Cell culture
Human ADSCs were obtained from Cellular Engineering Technologies (Coralville, IA). HL-7702 cells were supplied by American Type Culture Collection (Manassas). ADSCs and HL-7702 cells were cultured in 10% fetal bovine serum (FBS)-containing DMEM medium at 37°C, 5% CO2. The grown ADSCs were observed under a microscope.
Cell treatment
HL-7702 cells were grown in hypoxia condition (94% N2, 5% CO2, 1% O2) at 37 °C for 1 h, follow by normal condition for 8 h to establish the I/R injury model. We obtained ALOX5-overexpressed plasmid and empty vector from Hanbio (Shanghai, China), and negative control (NC), miR-183 mimics from Ribobio (Guangzhou, China). ADSCs or HL-7702 cells were transfected with NC (100 nM), miR-183 mimics (100 nM), ALOX5-overexpressed plasmid (1μg) or empty vector (1μg) using Lipofectamine 3000 (Invitrogen) in line with manufacturer’s instructions.
Animal
Male Sprague-Dawley rats (180-200g) were obtained from Laboratory Animal Center of Sun Yat-Sen University. The experiment was carried out after clinical examination of health under the same conditions (temperature (20 ± 2°C), 50% humidity, 12 h light/dark cycle). All animal experiments have been approved by the Committee of the Ethics Committee of Southwest Hospital, Third Military Medical University (Army Medical University).
Rat model of hepatic I/R
On account of the previous researches[22, 23], Briefly, after anesthesia, a traumatic clip was applied to occlude liver arterial/portal venous blood for 60 min, then the clip was removed for reperfusion.
Detection of ADSCs surface antigen
ADSCs were collected and counted (1×106/mL). And 100 μL cell suspension was added with FITC-CD31, FITC-CD34, FITC-CD45, FITC-CD90 and FITC-CD105 at 4°C for 60 min in dark.
After washing with PBS, cells were resuspended with 500 μL PBS. Then the positive rate of cell surface antigen in each group was examined by flow cytometry.
Oil red staining
The harvested ADSCs (1×105/mL) were incubated with adipogenic differentiation medium (Millipore) for 16 days. After washing using PBS, ADSCs were fixed using 4% formaldehyde for 30 min and treated with Oil red dye for 30 min. The staining effect was observed under a microscope.
Alizarin Red Staining
The harvested ADSCs (1×105/mL) were grown with osteogenic differentiation medium (Millipore) for 2 weeks. After washing and fixing, ADSCs were stained with alizarin red dye for 5 min. The staining effect was observed under a microscope.
Exosome extraction
As described in previous study[24], ADSCs were transfected with miR-183 mimics for 48 h, and the medium was harvested and filtered using 0.22 μm filter (Merck–Millipore). ExoQuick-TCExosome Precipitation Solution (System Biosciences) was applied to extract exosomes from ADSCs.
RT-qPCR analysis
The extraction of total RNAs was completed using TRIzol reagent (Invitrogen). cDNAs were synthesized using SuperScript II (Vazyme, Nanjing, China) with total RNAs. Amplification reaction was conducted using SYBR Green Mix (Promega). Amplification data was obtained through ABI Prism 7900 (Applied Biosystems) and counted using 2−ΔΔCt method.
Cell proliferation detection
The treated HL-7702 cells (3×104 cells) were seeded into a 24-well plate and added with 200 μl Edu (Invitrogen, Cat. No. A10044)/medium (1:1000) for 2 h incubation at 37°C with 5% CO2. Then the cells were harvested and resuspended with PBS. Besides, the cells were fixed with 4% formaldehyde for 30 min, neutralized with 2mg/ml glycine for 5 min, and permeated by 0.5% Triton X-100 for 10 min. Edu-positive cells were identified by flow cytometry (Applied Biosystems).
Hoechest staining
The treated HL-7702 cells (3×104 cells) were seeded into a 24-well plate for 48 h incubation at 37°C with 5% CO2, and added with 1 ml diluent Hoechest 33342 (Beyotime Institute of Biotechnology) at 37°C for 30 min. The stained apoptotic cells were observed using a fluorescence microscope (Nikon).
Western blotting analysis
The total protein was extracted from the treated HL-7702 cells, rat liver tissues and exosomes using RIPA buffer (Beyotime Biotechnology, Jiangsu, China) supplementing with 1% protease inhibitors (Pierce). After quantification with bicinchoninic acid (BCA, Thermo Fisher Scientific), the protein (40 μg) was separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore). After blocking, the membranes including the target protein were processed with primary antibodies against Bax (Abcam), Bcl2 (Abcam), caspase 3 (Abcam), TSG101 (Abcam), CD9 (Abcam), CD63 (Abcam), ALOX5 (Abcam), p-p65 (Abcam), p-JNK (Abcam), p-p38 (Abcam), p-ERK (Abcam) and GAPDH (Abcam) overnight at 4°C, and then secondary antibody (Abcam) for 1 h. Enhanced chemiluminescence (ECL, Millipore) was applied for visualization.
Transmission electron microscopy (TEM)
The extracted exosomes from ADSCs were suspended in 0. 2% paraformaldehyde (Sigma-Aldrich), and 3 μL suspension was added onto Electron Microscopy Sciences coated with Formvar-Carbon at room temperature for 5 min. Then the samples were dyed with 1.75% uranyl acetate. After drying, the exosomes were observed using TEM (HITACHI H-7500, Hitachi, Tokyo, Japan).
Dynamic light scattering (DLS)
Nanosizer™ technology (Malvern Instruments, Malvern, UK) and Zetasizer software (Malvern) were applied for the detection of exosome size.
Isolation of RNA and protein from exosomes
Based on the instructions, total Exosome RNA & Protein Isolation Kit (Invitrogen) was adopted to extract the RNA and protein from exosomes.
Exosome uptake
Referring to previous researches[25, 26], exosome uptake was analyzed using Vybrant® DiD (Life Technologies) at room temperature for 10 min.
ELISA analysis
In accordance with the kit instructions, AST, ALT and LDH levels were examined using respective kits. AST ELISA kit (Cat. No. C0010-2-1) and ALT ELISA kit (Cat. No. C009-2-1) were obtained from Jiancheng Bioengineering Institute (Nanjing, China); LDH ELISA kit (Cat. No. C0016) was purchased from Beyotime Biotechnology (Beijing, China).
TUNEL staining
Liver tissue section of rats in each group was dewaxed, and Apoptosis was examined using DeadEnd™ Fluorometric TUNEL System kit (Promega, Cat. no. G3250) in line with the manufacturer’s instructions.
Immunohistochemistry (IHC) assay
After routine dewaxing and hydration, liver tissue sections were added with 3% H202 at room temperature for 10 min. After thermal antigen repair, the sections were sealed using 5% BSA at 37°C for 30 min, incubated with ALOX5 antibody (1:200, Abcam) overnight at 4 °C, followed by secondary antibody for 30 min. After DAB coloring for 10 min, the samples were dehydrated, transparent and then sealed with neutral gum. The results were photographed using a microscope.
Dual-luciferase reporter assay
ALOX5 luciferase reporter plasmid was obtained from VIPOTION Biotechnology (Guangzhou, China). HL-7702 cells were co-transfected with ALOX5 luciferase reporter plasmid and miR-183 mimics, and the experiment was conducted using the Dual-Luciferase® Reporter Assay System (Promega) based on the specification.
Statistical analysis
Measurement data with normal distribution were expressed as mean ± SD. SPSS 21.0 statistical software was used for data processing and analysis using t-test or one-way analysis of variance (ANOVA) with Duncan’s test. P<0.05 signified that the data was statistically significant.