**2. Results**

#### *2.1. Bioinformatics Analysis of P. americana Thymosin*

Based on the genome and transcriptome databases of *P. americana* which were established by our laboratory, we identified the genome and transcript sequences of Pa-THYs by bioinformatics analysis. There are one genome sequence and three transcript sequences of Pa-THYs. The genome sequence, which contained six exons: exon 1 (139 bp), exon 2 (114 bp), exon 3 (114 bp), exon 4 (114 bp), exon 5 (114 bp), and exon 6 (26 bp) and five introns: intron 1 (4350 bp), intron 2 (7658 bp), intron 3 (6807 bp), intron 4 (2311 bp), and intron 5 (4419 bp) (Figure 1a).

**Figure 1.** The gene structure of β-thymosin in *P. americana*. (**a**) The structure and composition of β-thymosin in *P. americana*. (**b**) The exon skipping splicing process of *P. americana* thymosin (Pa-THYs).

Due to skipping splicing of exons, three different transcripts were formed, which named as THY1, THY2 and THY3. THY3 contains all the exon sequences, while exon 4 is absent in THY1 and exon 2 and 4 are absent in THY2 (Figure 1b). These three transcript sequences were deposited in GenBank. They contain 507, 393, and 621 base pairs and encode 168, 130, and 206 amino acids respectively. The molecular weight of the THYs was 19 kDa, 14.6 kDa, and 23.4 kDa, and PI (isoelectric point) was 6.15, 5.70, and 5.95, respectively. The corresponding proteins were named as Pa-THY1, Pa-THY2, and Pa-THY3. No signal peptide was detected in Pa-THYs by the SignalP 3.0 software. These three proteins are hydrophilic non-transmembrane proteins. The secondary structure of the protein is mainly composed of an alpha helix and irregular curl. According to sequence BLAST (Basic Local Alignment Search Tool) results, the longest sequence of THY3 was wholly contained THY1 and THY2. By using motif scan software, we found that THY3 have five "THY" domains, THY1 and THY2 have four and three "THY" domains, respectively (Table 1).

**Table 1.** Results of bioinformatics analysis of Pa-THYs.


#### *2.2. Function Domain Analysis of P. americana Thymosin*

According to the results of sequence alignment, Pa-THY isoforms 1, 2, and 3 resembled the "assembly-promoting form" like other multimeric thymosin, which can promote a free barbed-end filament elongation [6]. This is because they have a long N-terminal helix (green rectangle) and unstable C-terminal structure which was decided by two function residues (red rectangle) [20,21] (Figure 2). Additionally, multimeric thymosin contains more than two "THY" domains, which can bind at least two G-actins [17]. The "THY" domain has a highly conserved region. In vertebrates, the conserved motif sequence is LKKTET [13], while in *P. americana* and other invertebrates the motif sequences are LKH(R)TET, MKKAET, and MKPTQT (black rectangle), respectively (Figure 2). Pa-THY isoforms 1, 2, and 3 have 4, 3, and 5 domains, respectively, which means that they can combine more and different numbers of G-actin, hence their properties may be different from each other.


**Figure 2.** Function domains analysis of thymosins. Alignment of five Pa-THYs domains with the other four thymosins from different species (Ciboulot (Cib D, three), Actobidin (Act D, two), tetra thymosinβ (Ce D, four), and human thymosin β4 (HsTβ4 D, one)). α-helix, motif sequences, and function residues were marked by green, black and red rectangle, respectively.

#### *2.3. Expression and Purification of Recombinant Protein Pa-THYs*

Recombinant plasmids (PET-THYs) were transformed into *E coli* BL21 (DE3) (Novagen, USA). The results were confirmed by bacterial PCR and Sanger sequencing (TSINGKE, Beijing). Then, the *E. coli* was induced to express the THYs protein and Ni-affinity chromatography was used to purify the proteins. As expected, after being analyzed by SDS-PAGE, recombinant protein Pa-THY1, Pa-THY2 and Pa-THY3 with a molecular mass of approximately 25 kDa, 20 kDa and 30 kDa were detected (Figure 3). All of the above proteins were soluble. In order to ge<sup>t</sup> higher concentration protein, the Millipore was used to concentrate these proteins. After that, the concentration of protein was about 3 μg/μ<sup>L</sup> (Pa-THY1), 2 μg/μ<sup>L</sup> (Pa-THY2), and 3 μg/μ<sup>L</sup> (Pa-THY3), respectively.

#### *2.4. Pa-THYs Promoted Cell Migration of Fibroblasts*

To confirm the bioactivity and the ability for wound healing of purified proteins (Pa-THYs), the fibroblasts (NIH/3T3) were used to evaluate the ability of Pa-THYs on cell migration and proliferation. An in vitro cell scratch was performed to investigate the effect of Pa-THYs on the migration of fibroblasts. The results showed that Pa-THYs proteins promoted migration of fibroblasts with different concentrations. Based on the quantitative analysis of cell migration rates, Pa-THYs and Tβ4 obviously accelerated the migration of fibroblasts compared to PBS (phosphate buffer saline), and Pa-THY3 had a stronger effect than others during cell migration in a low concentration (0.1 μg/mL). For Tβ4, Pa-THY1 and Pa-THY2, the best concentration for cell migration was 1 μg/mL, while for Pa-THY3 was 0.1 μg/mL. Increasing protein concentration revealed a negative effect for cell migration in Tβ4 and all Pa-THYs proteins (Figure 4a,b).

**Figure 3.** The purification of recombinant protein Pa-THYs: lane 1, marker (M) proteins and their corresponding molecular masses; lane 2, the concentrated Pa-THY1 protein; lane 3, the concentrated Pa-THY2 protein; lane 4, the concentrated Pa-THY3 protein.

**Figure 4.** Effects of Pa-THYs on the migration and proliferation of fibroblasts. (**a**) Representative images of NIH/3T3 cells treated by Tβ4 and Pa-THYs proteins (0, 0.1 μg/mL, 1 μg/mL and 10 μg/mL). Magnification, ×200. (**b**) Quantification of wound-healing assays in NIH/3T3 cells treated with Tβ4 and Pa-THYs proteins. The wound healing percentage was compared with control group. (**c**) Cell viability of NIH/3T3 cells treated by Tβ4 and Pa-THYs proteins with different concentrations (0, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, and 10 μg/mL).

MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide) assays were employed to determine the e ffects of Pa-THYs on the proliferation of fibroblasts. Compared to the control group, all Pa-THYs and Tβ4 with di fferent concentrations had no e ffect on the proliferation of fibroblasts (Figure 4c).

#### *2.5. Recombinant Protein Pa-THYs Promoted Wound Healing*

Previous data showed considerable migration e ffects of Pa-THYs on fibroblasts and initially proved that Pa-THYs were involved in wound healing. To observe the e ffect of Pa-THYs on wound healing, a full-thickness skin wound model was established on the dorsal region of mice. Mice were treated with Pa-THYs proteins every day (24 h interval), with PBS treatment as a negative control and Tβ4 treatment as a positive control. The body weight and area of wounds were measured every two days. The surface changes of dermal wounded skin and the images of the wound appearance were observed and obtained every two days after treatment. During the wound healing process, all wounds were dry and had a large scar at first, then the scar of Tβ4 and Pa-THYs groups became moist and granulation tissue appeared while those in the PBS group were about to fall o ff (Figure 5a). Compared to PBS and Tβ4 groups, Pa-THYs groups showed obvious wound contraction since day 3. Based on the statistical data, Pa-THYs groups significantly accelerated wound healing compared with the PBS group at day 3, Pa-THYs and Tβ4 treatment significantly accelerated wound healing compared with PBS group at day 9 and 11 (Figure 5b). The results also showed that the mice's body weight was not a ffected after treatment (Figure 5c). Obvious abnormal behavior or noticeable toxicity were not observed.

**Figure 5.** Effects of Pa-THYs proteins on wound healing in mouse models. (**a**) Representative images of skin wounds treated by Tβ4 and Pa-THYs proteins at indicated time points (0 d, 3 d, 5 d, 7 d, 9 d, and 11 d). (**b**) Quantification data of wound-healing closure in mice treated with Tβ4 and Pa-THYs proteins at indicated time points (0 d, 3 d, 5 d, 7 d, 9 d, and 11 d). \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001. (**c**) The body weight of mice treated with Tβ4 and Pa-THYs proteins at indicated time points (0 d, 3 d, 5 d, 7 d, 9 d, and 11 d).

#### *2.6. Pa-THYs Promoted Wound Healing by Accelerating Dermal Regeneration*

To examine the e ffect of Pa-THYs on dermal regeneration, we collected the dermal tissues of mice treated by Tβ4 and Pa-THYs proteins at indicated time points (3 d, 5 d, 7 d, and 10 d). We analyzed the formation of granulation tissue and the proliferation of fibroblasts and inflammatory cells by H&E (hematoxylin-eosin) staining. Compared with the PBS group, our results indicated that there were a large number of fibroblasts and monocytes that migrated to the bottom of the wound area in Tβ4 and Pa-THYs treated groups on day 3 and few granulations were formed. On day 5, compared with the PBS group, there were a large number of granulation tissues were formed in Pa-THYs and Tβ4 treated groups. On day 7, as expected, the results clearly demonstrated that a high level of inflammation reaction happened in the PBS group. By contrast, the inflammatory response was milder in Tβ4 and Pa-THYs treated groups, and granulation tissues almost filled the whole wound area. On day 10, except for the PBS group, inflammatory cells nearly disappeared in the other treated groups and the wound area displayed better epithelialization, forming a complete epithelial structure (stratum corneum, hyaline layer, granular layer, spinous cell layer, basal layer). There was no obvious di fference between the treated groups (Figure 6).

**Figure 6.** The H&E (hematoxylin-eosin) staining of dermal tissues treated with Tβ4 and Pa-THYs proteins. a: adipose tissue; c: connective tissue; f: fibroblasts; g: granulation tissue; sc: stratumcorneum; tl: transparent layer; tg: granular layer; pc: prickle cell layer; bl: basal layer; D: dermis; monocytes: marked by black arrowhead (Scale bar = 100 μm, ×200).

#### *2.7. Pa-THYs Promoted Wound Healing Through Stimulating Angiogenesis*

Angiogenesis is a critical process for wound healing in that newly formed blood vessels supply nutrients, amino acids, and oxygen to stimulate wound repair [22]. To evaluate the neovascularization during wound healing, the blood vessels were observed at indicated time points by CD31 (Platelet endothelial cell adhesion molecule-1) immunohistochemistry. Compared to the PBS group, Pa-THYs could significantly promote angiogenesis in the early stages of wound healing. As is seen, there were many newly formed blood vessels at the bottom of the wound area on days 3 and 5. Compared to the PBS group, the CD31+ area in Tβ4 and Pa-THYs treated groups were significantly increased on day 3 and day 5. After that, the new blood vessels significantly increased on day 7 and day 10 in PBS group while the new blood vessels showed no obviously variation in Tβ4 and Pa-THYs treated groups, and a large number of stripe-like blood vessels remained in the PBS group on day 10 (Figure 7).

**Figure 7.** The CD31 staining of wound areas in dermal tissues treated with Tβ4 and Pa-THYs proteins (scale bar = 200 μm, ×100). All the groups compared to PBS group in certain time point, two-way ANOVA analysis,; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001.

#### *2.8. Pa-THYs Promoted Wound Healing Through Stimulating Collagen Deposition*

Collagen is an important component for reconstructing dermis tissues at wound sites [23]. Masson's trichrome staining was applied to describe collagen deposition (the blue shaded area) in dermal tissues treated with Tβ4 and Pa-THYs proteins. As showed in Figure 8, compared with the PBS group, significant accumulation of the collagen fibers was observed in the bottom of wound tissues when treated with Tβ4 and Pa-THYs proteins on day 3 and day 5. From day 5 to day 10, collagen gradually filled up with the whole wound tissue in all groups. Statistical analysis found that collagen fibers were slowly formed on day 10 in Pa-THYs groups compared to PBS and Tβ4 treated groups. These data indicated that Pa-THYs could promote wound healing through stimulating collagen deposition in early stage.

**Figure 8.** Masson staining of wound areas in dermal tissues treated with Tβ4 and Pa-THYs proteins (Scale bar = 500 μm, ×40). All the groups compared to PBS group in certain time point, two-way ANOVA analysis, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\*\* *p* < 0.0001.

#### *2.9. Pa-THYs Stimulating the Expression of Cytokines and Growth Factors*

To confirm the role of cytokines and growth factors to wound healing after Tβ4 and Pa-THYs proteins treatment, we examined the expression of relative factors (vascular endothelial growth factor (VEGF), fibroblast growth factor (b-FGF), transforming growth factor-β (TGF-β), matrix metallopeptidase 2 (MMP-2), and PDGF-BB at indicated time points (3 d, 5 d, 7 d, and 10 d). β-actin was used as a reference housekeeping gene to assess the di fferent expression of factors between each group. As seen in Figure 9, our results show that all the factors participated in wound repair and that di fferent Pa-THYs proteins may promote wound healing in di fferent ways. Compared with the PBS group, Pa-THY1 mainly stimulated the expressions of b-FGF, MMP-2, TGF-β and PDGF-BB, but no factors significantly increased. Pa-THY2 mainly stimulated the expressions of MMP-2, TGF-β and PDGF-BB, and TGF-β significantly increased in day 5 and day 10. Pa-THY3 mainly stimulated the expressions of MMP-2 and PDGF-BB to accelerate wound healing, MMP-2 significantly increased in day 7 and PDGF-BB significantly increased in day 10.

**Figure 9.** The expressions of vascular endothelial growth factor (VEGF), fibroblast growth factor (b-FGF), transforming growth factor-β (TGF-β), matrix metallopeptidase 2 (MMP-2), and platelet derived growth factor-BB (PDGF-BB) in mice treated with Tβ4 and Pa-THYs proteins were determined by qRT-PCR. All the factors were compared to PBS group in certain time point, Two-way ANOVA analysis, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.
