Animals
Male Sprague-Dawley (SD) rats were obtained from the Animal Experimental Center of Harbin Medical University (Harbin, Heilongjiang, China). All animal protocols strictly followed the guidelines of the Care and Use of Laboratory Animals of the United States National Institutes of Health, and our work received ethical approval from the appropriate committee (approval number Ky2018–135). Rats were randomly placed into one of three groups (6 rats/group), and were euthanized at 4, 8, and 15 days post surgery for the corresponding experiments. All the rats were housed in cages with constant humidity (60 ± 5%) and temperature (23 ± 2℃) with a 12-h/12-h light/dark cycle and free access to laboratory feed and water.
ADSCs and RPMCs harvest and identification
ADSCs were extracted as reporter earlier [12]. Male SD rats, with body weight between 100–150 g, were euthanized via 2% sodium pentobarbital (40 mg/kg) intraperitoneal injection. Following a 75% alcohol-mediated disinfection, the skin was incised diagonally at the groin to remove subcutaneous fat. The adipose tissue was rinsed thrice with sterile cold phosphate-buffered saline (PBS) and chopped. The extracellular matrix (ECM) was lysed in 0.2% COLΙ (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37℃, before undergoing centrifugation at 1000 × g for 10 min. ADSCs were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel) and 1% penicillin-streptomycin (Beyotime, Shanghai, China), and incubated at 37°C and 5% CO2. Following a 48 h incubation, the medium was changed twice a week, with care being taken to not remove the adherent cells. Phenotypic analysis was performed at passage 3 to begin generating ADSCs. Flow cytometry was employed to detect levels of different surface markers. The ADSCs were cultured separately in adipogenic, osteogenic, or chondrogenic differentiation medium (Cyagen, Santa Clara, CA, USA) to identify their differentiation potentials.
RPMCs were isolated as previously described [42]. Briefly, 25 ml of 0.25% trypsin and 0.02% EDTA-Na2 were injected into the intraperitoneal region of rats. Peritoneal effusions were collected 2 h later under aseptic conditions. The isolated RPMCs were grown in DMEM/F12 medium with 20% FBS at 37°C and 5% CO2. RPMCs were verified by certain characteristics, such as, a polygonal pebble morphology and presence of mesothelium-specific markers cytokeratin–19, vimentin, and E-cadherin. Cells between passages 2 and 3, grown as a monolayer to 80% confluence, were employed in subsequent examinations.
Isolation, identification, and analysis of ADSC-Exos
Once the ADSCs achieved 80% confluency, the old culture medium was removed and Exo-depleted medium was introduced. After 24 h, the culture medium was collected without ADSCs before sequential centrifugation at 300 × g for 10 min, 3000 × g for 10 min, 10,000 × g for 30 min, and 100,000 × g for 2 h for Exos extraction. The Exos were mixed in PBS and total protein concentrations in the Exo samples were determined via a bicinchoninic acid assay (Beyotime). Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting were then employed to characterize the isolated Exos. The abovementioned experiments were repeated thrice.
Cellular internalization of ADSC-Exos
ADSC-Exos were exposed to 1 μM PKH26 (Sigma-Aldrich) for 5 min, and ultracentrifugation was performed to remove excess dye. The labeled Exos were added into serum-free RPMC culture medium and maintained overnight in an incubator. Hoechst 33342 dye (UE, China, Suzhou) was used to stain the nuclei and subsequently, images were captured with a fluorescence microscope (Leica, Wetzlar, Germany).
MiRNAs high-throughput sequence and data analyses
The miRNAs in ADSC-Exos were sequenced at OE Biotech Company (Shanghai, China). In short, ADSC-Exos miRNAs were isolation with the RNA isolation Kit (Takara), and the extraction quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies). Next, 20 ng of Exo RNA was isolated for library construction. Finally, the libraries were sequenced with an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, USA). The top 10 miRNAs exhibiting the highest expression in ADSC-Exos were studied using the miRanda software to predict target genes. DAVID (https://david.ncifcrf.gov/) and KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/kobas3/) were employed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-enrichment analyses for the target genes.
Experimental design and surgical procedures
All animals were operated upon as described previously [43]. The rats were fasted for 8 h before the surgery. Male SD rats, with body weight ranging from 200–250 g, were anesthetized via intraperitoneal injection with 2% sodium pentobarbital (40 mg/kg). After disinfection with 75% alcohol, a median abdominal incision of approximately 2.0 cm was made on the skin. The cecum was extracted and rubbed gently with two pieces of dry gauze on the ventral and dorsal sides, until it lost luster and bleeding spots were visible. The cecum was then put back in place, and the layers were closed. Rats (n = 54) were arbitrarily separated into the following three groups: (1) Sham: only the abdominal incision was performed, which was stratified and closed after 3 min of exposure; (2) Control: 100 µL PBS was injected through the tail vein for three consecutive days after abdominal closure; (3) ADSC-Exos: 400 µg ADSC-Exos was dissolved in 100 µL PBS and administered via the tail vein for three consecutive days after abdominal closure. After 4, 8, or 15 days, animals from each group (n = 6) were euthanized, and adhesion was evaluated in all animals using a U-shaped incision. After adhesion grading was performed, the tissues and peritoneal fluid were collected for subsequent experiments.
Peritoneal adhesion scoring system
After euthanasia at 8 and 15 days after peritoneal scraping, the degree of postoperative intraperitoneal adhesion was assessed using the adhesion scoring criteria proposed by Nair et al. [44]. Nair’s score (Table 1) was used for evaluating the degree of postoperative intraperitoneal adhesion. The grade evaluators were third party contractors who were blinded to the research design.
Enzyme-linked immunosorbent assay (ELISA) for tissue plasminogen activator (t-PA) and plasminogen activator inhibitor–1 (PAI–1) levels in peritoneal fluid
Briefly, peritoneal fluids were collected from the abdominal cavities of rats 8 days after peritoneal scraping. The contents of liquid-related factors, t-PA, and PAI–1 in peritoneal fluid were determined using t-PA and PAI–1 ELISA kits (Shanghai Jianglai Biotechnology Inc., Shanghai, China), following kit operational guidelines.
Histological analysis of peritoneal adhesions
Rats were sacrificed at 8 and 15 days after scraping (n = 6 at each time point/group). When fibrous bands did not form, the cecal wall was taken as the specimen; otherwise, the entire fibrous zone was taken. The specimens were immersed in formalin for 24 h, embedded in paraffin, and sliced into 4-μm-thick sections. For histology, all specimens received hematoxylin and eosin (H&E) staining using routine procedures. Observation was done under a light microscope (Nikon Eclipse Ni-U, Tokyo, Japan) at 100× and 200× magnification. Five randomly selected areas in each section were evaluated by an independent pathologist at 200× magnification.
Immunohistochemical evaluation of peritoneal adhesions
For immunohistochemical analyses, the sections were exposed overnight at 4°C to antibodies against E-cadherin (rabbit monoclonal; 1:200; ab76319; Abcam), COL I (rabbit monoclonal; 1:200; ab270993; Abcam, Cambridge, UK), MMP–9 (rabbit monoclonal; 1:200; ab76003; Abcam), TIMP–1 (rabbit polyclonal; 1:200; Abcam), α-SMA (mouse monoclonal; 1:200; ab7817; Abcam), or fibronectin (rabbit monoclonal; 1:200; ab199056; Abcam), followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:500; 115–035–003; Jackson ImmunoResearch, Ely, UK)..
Immunofluorescence staining of peritoneal adhesions
Next, we stained sections with immunoflourescent markers for inflammation and apoptosis as described earlier [45]. In short, following a blocking step, the sections were exposed overnight at 4°C to antibodies against CD163 (rabbit monoclonal; 1:100; ab182422; Abcam), C-C chemokine receptor type 7 (CCR7; rabbit monoclonal; 1:200; ab32527; Abcam), interleukin (IL)–10 (rabbit monoclonal; 1:100; ab33471; Abcam), IL–6 (mouse monoclonal; 1:100; TA500067; Origene), COX–2 (rabbit monoclonal; 1:100; ab179800 Abcam), and cleaved caspase–3 (rabbit monoclonal; 1:100; ab32042; Abcam). Following a PBS-wash, the sections were exposed to a secondary antibody (1:200; SA00003 and SA00009; Proteintech) without light for 1 h, before counterstaining with 4,6-diamidino–2-phenylindole (DAPI, Beyotime) for 10 min. Image capture was done with an Eclipse Ni-U microscope (Nikon) and analysis with the ImageJ software.
Treating RPMCs with ADSC-Exos
To establish peritoneal injury healing model in vitro, 1 × 106 RPMCs were incubated in 6-well culture plates for 18 h, after which they were randomly divided into four groups. ADSC-Exos were added at concentrations of 0, 25, 50, or 100 μg/mL after replacing the RPMC medium with Exo-free medium. Next, to further investigate the associated mechanisms, RPMCs inoculated on a 6-well culture plate were arbitrarily separated into four groups: (1) Control group: the RPMC medium was replaced with Exo-free medium; (2) ADSC-Exos group: the RPMC medium was replaced with Exo-free medium containing 100 μg/mL ADSC-Exos; (3) ADSC-Exos + LY group: RPMCs were treated with 10 nM LY294002 (a PI3K/Akt inhibitor; MedChemExpress, Monmouth Junction, NJ, USA) for 30 min prior to adding 100 μg/mL ADSC-Exos; (4) ADSC-Exos + PD group: RPMCs were treated with 50 nM MAPK/ERK1/2 inhibitor PD98059 (MedChemExpress) for 30 min prior to adding 100 μg/mL ADSC-Exos. The RPMCs in each group were collected after 30 min or 24 h later and analyzed using western blotting. In addition, 5-ethynyl–2-deoxyuridine (EdU), scratch-wound, and transwell assays were conducted 24 h later.
Proliferation analysis of RPMC
RPMC proliferation was assessed with the EdU Assay Kit (UE), following kit operational guidelines. Briefly, RPMCs (1 × 104/group) were exposed to 50 μM EdU for 1 h, then fixed in 4% paraformaldehyde and stained using the EdU kit. Hoechst dye (UE) for 20 min stained the cell nuclei. At last, quantification of EdU-positive cells was performed manually using a fluorescence microscope.
Migration analysis of RPMC
To examine cell migratory patterns, we selected scratch-wound and transwell assays. In the scratch-wound experiments, ADSCs (1.5 × 105 cells/well) were plated in 6-well plates and allowed to attach overnight. A linear wound was generated in the monolayer using a sterile 200-μL pipette tip. Serum-free medium containing ADSC-Exos was then introduced to each well. Image capture was done at 0 and 24 h following ADSC-Exos exposure with the help of an inverted microscope.
For the transwell experiments, RPMCs (1 × 104/group) were plated in the upper chamber of the transwell plate (Corning Inc., NY, USA). Then, medium with 10% free-Exos serum and different concentrations of ADSC-Exos was introduced to the lower chamber. Following 24 h, RPMCs on the permeable membrane of the upper chamber underwent fixation in paraformaldehyde, staining with crystal violet. Finally, cells that travelled to the other side of the membrane were counted under a light microscope.
Western blotting analysis
Briefly, ADSCs lysis was done in RIPA buffer (Beyotime) for protein extraction. The protein concentrations in the lysates were estimated by performing dipicolinic acid assays (Beyotime). Immunoblotting was done with primary rabbit antibodies against TSG101 (monoclonal; 1:2000; ab125011; Abcam), CD9 (monoclonal, 1:2000; ab92726; Abcam), HSP70 (monoclonal; 1:1000; ab2787; Abcam), ERK1/2 (1:1000; Cell Signaling Technology), phospho-ERK1/2 (1:2000; Cell Signaling Technology), Akt (1:1000; Cell Signaling Technology), and phospho-Akt (1:2000; Cell Signaling Technology). A horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5000; Boster, China) was employed as the secondary antibody. The ImageJ software was used for densitometric analysis of the final protein bands.
Statistical analyses
All data are presented as mean ± standard deviation. Quantitative data across all groups were compared using one-way analysis of variance and subsequently, the Tukey’s test. P < 0.05 was set as significance threshold.