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Open Access Research article

Tanshinone IIA pretreatment protects free flaps against hypoxic injury by upregulating stem cell-related biomarkers in epithelial skin cells

Zihan Xu1, Lijun Wu2, Yaowen Sun1, Yadong Guo1, Gaoping Qin1, Shengzhi Mu1, Ronghui Fan1, Benfeng Wang1, Wenjie Gao1 and Zhenxin Zhang1*

Author Affiliations

1 Department of Burns and Plastic Surgery, Shaanxi Provincial People’s Hospital, 256 West Youyi Road, Xi’an 710068, China

2 Department of Plastic Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China

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BMC Complementary and Alternative Medicine 2014, 14:331  doi:10.1186/1472-6882-14-331

Published: 4 September 2014

Abstract

Background

Partial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. The purpose of this study was to determine whether Tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability.

Methods

Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for 2 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. Polymerase chain reaction and western blot analysis were used to assess the expression of β-catenin, vascular endothelial growth factor (VEGF), sex determining region Y-box 2 (SOX2), OCT4 (also known as POU domain class 5 transcription factor 1), Nanog, and glycogen synthase kinase-3 beta (GSK-3β) in TSA-treated cells. In other experiments, after mice were pretreated with TSA for 2 weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was conducted to examine Wnt signaling as well as stem cell- and angiogenesis-related biomarkers in epithelial tissue in vivo.

Results

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, Nanog, and OCT4 were also higher in TSA-pretreated epithelial cells than in control cells. In the reproducible ischemic 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 VEGF and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue.

Conclusions

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.

Keywords:
Free flap; Epithelial cell; Traditional Chinese medicine; Wnt; Stem cell