Porcine placenta extract promotes keratinocyte and fibroblast activities via ERK AKT and JNK growth signaling pathways

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The fibroblast and keratinocyte are epidermal cells involve in granulation tissue formation and re-epithelialization. The homeostasis of extracellular matrix (ECM) deposition/degradation also attributes wound completion by regulation of ECM protein constitution and matrix metalloprotease (MMP) activities. This study aims to investigate the stimulatory effect of PPE on keratinocyte/fibroblast proliferation and migration including MMPs and ECM protein expression. Methods: The human keratinocyte cell line (HaCaT) and human fibroblast cell line (Hs 895.Sk) were cultured with various PPE concentration and investigated cell proliferation, cell migration, MMP gene expression and ECM proteins’ gene expression by MTT assay, scratching wound assay, and qRT-PCR, respectively. ERK1/2, p-ERK1/2, Akt, p-Akt, JNK, p-JNK, and cyclin-D1 were also investigated by Western blot analysis. LY294002, PD98059, and SP600125, in which inhibitors were utilized to confirm the underlying signaling pathway of PPE stimulation. Jcl:SD rat were generated a wound and applied topical PPE with various concentration. The wound biopsy and histological staining with Masson’s trichrome were performed to investigate granulation tissue appearance. All In Vivo procedures were approved by the Ethics Committee for the Use of Animals of the Naresuan University (NU-AE630609). Results: PPE statistically significantly increased keratinocyte/fibroblast proliferation and migration with dose dependent manner. PPE 10 µg/mL statistically significantly enhanced MMP-1, MMP-2, MMP-9, MMP-10, and MMP-14 gene expression in keratinocyte. PPE 50 µg/mL also increased MMP-2, MMP-20, and MMP-14 including α-SMA, fibronectin, collagen I, and collagen III gene expression with statistically significant difference. PPE can activate phosphorylation of ERK1/2, Akt, and JNK including cyclin-D1 expression. Interestingly, PPE also stimulate granulation tissue appearance. Conclusion : PPE stimulated fibroblast/keratinocyte proliferation and migration through JNK, ERK1/2, and Akt pathway. PPE also activated MMP gene expression either keratinocyte or fibroblast and stimulated ECM protein gene expression in fibroblast. Consequently, PPE provides the potential as a complementary treatment to improve delayed wound healing and prevent scar/keloid formation. Porcine Placenta Extract Keratinocyte and fibroblast activities granulation tissue formation Wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The process of wound healing is a multifaceted and ever-changing biological phenomenon that holds significant importance in the restoration and rejuvenation of tissues. Fibroblast and keratinocyte are dermal cells dominate in granulation tissue formation and re-epithelialization, which involve in the late stage of wound healing [1]. Extracellular matrix (ECM) acts a structural network for cellular organization and cell-cell communication facilitator [2]. In the healing process, ECM has been overwhelmingly reconstituted by fibroblast-derived ECM proteins (e.g. collagen, fibronectin) [3, 4]. Meanwhile, ECM protein overproduction has been impeded by matrix metalloprotease (MMP), in which degrades ECM proteins [5]. Therefore, the equilibrium of ECM deposition/degradation is then essential in wound healing process to facilitate rapid healing process and prevent scar/keloid production. Numerous researchers emphasize in novel treatment approaches that has grown for the last few years, with particular attention paid to the possibility that bioactive compounds derived from natural sources could improve the efficiency of wound healing. The extract derived from porcine placenta has been recognized as a viable option with a wide range of therapeutic properties that engage with signal transduction pathways. This characteristic renders it a potential contribution to the domain of regenerative medicine [6]. Porcine Placenta Extract (PPE), rich in growth factors, cytokines, and extracellular matrix components, has shown remarkable potential in promoting cell proliferation, modulating inflammation, and enhancing tissue regeneration. Basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGFβ-1) were introduced as important substances in PPE, that played a role in thermal-induced wound healing acceleration in rodents [7]. The intricate interplay between the bioactive constituents of PPE and cellular signaling pathways offers a unique perspective on how natural compounds can influence key processes in wound healing, ultimately improving outcomes for patients. At the molecular level, signal transduction pathways play a critical role in regulating cellular responses during wound healing. By modulating these signaling cascades, PPE can potentially optimize the wound microenvironment, facilitating efficient tissue repair and regeneration [8, 9]. In previous studies, PPE has been illustrated the stimulatory effect on cellular proliferation and migration through Akt, ERK1/2, and JNK pathway in human osteoblast [10]. Even though the stimulatory effects of PPE on wound healing have been initiated both In Vitro and In Vivo for a while, the well-rounded perspective of cellular activities should be further investigated to clarify the underlying mechanism of PPE, especially influencing on epidermal cell activities in the late stage of wound healing, in which many previous studies have been seemingly omitted. This current study aims to investigate the stimulatory effect of PPE on keratinocyte/fibroblast proliferation and migration including gene expression of MMP and ECM proteins. ERK1/2, Akt, and JNK pathway have been also elucidated to clarity the underlying mechanism of PPE on keratinocyte and fibroblast. The stimulatory effects of PPE In Vivo have been also investigated with a wound biopsy and histological staining to observe granulation tissue appearance. Methods Porcine Placenta Extract (PPE) Preparation The formulation of the PPE solution was undertaken by Faculty of Sciences at Mahidol University, Bangkok, Thailand. The process involved initial cleaning and mechanical homogenization of porcine placenta in a phosphate-buffered saline (PBS) solution. Subsequently, the homogenate underwent sonication followed by centrifugation at 4°C for a duration of one hour. The resulting supernatant was then carefully collected and subjected to sterile filtration by using 0.2 µm filters. The protein concentration within the solution was quantified utilizing the Bradford assay method and denoted as the concentration of the PPE solution. Cell culture The human fibroblasts cell lines Hs 895.Sk was obtained from ATCC, the human keratinocytes cell lines HaCaT was a kind gift from Assoc. Prof. Thaned Kangsamaksin (Mahidol University). Cells were maintained according to international guidelines on good cell culture practice in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, MA, USA) at 37 °C humidified atmosphere with 5% CO 2 , supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B. Determination of cell proliferation Cell proliferation was evaluated by utilizing the MTT assay. Initially, cells were plated at a concentration of 5×10 3 cells per well in a 96-well plate and allowed to adhere overnight. Following this, various concentrations of PPE were introduced and incubated for the specified duration. Upon completion of the incubation period, MTT reagent at a concentration of 0.5 mg/mL was added and incubated for an additional 4 hours. The resultant formazan product was solubilized using DMSO, and the absorbance was measured at 570 nm by using a microplate spectrophotometer. The percentage of cell proliferation was determined by using the following formula: ((Absorbance at 570 nm of PPE treated cells) / (Absorbance at 570 nm of control cells)) × 100 (%). Determination of cell migration Human keratinocyte and fibroblast cell lines were seeded into 6-well plates and incubated in a humidified atmosphere at 37°C with 5% CO 2 until reaching 90-100% confluence. Subsequently, the monolayer cell was scratched using a 200 µL pipette tip, followed by washing with PBS. The culture medium was then replaced with complete medium containing various concentrations of PPE. The scratched wound was periodically photographed under a microscope at a 10X objective lens for 24 hours. The area of the scratched wound was quantified using the ImageJ program, and the percentage of wound recovery was calculated using the formula: (initial area - area at 24 hours) / initial area) × 100%. RNA extraction and Real-time PCR Human keratinocyte and fibroblast cells were treated to different concentrations of PPE for 6 hours. The extraction of total cellular RNA was carried out by utilizing the total RNA Mini Kit (BioRad, CA, USA) as manufacturer’s instruction. Subsequently, first-strand cDNA synthesis was performed by using the Tetro cDNA synthesis kit (Bioline, UK), followed by real-time PCR analysis. The corresponding primer sequences of extracellular matrix-related genes and matrix metalloprotease genes that used in the study are detailed in Table 1. The relative quantification of gene expression was conducted by employing the 2 -ΔΔCT method, with normalization against the expression level of GAPDH. Table 1 Primer sequences for quantitative real-time PCR. Gene name Primer Sequences Metalloproteinase-1 (MMP-1) F: 5’-GATGTGGAGTGCCTGATGTG-3’ R: 5’-CTGCTTGACCCTCAGAGACC-3’ Metalloproteinase-2 (MMP-2) F: 5’-GCAGTGAATCTACAGGGACGC-3’ R: 5’-ATCCTGATCCAACCAATCACC-3’ Metalloproteinase-9 (MMP-9) F: 5’-CCTTCCTTATCGCCGACAAG-3’ R: 5’-TGAACAGCAGCATCTTCCCC-3’ Metalloproteinase-10 (MMP-10) F: 5’-CATTCCTTGTGCTGTTGTGTC-3’ R: 5’-TGTCTAGCTTCCCTGTCACC-3’ Metalloproteinase-14 (MMP-14) F: 5’-ATAAACCCAAAAACCCCACC-3’ R: 5’-AAACACCCAATGCTTGTCTC-3’ Type 1 Collagen ( Col1 ) F: 5’- GTGCTAAAGGTGCCAATGGT-3’ R: 5’- ACCAGGTTCACCGCTGTTAC-3’ Type 3 Collagen ( Col3 ) F: 5’- CCAGGAGCTAACGGTCTCAG-3’ R: 5’- CAGGGTTTCCATCTCTTCCA-3’ Fibronectin F: 5’- AATGGCCAGATGATGAGCTG-3’ R: 5’- TGGCACCGAGATATTCCTCC-3’ 18S ribosome F: 5’- CCATCCAATCGGTATGTAGCG-3’ R: 5’- GTAACCCGTTGAACCCCATT-3’ Immunoblotting Cell lines were subjected to treatment with PPE, both with and without 15 µM of inhibitors (LY294002 or PD98059 or SP600125) (Med Chem Express, NJ, USA). Following treatment, the cells were rinsed with ice-cold PBS and subsequently lysed using RIPA lysis buffer supplemented with a protease inhibitor cocktail. The resultant protein lysates were obtained through centrifugation and their concentrations were determined using the Bradford assay. The proteins were then separated via SDS-gel electrophoresis and transferred into PVDF membranes. To prevent nonspecific binding, the membranes were incubated in a 5% skim milk buffer for 1 hour, followed by washing with TBST buffer. Then, the membranes were exposed to specific primary antibodies (anti-pAkt, anti-Akt, anti-pERK, anti-ERK, anti-pJNK, anti-JNK, anti-β-actin, and anti-cyclin D1) (all antibodies procured from Cell Signaling, MA, USA), and allowed to incubate overnight at 4°C with gentle shaking. After washing with TBST, the membranes were incubated with an HRP-linked anti-rabbit antibody (Cell Signaling, MA, USA) for 1 hour at room temperature, washed again, and then subjected to detection reagent incubation. The resulting images were developed using the Chimidoc™ XRS system and analyzed utilizing Image Lab software (Bio-Rad, CA, USA). In Vivo Wound healing model The 10 weeks Jcl:SD rat were introduced to full thickness excision wound by tissue biopsy at 8 mm width and 2 mm dept. Wounds were treated once a day for 14 days with topical application by various concentration of PPE (20, 200 and 2,000 µg/mL), and 200 ng/mL of basic fibroblast growth factor as a positive control (Thermo Fisher Scientific, MA, USA), and PBS as vehicle control. Until the complete treatment cycle, rats were anesthetized and collected wound tissues using tissue biopsy. The histological samples underwent fixation in formalin, followed by embedding in paraffin. Subsequently, they were transversely sectioned at a thickness of 4 µm and subjected to examination utilizing Masson's Trichrome staining method. All procedures described were approved by the Ethics Committee for the Use of Animals of the Naresuan University (NU-AE630609). Statistical analysis All data are shown as mean ± SEM. The statistically significant difference was analyzed by using ANOVA with appropriate post-hoc comparison analysis ( p -value < 0.05 was considered as statistically significant difference). The statistical analysis was performed by using commercially available software (GraphPad Prism version 9, San Diego, CA, USA). Results Porcine placenta extract enhanced human keratinocytes and fibroblast proliferation and migration. To evaluate the activities of PPE on human keratinocytes and fibroblasts. Cells were seeded into 96-well cell cultures plates and treated with or without medium supplemented with different concentration of PPE. Results showed that PPE promotes keratinocytes and fibroblasts proliferation, especially at 72 hours of incubation time (Figure 1A). Moreover, the effect of PPE on cell migration was evaluated by cell scratch assay. Likewise, PPE significantly enhanced keratinocytes and fibroblasts migration at 24 hours incubation compared to untreated group (Figure 1B). At 10 µg/mL of PPE dramatically increased keratinocytes proliferation and migration, but not in fibroblasts. Seemingly, human keratinocyte is more susceptible to inducing proliferation and migration than fibroblast. Porcine placenta extract induced the up-regulation of extracellular matrix-related genes To determine the PPE activities on would healing mechanisms, the functionality of PPE on extracellular content was accessed by genes expression of human keratinocytes and fibroblast. After cell treatment with PPE, mRNA level of extracellular matrix-related genes was measured using qRT-PCR. The expression of metalloproteinase enzyme genes including MMP-1 /-2 /-9 /-10 /-14 were significantly increased either human keratinocyte or fibroblasts compared to control group with dose dependent manner (Figure 2A). Additionally, PPE significantly up-regulated the expression of collagens, fibronectin, and alpha-smooth muscle actin (α-SMA) in human fibroblast cell lines (Figure 2B). Consequently, PPE potentially promotes the extracellular matrix-related genes. Porcine placenta extract promoted human keratinocytes/fibroblasts proliferation and migration mediated by ERK/AKT and JNK signaling pathways To determine the molecular underlying mechanism of PPE on human keratinocytes and fibroblasts, cells were treated with 10 and 50 µg/mL of PPE for human keratinocytes and fibroblasts, respectively. Intracellular protein was collected and measured protein expression by using immunoblotting. Results showed that PPE dramatically induced phosphorylation of ERK/AKT and JNK signaling pathways in early time activation (within 60 minutes) (Figure 3A). These were confirmed by adding LY294002 (ERK inhibitor), PD58059 (PI3K inhibitor) and PD600125 (JNK inhibitor) that attenuated the overexpression of signaling proteins by PPE (Figure 3B). Not only signaling proteins, but PPE also upregulated the expression of cell cycle-derived protein, cyclin D1 (Figure 3A). According to the phenotype studies, PPE-treated cell with or without specific signaling protein inhibitors and subsequently determined cell proliferation and migration. We demonstrated that activation of cell proliferation by PPE on keratinocytes is involved with ERK, AKT and JNK, whereas on fibroblasts is involved with only JNK signaling (Figure 4A). For activation of cell migration, PPE is associated with ERK, AKT and JNK activation in keratinocytes, and associated with ERK and JNK activation in fibroblasts (Figure 4B). These suggest that the activation of cell proliferation and migration of PPE on these cells are involved with ERK, AKT and JNK signaling pathways. Porcine placenta extract enhanced generation of keratin and collogen in wound-healing mice model To determine the effectiveness of PPE on keratinocyte and fibroblast activities as clinical setting, full thickness excision wound in rats was tropically applied by PPE with various concentration for 14 days. Wound biopsy was collected and determined the generation of keratin and collagen by Masson's trichrome staining. Results showed that at low concentration of PPE (20-200 µg/mL) seemingly induce generation of granulation tissue by thicker layer of keratin when compared to PBS control. Moreover, at high concentrations of PPE (2,000 µg/mL) showed regenerated of collagen fiber and some are arranged as collagen bundles as shown in positive treatment with fibroblast growth factor (Figure 5). Hence, these indicated that PPE promote keratinocyte and fibroblast activities and is potential compound for wound healing treatment. Discussion Porcine placenta extract (PPE) has been emphasized as one of the growth factor-enriched compound that derived by agricultural waste-product. PPE in this study was performed proteomics and searched the protein sequence against the domestic porcine Sus scrofa proteome database. The analysis of DAVID bioinformatics elucidated signal-involved proteins, phosphoproteins, and disulfide-bond proteins mostly expressed in PPE in this study [11, 12]. PPE may consist of essential growth factors (EGF, FGF, IGF, VEGF, PDGF etc.), chemokines, antioxidants, etc. Many studies concluded its efficiency and efficacy on cell proliferation, cell migration, cell stress reduction, and anti-inflammation [10, 12-14]. Keratinocyte and fibroblast have contributed to complete wound healing as main responders in the overlapping late process; proliferation/migration phase and remodeling phase. The proliferation and migration phase particularly categorizes to granulation tissue formation, angiogenesis, wound contraction, and re-epithelialization. Fibroblast is one of dominant cells in granulation tissue formation, in which fibroblast proliferates and produces collagen, fibronectin, and α-Smooth muscle actin (α-SMA) to constitute a new extracellular matrix (ECM) [15]. Matrix metalloprotease (MMP) is the ECM degradation enzymes that regulates ECM deposition/degradation. MMP has been categorized to various functional subtypes, for example, MMP-1 (Interstitial collagenase), MMP-2 (Gelatinase-A), MMP-9 (Gelatinase-B), MMP-10 (Stromelysin 2), MMP-14, etc. In case the imbalance of ECM deposition/degradation is existed, the delayed wound healing or scar/keloid formation can be prescribed [5]. The re-epithelialization is mostly dominated by keratinocyte, whereby keratinocyte proliferates and migrates from nearby intact epidermis surrounding wound edge for covering the developed granulation tissue [1, 16]. In this study, PPE can stimulate the proliferation and migration of keratinocyte and fibroblast with dose dependent manner as shown in Figure 1. PPE also activated MMP-2, MMP-9, MMP-10, and MMP-14 gene expression in keratinocyte and fibroblast as shown in Figure 2A. These all MMPs could be encoded to ECM degradation enzymes, in which degrades overproduced ECM resulting in scar/keloid minimizing. Moreover, PPE can stimulate α-SMA, fibronectin, collagen II, and collagen III gene expression in fibroblast as shown in Figure 2B. These are the major components of extracellular matrix. According to In Vivo assay, the Masson’s trichrome staining of rodent full-thickness wound tissue with PPE topical application elucidated granulation tissue appearance, collagen fiber regeneration, and thickening of keratin layer as shown in Figure 5. Moreover, the wound healing duration In Vivo was seemingly shortened in PPE treatment group compared to control group (data not shown). These findings suggested PPE could promote wound healing by increasing fibroblast/keratinocyte proliferation and migration including stimulating MMPs, α-SMA, fibronectin, and collagen production. ERK1/2 and PI3K/Akt pathway have been illustrated as the associated signaling pathway of proliferation and migration [17]. In this study, PPE induced phosphorylation of ERK1/2 and Akt both keratinocyte and fibroblast as shown in Figure 3A. PPE also upregulated Cyclin-D1, which is a protein regulator of cell cycle. Moreover, PPE co-cultured with LY294002 and PD58059 showed the suppression of ERK1/2 and Akt phosphorylation resulting in proliferative and migrative reduction both in keratinocyte and fibroblast as shown in Figure 3B and Figure 4. These findings suggested PPE could promote keratinocyte and fibroblast proliferation/migration through ERK1/2 and Akt pathway. JNK pathway has been also elucidated as the associated pathway of cell growth and differentiation, which is a regulator of transcription factor. In this study, PPE also induce phosphorylation of JNK, which JNK can be suppressed by PPE co-cultured with SP600125, resulting in proliferative and migrative reduction both in keratinocyte and fibroblast as shown in figure 3 and Figure 4. Recent study indicated JNK-gene knockout in keratinocyte and fibroblast attenuated migration and proliferation ability. On another hand, the activation of JNK in fibroblast stimulated collagen synthesis [18, 19]. JNK also displays a downstream signaling of TGFβ and PDGF to regulate expression of MMPs, ECM synthesis (collagen, fibronectin, etc.), growth factor secretion, and ECM remodeling in human dermal fibroblast [20]. These finding suggested PPE could also promote keratinocyte and fibroblast proliferation and migration through JNK pathway, whereby it is seemingly a dominant pathway of PPE on the proliferative and migrative stimulation in keratinocyte and fibroblast. Moreover, PPE may activate MMPs and ECM synthesis via JNK signaling pathway, in which PPE may contains PDGF and TGFβ. The limitation in this study is cell line performing in all In Vitro assay, in which may not truly reflect human primary dermal cells. However, the cellular activities and morphology is not different between primary keratinocyte and HaCaT cell (Cell lines) including between primary fibroblast and fibroblast cell line [21]. Remarkably, this is the first study that evaluates the stimulatory effect of PPE on MMP and ECM gene expression in keratinocyte and fibroblast in addition to proliferation, migration, and their signaling pathway. PPE greatly composes of various growth factors, nutrients, chemokines, etc. The purification into single substance may deprive the integrated bioactive functions. The quantitative data of rodent granulation tissue appearance by Masson’s trichrome staining indicated insignificant difference even performing with sufficient sample size. This insignificant difference of granulation tissue appearance may be a consequence of topical PPE applying. Topical PPE applying in this study is a clear soluble liquid, in which can not securely adhere on wound bed resulting in dissensus data from each rodent sample. Topical PPE should be developed as a hydrogel or cream to provide strong adhesion on wound bed. Since PPE in this study showed the potential ability in wound healing especially keratinocyte/fibroblast proliferative and migrative enhancement including ECM deposition/degradation balancing. Interestingly, Topical PPE on excision wound in rodents illustrated the collagen fiber regeneration and keratin layer thickening as summarized in Figure 6. Topical PPE applying may convey to complementary or alternative clinical treatment to improve delayed wound healing in diabetic foot ulcer patient or reduce a scar/keloid formation. Conclusion PPE provides the stimulatory effect on keratinocyte/fibroblast proliferation and migration through JNK, ERK1/2, and PI3K/Akt pathway. Concomitantly, this is the first study to demonstrate the stimulatory effect of PPE on MMPs and ECM gene expression in keratinocyte and fibroblast. PPE activates MMP-1, MMP-2, MMP-9, MMP-10, and MMP-14 gene expression. PPE also stimulates α-SMA, fibronectin, and collagen production. This indicates the potential of PPE on ECM deposition/degradation equilibrium. Abbreviations PPE: Porcine placenta extract; MMP: Matrix metalloprotease; ECM: Extracellular matrix; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-PCR: Reverse transcription polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LC MS/MS: Liquid Chromatography with tandem mass spectrometry; bFGF: Basic fibroblast growth factor; EGF: Epidermal growth factor; VEGF: Vascular endothelial growth factor; IGF: Insulin-like growth factor; PDGF: Platelet-derived growth factor; JNK: c-Jun N-terminal kinases; Akt: Protein kinase B; ERK1/2: Extracellular signal-regulated kinases Declarations Author’s Contributions C.N., W.S., and A.J. conceived and designed the experiments; W.S., J.J., C.N., and N.K. performed the experiments; C.N., W.S., A.J. analyzed the data; A.J., T.S., and R.T. contributed reagents/materials/analysis tools; S.P., P.R., P.M., T.J., P.M., K.S., and all authors wrote, read, prepared, and approved manuscript. Funding This work was supported by Program Management Unit for National Competitiveness Improvement (PMU-C), CCF Energy Supplement (Thailand) Limited, and Mahidol University (Grant number C10F640021). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable requests. Ethics approval and consent to participates Not applicable Consent for publication Not applicable Competing interests The authors declare that there is no competing interest. Author details 2 Department of Cardio-Thoracic Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (C.N) 3 Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (W.S.) 4 Clinical Research Unit, Faculty of Medicine, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (J.J.) 5 Department of Biochemistry, Faculty of Medical Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (S.P.); [email protected] (N.K.) 6 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; [email protected] (R.T.); [email protected] (T.S.); [email protected] (T.J.) 7 Faculty of Medical Technology, Huachiew Chalermprakiet University, Bangkok 10540, Thailand; [email protected] (P.R.); [email protected] (P.M.); [email protected] (K.S.) 8 Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand; [email protected] (P.M.) 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Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay. Arzneimittelforschung. 2009;59(3):146-52. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1FulllenghtBlots1.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4293166","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309921944,"identity":"32c80e38-a2af-4515-a084-e497ea42c2fa","order_by":0,"name":"Chatchai Nensat","email":"","orcid":"","institution":"Naresuan University","correspondingAuthor":false,"prefix":"","firstName":"Chatchai","middleName":"","lastName":"Nensat","suffix":""},{"id":309921945,"identity":"74efdd95-432a-45eb-a1d2-c5431d9b73e1","order_by":1,"name":"Worawat Songjang","email":"","orcid":"","institution":"Naresuan University","correspondingAuthor":false,"prefix":"","firstName":"Worawat","middleName":"","lastName":"Songjang","suffix":""},{"id":309921946,"identity":"553033b9-74d4-410b-9e45-9e122e56bc10","order_by":2,"name":"Jeranan Jantra","email":"","orcid":"","institution":"Naresuan University","correspondingAuthor":false,"prefix":"","firstName":"Jeranan","middleName":"","lastName":"Jantra","suffix":""},{"id":309921949,"identity":"47a7faf0-b622-4ffd-ada0-2421cbb94754","order_by":3,"name":"Nutthamon Kijchalao","email":"","orcid":"","institution":"Naresuan University","correspondingAuthor":false,"prefix":"","firstName":"Nutthamon","middleName":"","lastName":"Kijchalao","suffix":""},{"id":309921951,"identity":"d4704dc9-29a9-42ab-95d3-8c2c1fcca65a","order_by":4,"name":"Rutaiwan Tohtong","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Rutaiwan","middleName":"","lastName":"Tohtong","suffix":""},{"id":309921952,"identity":"24128384-0ea6-420b-9405-95e0f2becd54","order_by":5,"name":"Tuangporn Suthiphongchai","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Tuangporn","middleName":"","lastName":"Suthiphongchai","suffix":""},{"id":309921953,"identity":"e4b84949-b045-416d-893e-4dbe083b9e75","order_by":6,"name":"Suchada Phimsen","email":"","orcid":"","institution":"Naresuan University","correspondingAuthor":false,"prefix":"","firstName":"Suchada","middleName":"","lastName":"Phimsen","suffix":""},{"id":309921954,"identity":"620f21c9-7dd0-450f-b44c-f655a3bfa4f5","order_by":7,"name":"Panthip Rattanasinganchan","email":"","orcid":"","institution":"Huachiew Chalermprakiet University","correspondingAuthor":false,"prefix":"","firstName":"Panthip","middleName":"","lastName":"Rattanasinganchan","suffix":""},{"id":309921955,"identity":"10e9611f-35e4-422c-91c7-aed86075a438","order_by":8,"name":"Pornphimon Metheenukul","email":"","orcid":"","institution":"Kasetsart University","correspondingAuthor":false,"prefix":"","firstName":"Pornphimon","middleName":"","lastName":"Metheenukul","suffix":""},{"id":309921956,"identity":"6c3591c6-a156-4fc7-8bc3-297d5f25b04b","order_by":9,"name":"Tavan Janvilisri","email":"","orcid":"","institution":"Mahidol University","correspondingAuthor":false,"prefix":"","firstName":"Tavan","middleName":"","lastName":"Janvilisri","suffix":""},{"id":309921959,"identity":"57999abb-8653-45f2-b222-15c0216330c2","order_by":10,"name":"Penpak Moolthiya","email":"","orcid":"","institution":"Huachiew Chalermprakiet University","correspondingAuthor":false,"prefix":"","firstName":"Penpak","middleName":"","lastName":"Moolthiya","suffix":""},{"id":309921964,"identity":"659375b8-6906-4902-af7b-4c03f269d969","order_by":11,"name":"Kittipat Sopitthummakhun","email":"","orcid":"","institution":"Huachiew Chalermprakiet University","correspondingAuthor":false,"prefix":"","firstName":"Kittipat","middleName":"","lastName":"Sopitthummakhun","suffix":""},{"id":309921967,"identity":"5e46fa9b-f582-4173-b852-e2fbd123f60b","order_by":12,"name":"Arunya Jiraviriyakul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYPACNjkYK4FoLcZQhgHRWhgSG4jWwj/7jNljnhq+9O0SCYwffjD8ySOoReJcjrkxzzG23J0zEpglexgMigm76AyPmTQPG1vuhhsJDNJAh8FciBvIg7X8Y0s3uJHA/JsoLQYgLbxtbAlALWzE2WJ4hq1Mcm4fm+GGMw/bLHsMjAlrkTvDvE3izbdj8gbHkw/f+FEhR1gLCDDxMBwDUoxAxQbEqAep/cFQQ6TSUTAKRsEoGJEAALtUNW7gxgIhAAAAAElFTkSuQmCC","orcid":"","institution":"Naresuan University","correspondingAuthor":true,"prefix":"","firstName":"Arunya","middleName":"","lastName":"Jiraviriyakul","suffix":""}],"badges":[],"createdAt":"2024-04-19 12:30:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4293166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4293166/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58109964,"identity":"46b7b2d7-3bc1-4495-84da-41f3193884ce","added_by":"auto","created_at":"2024-06-11 09:02:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4301046,"visible":true,"origin":"","legend":"\u003cp\u003ePPE enhanced human keratinocytes and fibroblast proliferation and migration. \u003cstrong\u003e(A)\u003c/strong\u003e HaCat and \u003cstrong\u003e(B)\u003c/strong\u003e human skin fibroblast cells were seeded at density 5×10\u003csup\u003e3\u003c/sup\u003e cells into 96 well plate before exposing with PPE with indicated concentrations. Cell viability was accessed using MTT assay at 24-72 h and calculated as % cell proliferation\u003cstrong\u003e. (B)\u003c/strong\u003e cells were cultured in 6 well plates, scratched and incubated as various concentrations of PPE. At 24 h, wound width was photographed, measured, and calculated as %wound recovery. Data are shown as the mean ± SEM (n=3).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/13f61829f91f1e6846253dd7.png"},{"id":58109965,"identity":"5b9e4eb0-18b5-429b-962c-865a40ebd630","added_by":"auto","created_at":"2024-06-11 09:02:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":537680,"visible":true,"origin":"","legend":"\u003cp\u003ePPE up regulated the expression of MMPs, and extracellular matrix associated genes. Human keratinocyte and fibroblast were incubated with PPE for 6 h. The cellular mRNA was extracted and measured the expression of \u003cstrong\u003e(A) \u003c/strong\u003eMMPs genes and \u003cstrong\u003e(B)\u003c/strong\u003efibroblast extracellular matrix associated genes using real-time PCR. Data was calculated as relative mRNA expression to control. Data are shown as the mean ± SEM (n=3).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/3efbe130cbbeead057cb5e3a.png"},{"id":58110325,"identity":"7cb41dcf-79b3-4217-bd5e-bc8ad1c99277","added_by":"auto","created_at":"2024-06-11 09:10:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3563786,"visible":true,"origin":"","legend":"\u003cp\u003ePPE induced ERK1/2, AKT and JNK phosphorylation in keratinocyte and human skin fibroblast cells. \u003cstrong\u003e(A) \u003c/strong\u003eCells were treated 10 µg/ml of PPE as indicated times, then the protein extracts were used in Western blot analysis. Beta-actin was used as a control. ERK1/2, AKT and JNK were detected. \u003cstrong\u003e(B)\u003c/strong\u003eAttenuated of selected proteins was demonstrated by adding of LY294002 (ERK inhibitor), PD98059 (AKT inhibitor) and SP600125 (JNK inhibitor) and measured the expression of signaling proteins. Full-length blots are presented in Additional file 1 (Full-length-blots).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/65a45550d76876948587f0c6.png"},{"id":58109970,"identity":"3ab7af4a-f1a0-4b8c-8b70-804e48236335","added_by":"auto","created_at":"2024-06-11 09:02:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7716323,"visible":true,"origin":"","legend":"\u003cp\u003ePPE promotes proliferation and migration of human keratinocyte and fibroblast through the activation of candidate growth signaling proteins. PPE treated-HaCaT and PPE treated-Hs 895.Sk were incubated with/without LY294002 (ERK inhibitor), PD98059 (AKT inhibitor) and SP600125 (JNK inhibitor). \u003cstrong\u003e(A)\u003c/strong\u003e Cell viability was accessed using MTT assay at 48 h and calculated as % cell proliferation. \u003cstrong\u003e(B)\u003c/strong\u003ecells were scratched and incubated for 24 h, wound width was photographed, measured, and calculated as %wound recovery. Data are shown as the mean ± SEM (n=3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/27e82909fc55523a79970bf3.png"},{"id":58110326,"identity":"38c90fe2-7383-4fa0-b6d9-79e021454a14","added_by":"auto","created_at":"2024-06-11 09:10:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3932329,"visible":true,"origin":"","legend":"\u003cp\u003ePPE induced the production of keratinocytes \u003cem\u003eIn Vivo \u003c/em\u003ewound healing model. Full thickness excision wound was tropical applied with PPE with different concentration for 14 days. Wound biopsy was collected and determined the generation of keratin and collagen by Masson's trichrome staining. The keratin layer was shown as red color, whereas newly formed collagen fibers in the dermal layer were indicated by the blue color. Scale bar is equal 10 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/3c845c453c68b232e1f12a5f.png"},{"id":58109969,"identity":"41263d19-ed3b-41e1-b029-a3af10bf7fdf","added_by":"auto","created_at":"2024-06-11 09:02:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":984947,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic diagram of stimulatory effect of PPE on keratinocyte and fibroblast activities including granulation tissue appearance \u003cem\u003eIn Vivo\u003c/em\u003e. PPE activated keratinocyte/fibroblast proliferation and migration through JNK, ERK1/2, and Akt pathway. PPE also stimulated MMPs gene expression either keratinocyte or fibroblast and ECM protein gene expression in fibroblast. Topical PPE treatment on excision wound in rodents for 14 days initiates collagen fiber formation and keratin layer thickening compared to non-treatment. The stimulatory effect of PPE on keratinocyte and fibroblast activities could drive granulation tissue formation, collagen fiber regeneration, ECM deposition/degradation balancing, and re-epithelialization.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/80894721f6c3ca70f8e9c4ad.png"},{"id":73761137,"identity":"c8a86587-03a2-45bc-975f-22c0e8e9856b","added_by":"auto","created_at":"2025-01-14 11:32:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29450783,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/3d05e3f7-30a9-4f5b-8600-644ea39f479c.pdf"},{"id":58109971,"identity":"f26c666f-3020-4e36-82a0-ee1ba2452025","added_by":"auto","created_at":"2024-06-11 09:02:28","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":88584551,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1FulllenghtBlots1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4293166/v1/c9e62f210b99418d9cdd71fd.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Porcine placenta extract promotes keratinocyte and fibroblast activities via ERK AKT and JNK growth signaling pathways","fulltext":[{"header":"Background","content":"\u003cp\u003eThe process of wound healing is a multifaceted and ever-changing biological phenomenon that holds significant importance in the restoration and rejuvenation of tissues. Fibroblast and keratinocyte are dermal cells dominate in granulation tissue formation and re-epithelialization, which involve in the late stage of wound healing [1]. Extracellular matrix (ECM) acts a structural network for cellular organization and cell-cell communication facilitator [2]. In the healing process, ECM has been overwhelmingly reconstituted by fibroblast-derived ECM proteins (e.g. collagen, fibronectin) [3, 4]. Meanwhile, ECM protein overproduction has been impeded by matrix metalloprotease (MMP), in which degrades ECM proteins [5]. Therefore, the equilibrium of ECM deposition/degradation is then essential in wound healing process to facilitate rapid healing process and prevent scar/keloid production. Numerous researchers emphasize in novel treatment approaches that has grown for the last few years, with particular attention paid to the possibility that bioactive compounds derived from natural sources could improve the efficiency of wound healing. The extract derived from porcine placenta has been recognized as a viable option with a wide range of therapeutic properties that engage with signal transduction pathways. This characteristic renders it a potential contribution to the domain of regenerative medicine [6].\u003c/p\u003e\n\u003cp\u003ePorcine Placenta Extract (PPE), rich in growth factors, cytokines, and extracellular matrix components, has shown remarkable potential in promoting cell proliferation, modulating inflammation, and enhancing tissue regeneration. Basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF\u0026beta;-1) were introduced as important substances in PPE, that played a role in thermal-induced wound healing acceleration in rodents [7]. The intricate interplay between the bioactive constituents of PPE and cellular signaling pathways offers a unique perspective on how natural compounds can influence key processes in wound healing, ultimately improving outcomes for patients. At the molecular level, signal transduction pathways play a critical role in regulating cellular responses during wound healing. By modulating these signaling cascades, PPE can potentially optimize the wound microenvironment, facilitating efficient tissue repair and regeneration [8, 9]. In previous studies, PPE has been illustrated the stimulatory effect on cellular proliferation and migration through Akt, ERK1/2, and JNK pathway in human osteoblast [10]. Even though the stimulatory effects of PPE on wound healing have been initiated both \u003cem\u003eIn Vitro\u003c/em\u003e and \u003cem\u003eIn Vivo\u003c/em\u003e for a while, the well-rounded perspective of cellular activities should be further investigated to clarify the underlying mechanism of PPE, especially influencing on epidermal cell activities in the late stage of wound healing, in which many previous studies have been seemingly omitted. \u003c/p\u003e\n\u003cp\u003eThis current study aims to investigate the stimulatory effect of PPE on keratinocyte/fibroblast proliferation and migration including gene expression of MMP and ECM proteins. ERK1/2, Akt, and JNK pathway have been also elucidated to clarity the underlying mechanism of PPE on keratinocyte and fibroblast. The stimulatory effects of PPE \u003cem\u003eIn Vivo\u003c/em\u003e have been also investigated with a wound biopsy and histological staining to observe granulation tissue appearance. \u003c/p\u003e\n"},{"header":"Methods ","content":"\u003cp\u003e\u003cstrong\u003ePorcine Placenta Extract (PPE) Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe formulation of the PPE solution was undertaken by Faculty of Sciences at Mahidol University, Bangkok, Thailand. The process involved initial cleaning and mechanical homogenization of porcine placenta in a phosphate-buffered saline (PBS) solution. Subsequently, the homogenate underwent sonication followed by centrifugation at 4\u0026deg;C for a duration of one hour. The resulting supernatant was then carefully collected and subjected to sterile filtration by using 0.2 \u0026micro;m filters. The protein concentration within the solution was quantified utilizing the Bradford assay method and denoted as the concentration of the PPE solution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human fibroblasts cell lines Hs 895.Sk was obtained from ATCC, the human keratinocytes cell lines HaCaT was a kind gift from\u0026nbsp;Assoc. Prof. Thaned Kangsamaksin\u0026nbsp;(Mahidol University). Cells were maintained according to international guidelines on good cell culture practice in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, MA, USA) at 37 \u0026deg;C humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e, supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, 100 \u0026micro;g/mL of streptomycin, and 0.25 \u0026micro;g/mL of amphotericin B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was evaluated by utilizing the MTT assay. Initially, cells were plated at a concentration of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well in a 96-well plate and allowed to adhere overnight. Following this, various concentrations of PPE were introduced and incubated for the specified duration. Upon completion of the incubation period, MTT reagent at a concentration of 0.5 mg/mL was added and incubated for an additional 4 hours. The resultant formazan product was solubilized using DMSO, and the absorbance was measured at 570 nm by using a microplate spectrophotometer. The percentage of cell proliferation was determined by using the following formula: ((Absorbance at 570 nm of PPE treated cells) / (Absorbance at 570 nm of control cells)) \u0026times; 100 (%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of cell migration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman keratinocyte and fibroblast cell lines were seeded into 6-well plates and incubated in a humidified atmosphere at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e until reaching 90-100% confluence. Subsequently, the monolayer cell was scratched using a 200 \u0026micro;L pipette tip, followed by washing with PBS. The culture medium was then replaced with complete medium containing various concentrations of PPE. The scratched wound was periodically photographed under a microscope at a 10X objective lens for 24 hours. The area of the scratched wound was quantified using the ImageJ program, and the percentage of wound recovery was calculated using the formula: (initial area - area at 24 hours) / initial area) \u0026times; 100%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and Real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman keratinocyte and fibroblast cells were treated to different concentrations of PPE for 6 hours. The extraction of total cellular RNA was carried out by utilizing the total RNA Mini Kit (BioRad, CA, USA) as manufacturer\u0026rsquo;s instruction. Subsequently, first-strand cDNA synthesis was performed by using the Tetro cDNA synthesis kit (Bioline, UK), followed by real-time PCR analysis. The corresponding primer sequences of extracellular matrix-related genes and matrix metalloprotease genes that used in the study are detailed in Table 1. The relative quantification of gene expression was conducted by employing the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method, with normalization against the expression level of GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Primer sequences for quantitative real-time PCR.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer Sequences\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloproteinase-1 \u003cem\u003e(MMP-1)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-GATGTGGAGTGCCTGATGTG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-CTGCTTGACCCTCAGAGACC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloproteinase-2 \u003cem\u003e(MMP-2)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-GCAGTGAATCTACAGGGACGC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-ATCCTGATCCAACCAATCACC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloproteinase-9 \u003cem\u003e(MMP-9)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-CCTTCCTTATCGCCGACAAG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-TGAACAGCAGCATCTTCCCC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloproteinase-10\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e(MMP-10)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-CATTCCTTGTGCTGTTGTGTC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-TGTCTAGCTTCCCTGTCACC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloproteinase-14\u003cbr\u003e\u003cem\u003e(MMP-14)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;-ATAAACCCAAAAACCCCACC-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;-AAACACCCAATGCTTGTCTC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eType 1 Collagen (\u003cem\u003eCol1\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;- GTGCTAAAGGTGCCAATGGT-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;- ACCAGGTTCACCGCTGTTAC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eType 3 Collagen (\u003cem\u003eCol3\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;- CCAGGAGCTAACGGTCTCAG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;- CAGGGTTTCCATCTCTTCCA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003eFibronectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;- AATGGCCAGATGATGAGCTG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;- TGGCACCGAGATATTCCTCC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.909297052154194%\" valign=\"top\"\u003e\n \u003cp\u003e18S ribosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"60.090702947845806%\" valign=\"top\"\u003e\n \u003cp\u003eF: 5\u0026rsquo;- CCATCCAATCGGTATGTAGCG-3\u0026rsquo;\u003c/p\u003e\n \u003cp\u003eR: 5\u0026rsquo;- GTAACCCGTTGAACCCCATT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lines were subjected to treatment with PPE, both with and without 15 \u0026micro;M of inhibitors (LY294002 or PD98059 or SP600125) (Med Chem Express, NJ, USA). Following treatment, the cells were rinsed with ice-cold PBS and subsequently lysed using RIPA lysis buffer supplemented with a protease inhibitor cocktail. The resultant protein lysates were obtained through centrifugation and their concentrations were determined using the Bradford assay. The proteins were then separated via SDS-gel electrophoresis and transferred into PVDF membranes. To prevent nonspecific binding, the membranes were incubated in a 5% skim milk buffer for 1 hour, followed by washing with TBST buffer. Then, the membranes were exposed to specific primary antibodies (anti-pAkt, anti-Akt, anti-pERK, anti-ERK, anti-pJNK, anti-JNK, anti-\u0026beta;-actin, and anti-cyclin D1) (all antibodies procured from Cell Signaling, MA, USA), and allowed to incubate overnight at 4\u0026deg;C with gentle shaking. After washing with TBST, the membranes were incubated with an HRP-linked anti-rabbit antibody (Cell Signaling, MA, USA) for 1 hour at room temperature, washed again, and then subjected to detection reagent incubation. The resulting images were developed using the Chimidoc\u0026trade; XRS system and analyzed utilizing Image Lab software (Bio-Rad, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Wound healing model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 10 weeks Jcl:SD rat were introduced to full thickness excision wound by tissue biopsy at 8 mm width and 2 mm dept. Wounds were treated once a day for 14 days with topical application by various concentration of PPE (20, 200 and 2,000 \u0026micro;g/mL), and 200 ng/mL of basic fibroblast growth factor as a positive control (Thermo Fisher Scientific, MA, USA), and PBS as vehicle control. Until the complete treatment cycle, rats were anesthetized and collected wound tissues using tissue biopsy. The histological samples underwent fixation in formalin, followed by embedding in paraffin. Subsequently, they were transversely sectioned at a thickness of 4 \u0026micro;m and subjected to examination utilizing Masson\u0026apos;s Trichrome staining method. All procedures described were approved by the Ethics Committee for the Use of Animals of the Naresuan University (NU-AE630609).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are shown as mean \u0026plusmn; SEM. The statistically significant difference was analyzed by using ANOVA with appropriate post-hoc comparison analysis (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 was considered as statistically significant difference). The statistical analysis was performed by using commercially available software (GraphPad Prism version 9, San Diego, CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePorcine placenta extract enhanced human keratinocytes and fibroblast proliferation and migration.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the activities of PPE on human keratinocytes and fibroblasts. Cells were seeded into 96-well cell cultures plates and treated with or without medium supplemented with different concentration of PPE. Results showed that PPE promotes keratinocytes and fibroblasts proliferation, especially at 72 hours of incubation time (Figure 1A). Moreover, the effect of PPE on cell migration was evaluated by cell scratch assay. Likewise, PPE significantly enhanced keratinocytes and fibroblasts migration at 24 hours incubation compared to untreated group (Figure 1B). At 10 µg/mL of PPE dramatically increased keratinocytes proliferation and migration, but not in fibroblasts. Seemingly, human keratinocyte is more susceptible to inducing proliferation and migration than fibroblast.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePorcine placenta extract induced the up-regulation of extracellular matrix-related genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the PPE activities on would healing mechanisms, the functionality of PPE on extracellular content was accessed by genes expression of human keratinocytes and fibroblast. After cell treatment with PPE, mRNA level of extracellular matrix-related genes was measured using qRT-PCR. The expression of metalloproteinase enzyme genes including MMP-1 /-2 /-9 /-10 /-14 were significantly increased either human keratinocyte or fibroblasts compared to control group with dose dependent manner (Figure 2A). Additionally, PPE significantly up-regulated the expression of collagens, fibronectin, and alpha-smooth muscle actin (α-SMA) in human fibroblast cell lines (Figure 2B). Consequently, PPE potentially promotes the extracellular matrix-related genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePorcine placenta extract promoted human keratinocytes/fibroblasts proliferation and migration mediated by ERK/AKT and JNK signaling pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine\u0026nbsp;the molecular underlying mechanism of PPE on human keratinocytes and fibroblasts, cells were treated with 10 and 50 µg/mL of PPE for human keratinocytes and fibroblasts, respectively. Intracellular protein was collected and measured protein expression by using immunoblotting. Results showed that PPE dramatically induced phosphorylation of ERK/AKT and JNK signaling pathways in early time activation (within 60 minutes) (Figure 3A). These were confirmed by adding LY294002 (ERK inhibitor), PD58059 (PI3K inhibitor) and PD600125 (JNK inhibitor) that attenuated the overexpression of signaling proteins by PPE (Figure 3B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Not only signaling proteins, but PPE also upregulated the expression of cell cycle-derived protein, cyclin D1 (Figure 3A). According to the phenotype studies, PPE-treated cell with or without specific signaling protein inhibitors and subsequently determined cell proliferation and migration. We demonstrated that activation of cell proliferation by PPE on keratinocytes is involved with ERK, AKT and JNK, whereas on fibroblasts is involved with only JNK signaling (Figure 4A). For activation of cell migration, PPE is associated with ERK, AKT and JNK activation in keratinocytes, and associated with ERK and JNK activation in fibroblasts (Figure 4B). These suggest that the activation of cell proliferation and migration of PPE on these cells are involved with ERK, AKT and JNK signaling pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePorcine placenta extract enhanced generation of keratin and collogen in wound-healing mice model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the effectiveness of PPE on keratinocyte and fibroblast activities as clinical setting, full thickness excision wound in rats was tropically applied by PPE with various concentration for 14 days. Wound biopsy was collected and determined the generation of keratin and collagen by Masson's trichrome staining. Results showed that at low concentration of PPE (20-200 µg/mL) seemingly induce generation of granulation tissue by thicker layer of keratin when compared to PBS control. Moreover, at high concentrations of PPE (2,000 µg/mL) showed regenerated of collagen fiber and some are arranged as collagen bundles as shown in positive treatment with fibroblast growth factor (Figure 5). Hence, these indicated that PPE promote keratinocyte and fibroblast activities and is potential compound for wound healing treatment.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePorcine placenta extract (PPE) has been emphasized as one of the growth factor-enriched compound that derived by agricultural waste-product. PPE in this study was performed proteomics and searched the protein sequence against the domestic porcine \u003cem\u003eSus scrofa\u003c/em\u003e proteome database. The analysis of DAVID bioinformatics elucidated signal-involved proteins, phosphoproteins, and disulfide-bond proteins mostly expressed in PPE in this study [11, 12]. PPE may consist of essential growth factors (EGF, FGF, IGF, VEGF, PDGF etc.), chemokines, antioxidants, etc. Many studies concluded its efficiency and efficacy on cell proliferation, cell migration, cell stress reduction, and anti-inflammation [10, 12-14]. Keratinocyte and fibroblast have contributed to complete wound healing as main responders in the overlapping late process; proliferation/migration phase and remodeling phase. The proliferation and migration phase particularly categorizes to granulation tissue formation, angiogenesis, wound contraction, and re-epithelialization. Fibroblast is one of dominant cells in granulation tissue formation, in which fibroblast proliferates and produces collagen, fibronectin, and \u0026alpha;-Smooth muscle actin (\u0026alpha;-SMA) to constitute a new extracellular matrix (ECM) [15]. Matrix metalloprotease (MMP) is the ECM degradation enzymes that regulates ECM deposition/degradation. MMP has been categorized to various functional subtypes, for example, MMP-1 (Interstitial collagenase), MMP-2 (Gelatinase-A), MMP-9 (Gelatinase-B), MMP-10 (Stromelysin 2), MMP-14, etc. In case the imbalance of ECM deposition/degradation is existed, the delayed wound healing or scar/keloid formation can be prescribed [5]. The re-epithelialization is mostly dominated by keratinocyte, whereby keratinocyte proliferates and migrates from nearby intact epidermis surrounding wound edge for covering the developed granulation tissue [1, 16].\u003c/p\u003e\n\u003cp\u003eIn this study, PPE can stimulate the proliferation and migration of keratinocyte and fibroblast with dose dependent manner as shown in Figure 1. PPE also activated MMP-2, MMP-9, MMP-10, and MMP-14 gene expression in keratinocyte and fibroblast as shown in Figure 2A. These all MMPs could be encoded to ECM degradation enzymes, in which degrades overproduced ECM resulting in scar/keloid minimizing. Moreover, PPE can stimulate \u0026alpha;-SMA, fibronectin, collagen II, and collagen III gene expression in fibroblast as shown in Figure 2B. These are the major components of extracellular matrix. According to \u003cem\u003eIn Vivo \u003c/em\u003eassay, the Masson\u0026rsquo;s trichrome staining of rodent full-thickness wound tissue with PPE topical application elucidated granulation tissue appearance, collagen fiber regeneration, and thickening of keratin layer as shown in Figure 5. Moreover, the wound healing duration \u003cem\u003eIn Vivo\u003c/em\u003e was seemingly shortened in PPE treatment group compared to control group (data not shown). These findings suggested PPE could promote wound healing by increasing fibroblast/keratinocyte proliferation and migration including stimulating MMPs, \u0026alpha;-SMA, fibronectin, and collagen production.\u003c/p\u003e\n\u003cp\u003eERK1/2 and PI3K/Akt pathway have been illustrated as the associated signaling pathway of proliferation and migration [17]. In this study, PPE induced phosphorylation of ERK1/2 and Akt both keratinocyte and fibroblast as shown in Figure 3A. PPE also upregulated Cyclin-D1, which is a protein regulator of cell cycle. Moreover, PPE co-cultured with LY294002 and PD58059 showed the suppression of ERK1/2 and Akt phosphorylation resulting in proliferative and migrative reduction both in keratinocyte and fibroblast as shown in Figure 3B and Figure 4. These findings suggested PPE could promote keratinocyte and fibroblast proliferation/migration through ERK1/2 and Akt pathway. JNK pathway has been also elucidated as the associated pathway of cell growth and differentiation, which is a regulator of transcription factor. In this study, PPE also induce phosphorylation of JNK, which JNK can be suppressed by PPE co-cultured with SP600125, resulting in proliferative and migrative reduction both in keratinocyte and fibroblast as shown in figure 3 and Figure 4. Recent study indicated JNK-gene knockout in keratinocyte and fibroblast attenuated migration and proliferation ability. On another hand, the activation of JNK in fibroblast stimulated collagen synthesis [18, 19]. JNK also displays a downstream signaling of TGF\u0026beta; and PDGF to regulate expression of MMPs, ECM synthesis (collagen, fibronectin, etc.), growth factor secretion, and ECM remodeling in human dermal fibroblast [20]. These finding suggested PPE could also promote keratinocyte and fibroblast proliferation and migration through JNK pathway, whereby it is seemingly a dominant pathway of PPE on the proliferative and migrative stimulation in keratinocyte and fibroblast. Moreover, PPE may activate MMPs and ECM synthesis via JNK signaling pathway, in which PPE may contains PDGF and TGF\u0026beta;.\u003c/p\u003e\n\u003cp\u003eThe limitation in this study is cell line performing in all \u003cem\u003eIn Vitro\u003c/em\u003e assay, in which may not truly reflect human primary dermal cells. However, the cellular activities and morphology is not different between primary keratinocyte and HaCaT cell (Cell lines) including between primary fibroblast and fibroblast cell line [21]. Remarkably, this is the first study that evaluates the stimulatory effect of PPE on MMP and ECM gene expression in keratinocyte and fibroblast in addition to proliferation, migration, and their signaling pathway. PPE greatly composes of various growth factors, nutrients, chemokines, etc. The purification into single substance may deprive the integrated bioactive functions. The quantitative data of rodent granulation tissue appearance by Masson\u0026rsquo;s trichrome staining indicated insignificant difference even performing with sufficient sample size. This insignificant difference of granulation tissue appearance may be a consequence of topical PPE applying. Topical PPE applying in this study is a clear soluble liquid, in which can not securely adhere on wound bed resulting in dissensus data from each rodent sample. Topical PPE should be developed as a hydrogel or cream to provide strong adhesion on wound bed. Since PPE in this study showed the potential ability in wound healing especially keratinocyte/fibroblast proliferative and migrative enhancement including ECM deposition/degradation balancing. Interestingly, Topical PPE on excision wound in rodents illustrated the collagen fiber regeneration and keratin layer thickening as summarized in Figure 6. Topical PPE applying may convey to complementary or alternative clinical treatment to improve delayed wound healing in diabetic foot ulcer patient or reduce a scar/keloid formation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePPE provides the stimulatory effect on keratinocyte/fibroblast proliferation and migration through JNK, ERK1/2, and PI3K/Akt pathway. Concomitantly, this is the first study to demonstrate the stimulatory effect of PPE on MMPs and ECM gene expression in keratinocyte and fibroblast. PPE activates MMP-1, MMP-2, MMP-9, MMP-10, and MMP-14 gene expression. PPE also stimulates \u0026alpha;-SMA, fibronectin, and collagen production. This indicates the potential of PPE on ECM deposition/degradation equilibrium.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePPE: Porcine placenta extract; MMP: Matrix metalloprotease; ECM: Extracellular matrix; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-PCR: Reverse transcription polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LC MS/MS: Liquid Chromatography with tandem mass spectrometry; bFGF: Basic fibroblast growth factor; EGF: Epidermal growth factor; VEGF: Vascular endothelial growth factor; IGF: Insulin-like growth factor; PDGF: Platelet-derived growth factor; JNK: c-Jun N-terminal kinases; Akt: Protein kinase B; ERK1/2: Extracellular signal-regulated kinases\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor’s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.N., W.S., and A.J. conceived and designed the experiments; W.S., J.J., C.N., and N.K. performed the experiments; C.N., W.S., A.J. analyzed the data; A.J., T.S., and R.T. contributed reagents/materials/analysis tools; S.P., P.R., P.M., T.J., P.M., K.S., and all authors wrote, read, prepared, and approved manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Program Management Unit for National Competitiveness Improvement (PMU-C), CCF Energy Supplement (Thailand) Limited, and Mahidol University (Grant number C10F640021). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable requests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Cardio-Thoracic Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (C.N) \u003csup\u003e3\u003c/sup\u003eDepartment of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (W.S.) \u003csup\u003e4\u003c/sup\u003eClinical Research Unit, Faculty of Medicine, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (J.J.) \u003csup\u003e5\u003c/sup\u003eDepartment of Biochemistry, Faculty of Medical Sciences, Naresuan University, Phitsanulok 65000, Thailand; [email protected] (S.P.); [email protected] (N.K.) \u003csup\u003e6\u003c/sup\u003eDepartment of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; [email protected] (R.T.); [email protected] (T.S.); [email protected] (T.J.) \u003csup\u003e7\u003c/sup\u003eFaculty of Medical Technology, Huachiew Chalermprakiet University, Bangkok 10540, Thailand; [email protected] (P.R.); [email protected] (P.M.); [email protected] (K.S.) \u003csup\u003e8\u003c/sup\u003eDepartment of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand; [email protected] (P.M.) \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmiri N, Golin AP, Jalili RB, Ghahary A. 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Antioxidants. 2022;11(8):1545.\u003c/li\u003e\n\u003cli\u003eTansathien K, Ngawhirunpat T, Rangsimawong W, Patrojanasophon P, Opanasopit P, Nuntharatanapong N. In Vitro Biological Activity and In Vivo Human Study of Porcine-Placenta-Extract-Loaded Nanovesicle Formulations for Skin and Hair Rejuvenation. Pharmaceutics. 2022;14(9):1846.\u003c/li\u003e\n\u003cli\u003eAlhajj M, Goyal A. Physiology, Granulation Tissue: StatPearls Publishing, Treasure Island (FL); 2023 2023.\u003c/li\u003e\n\u003cli\u003eBartolo I, Reis RL, Marques AP, Cerqueira MT. Keratinocyte Growth Factor-Based Strategies for Wound Re-Epithelialization. Tissue Eng Part B Rev. 2022;28(3):665-76.\u003c/li\u003e\n\u003cli\u003eSamson SC, Khan AM, Mendoza MC. ERK signaling for cell migration and invasion. Frontiers in Molecular Biosciences. 2022;9.\u003c/li\u003e\n\u003cli\u003eNikoloudaki G, Brooks S, Peidl AP, Tinney D, Hamilton DW. JNK Signaling as a Key Modulator of Soft Connective Tissue Physiology, Pathology, and Healing. International Journal of Molecular Sciences. 2020;21(3):1015.\u003c/li\u003e\n\u003cli\u003eZhao B, Liu JQ, Zheng Z, Zhang J, Wang SY, Han SC, et al. Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways. Cell Tissue Res. 2016;365(1):85-99.\u003c/li\u003e\n\u003cli\u003eHammouda MB, Ford AE, Liu Y, Zhang JY. The JNK Signaling Pathway in Inflammatory Skin Disorders and Cancer. Cells. 2020;9(4):857.\u003c/li\u003e\n\u003cli\u003eOlschl\u0026auml;ger V, Schrader A, Hockertz S. Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay. Arzneimittelforschung. 2009;59(3):146-52.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Porcine Placenta Extract, Keratinocyte and fibroblast activities, granulation tissue formation, Wound healing","lastPublishedDoi":"10.21203/rs.3.rs-4293166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4293166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003ePorcine Placenta Extract (PPE) has been disclosed as a biological protein-enriched compound, whereas it has been declared the stimulatory effects on cell proliferation, cell migration, angiogenesis, and various cellular mechanisms. The fibroblast and keratinocyte are epidermal cells involve in granulation tissue formation and re-epithelialization. The homeostasis of extracellular matrix (ECM) deposition/degradation also attributes wound completion by regulation of ECM protein constitution and matrix metalloprotease (MMP) activities. This study aims to investigate the stimulatory effect of PPE on keratinocyte/fibroblast proliferation and migration including MMPs and ECM protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The human keratinocyte cell line (HaCaT) and human fibroblast cell line (Hs 895.Sk) were cultured with various PPE concentration and investigated cell proliferation, cell migration, MMP gene expression and ECM proteins’ gene expression by MTT assay, scratching wound assay, and qRT-PCR, respectively. ERK1/2, p-ERK1/2, Akt, p-Akt, JNK, p-JNK, and cyclin-D1 were also investigated by Western blot analysis. LY294002, PD98059, and SP600125, in which inhibitors were utilized to confirm the underlying signaling pathway of PPE stimulation. Jcl:SD rat were generated a wound and applied topical PPE with various concentration. The wound biopsy and histological staining with Masson’s trichrome were performed to investigate granulation tissue appearance. All \u003cem\u003eIn Vivo \u003c/em\u003eprocedures were approved by the Ethics Committee for the Use of Animals of the Naresuan University (NU-AE630609).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e PPE statistically significantly increased keratinocyte/fibroblast proliferation and migration with dose dependent manner. PPE 10 µg/mL statistically significantly enhanced MMP-1, MMP-2, MMP-9, MMP-10, and MMP-14 gene expression in keratinocyte. PPE 50 µg/mL also increased MMP-2, MMP-20, and MMP-14 including α-SMA, fibronectin, collagen I, and collagen III gene expression with statistically significant difference. PPE can activate phosphorylation of ERK1/2, Akt, and JNK including cyclin-D1 expression. Interestingly, PPE also stimulate granulation tissue appearance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: PPE stimulated fibroblast/keratinocyte proliferation and migration through JNK, ERK1/2, and Akt pathway. PPE also activated MMP gene expression either keratinocyte or fibroblast and stimulated ECM protein gene expression in fibroblast. Consequently, PPE provides the potential as a complementary treatment to improve delayed wound healing and prevent scar/keloid formation.\u003c/p\u003e","manuscriptTitle":"Porcine placenta extract promotes keratinocyte and fibroblast activities via ERK AKT and JNK growth signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 09:02:23","doi":"10.21203/rs.3.rs-4293166/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e00f11d-f670-4709-b8c9-1feef44e314a","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-14T11:24:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-11 09:02:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4293166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4293166","identity":"rs-4293166","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}