Xiang Fei, Wang Pei, Gong Hao, Luo Jia, Zhou Xin, Zhan Chenglin, Hu Tianxing, Wang Mengru, Xing Yizhan, Guo Haiying, Luo Gaoxing, Li Yuhong
Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Army Medical University, Chongqing 400038, PR China.
Department of Cell Biology, Army Medical University, Chongqing 400038, PR China.
Burns Trauma. 2023 Jul 3;11:tkac053. doi: 10.1093/burnst/tkac053. eCollection 2023.
Burn wound healing is a complex process and the role of Wnt ligands varies in this process. Whether and how Wnt4 functions in burn wound healing is not well understood. In this study, we aim to reveal the effects and potential mechanisms of Wnt4 in burn wound healing.
First, the expression of Wnt4 during burn wound healing was determined by immunofluorescence, Western blotting and qPCR. Then, Wnt4 was overexpressed in burn wounds. The healing rate and healing quality were analysed by gross photography and haematoxyline and eosin staining. Collagen secretion was observed by Masson staining. Vessel formation and fibroblast distribution were observed by immunostaining. Next, Wnt4 was knocked down in HaCaT cells. The migration of HaCaT cells was analysed by scratch healing and transwell assays. Next, the expression of β-catenin was detected by Western blotting and immunofluorescence. The binding of Frizzled2 and Wnt4 was detected by coimmunoprecipitation and immunofluorescence. Finally, the molecular changes induced by Wnt4 were analysed by RNA sequencing, immunofluorescence, Western blotting and qPCR in HaCaT cells and burn wound healing tissues.
The expression of Wnt4 was enhanced in burn wound skin. Overexpression of Wnt4 in burn wound skin increased the thickness of epidermis. Collagen secretion, vessel formation and fibroblast distribution were not significantly impacted by Wnt4 overexpression. When Wnt4 was knocked down in HaCaT cells, the ratio of proliferating cells decreased, the ratio of apoptotic cells increased and the ratio of the healing area in the scratch healing assay to the number of migrated cells in the transwell assay decreased. The nuclear translocation of β-catenin decreased in shRNA of Wnt4 mediated by lentivirus-treated HaCaT cells and increased in Wnt4-overexpressing epidermal cells. RNA-sequencing analysis revealed that cell junction-related signalling pathways were significantly impacted by Wnt4 knockdown. The expression of the cell junction proteins was decreased by the overexpression of Wnt4.
Wnt4 promoted the migration of epidermal cells. Overexpression of Wnt4 increased the thickness of the burn wound. A potential mechanism for this effect is that Wnt4 binds with Frizzled2 and increases the nuclear translocation of β-catenin, thus activating the canonical Wnt signalling pathway and decreasing the cell junction between epidermal cells.
烧伤创面愈合是一个复杂的过程，Wnt配体在这个过程中的作用各不相同。Wnt4在烧伤创面愈合中的功能以及作用方式尚不清楚。在本研究中，我们旨在揭示Wnt4在烧伤创面愈合中的作用及潜在机制。
首先，通过免疫荧光、蛋白质免疫印迹法和定量聚合酶链反应确定烧伤创面愈合过程中Wnt4的表达。然后，在烧伤创面过表达Wnt4。通过大体摄影以及苏木精-伊红染色分析愈合率和愈合质量。通过Masson染色观察胶原蛋白分泌情况。通过免疫染色观察血管形成和成纤维细胞分布。接下来，在HaCaT细胞中敲低Wnt4。通过划痕愈合实验和Transwell实验分析HaCaT细胞的迁移情况。随后，通过蛋白质免疫印迹法和免疫荧光检测β-连环蛋白的表达。通过免疫共沉淀和免疫荧光检测卷曲蛋白2（Frizzled2）与Wnt4的结合情况。最后，在HaCaT细胞和烧伤创面愈合组织中，通过RNA测序、免疫荧光、蛋白质免疫印迹法和定量聚合酶链反应分析Wnt4诱导的分子变化。
烧伤创面皮肤中Wnt4的表达增强。烧伤创面皮肤中Wnt4的过表达增加了表皮厚度。Wnt4过表达对胶原蛋白分泌、血管形成和成纤维细胞分布没有显著影响。当在HaCaT细胞中敲低Wnt4时，增殖细胞比例降低，凋亡细胞比例增加，划痕愈合实验中的愈合面积与Transwell实验中迁移细胞数量的比值降低。慢病毒处理的HaCaT细胞中，Wnt4的短发夹RNA（shRNA）介导的β-连环蛋白核转位减少，而在Wnt4过表达的表皮细胞中增加。RNA测序分析表明，细胞连接相关信号通路受到Wnt4敲低的显著影响。Wnt4的过表达降低了细胞连接蛋白的表达。
Wnt4促进表皮细胞迁移。Wnt4的过表达增加了烧伤创面的厚度。这种作用的潜在机制是Wnt4与Frizzled2结合并增加β-连环蛋白的核转位，从而激活经典Wnt信号通路并减少表皮细胞之间的细胞连接。
