Abstract
Partial or total flap necrosis after flap transplantation is sometimes clinically encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. In this study, we determine whether tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for two weeks. Cell counting kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Then, Polymerase chain reaction and Western blot analysis were used to determine the expression of β-catenin, GSK-3β, SOX2, and OCT4 in TSA-treated cells. In vivo, after mice were pretreated with TSA for two weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was also conducted to examine the related biomarkers mentioned above. Results show that epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2 and OCT4 controlled by Wnt signaling were also found higher in TSA pretreated epithelial cells. In the reproducible ischaemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. The results show that TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.
Highlights
In plastic surgery, flaps are routinely used for coverage of tissue defects resulting from trauma, ablative surgery, or congenital malformation
The viability of control epithelial cells declined after 72 h of treatment with CoCl2, whereas epithelial cells pretreated with tanshinone IIA (TSA) showed higher viability
To investigate the mechanism by which TSA protects against ischemia-induced flap necrosis, we examined components of the Wnt signaling pathway and stem cell-related biomarkers in epithelial skin tissue using immunohistochemistry
Summary
Flaps are routinely used for coverage of tissue defects resulting from trauma, ablative surgery, or congenital malformation. Nowadays, understanding of the epithelial stem cells, together with the exploration of stem cell related signaling pathways that control the reconstruction of skin, open a new door to the flap surgery. When Wnt signaling is reduced, its associated biomarkers, such as OCT4, SOX2, and β-catenin, are downregulated, and cell resistance to hypoxia and capacity for regeneration. If Wnt signaling is required for tissue regeneration, can we find a convenient and effective way to elevate Wnt signaling in epithelial skin cells with limited regenerative capacity to improve the healing response?. We examined the regulation of the Wnt signaling pathway and stem cell-related biomarkers in epithelial cells and tissue. We tested whether TSA therapy activates the Wnt signaling pathway and upregulates stem cell-related biomarkers in epithelial cells
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