Animals
C57BL/6 male mice, 8 weeks of age, had been utilized for this examine. These mice had been obtained from the Laboratory Animal Heart of the Fourth Navy Medical College. The mice had been group-housed and maintained beneath particular pathogen-free circumstances, with a regular 12-hour mild/darkish cycle and advert libitum entry to meals and water. All animal experiments had been performed in compliance with related legal guidelines and moral rules, adhering to the Pointers of Intramural Animal Use and Care Committees of the Fourth Navy Medical College, authorised by the Ethics Committee of the Fourth Navy Medical College (Approval No. 2020-003), and in accordance with the ARRIVE pointers.
PHx surgical procedure
The PHx process was carried out beneath sterile circumstances [22]. Animals had been anesthetized utilizing isoflurane inhalation (R500IP, RWD Life Science, USA). For the two/3 PHx, the median and left liver lobes had been sequentially resected. The Sham operation concerned a laparotomy. Sampling time factors had been established at 72 h all through the examine.
LT-EV assortment and Hep-EV isolation
Tissue EVs had been collected in line with our beforehand printed protocol [54]. Following 72 h post-Sham or PHx surgical procedures, mice had been sacrificed, and liver tissues had been gently dissociated into tiny items after which digested with Liberase TM enzyme (LIBTM-RO, Sigma-Aldrich, USA) for 30 min at 37 °C. This was adopted by filtration by way of a 0.70-µm pore dimension filter. The filtrate was then centrifuged at 300 g for 10 min and at 2,000 g for 20 min at 4 °C to take away cells and tissue particles. The supernatant was additional centrifuged at 16,800 g for 30 min at 4 °C to gather LT-EVs. For added processing, LT-EVs had been stained at the hours of darkness with ASGPR-PE antibody (sc-166633 PE, Santa Cruz Biotechnology, USA; diluted 1:100) at 4 °C for 4 h. Following staining, the LT-EVs had been incubated with anti-PE magnetic microbeads (130-048-801, Miltenyi Biotec, USA) diluted 1:16 in MACS buffer (130-091-221, Miltenyi Biotec, USA) at 4 °C for 15 min. The combination was then handed by way of an LD column (130-042-901, Miltenyi Biotec, USA) to seize ASGPR+ LT-EVs (i.e., Hep-EVs). Captured Hep-EVs had been transferred into contemporary sterile tubes and subjected to 2 PBS washes. Lastly, Hep-EVs had been collected after centrifugation at 16,800 g for 30 min at 4 °C.
Hep-EV inhibition and replenishment in vivo
The liver-targeting AAV serotype 8 (AAV8), that includes a hepatocyte-specific promoter, human α1-antitrypsin promoter (hAATp), and an apolipoprotein E (ApoE) enhancer, was utilized to ship shRNA particularly to knock down Rab27a in hepatocytes in vivo. Mice had been injected by way of the tail vein with both an AAV vector carrying the Rab27a shRNA (pAAV-ApoE/hAATp-Rab27a-shRNA, Genechem, China) or a adverse management vector (pAAV-ApoE/hAATp-null, Genechem, China) at a dose of 1 × 1011 v.g per 200 µl per mouse. These AAV injections occurred 4 weeks previous to the PHx and Hep-EV administration. For Hep-EV administration, Hep-EVs suspended in filtered PBS had been injected by way of the caudal vein of the mouse at a dose of 5 × 1010 particles per 200 µl per mouse for twenty-four h, adopted by the PHx surgical procedure. For Hep-EV biodistribution evaluation, Hep-EVs had been stained by 5 µM DiR (Invitrogen, USA) earlier than intravenous injection. Mice had been euthanized after 24 h, and the tissues had been examined utilizing an in vivo imaging system (IVIS Lumina II, Caliper Life Science, USA).
Liver bulk RNA-seq evaluation
The entire RNA from your complete liver was extracted utilizing the Trizol reagent equipment (Invitrogen, USA) following the producer’s protocol. RNA high quality was assessed utilizing an Agilent 2100 Bioanalyzer (Agilent Applied sciences, USA) and verified by way of RNase free agarose gel electrophoresis. Subsequently, eukaryotic mRNA was enriched utilizing Oligo (dT) beads. After whole RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads. The enriched mRNA was then fragmented into brief fragments utilizing fragmentation buffer and reversely transcribed into cDNA utilizing NEBNext Extremely RNA Library Prep Equipment for Illumina (NEB #7530, New England Biolabs, USA). The purified double-stranded cDNA fragments had been finish repaired, addition of a base, and ligation to Illumina sequencing adapters. The ligation response was purified utilizing the AMPure XP Beads (1.0X) and amplified by way of polymerase chain response (PCR). The ensuing cDNA library was sequenced utilizing Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Genes/transcripts exhibiting false discovery price (FDR) parameter beneath 0.05 and an absolute fold change ≥ 2 had been thought of differentially expressed genes/transcripts. Subsequent purposeful evaluation was carried out using the GO and KEGG databases, or by way of GSEA enrichment evaluation.
scRNA-seq microarray dataset
scRNA-seq microarray datasets had been screened and downloaded from the Gene Expression Omnibus (GEO). The GSM4572241 and GSM4572243 collection had been obtained from the GEO database for evaluation of DEGs. Additional purposeful evaluation was carried out based mostly on the GO and KEGG databases.
Cell clustering
The cell-by-gene matrices for every pattern had been individually imported to Seurat model 3.1.1 for subsequent evaluation. Seurat was utilized to conduct expression high quality examine, information normalization, t-distributed stochastic neighbor embedding (tSNE) plot evaluation, cell clustering, cluster visualization, and cell sort annotation.
Pseudotemporal trajectory evaluation
The only-cell pseudotemporal trajectory [55, 56] was analyzed utilizing matrices of cells and gene expressions by Monocle (Model 2.10.1). The house was lowered with two dimensions, and cells had been ordered (sigma = 0.001, lambda = NULL, param.gamma = 10, tol = 0.001). The trajectory was visualized utilizing a tree-like construction, together with suggestions and branches.
Proteomic evaluation
Protein lysates of Hep-EVs from Sham and PHx teams had been ready and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) evaluation on an Orbitrap Exploris™ 480 mass spectrometer with a NanoSpray III ion supply. The uncooked information had been analyzed utilizing the Proteome Discoverer system (v2.4.1.15). Proteins had been recognized by comparability with the Uniport database, with a false discovery price set at 0.01 for each peptides and proteins. Quantification of proteins was carried out utilizing the default parameters in MaxQuant. Among the many recognized proteins, 516 proteins had been DEPs (fold change > 1.5 and p-value < 0.05). These proteins had been chosen for additional purposeful evaluation based mostly on GO and KEGG databases.
Cell tradition and assays
The murine AML12 hepatocytes (ATCC CRL-2254) had been cultured in Dulbecco’s modified Eagle’s medium with F-12 (DMEM F-12, Gibco, USA) at 37 °C with 5% CO2. The medium was supplemented with 10% fetal bovine serum (FBS; HyClone, USA), 1% ITS liquid medium complement (Sigma Aldrich, USA), 1% 100 µg/ml of penicillin and streptomycin (HyClone, USA), and 40 ng/ml of dexamethasone (Sigma-Aldrich, USA). Hep-EVs had been used to deal with AML12 cells for 48 h at a protein focus of 10 µg/ml. The RO-3306 compound was dissolved in dimethyl sulfoxide (DMSO) (D8371, Solarbio, China) and added to Hep-EVs at a focus of 5 µM for a period of 24 h, then washed previous to Hep-EVs remedy.
For the wound closure assay, hepatocytes had been seeded into 6-well plates at 5 × 105 cells/properly. When the cells reached 90% confluence, sterile 200 µl pipette suggestions had been used to create cell wounds on the plates. After washing with PBS, the medium was became tradition medium with out FBS and added with 10 µg/ml of Hep-EVs dissolved in PBS to particular wells for a period of 48 h. Cells had been photographed at 0 h, 6 h, and 12 h with an inverted microscope (Leica, Germany). Quantification of the wound space was carried out utilizing the ImageJ software program (NIH, USA). For the EdU labeling assay, hepatocytes had been seeded into 6-well plates at 5 × 105 cells/properly. DNA synthesis was examined by 2-hour EdU labeling utilizing a business kFluor488 Click on-iT EdU equipment (KGA331, KeyGEN, China). Quantification of the positively labeled cell percentages was carried out utilizing the ImageJ software program (NIH, USA).
Built-in evaluation of associations between genes and proteins
The genes/proteins and DEGs/DEPs detected within the scRNA-seq and proteomic analyses had been enumerated. Subsequently, correlation evaluation was performed by R (model 3.5.1). A nine-quadrant map was generated based mostly on the alternations in gene expression inside the hepatocyte transcriptome and the Hep-EV proteome. Quantitative and enrichment evaluation of genes in every quadrant was carried out.
TEM evaluation
TEM was utilized to substantiate the presence of EVs within the livers. Put up-perfusion liver from the PHx and Sham mice had been minimize into items of roughly 1 mm × 1 mm × 1 mm, quickly fastened in 3% glutaraldehyde, and postfixed in a 1% OsO4 resolution at 4 °C. Following fixation, the samples had been dehydrated with gradient acetone and embedded in araldite (EM TP, Leica, Germany). The sliced sections, ready utilizing an ultramicrotome (EM UC7, Leica, Germany), had been stained with uranyl acetate and lead citrate after which examined with 120 KV TEM (JEM-1400FLASH, JEOL, Japan).
The morphology of LT-EVs was additionally characterised utilizing TEM. A complete of 4 µl of the EV resolution, with a protein focus of 1 mg/ml, was deposited onto a carbon-coated 400-square mesh copper grid. Ten minutes after the pattern was deposited, the grid was rinsed with 10 drops of deionized water. Subsequently, a drop of 1% phosphotungstic acid (12501-23-4, RHAWN, China) was added to the grid to conduct the adverse staining. The grid was then naturally dried and visualized utilizing the 120 KV FEI TEM (TECNAI Spirit, FEI, USA). TEM-EDS was performed utilizing a discipline emission TEM (JEOL, Japan).
NTA experiments
The focus and dimension distribution of LT-EVs and sorted Hep-EVs had been analyzed by the Nanosight NS300 system (Malvern Panalytical, UK). Information had been analyzed by Nanosight NTA 2.3 Analytical Software program with the detection threshold optimized for every pattern and display screen acquire at 10 to trace as many particles as doable whereas sustaining minimal background interference. Moreover, a clean 0.2 μm-filtered PBS was run as a adverse management for comparative evaluation.
Western blot evaluation
LT-EV and Hep-EV proteins had been extracted utilizing the RIPA lysis buffer (P0013B, Beyotime, China). The protein focus of every pattern was decided utilizing the BCA protein assay equipment (PA115, TIANGEN, China). Subsequently, all samples had been ready at a remaining focus of 1 µg/µl in a loading buffer (CW0027S, CwBio, China). For protein separation based mostly on molecular weight, 20 µg of protein samples had been loaded right into a 4–20% SDS-polyacrylamide gel (LK206, Epizyme, USA) inside the Bio-Rad Electrophoresis System. The proteins inside the gel had been then transferred to polyvinylidene difluoride (PVDF) membranes. Following blocking in 5% bovine serum albumin (BSA) resolution (218072801, MP Biomedical, USA), the membranes had been subjected to in a single day incubation at 4 °C with main antibodies together with CD63 (SC5275, Santa Cruz Biotechnology, USA), TSG101 (ab125011, Abcam, UK), Flotilin-1 (18634 S, Cell Signaling Know-how, USA), Golgin84 (NBP1-83352, Novus Biologicals, USA), Dnmt1 (5032T, Cell Signaling Know-how, USA), Ncapg2 (24563-1-AP, Proteintech, China), Mcm3 (A1060, Abclone, China), Prg4 (PA3-118, Invitrogen, USA), or Cdk1 (ab18, Abcam, UK). Subsequent to this, the PVDF membranes had been subjected to incubation with corresponding secondary antibodies (Jackson ImmunoResearch, USA) for 1 h at room temperature. Lastly, the PVDF membranes had been imaged utilizing Western chemiluminescent horseradish peroxidase (HRP) substrate (Millipore, USA) with an imaging system (Tanon 4600, Shanghai, China).
Stream cytometric evaluation
The collected LT-EVs had been subjected to staining for ASGPR (sc-166633, Santa Cruz Biotechnology, USA), F4/80 (ab6640, Abcam, UK), CD11b (101201, BioLegend, USA), CD31 (102407, BioLegend, USA), GFAP (38014, SAB, China), CK19 (60187-1, Proteintech, China), CD3 (100235, BioLegend, USA), or CD19 (152420, BioLegend, USA) utilizing main antibodies and their respective isotype management, together with PE Mouse IgG2b,κ Isotype Ctrl Antibody (400312, BioLegend, USA), APC Mouse IgG2b,κ Isotype Ctrl Antibody (981906, BioLegend, USA), or Biotin Mouse IgG2b,κ Isotype Ctrl Antibody (401203, BioLegend, USA), at a focus of 1:100 for 1 h at 4ºC. Subsequently, fluorescence-conjugated secondary antibodies had been utilized. Following PBS washing, the odds of positively stained LT-EVs had been decided utilizing a circulate cytometer (NovoCyte; ACEA Biosciences, USA) and analyzed utilizing NovoExpress software program.
IF staining
Contemporary liver tissue samples had been fastened in 4% paraformaldehyde (PFA) (Biosharp, China) at 4 °C for 4 h, washed with PBS, and dehydrated with 30% sucrose for twenty-four h. After dehydration, the samples had been embedded in an optimum slicing temperature (OCT) compound (Leica, Germany), and 10 μm cryosections had been ready utilizing a Cryostat (CM1950, Leica, Germany). The air-dried cryosections had been permeabilized by 0.3% Triton X-100 (Sigma-Aldrich, USA) for 20 min at room temperature, following by blocking in goat serum (Boster, China) for 30 min at room temperature. The cryosections had been then incubated with the next main antibodies in a single day at a focus of 1:100 at 4 °C: rabbit anti-mouse Ki67 main antibody (ab15580, Abcam, UK), rat anti-mouse F4/80 main antibody (ab6640, Abcam, UK), mouse anti-mouse TNF-α main antibody (ab1793, Abcam, UK), rabbit anti-mouse VCAM-1 main antibody (A19131, Abclonal, China), rat anti-mouse Stabilin2 main antibody (D17-3, Medical & Organic Laboratories, Japan), mouse anti-mouse GFAP main antibody (38014, SAB, China), rabbit anti-mouse α-SMA main antibody (ab124964, Abcam, UK) in a single day at a focus of 1:100 at 4 °C. After washing with PBS, sections had been then stained with the next fluorescence-conjugated secondary antibodies at a focus of 1:200 at 4 °C for 1 h at room temperature: Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (R37119, Invitrogen, USA), Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (A-11001, Invitrogen, USA), Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (A-11005, Invitrogen, USA), Alexa Fluor 488-conjugated rooster anti-rat secondary antibody (A-21470, Invitrogen, USA). Moreover, for F-actin staining of hepatocyte borders, after washing with PBS, sections had been probed with phalloidin conjugated to Alexa Fluor 488 (R37110, Invitrogen, USA) in line with the producer’s directions, and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (ab104139, Abcam, UK). The liver tissues had been imaged by CLSM (A1plus, Nikon, Japan) and analyzed utilizing the ImageJ software program (NIH, USA).
Cultured AML12 hepatocytes had been washed with PBS for 3 times, and glued with 4% PFA for 30 min at room temperature, adopted by washing with PBS and blocking in goat serum for 30 min at room temperature. Cells had been incubated with the next main antibodies in a single day at a focus of 1:100 at 4 °C: rabbit anti-mouse Ki67 main antibody (ab15580, Abcam, UK), rat anti-mouse PHH3 main antibody (66863-1-lg, Proteintech, China. After washing with PBS, the cells had been stained with the next fluorescence-conjugated secondary antibodies at room temperature for 1.5 h: Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (R37119, Invitrogen, USA), Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (A-11001, Invitrogen, USA. Subsequently, the cells had been washed with PBS and their nuclei had been counterstained with DAPI (ab104139, Abcam, UK). Fluorescence imaging was carried out by CLSM (A1plus, Nikon, Japan) and analyzed utilizing the ImageJ software program (NIH, USA).
Histological staining
Contemporary liver tissue samples had been fastened in 4% PFA for twenty-four h after which washed with operating water to take away extra PFA. Then, the samples underwent dehydration by way of graded ethanol and had been embedded in paraffin. They had been then sectioned at 5 μm per slice. H&E staining was carried out with a business staining equipment (Baso Know-how, China). Pictures of the stained samples had been captured utilizing the SLIDEVIEW VS200 (Olympus, Japan).
Statistical evaluation
All information had been expressed as imply ± commonplace deviation (SD). Statistical comparisons between information units had been performed with an evaluation of normality and variance, adopted by a two-tailed unpaired Scholar’s t check for two-group comparisons and one-way ANOVA with Turkey’s post-hoc checks for a number of group comparisons utilizing the GraphPad Prism 9.0.0 software program. Survival charges had been analyzed utilizing the Log-rank check. A distinction was thought of statistically important when p < 0.05.