Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
College of Pharmacy, Jinan University, Guangzhou, 518020, China.
Biochem Biophys Res Commun. 2024 Nov 19;734:150462. doi: 10.1016/j.bbrc.2024.150462. Epub 2024 Jul 29.
The first barrier of the human body is the skin, and more serious harm may occur when skin wound healing is delayed. One of the components of enamel matrix proteins is amelogenin, which inhibits inflammation and promotes periodontal tissue regeneration. However, its role in skin wound healing and angiogenesis is inconclusive. Thus, this study aimed to assess the therapeutic effect of recombinant human amelogenin (rhAM) on mouse skin wounds and to determine its effect on angiogenesis and its underlying mechanism. rhAM was expressed in Escherichia coli and purified using the optimized acetic acid method. A skin injury mouse model was established to explore the effects of rhAM on skin wound healing. After treatment with rhAM for 7 days, the wound healing rate was calculated, and the therapeutic effect of rhAM on skin wounds was assessed using hematoxylin & eosin (HE), Masson, and CD31 immunofluorescence staining. The expression of growth and inflammatory factors in wound tissues were detected using Western Blot. In addition, the rhAM effects on the proliferation and migration of human umbilical vein endothelial cells (HUVEC) and mouse fibroblasts (NIH 3T3) were studied in vitro using the Cell Counting Kit-8, cell scratch, cytoskeleton staining, and qPCR. The rhAM effect on HUVEC angiogenesis and its potential mechanism was studied using tube formation and Western Blot. The results showed that the purity of the obtained rhAM was more than 90 % using the optimized acetic acid method, and high-dose rhAM treatment could improve wound healing rate in mice. Additionally, more blood vessels and collagen were produced in the skin wound, and the expression of angiopoietin-related protein 2 (ANGPTL2) and transforming growth factor (TGF)-β1 was upregulated; however, that of interleukin-6 was down-regulated. We also found that rhAM promoted the proliferation and migration of HUVEC and NIH 3T3, the mRNA levels of vascular endothelial growth factor (VEGF), fibroblast growth factor, TGF-β1 and ANGPTL2 in HUVEC cells were upregulated, and expression of VEGF and phosphorylation of the p38 mitogen-activated protein kinase were activated. Therefore, rhAM could promote skin wound healing by upregulating angiogenesis and inhibiting inflammation.
人体的第一道屏障是皮肤，当皮肤伤口愈合延迟时，可能会造成更严重的伤害。釉基质蛋白的一种成分是釉原蛋白，它可以抑制炎症并促进牙周组织再生。然而，其在皮肤伤口愈合和血管生成中的作用尚不确定。因此，本研究旨在评估重组人釉原蛋白(rhAM)对小鼠皮肤伤口的治疗效果，并确定其对血管生成的作用及其潜在机制。rhAM 在大肠杆菌中表达，并使用优化的醋酸法进行纯化。建立皮肤损伤小鼠模型，以探讨 rhAM 对皮肤伤口愈合的影响。rhAM 治疗 7 天后，计算伤口愈合率，并通过苏木精和伊红(HE)、Masson 和 CD31 免疫荧光染色评估 rhAM 对皮肤伤口的治疗效果。通过 Western Blot 检测伤口组织中生长和炎症因子的表达。此外，还通过细胞计数试剂盒-8、细胞划痕、细胞骨架染色和 qPCR 研究了 rhAM 对人脐静脉内皮细胞(HUVEC)和小鼠成纤维细胞(NIH 3T3)的增殖和迁移的影响。通过管形成和 Western Blot 研究了 rhAM 对 HUVEC 血管生成的影响及其潜在机制。结果表明，使用优化的醋酸法获得的 rhAM 的纯度超过 90%，高剂量 rhAM 治疗可提高小鼠伤口愈合率。此外，皮肤伤口中产生了更多的血管和胶原，血管生成素相关蛋白 2(ANGPTL2)和转化生长因子(TGF)-β1 的表达上调，而白细胞介素-6 的表达下调。我们还发现 rhAM 促进了 HUVEC 和 NIH 3T3 的增殖和迁移，HUVEC 细胞中血管内皮生长因子(VEGF)、成纤维细胞生长因子、TGF-β1 和 ANGPTL2 的 mRNA 水平上调，VEGF 的表达和 p38 丝裂原活化蛋白激酶的磷酸化被激活。因此，rhAM 可以通过上调血管生成和抑制炎症来促进皮肤伤口愈合。
