesterol myristate, berberine, borneol, Wnt/β-catenin signal pathway, hair follicle stem cells, acute skin injury

CLC Number:

Shen Qian, Hu Peixin, Zhong Shuxian, Yang Yalan, Zhang Saixia, Li Chun. Tortoise shell ointment for acute skin wound in rats via Wnt/beta-catenin signal pathway in hair follicle stem cells Tortoise shell ointment for acute skin wound in rats via Wnt/beta-catenin signal pathway in hair follicle stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4668-4674.

Figures/Tables 3

2.3 组织病理学检查结果 2.3.1 苏木精-伊红染色结果如图3所示，根据不同时间点描述各组典型表现如下。第3天，各组均出现大量炎性细胞浸润。第5天，龟板软膏组表皮增厚，炎症细胞浸润减少；空白对照组仍有炎性浸润，开始出现表皮组织。第7天，龟板软膏组创面基本愈合，表皮与真皮连接紧密，皮肤附属器增多；S8组见较厚表皮组织覆盖；空白对照组仍有炎性浸润，表皮组织较薄。第10天，龟板软膏组和S8组表皮修复接近正常表皮，表皮分层明显，结构致密；空白对照组仍有少许结痂覆盖。 2.3.2 Masson染色结果如图4所示，根据不同时间点描述各组典型表现如下。第3天，各组胶原纤维生成较少。第5天，各组胶原纤维厚薄不一，龟板软膏组胶原纤维数量较其他组多。第7天，龟板软膏组和S8组胶原纤维排列致密；空白对照组较稀疏。第10天，龟板软膏组较其他组更趋向正常皮肤组织胶原纤维的特性。 2.4 不同时间点各组皮肤创面β-catenin、LEF1、C-myc、CD34、Integrin β1蛋白表达水平 Western blot结果如图5所示，同一时间点龟板软膏组和S8组的β-catenin、LEF1、C-myc、CD34、Integrin β1表达水平均大于空白对照组。创面在修复过程中，龟板软膏组和S8组β-catenin、CD34和Integrin β1的表达水平均呈上升趋势，LEF1和C-myc的表达水平呈“抛物线式”变化，在修复的早中期上调，在修复后期下降。各组差异均具有统计学意义(P < 0.05)。 2.5 各组皮肤创面β-catenin、LEF1、C-myc、CD34、Integrin β1的免疫荧光表达第3天各组免疫荧光表达如图6所示，β-catenin主要表达于表皮棘层和基底层，LEF1和C-myc主要表达于基底层，龟板软膏组和S8组的β-catenin、LEF1、C-myc、CD34和Integrin β1的阳性表达均强于空白对照组。如图7所示，龟板软膏组BrdU标记的增殖细胞数量较S8组和空白对照组多，且同时表达CD34、LEF1阳性。第5，7和10天，龟板软膏组和S8组β-catenin、LEF1、C-myc、CD34和Integrin β1的阳性表达强度与第3天的结果类似，均大于空白对照组，结果未显示。"

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