**3. Discussion**

In vertebrates, β-thymosins usually have one conserved "THY" domain, while in invertebrates, the number of "THY" domains ranges from 2 (*Drosophila melanogaster*, NP\_726909.1) to 27 (*Hydra vulgaris*, AAW82079.1) [16]. Di fferent splicing methods could produce a variety of β-thymosin isoforms, and it can significantly increase the complexity of thymosin in invertebrates [15]. For example, there are two thymosin isoforms (HaTHY1 and HaTHY2) in *Helicoverpa armigera*, which are expressed di fferently in di fferent organs and co-regulated growth and immune reaction [18]. In this study, due to exons alternative splicing, three *P. americana* thymosin isoforms were formed. The 4, 3, and 5 "THY" domains were identified in THY1, THY2 and THY3, respectively. A similar phenomenon was also found in other species such as the fruit fly [24], cotton bollworm [18], and termite [25]. Thus, the functions of these three isoforms may be di fferent.

Multimeric thymosin could bind to more than one G-actin and enhance motility of filaments by promoting assembly in the barbed end (+). Its function was similar to profilin: G-actin complex was only occurring in multimeric thymosin [6,26]. The study found that the N-terminal a-helix amphipathic (M6, I9 and F12) and N-terminal a-helix length in β-thymosin/WH2 control the a ffinity of these peptides for actin, the elongation of N-terminal a-helix of β-thymosin may lead to the loss of actin sequester function [27,28]. Recent research confirmed that it is also relative to the stability of the C-terminal helix, which is mainly due to the two sites of Tβ4 (Ser31 and Thr34) were substitution [21,29]. Through sequence alignment, we found that thymosin from *P. americana* have a long N-terminal a-helix and unstable C-terminal helix. We observed that Pa-THYs not only combine more than one G-actin, but also promote G-actin assembly in the barbed end (+) of filament.

Cell migration involves dynamic change of the cytoskeleton. Multimeric β-thymosin could combine more than one G-actin to regulate assemble and disassemble of microfilaments. For example, Ciboulot (*Drosophila melanogaster*) have three "THY" domains and can bind two G-actin monomers; tetraThymosin (*Caenorhabditis elegans*) have four "THY" domains and can bind three G-actin monomers [17]. Pa-THYs could promote fibroblasts migration but have no e ffect on proliferation. It also demonstrated that the ability of Pa-THYs to stimulate fibroblasts migration was Pa-THY3 > Pa-THY1 > Pa-THY2. Therefore, we speculate that the di fferent ability on cell migration may be related to the number of "THY" domains.

Wound healing is a complex physiological process of organisms, and is regulated by various cells and some intra- and intercellular signaling pathways derived from the epidermis and dermis [30]. It involves several interrelated phases, including hemostasis or coagulation (platelet aggregation and vasoconstriction), inflammation (release cytokines and remove debris), tissue regeneration (angiogenesis and granulation tissue formation) and tissue remolding (collagen deposition) [31]. Tβ4 could accelerate wound healing either directly applying to the surface of full-thickness dermal wound or giving intraperitoneal by stimulating angiogenesis, keratinocyte migration, collagen deposition and wound contraction [7,32–34]. It has also been found that Tβ4 have anti-inflammation properties in corneal wound healing [35], and cascade four Tβ4 (4xTβ4) was more e fficient than standard Tβ4 in wound healing [36]. Researchers sugges<sup>t</sup> that granulation tissue, which is mainly composed of fibroblasts, macrophages, and new blood capillaries, invades the wound space on the fourth day after injury [37]. Neovascularization occurs under hypoxic conditions, with the aim of transporting and utilizating adequate oxygen when tissue is destroyed [22]. In our research, at the early stage of wound healing, a large amount of fibroblasts, a few granulation tissues, and a few new blood vessels were observed in Pa-THYs and Tβ4 treatments. On day 7, granulation tissues were nearly filled in the whole wound, but there were still lots of inflammatory infiltrations in the PBS group. This may be one reason for delayed wound repair. Once the wound is filled up with new granulation tissue, the new blood vessels begin apoptosis [38]. On day 10, the wound in treatment groups had completely epithelialized, the inflammatory reactions and striate vessels disappeared compared with control group. Collagen is the major component for the improved strength of wound, and increasing collagen deposition can accelerate the epithelialization [39]. In this experiment, compared with the control group, the treatment groups present more collagen deposition at days 3 and 5. All of this confirmed that Pa-THYs could accelerate wound healing by promoting fibroblast migration, neovascularization, collagen deposition, and by inhibiting inflammation.

Researchers found that many growth factors are important in wound healing, such as VEGF, b-FGF, TGF-β, MMP-2, and PDGF-BB [40]. VEGF, b-FGF, and TGF-β are the most potent cytokines in promoting wound angiogenesis [41,42]. The expression of VEGF can be directly up-regulated by b-FGF, thus they have a synergistic effect [43]. MMPs were reported to be upregulated during wound healing when treated with Tβ4 [5]. PDGF-BB was approved for treatment of diabetic foot ulcers by the FDA [44], and it was reported that there is a more pronounced effect on myocardial angiogenesis when combined with PDGF-BB and b-FGF [45]. Previously, Tβ4 was reported to up-regulate the expression of VEGF and MMPs during dermal wound repair [46,47], which is in accordance with the results of our experiment. Most interestingly, researchers found that the new blood vessels were not formed after long time use Tβ4 to cure injured corneal [48]. This indicated that VEGF was not the major factor to promote angiogenesis. In this study, after treatment with protein Pa-THY1, Pa-THY2, and Pa-THY3, the expression changes of above factors were different. Pa-THY1 can obviously enhance the expression of b-FGF, MMP-2, TGF-β, and PDGF-BB. Pa-THY2 enhanced the expression of MMP-2, TGF-β, and PDGF-BB. Pa-THY3 up-regulated the expression of MMP-2 and PDGF-BB. Therefore, we suspect that different isoforms of thymosin from *P. americana* may regulate wound healing through different signal pathways. Further studies are required to evaluate the molecular mechanisms of Pa-THYs for accelerating wound healing.
