Wound-healing acceleration of mice skin by Sipunculus nudus extract and its mechanism
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摘要: 为探讨方格星虫水提物（SNE）促进小鼠皮肤创伤愈合的活性及其机制，测定 SNE 的止血作用，建立小鼠全皮层创伤模型，观察伤口愈合形态并测定愈合率；采用苏木素-伊红（HE）、马森（Masson）染色、透射电镜（TEM）观察创面的组织结构；实时荧光定量聚合酶链式反应（qRT-PCR）检测细胞因子及其相关蛋白的表达水平。结果显示，SNE 具有止血作用，能明显提高小鼠创伤愈合率并缩短掉痂时间（P < 0.05）。研究中，SNE 组小鼠表皮生长完全、创面毛细血管和胶原纤维增生明显优于空白（NT）组；第 7 d，SNE 明显降低白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和转化生长因子-β1（TGF-β1）的表达水平（ P < 0.05）；第 28 d，相比于 NT 组，SNE 组的 Smad7 表达水平上调，TGF-β II 型受体（TGF-βRII）、I 型胶原蛋白（COL1A1）和 α-平滑肌肌动蛋白（α-SMA）的表达水平下调，差异具有统计学意义（ P < 0.05），而与云南白药组（PC 组）的差异无统计学意义（ P > 0.05）。结果表明，SNE 具有促进伤口愈合活性和抑制增生性瘢痕生成作用，其作用机制与止血、调节炎症因子、促进胶原纤维重塑以及调控 TGF-β/Smads 信号通路传导有密切关联。SNE 作为皮肤创伤修复和抑制瘢痕制剂，具有潜在的临床应用价值。Abstract: In order to explore the effect of Sipunculus nudus extract (SNE) on skin wound healing in mice and its mechanism, hemostasis effect of SNE was measured, the mouse skin wound model was established by full-thickness excision. The morphological changes of the wound were observed after the treatment with SNE and the healing rate was measured. The changes of wound histology were observed by hematoxylin eosin (HE) staining, Masson staining and transmission electron microscope (TEM). The expression of cell factors and related proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that the SNE possessed hemostatic function. SNE could obviously improve the healing rate of wound in mouse and shorten time of scab removal compared with the none-treatment (NT) group (P < 0.05).The pathological histology analysis results showed complete epidermal regeneration, with remarkable capillary and collagen fiber observed in the SNE group. The expression level of tumor necrosis factor-α (TNF -α), interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) in SNE group was significantly lower than that of the NT group on 7 d ( P < 0.05). Moreover, compared with the NT group, the gene expressions level of Smad7 was significantly increased and the level of type II TGF-β receptors (TGF-βRII), collagen I (COL1A1) and α-smooth muscle actin (α-SMA) were significantly reduced in the SNE group on 28 d ( P < 0.05), but the difference was not statistically significant compared to Yunnanbaiyao group (PC group) ( P > 0.05). These results indicated that SNE possessed obvious activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to hemostatic function, regulation of inflammatory factors, collagen deposition, collagen fiber remodeling and intervening TGF-β/Smads signal pathway. Therefore, SNE may have promising clinical applications in skin wound repair and scar inhibition.
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Key words:
- Sipunculus nudus extract /
- wound healing /
- anti-scar /
- hemostasis
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图 1 SNE 对小鼠尾部出血时间的影响（n = 6，* P < 0.05， ** P < 0.01）
Figure 1. Effects of SNE treatment on tail bleeding time in mice (n = 6, * P < 0.05, ** P < 0.01)
图 3 SNE 对创伤愈合率的影响（n = 7，* P < 0.05 ** P < 0.01，SNE 组与 NT 组比）
Figure 3. Effect of SNE on wound closure (n = 7, Compared with the NT group, * P < 0.05, ** P < 0.01)
图 4 掉痂所用的时间（n = 17，* P < 0.05，** P < 0.01）
Figure 4. Time of scab removal (n = 17, * P < 0.05, ** P < 0.01)
图 5 小鼠皮肤创面 TNF-α、IL-1β 和 TGF-β1 的表达水平（n = 4，* P < 0.05，** P < 0.01）
Figure 5. Expression of TNF-α，IL-1β and TGF-β1 in wound of mice skin (n = 4, * P < 0.05, ** P < 0.01)
图 6 组织学分析创面表皮的愈合质量
Figure 6. Histological analysis of the healing quality of wound epidermis
图 7 创面的表皮厚度指数（n = 4，* P < 0.05，** P < 0.01）
Figure 7. Epidermal thickness index of wound epidermis (n = 4, * P < 0.05, ** P < 0.01)
图 8 创面的表皮薄厚比（n = 4，* P < 0.05，** P < 0.01）
Figure 8. Thin/thick ratio of epithelial thickness of wound epidermis (n = 4, * P < 0.05, ** P < 0.01)
图 9 Masson 染色切片分析创口真皮位置胶原沉积和重组情况
Figure 9. Analysis of Masson-stained sections for collagen deposition and recombination at the wound dermal
图 10 小鼠皮肤创面的 COL1A1 的表达（n = 4，* P < 0.05， ** P < 0.01）
Figure 10. Expression of type I collagen in wound of mice skin (n = 4, * P < 0.05, ** P < 0.01)
图 11 伤后 21 d 创口胶原纤维分布
Figure 11. Distribution of collagen fibers at 21 days post-wounding
图 12 TGF-β/Smads 信号通路几种相关的因子的表达（n = 4，* P < 0.05，** P < 0.01）
Figure 12. Expression of several factors related to TGF-β / Smads signaling pathway (n = 4, * P < 0.05, ** P < 0.01)
表 1 qRT-PCR 引物
Table 1. Primers used for qRT-PCR analysis
基因 上游引物 下游引物 GADPH CAGGAGGCATTGCTGATGAT GAAGGCTGGGGCTCATTT TNF-α GGTCAATCTGCCCAAGTA CACCCATTCCCTTCACAG IL-1β TATGGGCTGGACTGTTTCTAATGC TTCTTGTGACCCTGAGCGACCT TGF-β1 CCGCAACAACGCCATCTAT CCAAGGTAACGCCAGGAAT COL1A1 ACGCCATCAAGGTCTACTGC GAATCCATCGGTCATGCTCT α-SMA AGACATCAGGGAGTAATGGTTG GAAGCTCGTTATAGAAAGAGTGG TGF-βRII TGAGAAGCCGCATGAAGT AGAGTGAAGCCGTGGTAGGT Smad7 TTTACAACCGCAGCAGTTAC GGCTGTAGGCTTTCTCATAGT 
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