Animal experiments
All animal studies followed the guidelines of the National Institutes of Health (NIH) and were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine. Male wild-type (NLRP3+/+) C57BL/6N mice (8–10 weeks) were purchased from Vital River Laboratory (Beijing, China). NLPR3L351PneoR mice (NLRP3-/-) were purchased from Jackson Laboratory (Sacramento, CA, USA), bred, and reproduced. NLRP3+/+ and NLRP3-/- mice were genotyped following the protocol by the vendor. Mice were housed in a temperature-controlled facility with a 12-h light/dark cycle and were given free access to food and water. In the first NAFLD model, mice were fed a methionine-choline-deficient (MCD) diet (Trophic Animal Feed High-tech Co., Nantong, China) or a methionine-choline-supplemented (MCS) diet for 4 weeks. In the second NAFLD model, mice were fed a 60 kcal% high-fat diet (HFD) (Research Diets Inc., Brogaarden, Denmark) or normal diet (ND) for 16 weeks. For the in vivo hepatic sEV function experiments, mice were injected with 20 mg/kg hepatic sEVs via the caudal vein for 2 days. For euthanasia, mice were anaesthetized by 3 vol% isoflurane and sacrificed by exsanguination.
Cell cultures and treatments
Mouse microvascular endothelial cell (MVEC) line EOMA and human hepatocyte cell line HepG2 were obtained from ATCC (Manassas, VA, USA). Human microvascular endothelial cell line-1 (HMEC-1) and human hepatocyte cell line L02 were obtained from BeNa Culture Collection (Beijing, China). Human hepatocyte cell line Huh7 was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). HMEC-1 was maintained in Endothelial Culture Media (ScienCell, Carlsbad, CA, USA), L02 was maintained in RPMI 1640 culture media (Gibco, Carlsbad, CA, USA), and other cell lines were maintained in Dulbecco's modified eagle medium (Gibco, Carlsbad, CA, USA) in a humidified incubator with 5% CO2 at 37°C. Dynasore, MCC950, and CA-074 were purchased from MedChemExpress (Monmouth Junction, NJ, USA) and dissolved in DMSO. Palmitic acid (PA) was dissolved in 0.1 mol/L NaOH by heating at 75°C for 30 min and conjugated with fatty acid-free BSA solution. Cells were pretreated with or without 20 μmol/L dynasore for 30 min, followed by incubation with 120 μg/mL hepatic sEVs in EV-free FBS-supplemented media (SBI System Biosciences, Palo Alto, CA, USA) for 24 h, with the presence or absence of 100 μmol/L MCC950 or 5 μmol/L CA-074.
Histology
Liver paraffin sections (4-μm-thick) were stained with hematoxylin and eosin (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's protocol. Frozen liver sections (8-μm-thick) were stained with Oil red O (Biotopped, Beijing, China) according to the manufacturer's instructions. Sections were observed and photographed at 400× magnification using a CX31 microscope (Olympus, Tokyo, Japan).
Doppler ultrasound imaging
Doppler ultrasound studies were performed using a Vevo 2100 Imaging System (VisualSonics, Toronto, Canada) with a MS400 linear-array transducer. Liver echogenicity were evaluated in the B-mode.
Measurement of aminotransferase activity and lipid content
The aminotransferase activity (aspartate aminotransferase and alanine aminotransferase) and lipid content (total cholesterol, triglyceride and low-density lipoprotein cholesterol) in plasma samples, liver samples or hepatocytes were determined using commercial kits according to the manufacturer's protocol (Nanjing Jiancheng Biology, Nanjing, China).
Immunofluorescence analysis
For tissue immunofluorescence staining, frozen cardiac sections (8-μm-thick) were fixed in pre-cooled acetone or 4% paraformaldehyde and blocked with 10% donkey serum for 1 h at room temperature, then incubated overnight at 4°C for 1 h at room temperature with the primary antibodies. After washing with PBS, the slides were incubated with the corresponding Alexa Fluor 488 or Alexa Fluor 555-labeled secondary antibodies for 2 h at room temperature. The sections were visualized using a LSM 800 laser confocal scanning microscope (Carl Zeiss, Oberkochen, Germany). Image analysis was performed using the Image Pro-Plus 6.0 software. Anti-cleaved-caspase-1 primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-IL-1β and vWF primary antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-ZO-1/2 primary antibodies and fluorescent secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).
Microvascular endothelial permeability assay
The microvascular endothelial permeability assay was performed as previously described [4]. Briefly, mice were injected with 80 mg/kg Evans blue solution (Sigma-Aldrich, St. Louis, MO, USA) via the caudal vein. Two hours later, mice were sacrificed and injected with PBS through the left ventricle to remove intravascular Evans blue solution. Tissues were collected and incubated in formamide at 60°C for 2 days. The Evans blue content was quantitated by spectrophotometry at 620 nm and normalized to tissue weight.
Screening of human disease target and GO enrichment analysis
The GeneCards database (https://www.genecards.org/) was used to gather the information of human targets related to microvascular hyperpermeability. The keyword ‘microvascular hyperpermeability’ was used to screen with a correlation score of ≥1.5 as candidate target genes. KEGG pathway enrichment analysis was performed using the clusterProfiler R package. The clusterProfiler R package was used to perform Gene Ontology (GO) enrichment analysis and to visualize the possible biological processes (BP), cellular component (CC) and molecular function (MF) that were involved [34].
Purification and characterization of sEV
Mouse liver tissues were minced in PBS and the hepatic sEVs were collected according to method of Loyer et al [35] with minor modifications. HepG2 were treated with 100 μmol/L palmitic acid (PA) or vehicles in EV-free FBS-supplemented media for 18 hours, and hepatocyte sEVs were collected from the cell culture media. Briefly, liver samples or cell culture media were centrifuged at 2,000 ×g for 30 min at 4°C to remove dead cells, and 12,000 ×g for 30 min for twice to remove cell debris and large extracellular vesicles. Supernatants were then collected and filtered through 0.22 μm microporous membranes, followed by ultracentrifugation at 110,000 ×g for 70 min at 4 °C twice. Finally, the sEV sediment was resuspended in PBS and filtered through 0.22 μm microporous membranes. Protein concentration was determined using bicinchoninic acid protein assay kits (Beyotime, Beijing, China). The sEV particle sizes were determined using nanoparticle tracking analysis with a Nanosight NS 300 (Malvern, Malvern, UK), and the morphology of sEV was identified using a JEM-1200EX transmission electronic microscope (JEOL, Tokyo, Japan).
In vivo and in vitro sEV internalization assay
Hepatic sEVs were labelled with 10 μmol/L DiR (US Everbright Inc., Suzhou, China) for 30 min at 37 °C or 5 μmol/L DiI (Beyotime, Beijing, China) for 20 min at room temperature. Subsequently, sEVs were collected by ultracentrifugation at 110,000 ×g for 70 min at 4 °C. The DiR or DiI-labeled sEV sediments were resuspended in PBS and filtered through 0.22 μm microporous membranes. The pellets derived from the ultracentrifugation of DiR or DiI alone was used as vehicle controls. For the in vivo internalization assay, hepatic sEVs were administrated to naive mouse via caudal vein injection. Twelve hours later, tissues were harvested and imaged by LB983 in vivo optical imaging system (Berthold, Bad Wildbad, Germany). For the in vitro internalization assay, MVECs were pretreated with or without dynasore for 30 min and incubated with DiI-labeled hepatic sEVs. Eight hours later, cells were harvested, fixed with 4% paraformaldehyde, counterstained with DAPI and visualized using a LSM 800 laser confocal scanning microscope (Carl Zeiss, Oberkochen, Germany).
Western blotting
The Western blotting assay was performed as previously described [36]. Briefly, hepatic sEVs, cell lysates or supernatants were boiled at 100 °C for 5 min, separated on 8%-15% SDS-polyacrylamide gel and transferred to a 0.22 μm PVDF membrane. Membranes were then blocked with 5% non-fat milk for 1 h at room temperature and incubated with the primary antibodies overnight at 4°C, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies. The protein bands were detected using a chemiluminescence imaging system (Tanon, Shanghai, China) and analyzed using Image J software. Densitometric analysis was used to determine the relative protein expression. The bands of caspase-1 p20 were normalized to that of pro-caspase-1. The bands of cleaved-IL-1β were normalized to that of pro-IL-1β. The cell culture supernatant HMGB1 were collected from the same numbers of cells and the bands were normalized to that of cellular GAPDH or β-actin. The rest of bands were normalized to that of GAPDH or β-actin. Anti-caspase-1 primary antibody was purchased from Wanlei Biology (Shenyang, China). Anti-HMGB1, LAMP1, and HSP70 primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ZO-1/2 primary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Anti-IL-1β, NLRP3, TSG101, and CD63 primary antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin and GAPDH primary antibodies were purchased from Affinity Biologicals (Ancaster, ON, Canada). Anti-mouse and rabbit secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Analysis of caspase-1 activity
The caspase-1 activity in cells was measured using flow cytometry in strict accordance with the instructions of the FAM-FLICA Caspase Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA). Briefly, cells were stained with FAM-YVAD-FMK for 1 hour, collected and detected by an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). The results were analyzed using FlowJo 10.0 Software.
Measurement of microvascular endothelial permeability
Endothelial barrier function was assessed by measuring the permeability of MVECs and HMEC-1 to fluorescein isothiocyanate-conjugated dextran (FITC-dextran) as described previously [37]. Briefly, microvascular endothelial cells were seeded on transwell chambers and incubated with hepatic sEVs or hepatocyte sEVs. Twenty-four hours later, the culture medium was replaced with PBS in the lower chamber and 1 mg/mL FITC–dextran 4 kDa (Sigma-Aldrich, St. Louis, MO, USA) in the upper chamber. One hour later, samples from the upper and lower compartments were measured by a fluorescent plate reader using excitation and emission wavelengths of 485 and 535 nm, respectively.
Analysis of lysosome membrane permeability
Lysosome membrane permeability was determined by acridine orange (AO) staining. The cells were then incubated with AO (Solarbio, Beijing, China) for 15 min at 37°C according to the manufacturer's guidelines. Live cells were immediately visualized using a LSM 800 laser confocal scanning microscope (Carl Zeiss, Oberkochen, Germany).
Analysis of cathepsin B activity
The activity of cathepsin B from disrupted lysosomes was determined using the Magic Red (MR) Cathepsin B Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA). Briefly, cells were stained with a fluorescent cell-permeable selective cathepsin B substrate MR-(RR)2 for 1 h, fixed with 4% paraformaldehyde, counterstained with DAPI, and visualized using a LSM 800 laser confocal scanning microscope (Carl Zeiss, Oberkochen, Germany).
MiRNA deep-sequencing
Hepatic sEV miRNAs were profiled by the small RNA sequencing analysis in a v3 flowcell on an Illumina HiSeq 2500 sequencer (Illumina Inc., San Diego, CA, USA). Differential expression analysis of the MCS and the MCD groups was performed using the DESeq R package. Prediction of miRNA targets and identification of miRNA binding sites were performed using RNA22 (version 2.0).
MiRNA real-time quantitative PCR
For the measurement of novel-miR-7 level, plasmatic sEVs were collected using an ExoQuick reagent (SBI System Biosciences, Palo Alto, CA, USA), and hepatocyte sEVs were collected from the cell culture media using a Ribo Exosome Isolation Reagent (Ribobio, Guangzhou, China). Total RNAs in sEVs or MVECs were isolated using TRIzol reagent and reverse transcribed using a miRNA first-strand cDNA synthesis kit (Sangon, Shanghai, China) according to the manufacturer's guidelines. RT-qPCR was performed using a TB Green™ Premix Ex Taq™ (Takara, Kyoto, Japan) according to the manufacturer's guidelines. Relative miRNA expression levels were calculated using the comparative threshold (CT) method. Results were normalized to U6 in MVECs or total RNA in sEVs. The primers were purchased from Sangon Biotech, and the sequences are listed in Supplementary Table 1.
Transfection of a novel-miR-7 mimic and inhibitor
For transfection of the novel-miR-7 mimic, cells were transfected with either a 5-FAM-labelled novel-miR-7 mimic or a mimic negative control (GenePharma, Shanghai, China) at a final concentration of 50 nM using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Transfection efficiencies were determined by confocal microscopy. For transfection of the novel-miR-7 inhibitor, cells were transfected with either a novel-miR-7 inhibitor or inhibitor-negative control at a final concentration of 100 nM using Lipofectamine 3000 according to the manufacturer's instructions. The sequences are listed in Supplementary Table 1.
Dual-luciferase reporter assay
The 3’-UTR fragment of LAMP1 was generated by PCR and cloned into pGL3 Luciferase Reporter Vector (Promega, Madison, WI, USA), which contained the putative novel-miR-7 binding sequence. pRL-TK vector was co-transfected in each experiment as an internal control. Luciferase activity assays were performed using a dual-luciferase reporter assay system (Promega) and normalized relative to Renilla luciferase activity according to the manufacturer’s instructions. The sequence of pGL3-LAMP1-3’UTR plasmid is listed in Supplementary Table 1.
Measurement of cell viability and LDH leakage
Cell viability was assessed by CCK-8 assays according to the manufacturer's protocol. The leakage of lactate dehydrogenase (LDH) was assessed using a LDH assay kit (Nanjing Jiancheng Biology, Nanjing, China) according to the manufacturer's protocol.
Measurement of lipid accumulation in hepatocytes
Hepatocytes were fixed with 4% paraformaldehyde and stained with oil red o (Biotopped, Beijing, China) for 30 min. Subsequently, the oil red o was extracted by isopropanol and quantitated by spectrophotometry at 510 nm. Cellular triglyceride content was measured according to the manufacturer's protocol (Nanjing Jiancheng Biology, Nanjing, China).
Statistical analysis
Data are presented as the mean with the standard error of the mean (SEM). Significant differences were examined using unpaired Student t test or one-way ANOVA. Statistical analysis was performed using SPSS software. P < 0.05 was considered statistically significant.