Isolation and identification of LDEVs
L. druckerii was cultured in MRS growth medium (Solarbio Science & Technology, Beijing, China) supplemented with 0.5% glucose. EVs were obtained from the L. druckerii culture supernatant. Briefly, L. druckerii was grown at 37 ° C for 12 h until the OD 600 nm reach to 0.9-1.0 which the start OD 600 nm was 0.2. The cell culture was centrifugated for 20 min (12,000×g) at 4 °C. Large particles in the supernatant was removed by filtered through a 0.45-μm membrane. Then, the solution was filtered through a 0.22-μm membrane and centrifugation for 10 min (2000×g) at 4 °C. The supernatant was transferred to a new clean centrifuge tube and centrifuged 10 min at 4 °C (10000×g). Then, the supernatant was moved to another centrifuge tube and ultracentrifuged for 75 min at 110,000×g at 4 °C. 1 mL sterile phosphate buffer saline was used to resuspend the precipitate and filtered through 0.22-μm membrane. Then the solutions were ultracentrifuged for 75 min (110,000×g) at 4 °C. 1 mL sterile phosphate buffer saline to resuspend the precipitate and store at -80°C. Nanoparticle tracking analysis (NTA) was used to determine the diameter size and particle number of LDEVs (ZetaVIEW S/N 17-310, PARTICLE METRIX). Morphological characteristics of LDEVs were detected with transmission electron microscopy (TEM) using Tecnai G2 Spirit BioTwin at 80Kv (FEI).
Labeling of LDEVs and imaging observation
PKH26 kit was used to label the Fluorescent of LDEVs (Sigma, St. Louis, MO, USA) according to product description. PKH26-marked LDEVs were co-cultured with hypertrophic scar derived fibroblasts (HFBs) for 24 h. Then, HFBs were fixed and stained with 4’, 6-diamidino-2-phenylindole (DAPI) and the stained images were observed under confocal microscopy.
Cell culture and treatment
Human tissues were gained from 8 volunteer patients which average age was 20 years and ranging from 7 years old to 42 years. All patients were from Department of Burns and Cutaneous Surgery, Xijing Hospital, Air Force Medical University (Xi'an, China) who was underwent surgical excision. For each patient, the nature of the HS was established by three clinicians. HFBs were isolated according to previous methods[44]. NFBs were isolated from skin biopsy samples as previously described. HFBs and NFBs (P3-P6) were used for experimental studies. Cells were cultured in 6-well culture plates with 1х104 cells per well. LDEVs was used to treat HFBs and NFBs for 24h and 48h.
CCK8 assays
The influence of LDEVS on HFBs proliferation was measured by the cell counting kit-8 (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions. 2000 HFBs per well were seeded in 96-well plates and cultured with PBS or LDEVs for 24 h and 48 h. Then CCK8 solution were added and absorbance at 450 nm were determined through a microtiter plate reader (Infinite 200 PRO, Switzerland).
Mouse model and treatment
6-8 weeks old BALB/c mice weighing 22-25 g (n=20) were used in this study. A murine model of excisional wound healing was chosen to investigate the influence of LDEVs on wound healing. In brief, the backs of mice were shaved and cleaned after the mice were anesthetized. A 1.5 cm2 full-thickness wound was shaped on the mouse dorsum. Animals (n = 10 / group) were randomly divided into two groups and injected subcutaneously with LDEVs or PBS at four injection sites (25 μL each).
Scleroderma mouse model was made according to the protocol previous. Briefly, BALB/c mice (n=24) were shaved (approximately 2.0 cm×2.0 cm) from the same part of the central back. Each group was injected with bleomycin hydrochloride buffer (1 mg/mL), 0.1 mL once a day, for 28 consecutive days. Mice were randomly divided into three groups (n=8): control group (PBS injected), LDEVs treat group (25 μL and 50 μL injected).
qRT-PCR
Trizol was used to extract the Total RNA of tissues and cells (Takara, Japan). cDNA was obtained by PrimeScriptTM RT reagent Kit (Takara, Japan). The 2-ΔΔCT method was used to analyze the relative gene expression which GAPDH was used as internal controls. qRT-PCR was performed by qRT-PCR system (BioRad, Singapore). Primer sequences used for this study were as follows: GAPDH: forward, 5'-CACCATGGAGAAGGCCGGGG-3', and reverse, 5'-GACGGACACATTGGGGG TAG-3'; α-SMA: forward, GACAATGGCTCTGGGCTCTGTAA, and reverse, TGT GCTTCGTCACCCACGTA; Collagen I: forward, GAGGGCAACAGCAGG TTCACTTA, and reverse, TCAGCACCACCGATGTCCA; Collagen III: forward, CCACGGAAACACTGGTGGAC, and reverse, GCCAGCTGCACATCAAGGAC.
Western blot analysis
HFBs after different treatments were washed with ice-cold PBS three times. 80 μL RIPA buffer containing protease and phosphatase inhibitor was added to lyse the cells and centrifugated for 10 min (14,000×g) at 4 °C. 50 mg total protein determined by BCA was processed by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk and incubated with different antibodies anti-Collagen III (1:1000, Abcam, UK) anti-Collagen I (1:1000, Abcam, UK), anti-SMA (1:1000, CST, USA). The membranes were imagined with ECL detection system (Alpha Innotech, San Leandro, CA).
Histological, immunofluorescent and immunohistochemistry analyses
Skin tissue samples were fixed in 10% formalin, dehydrated with ethanol and embedded in paraffin. Then, samples were cut into 4 μm-thick sections. Sections go through the following steps: deparaffinized in xylene, rehydrated through decreasing concentrations of ethanol and distilled water. At last, the sections were subjected to further analysis. Hematoxylin and eosin (H&E) and Masson’s trichrome were used for histological analysis.
Sections were incubated with autofluorescence quencher for 5 minutes after antigen extraction by boiling the sections in EDTA-Tris buffer (pH 9.0), then blocked with 3% BSA for 30 min at room temperature, followed by incubated with anti-Ki67 primary antibody (1:100; CST, USA) at 4°C for 12 h. Sections was washed with PBS three times and then incubated with secondary antibody (1:250; Abcam). Nuclei were stained with DAPI (0.5 µg/mL; Invitrogen, Carlsbad, USA). The pictures were achieved by fluorescence microscope (FV1000, Olympus, Japan). Three random visual fields were used to examine the Ki67-postive cells of different sections.
Rehydrated sections which were used for immunohistochemistry (IHC) staining were heated in EDTA-Tris buffer (pH 9.0) for 15 min by microwave. Then samples were blocked with 3% hydrogen peroxide for 25 min. Then, the sections were blocked with 3% BSA for 30 min and incubated with the primary antibody anti-CD31 (1:50; CST) overnight at 4°C. The immunoactivity was explored by horseradish peroxidase IHC detection system (Servicebio) and then counterstained with hematoxylin. Three random fields were used in each section to count the number of CD31-positive vessels.
Protein Preparation and iTRAQ Labeling
HFBs with or without LDEVs treatment were washed with ice-cold PBS three times. Then cells were lysed with RIPA buffer containing with protease and phosphatase inhibitor and then centrifuged for 10 min (14,000×g) at 4 °C. Bradford method was used to determine the concentration of proteins. Protein samples were stored at -80 °C until used. The method of proteomics was supplied by BGI (Shen Zhen, China). First, each sample was digested with Trypsin Gold (Promega, Madison, WI, USA) for 12h at 37 °C. Second, samples were desalted and dried under vacuum conditions. Next, the resuspended peptides were marked with new iTRAQ Reagent 8-plex (AB Sciex, Foster City, CA, USA). The mixed peptides were separated on a Shimadzu LC-20AB HPLC Pump system, desalted and vacuum-dried. The peptides separated from nanoHPLC (Thermo Scientific™ UltiMate™ 3000 UHPLC system) were subjected into the tandem mass spectrometry QEXACTIVE HF X (Thermo Fisher Scientific, San Jose, CA) for DDA (data-dependent acquisition) detection by nano-electrospray ionization.
Statistical analysis
The experiment was repeated at least three times, and each experiment was presented as mean ± SD. Student’s t test was used for statistical differences between paired samples, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. All analyses were performed using Prism 8 software (GraphPad). A P-value < 0.05 was considered as statistically significant.