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% CO2, 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 5×103 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% CO2 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).