**1. Introduction**

Skin is the largest external organ of the human body and is vulnerable to injuries. Particularly, in elderly or diabetic patients, wound healing tends to be delayed and the risk of wound infection is increased due to vascular aging and the weakness of tissue repair ability, which may eventually lead to chronic wounds. In addition, wound treatment brings serious economic burdens and psychological pressure to society, for example, in the United States, wound treatment costs more than \$30 billion a year [1]. Therefore, wound repair is one of the hot topics in the field of dermal surgery. At present, few agents have been discovered to substantially promote wound repair in patients [2]. Current strategies mainly include small molecule compounds extracted from plants or growth factors (epidermal growth factor-like proteins (EGF), and human platelet-derived growth factor (PDGF-BB). However, small molecule compounds are unstable and less active, and growth factors are expensive, which restricts

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their clinical application [3,4]. Therefore, the development of new drugs for wound healing has become very important.

Thymosin β4 is a small (5 kDa) peptide, containing 43 amino acids and is found in many tissues and cell lines of vertebrates, and is also known to be rich in platelets [5]. Structurally, there is only one "THY" domain in human thymosin β4 (Tβ4), which usually contains a conserved motif "LKKTET" that can form a complex with G-actin in a 1:1 ratio and inhibits G-actin to polymerize into filaments [6]. Aside from the function in actin-sequestering of Tβ4, it participates in numerous biological activities, including wound healing [7], angiogenesis [8], cardiac repair [9], anti-inflammation [10,11], hair regrowth [12], and reproduction [13]. To date, β-thymosin has been well researched in vertebrates, but there are very limited studies on invertebrates, especially in insects. With the development of bioinformatics, more and more thymosin β4 homologues from invertebrates were identified. Compared with β-thymosin in vertebrates, β-thymosin from invertebrates has more than one "THY" domain which was categorized as a multimeric β-thymosin [14]. Although their homology is relatively high, some functions are different. For example, multimeric β-thymosin can promote actin polymerization, whereas β-thymosin is thought to be a sequester protein, which suggests that the function of these two kinds of thymosin behave differently [6]. In addition, owing to exon skip splicing, many varieties of thymosin in invertebrates have several isoforms, actually, they come from the same gene and the functions of different isoforms are different [15,16]. However, most researches about multimeric β-thymosin focused on its structural characterization, the style to connect with G-actin or the expression in mRNA level, and little information has been explored on its molecular function [16–18].

*P. americana*, is a traditional Chinese medicine. Its extract has a good effect on wound healing [19]. Yet the key effective medicinal composition is still unknown, which hinders the further clinical utilization and exploitation of *P. americana*. Considering that both vertebrate Tβ4 and the extract of *P. americana* have good effects on wound healing, it is surprising that there are no reports about *P. americana* thymosin. Therefore, the aim of this study is to ge<sup>t</sup> insights into the role of *P. americana* thymosin in wound healing using animal models. Herein, we obtained the DNA sequences of thymosin β4 homologue from *P. americana* genome and transcriptome by bioinformatics analysis. By in vitro and in vivo experiments, we evaluate the function of Pa-THYs in wound healing. The present study first demonstrates the role of multimeric β-thymosin protein for promoting wound healing in animal models and provides a potential drug for wound healing.
