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. 2023 Dec 8;7(1):274–284. doi: 10.1021/acsptsci.3c00263

Low-Dose Trypsin Accelerates Wound Healing via Protease-Activated Receptor 2

Yuxin Xiang †,‡, Yuhong Jiang †,*, Lei Lu ‡,*
PMCID: PMC10789143  PMID: 38230283

Abstract

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The management of wounds remains a significant healthcare challenge, highlighting the need for effective wound healing strategies. To address this, it is crucial to explore the molecular mechanisms underlying tissue repair as well as explore potential therapeutic approaches. Trypsin, as a serine protease, has been clinically utilized for wound healing for decades; however, it still lacks systemic investigation on its role and related mechanism. This study aimed to investigate the effects of low-dose trypsin on wound healing both in vitro and in vivo. While trypsin is an endogenous stimulus for protease-activated receptor 2 (PAR2), we discovered that both low-dose trypsin and synthesized PAR2 agonists significantly enhanced the migration, adhesion, and proliferation of fibroblasts and macrophages, similar to the natural repair mechanism mediated by mast cell tryptase. Moreover, such cell functions induced by trypsin were largely inhibited by PAR2 blockade, indicating the participation of trypsin via PAR2 activation. Additionally, low-dose trypsin notably expedited healing and regeneration while enhancing collagen deposition in skin wounds in vivo. Importantly, upon stimulation of trypsin or PAR2 agonists, there were significant upregulations of genes including claudin-7 (Cldn7), occludin (Ocln), and interleukin-17A (IL-17A) associated with proliferation and migration, extracellular matrix (ECM), tight junction, and focal adhesion, which contributed to wound healing. In summary, our study suggested that a low-dose trypsin could be a promising strategy for wound healing, and its function was highly dependent on PAR2 activation.

Keywords: protease-activated receptor 2 (PAR2), protease, trypsin, agonist, wound healing

Wound healing is a complex biological process that involves a series of interconnected cellular and molecular events aimed at restoring the structural integrity and functionality of damaged tissues.1 Despite the remarkable progress made in the field of wound management, such as using recombinant growth factors, living cells, and nanodelivery technologies.2 chronic and nonhealing wounds continue to pose a significant challenge to healthcare professionals worldwide.3 Therefore, there is a persistent need to explore new therapeutic approaches that can enhance the wound healing process.4,5

The protease-based wound healing therapies can be traced back to ancient times.6 Enzymatic debridement has emerged as a promising strategy in wound management since the mid-20th century, providing healthcare professionals with a safe and effective option for removing necrotic tissue.7 Over the years, our understanding of the specific mechanisms of proteases in wound healing has expanded rather than debridement. Studies have shown that certain proteases, such as tryptase, thrombin, and trypsin, have been demonstrated to facilitate cell migration, angiogenesis, and the release of growth factor.8 Moreover, these proteases can initiate fibrosis and promote extracellular matrix (ECM) deposition on fibroblasts, thereby stimulating collagen synthesis and activating profibrotic transforming growth factor-β (TGF-β).9,10 Besides wound healing, proteases also play a crucial role in immune responses. Specifically, trypsin, a digestive protease, has been widely acknowledged since ancient times, which has led to its clinical application in chronic wound care since the 1960s.6 The early reports mainly depicted its function as primarily to facilitate the debridement process, which involves removing nonviable tissue, foreign substances, and bacteria from wounds and promoting a clean environment for the healing process.11 Recently, it has been indicated that trypsin provides a better resolution of inflammatory symptoms, ensuring a more efficient recovery from acute tissue injury.12 However, the molecular mechanisms of trypsin in wound healing are still in their early stages and have not been extensively studied.

Trypsin acts as the primary endogenous agonist of protease-activated receptor 2 (PAR2).13 PAR2 is a subfamily of G protein-coupled receptors (GPCRs)14 that regulates intracellular signal transduction, including calcium, ERK, cAMP, and RhoA pathways.15−17 PAR2 signaling is implicated in various cellular processes such as inflammation, angiogenesis, cell adhesion, proliferation, and migration, all of which are closely associated with tissue repair.12,18 Cell migration at the leading edge comes first after wounding, followed by the displacement of cells in this region triggering orientated cell division of the cells. Increased proliferation can itself generate a surplus of migrating cells that later push the leading edge toward the wound center.19 Trypsin and PAR2 function in an autocrine loop, collectively promoting proliferation, invasion, and metastasis through diverse mechanisms.20 The process of wound healing involves intricate communication among different cellular components of the skin and its distinct compartments. During the inflammatory phase, macrophages are the predominant immune cells found at the wound site.21−23 They not only contribute to microbial clearance but also promote healing by secreting chemokines, growth factors, and enzymes that facilitate substrate breakdown. As healing progresses, fibroblasts, the main repair cells, become abundant in the wound site.1,19 Remarkably, tryptase, a trypsin-like protease mainly found in mast cells, contributes to immune responses, angiogenesis, and tissue repair. In the event of a wound, mast cells release tryptase into the surrounding tissues, facilitating the regulation of the inflammatory response by attracting other immune cells to the site of injury.24−26 Additionally, the released tryptase promotes migration and proliferation of cells involved in wound healing, with its effects partly controlled through PAR2 receptors.24,27,28 For instance, tryptase promotes the differentiation of the fibroblast phenotype through the activation of the PAR2/Akt/mTOR pathway.29 Moreover, both tryptase and trypsin induce macrophage polarization toward a profibrotic M2a phenotype through PAR2.10 Cheaper and more readily available than currently known wound healing treatments with fewer side effects, trypsin mimics the body’s natural repair process, better addresses inflammatory symptoms, and promotes faster recovery from acute tissue damage. Building on this information, we developed a hypothesis proposing that trypsin could enhance the healing of wounds by activating PAR2 receptors in both macrophages and fibroblasts. This activation would facilitate the regulation of inflammation and tissue repair, mimicking the natural repair mechanism mediated by mast cell tryptase (Scheme 1).

Scheme 1. Schematic Diagram of Trypsin-Mediated PAR2 Activation for Accelerating Wound Healing.

Scheme 1

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Dashed box: Trypsin-like protease, e.g., mast cell tryptase, promotes wound healing via PAR2 activation.

In this study, we aimed to investigate the role of trypsin in activating PAR2 and its involvement in promoting the proliferation and migration of fibroblasts and macrophages at the wound site. Both in vitro and in vivo experiments were conducted to confirm the effects of trypsin on cellular processes related to wound healing. To gain a deeper understanding of the underlying mechanism, transcriptome data analysis was performed. The results of KEGG pathway analysis revealed the upregulation in certain signaling pathways, including ECM, tight junction, and focal adhesion pathways, which are closely associated with wound healing. The ultimate goal of this study is to contribute to existing knowledge about the involvement of trypsin in PAR2-mediated wound healing. The insights gained from this study could potentially lead to the development of therapeutic strategies targeting PAR2 agonism with the aim of improving wound management outcomes.

2. Materials and Methods

2.1. Materials and Cell Culture

Trypsin was obtained from Solarbio (Beijing, China), and EDTA was purchased from Adamas-β (Shanghai, China). PAR2 agonists (2f-LIGRL-NH2 and SLIGRL-NH2, ≥98%) and PAR2 inhibitor (P2pal-18S, palmitate-RSSAMDENSEKKRKSAIK-NH2, ≥98%) were custom-made by Apeptides (Shanghai, China). The (RADA)4 peptides (≥95% purity) were purchased from Shanghai Bootech BioScience & Technology Co., Ltd. (Shanghai, China).

The mouse fibroblast NIH/3T3 cells and mouse macrophage RAW264.7 cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 U/mL penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO2.

2.2. Cell Adhesion Assay

Cells were cultured at 5 × 105 cells/well in 12-well plates overnight before dissociation with trypsin and EDTA, respectively. Then, cells (8 × 104 cells/well) with the PAR2 agonist and the PAR2 inhibitor were spread into 48-well plates and allowed to adhere for 2 h. The nonadherent cells were removed by washing 3 times with phosphate-buffered saline (PBS), and the number of adherent cells was imaged using an inverted microscope (IX71, Olympus, Japan) and quantified using ImageJ after fixation using 4% paraformaldehyde and DAPI staining.

2.4. Scratch Wound Assay

Cells were seeded at 3 × 105 cells/well in 24-well plates overnight in the presence of varying concentrations of trypsin, 10% FBS, PAR2 agonist peptides, and serum-free media. Cell monolayers were scratched with a 200 μL sterile tip, and wound closure was analyzed at 0 and 48 h using an inverted microscope (IX71, Olympus, Japan) after scratching.

2.5. Cell Viability Assay

Cells were cultured at 1 × 104 cells/well in 96-well plates for 24 h. For the cytotoxicity assay, cells were treated with different concentrations of trypsin for 24 h. For the proliferation assay, cells were treated with 10 nM trypsin for 48 h. After that, each well was supplemented with 10 μL of CCK-8 solution for another 45 min or 2 h. The absorbance was measured at 450 nm using a microplate reader (Synery H1, Bioteck, Vermont).

2.6. Cutaneous Wound Healing in a Rat Model

Male Sprague–Dawley (SD) rats (∼250 g) purchased from Chengdu Dashuo Experimental Animal Co., Ltd. were used in this study. All procedures were conducted according to international ethical guidelines and the National Institutes of Health Guide Concerning the Care and Use of Laboratory Animals. Rats were anesthetized, depilated, and disinfected. A tissue biopsy instrument (1 cm in diameter) was used to remove full-thickness skin on the back of the rat. Hydrogels have been extensively utilized in the field of tissue regeneration and wound management.30 After the wound model was established, the wound was applied with the (RADA)4 self-assembling peptide hydrogel, a well-established synthetic peptide-based hydrogel that mimics the extracellular matrix, containing a low-concentration trypsin (100 nM, 100 μL). The control group used a (RADA)4 self-assembling peptide hydrogel alone without trypsin. The hydrogel preparation has been described in previous works.31,32 Photographs of the wounds were taken on days 0, 7, and 14 and analyzed using ImageJ. Rats were euthanized after anesthesia on day 14, and the wound tissues were obtained and fixed in 4% paraformaldehyde at 4 °C. The tissue was sectioned for hematoxylin and eosin (H&E), Masson’s trichrome, and CD68 immunohistochemical staining after fixation and embedding, and panoramic scanning and local photography were performed.

2.7. Transcriptomics Analysis

Total RNA was extracted from the cell using Trizol reagent (Cat No, 15596–026, Invitrogen, Carlsbad, California) and frozen immediately in liquid nitrogen and then sent for RNA-sequencing and analysis (Shanghai Applied Protein Technology). Significant differentially expressed genes (DEGs) were set at |log2fold change| > 1.0 and padj < 0.05.

2.8. Statistics

Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA). Data were expressed as means ± standard deviation (SD). Statistical comparison of two groups was performed using an unpaired t test, and the differences among multiple groups were analyzed by using one-way ANOVA with post hoc Tukey tests. Significance was set to *p < 0.05, **p < 0.01, and ***p < 0.001, and ns represents no significance.

3. Results and Discussion

3.1. PAR2 Inhibition Suppressed Trypsin-Enhanced Cell Adhesion

Trypsin, as the classical cell dissociation reagent, has been commonly utilized in a cell culture experiment. Interestingly, we discovered that the adhesion capacities were quite different between trypsin-dissociated and EDTA-dissociated cells (Figure 1). Trypsin significantly enhanced the adhesion of RAW264.7 and NIH/3T3 cells compared to the EDTA group as the control. Moreover, trypsin as a serine protease can cleave PAR2 to expose a specific tethered ligand, SLIGKV (human) or SLIGRL (mouse, rat), and the tethered ligand can bind to the receptor, consequently inducing PAR2 activation and intracellular signaling.33 By mimicking tethered ligands, many PAR2 agonists, such as SLIGRL-NH2 and 2f-LIGRL-NH2, have been developed via chemical synthesis, which can specifically activate PAR2.34 As shown in Figure 1, 2f-LIGRL-NH2 as a PAR2 agonist induced a similar effect with trypsin in promoting the adhesion of RAW264.7 and NIH/3T3 cells, suggesting that trypsin may promote cell adhesion via PAR2. To further support this hypothesis, we investigated whether a PAR2 inhibitor, P2pal-18S, could block trypsin-induced cell adhesion. It has been reported that P2pal-18S as a PAR2 pepducin can specifically inhibit PAR2 activation and related inflammatory responses.35 Unsurprisingly, the PAR2 inhibitor resulted in a significant decrease in the number of adherent cells compared with the group treated with trypsin (Figure 1). Therefore, trypsin facilitated cell adhesion of RAW264.7 and NIH/3T3 cells probably through PAR2 activation. Moreover, the adhesion assay results of RAW246.7 macrophages indicate a significantly lower level of adhesion in the group treated with the Trypsin + PAR2 inhibitor, compared to the control group. This could be attributed to the fact that in the control group, RAW264.7 cells themselves produce various proteases, such as matrix metalloproteinases (MMPs),36 which can activate a portion of the PAR2 receptors present on the cell membrane.37 However, when PAR2 inhibitors are introduced, the activation of PAR2 receptors by self-produced proteases and trypsin is effectively inhibited. Consequently, the adhesion of cells in the Trypsin + PAR2 inhibitor group is lower than that observed in the control group. It is worth noting that similar results were not observed in the corresponding experiment with NIH3T3 cells. This discrepancy can possibly be attributed to the fact that NIH3T3 cells are unable to produce proteases autonomously in the absence of external stimuli. Importantly, it has been commonly considered that cell adhesions to ECM are essential for wound healing, tissue repair, and remodeling,38 indicating trypsin-induced cell adhesion might exert a therapeutic potential for promoting wound healing.

Figure 1.

Figure 1

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Trypsin promoted cell adhesion via PAR2. (A) The adherent RAW264.7 and NIH/3T3 cells were stained with DAPI and detected by fluorescence microscopy. (B) Quantitative analysis of RAW264.7 and NIH/3T3 cell adhesion. PAR2 agonist: 2f-LIGRL-NH2; PAR2 inhibitor: P2pal-18S. **p < 0.01, ***p < 0.001, ns: no significance.

3.2. Low-Dose Trypsin Facilitated Cell Migration

The excessive presence of trypsin is known to be detrimental to cells since it can trigger abnormal degradation of intracellular proteins, cell membrane damage, and disruption of normal cellular function, leading to cell death.39 The commonly employed trypsin concentration for cell digestion is 0.25%, which is about 3.4 μM. However, in order to prevent cell damage, we reduced the trypsin concentration to 1–3000 nM for the CCK-8 assay on RAW264.7 and NIH/3T3 cells to investigate its impact on cytotoxicity. The results demonstrated that trypsin concentrations of 500 nM and higher had a substantial detrimental impact on cells, whereas a concentration of 100 nM did not significantly affect cell viability. Notably, when the trypsin concentration was set at 1, 10, or 30 nM, the cytotoxicity was minimal and there was even an enhancement in cell viability (Figure S1). Therefore, in this study, a low dose of trypsin was used to minimize these potential negative effects. The impact of trypsin on the migratory capacity of RAW264.7 and NIH/3T3 cells was investigated using a scratch wound assay. After incubation of 48 h, trypsin at concentrations of 1, 10, and 30 nM notably enhanced promoted wound gap closure of RAW264.7 cells compared to the control group (**p < 0.01, Figure 2A,B). In particular, the migration ability induced by trypsin at 10 and 30 nM was comparable to the 10% FBS group. There was also significant scratch closure in NIH/3T3 cells upon trypsin stimulation (Figure 2C–D), supporting the efficiency of low-dose trypsin in cell migration. As shown in Figure 2D, 2f-LIGRL-NH2 (1 and 10 μM) and SLIGRL-NH2 (30 and 50 μM) exhibited strong promotion of wound healing, indicating PAR2-induced cell migration. Similar results were observed in RAW264.7 cells (Figure S2). Based on these results, we selected a more potent PAR2 agonist, 2f-LIGRL-NH2, for further investigation in our follow-up experiments. These results suggested that trypsin elicited similar effects to PAR2 agonists, leading us to tentatively speculate that low doses of trypsin may facilitate cell migration by activating PAR2.

Figure 2.

Figure 2

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Both low-dose trypsin and PAR2 agonists enhanced cell migration. (A, B) The scratch wounds were made in monolayers of RAW264.7 cells, and cells were then treated with different concentrations of trypsin. (C–D) NIH/3T3 cells were also treated with trypsin and two PAR2 agonists (2f-LIGRL-NH2, SLIGRL-NH2), detected by an in vitro wound healing assay. Representative images were provided at 0 and 48 h. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.

3.3. Low-Dose Trypsin Promoted Cell Proliferation via PAR2

Subsequently, the impact of low-dose trypsin on the proliferation of RAW264.7 and NIH/3T3 cells was investigated. The results revealed that a concentration of 10 nM trypsin significantly enhanced cell proliferation in both RAW264.7 and NIH/3T3 cells (Figure 3). Interestingly, the effect of the PAR2 agonist on RAW264.7 and NIH/3T3 cell proliferation was observed to be less pronounced compared to trypsin. This could be attributed to the ability of trypsin to activate other receptors besides PAR2, thereby influencing cell proliferation.40 It is plausible that NIH/3T3 cells exhibited higher sensitivity to PAR2 agonists, while RAW264.7 cells may possess decreased responsiveness, resulting in a less apparent effect on cell proliferation. Notably, the PAR2 inhibitor effectively suppressed the proliferative effect induced by trypsin. This suggested that PAR2 activation by low doses of trypsin played a role in facilitating the proliferation of macrophages and fibroblasts. These findings may suggest trypsin treatment as a new therapeutic strategy for promoting tissue regeneration and wound healing. Our studies on cells are limited to fibroblasts and macrophages; wound healing is the result of a combination of cells, and future studies should expand cell types, such as keratinocytes and endothelial cells.41 In addition, trypsin plays a crucial role in facilitating wound healing, as it activates the PAR2 receptor in macrophages, thereby promoting the regulation of inflammation. Inflammation constitutes a crucial stage in the wound healing process, and M2 macrophages are known to be associated with decreased inflammation and enhanced tissue repair. Research suggests that physiological levels of trypsin and thrombin can influence macrophages and monocytes, leading to a biased M2 phenotype.10,22 Future studies should further explore the influence of trypsin activation of the PAR2 receptor on macrophage phenotype.

Figure 3.

Figure 3

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Low-dose trypsin activated PAR2 to facilitate cell proliferation. (A) NIH/3T3 cells were treated with different groups for 48 h, and cell proliferation was detected by microscope. (B) CCK-8 assay was performed to assess the cell proliferation of RAW264.7 and NIH/3T3 cells. **p < 0.01, ***p < 0.001, ns: no significance.

3.4. Low-Dose Trypsin Promoted Wound Healing In Vivo

A full-thickness wound was created on the back of SD rats by using a tissue biopsy instrument (Figure 4A). Subsequently, a trypsin-loaded hydrogel was applied to the wound bed. Figure 4B displays images of untreated wounds and wounds treated with trypsin at three different time points. Over the course of time, the size of the wounds in all three groups decreased to varying extents, with the wounds in the trypsin group shrinking more compared with those in the untreated group (Figure 4C). By day 14, the wounds treated with trypsin were nearly healed. Quantitative analysis of wound closure revealed that the areas of wounds in the trypsin-treated group were smaller than those in the control groups at two different time points, indicating that the trypsin group exhibited the highest rate of wound healing (Figure 4D).

Figure 4.

Figure 4

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Evaluation of trypsin effects on wound healing in vivo. (A) The size of the back wound was observed 0, 7, and 14 days after surgery. (B) Change of wound size in different treatment groups. The deep-purple area is the size of the wound on day 0, and the light-purple area is the size of the wound at n (n = 0, 7, 14) days. (C) Wound closure rate of each group at 0, 7, and 14 days after surgery. *p < 0.05, **p < 0.01.

According to the results of H&E staining, a new epithelial tissue was observed in the wounds of both groups. As shown, the trypsin treatment group exhibited significantly better wound healing effects compared with the control groups on days 7 and 14 (the black arrow indicates the length of the new epithelium, Figure 5A). Histological analysis of Masson’s trichrome staining revealed that the trypsin-treated group had more extensive collagen deposition and thicker collagen fibers compared to the control group. Moreover, the trypsin-treated group also showed an improved arrangement of collagen fibers, resembling the structure of normal skin (Figure 5B). This observation indicates the positive role of trypsin in ECM deposition and collagen alignment. Moreover, the trypsin-treated group exhibited greater numbers of vascular structures on day 7 (indicated by the black arrow). Statistical analysis of re-epithelialization and histological analysis-assessed indicators of ECM formation and neo-angiogenesis are shown in Figure S3. Specific scoring rules can be found in a separate document.42 Furthermore, as the CD68 immunohistochemical staining shown in Figure S4, the trypsin-treated group showed moderate inflammatory responses, which were close to the control group on day 7, indicating that a low dose of trypsin treatment did not initiate uncontrolled inflammation. Therefore, trypsin treatment significantly promoted collagen deposition, reduced inflammation, and accelerated wound closure and skin tissue regeneration. However, wound healing is a complex and time-consuming process that comprises four distinct stages: coagulation, inflammation, repair, and maturity stages.43 Our in vitro experiment did not capture long-term data, and therefore, future experiments should focus on establishing a comprehensive and long-term wound healing model that encompasses all stages of the process. This will allow for a thorough examination of the effects of trypsin treatment on wound healing at each stage.

Figure 5.

Figure 5

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H&E (A) and Masson’s (B) histological staining for tissues at 7 and 14 days after surgery. The black arrows indicate neoepithelium in (A), and the black arrows indicate the blood vessels in (B).

3.5. Transcriptome Analysis of RAW264.7 Treated with Trypsin and PAR2 Agonist

In order to investigate the underlying mechanism of the profound healing effects of low-dose trypsin, transcriptomic analysis on RAW264.7 cells treated with low-dose trypsin and PAR2 agonists was conducted. Principal component analysis (PCA) (Figure S5) revealed significant changes in gene expression patterns in RAW264.7 cells following treatment with trypsin and PAR2 agonists. Interestingly, the trypsin group and the PAR2 agonist group showed distinct clustering from the control group, suggesting that trypsin and PAR2 agonists may induce changes through a common pathway, in which trypsin activates the PAR2 receptor, leading to alterations in the macrophage phenotype. Using a threshold of |log2fold change (FC)| > 1 and P < 0.05, we identified genes with significantly differential expression. Our results (Figure 6) demonstrated that after 24 h of incubation, 89 genes exhibited significant differential expression in RAW264.7 cells treated with trypsin, while 151 genes showed significant differential expression in cells treated with PAR2 agonists. Importantly, the majority of upregulated genes were associated with cell proliferation and migration, as shown in Table 1. Claudin-7 (Cldn7) and occludin (Ocln) were of particular importance as they contribute to the formation of tight junctions.44,45 Additionally, regenerator-islet-derived protein 3β (Reg3β), interleukin-17A (IL-17A), and the IL-17A-dependent gene, polymeric immunoglobulin receptor (Pijr), were significantly upregulated. These genes are implicated in regulating immunity and have the potential to impact the inflammatory response at the site of the wound.46,47 Notably, IL-17A has been strongly linked to PAR2 activation,48 and IL-17A-mediated EGFR activation was a crucial contributor to cell proliferation and migration, ultimately facilitating the process of wound healing. Therefore, these genes offer novel insights and evidence regarding the involvement of trypsin in the repair process. Additionally, they suggest substantial potential for trypsin and PAR2 receptors to enhance wound healing, indicating a correlation between trypsin and PAR2 receptors. Our initial exploration of the underlying mechanism through transcriptome data analysis presents new therapeutic possibilities for tissue trauma.

Figure 6.

Figure 6

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Transcriptome analysis of RAW264.7 cells. Differential gene expression heat maps of RAW264.7 cells after various treatments when compared with the control group. The gene expression levels for each set of comparison were normalized to the mean values within those two groups.

Table 1. Upregulated Genes by Trypsin or PAR2 Agonist Stimulation.

gene genotype cell types signaling pathway and functions refs
claudin-7 (Cldn7) tight junction protein epithelial cells proliferation and epithelial differentiation are regulated by Wnt/β-catenin signaling (44)
occluding (Ocln) tight junction protein fibroblast cells proliferation, migration, and wound healing are regulated by Ras/ERK signaling (45)
regenerating-islet derived (Reg3β) bactericidal C-type lectin macrophages, epithelial cells tissue repair and M2 macrophage differentiation are regulated by STAT3 and Akt signaling (46)
interleukin-17A (IL-17A) proinflammatory cytokines keratinocytes proliferation and wound healing are regulated by activating epidermal growth factor receptor (EGFR) (47,49)
polymeric immunoglobin receptor (Pigr) immune enhancer macrophages activation of positive macrophage migration is regulated by Wnt/β-catenin signaling (50)
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To gain a comprehensive understanding of how these modified genomic expression profiles affect cellular function, gene set enrichment analysis (GSEA) was performed to examine the major gene ontology (GO) enrichment pathways linked to these regulatory relationships (Figure S6). The effectiveness of both trypsin and PAR2 agonists in stimulating cell proliferation and growth highlighted their crucial contributions to the wound healing process. To gain further insights into the underlying pathways involved in the differential gene regulation, the KEGG pathway enrichment analyses for all groups were also conducted (Figure 7). Interestingly, the trypsin group and the PAR2 agonist group exhibited numerous coregulated pathways, including ECM–receptor interaction, focal adhesion, Hippo signaling pathway–multiple species, and the AGE-RAGE signaling pathway in diabetic complications. Notably, it seems that the most significant gene expression differences induced by the agonist do not align with the regions highlighted in the red boxes related to trypsin. The potential reason for this difference may be that PAR2 agonists only specifically activate the PAR2 receptor, whereas trypsin activates many other receptors in addition to the PAR2 receptor,51 so there will be differences in the signaling pathways they most significantly affect. However, among the top 20 signaling pathways with the most significant effects, there are four signaling pathways that are jointly affected by PAR2 agonists and trypsin, suggesting that trypsin may play a role in activating PAR2 receptors through these four signaling pathways. Studies have indicated that the activation of PAR2 can stimulate mucin production in cells, thereby impacting the formation and regulation of focal adhesions.52 Focal adhesion structures consist of structural proteins and signaling molecules that connect the cell cytoskeleton to ECM, governing the cell’s mechanical properties, migration, and signaling.53 This, in turn, affects the cellular response to the ECM and the process of wound healing. Furthermore, trypsin-induced activation of PAR2 resulted in the accumulation of YAP, an essential molecule in the Hippo pathway that plays a role in regulating cell proliferation, growth, and tissue remodeling.54 Consequently, this cascade of events further influences the effectiveness of wound healing. In summary, our findings suggest that trypsin exerts its effects by activating PAR2, thereby enhancing cell proliferation and migration functions. Moreover, our initial exploration of key genes and signaling pathways regulated by trypsin reaffirms its tremendous potential in promoting wound healing.

Figure 7.

Figure 7

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KEGG distribution map of differential genes after treatment with the PAR2 agonist and trypsin compared with the control group. P value refers to the significance of pathway enrichment, and the value range is [0, 1]. The closer it is to zero, the more significant the enrichment.

4. Conclusions

Although protease-based therapies have been applied for wound healing, the function and underlying mechanism of proteases, such as trypsin, are still unclear. This study probed the role of trypsin in wound healing in vitro and in vivo. The low-dose trypsin significantly enhanced the migration, adhesion, and proliferation of RAW264.7 and NIH/3T3 cells, which exhibited a similar promoting effect with a PAR2 agonist, 2f-LIGRL-NH2. Importantly, PAR2 inhibition largely attenuated such trypsin-induced effects, suggesting that trypsin-mediated cell functions were probably dependent on PAR2 activation. It is interesting to note that such trypsin-mediated cell functions have resemblance to the natural repair mechanism facilitated by mast cell tryptase, which is released by mast cells upon tissue damage or injury and plays a crucial role in wound healing, tissue remodeling, and inflammation regulation via PAR2. Moreover, the hydrogel loaded with a low dose of trypsin notably accelerated the healing and regeneration of skin wounds in vivo, supporting the active participation of trypsin in wound healing. Alternatively, transcriptome sequencing results discovered that trypsin treatment could upregulate ECM, tight junction, and focal adhesion signaling pathways to promote wound healing. Therefore, by unraveling the intricate interplay among trypsin, PAR2, and wound healing, we not only demonstrated trypsin treatment as a promising therapeutic strategy for expediting and enhancing wound healing but also identified PAR2 as a potential target for therapeutic interventions in wound healing management.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 82003784), the Fundamental Research Funds for the Central Universities (No. 2682022ZTPY037), and the Zhejiang Provincial Natural Science Foundation of China under Grant No. LTGY23H140001.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00263.

  • Cell viability; cell migration; histological analysis scores; immunohistochemistry data, and additional transcriptome analysis (PDF)

The authors declare no competing financial interest.

Supplementary Material

pt3c00263_si_001.pdf (494KB, pdf)

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