The China Meals and Drug Administration (Beijing, China) supplied particular pathogen free (SPF) SD rats. The rats had free entry to foods and drinks. All animals had been handled humanely in accordance with the necessities of the Nationwide Institutes of Well being Laboratory Animal Care and Use Tips (eighth Version). The animal experiments had been authorised by animal Ethics Committee of Nankai College (License No: 2021-SYDWLL-000331).
Preparation and identification of HO-1/BMMSCs
BMMSCs had been extracted and cultured based on our earlier strategies . Cells had been cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12(Gibco at ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Biowest, Nuaille, France) + 1% penicillin–streptomycin (Gibco). The third era of BMMSCs was chosen for Hmox1 (encoding heme oxygenase 1 (HO-1)) gene transfection, utilizing Advert/HO-1, which additionally carried the inexperienced fluorescent protein (GFP) (GeneChem, Shanghai, China). HO-1/BMMSCs had been induced to bear osteogenic and adipogenic differentiation. The HO-1/BMMSC phenotype was recognized utilizing move cytometry, with anti-cluster of differentiation (CD)29, CD90, CD34, CD45, RT1A, and RT1B antibodies (BioLegend, San Diego, CA, USA). Expression of Hmox1 was verified utilizing quantitative real-time reverse transcription PCR (qRT-PCR) and western blotting.
Exosome isolation and characterization
In response to earlier strategies , exosomes had been separated utilizing differential centrifugation. BMMSCs or HO-1/BMMSCs had been cultured in exosome-free FBS media (Sesh-biotech, Shanghai, China) for 72 h, and the supernatant was collected and centrifuged at 250×g for 15 min and 3000×g for 30 min, consecutively, to take away cell fragments. After 0.22 μm filtration and centrifugation at 110,000×g for 140 min at 4 ℃, the exosomes had been deposited on the backside of the ultracentrifuge tube. The exosomes had been re-suspended in sterile phosphate-buffered saline (PBS), and the supernatant was retained after centrifugation at 10,000×g for five min. The obtained exosomes had been quickly saved at − 80 ℃. Western blotting was used to detect exosomes markers, and antibodies towards CD9, tumor susceptibility 101 (TSG101), CD63, Calnexin (all ProteinTech, Wuhan, China), and CD81 (Abcam, Cambridge, UK) had been used. The morphology of the exosomes was noticed utilizing transmission electron microscopy (TEM, Hitachi-HT7700, Tokyo, Japan). The particle focus, dimension, and distribution had been detected utilizing nanoparticle monitoring evaluation (NTA, Malvern-NS300, Malvern, UK).
Institution of MCD mannequin in rats
SD rats aged 7–8 weeks (230 ± 20 g) had been chosen and fed with a methionine and choline poor (MCD) food plan with free entry to water. Liver tissue was collected 14 days later for hematoxylin and eosin (H&E) staining and oil pink staining.
Mannequin of LT with extreme steatotic donor liver and in vivo experimental design
The identical physician carried out all of the LTs in accordance with the formulation developed by Kamada and Calne . The steatotic livers had been transplanted into regular SD rats (230 ± 20 g). The length of the hepatic stage was (21 ± 1 min).
The animals had been divided into seven teams: Sham group (Sham), PBS group, BMMSC group, HO-1/BMMSC group, M-exo group, HM-exo group, and Fer-1 group, with six rats in every group at every time level. Within the Sham group, rats had been subjected to stomach opening and stomach closing solely. Within the PBS group, 500 μL of PBS was injected by way of the portal vein after transplantation. Within the BMMSC group, 2 × 106 BMMSCs had been injected via the portal vein after transplantation. Within the HO-1/BMMSC group, 2 × 106 HO-1/BMMSCs had been injected via the portal vein. Within the M-exo group, M-exos had been injected by way of the portal vein (2.5 × 1010 particles in 500 μL of PBS). Within the HM-exo group, HM-exos had been additionally injected by way of the portal vein (2.5 × 1010 particles in 500 μL of PBS). The Fer-1 group was injected with Ferrostatin-1 (Fer-1) (10 mg/kg/d) by way of intraperitoneal injection after surgical procedure (MCE, Shanghai, China). Rats in every group had been sacrificed on postoperative day (POD) 1, POD3, and POD7, and liver tissue and serum had been collected. An computerized biochemical analyzer was used to measure the serum ranges of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and whole bilirubin (TBIL). The ultrastructure of the liver tissue was noticed utilizing transmission electron microscopy.
In vivo biodistribution of exosomes
Exosomes had been first labeled with 2 μL of chloromethyl (CM)-1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) dye (Invitrogen, Waltham, MA, USA). The labeled exosomes had been incubated at 4 °C for 15 min at nighttime, then chilly PBS was added and the exosomes had been centrifuged at 110,000×g for 70 min at 4 °C. After washing with PBS, the labeled exosomes had been centrifuged once more for 70 min. The exosomes (2.5 × 1010 particles in 500 μL of PBS) had been injected into the rats. Liver tissue was taken for frozen sectioning on POD3 and nuclear stained utilizing 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China). The distribution of the exosomes was noticed below a fluorescence microscope.
RNA sequencing (RNA-Seq)
The PBS group and the HM-exo liver tissue had been chosen for detection (n = 3). Complete RNA of the pattern was extracted, and recognized and quantified utilizing Nano Drop expertise and an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). The mRNA library was then constructed and amplified with Phi29 to provide 100-base pair reads on the BGIseq500 platform (BGI, Shenzhen, China). SOAPnuke (V1.5.2) was used to filter the sequencing knowledge, and Bowtie2 (V2.2.5) was used to match the clear reads with the gene database established by Shenzhen Beijing Genomics Institute to calculate gene expression ranges and determine differentially expressed genes (DEGs) (Fold change > 1.5; q-value < 0.05). The annotated DEGs had been analyzed utilizing Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based mostly on Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) evaluation. Gene set enrichment evaluation (GSEA) was used to judge DEGs enriched for both negatively or positively correlated genes . The RNA-Seq knowledge had been deposited within the NCBI Sequence Learn Archive (PRJNA795289).
Steatotic hepatocytes and hepatocytes hypoxia and reoxygenation (H/R) mannequin
IAR20 cells had been cultured in minimal important medium (MEM) (Gibco) containing 10% FBS (Biowest); LO2 cells had been cultured in 1640 medium (Gibco) containing 10% FBS. Cells had been inoculated into 12-well plates and incubated with 20 μL of oleic acid and 5 μL of palm oil (Sigma-Aldrich, St. Louis, MO, USA) for twenty-four h to induce steatosis.
In response to a beforehand revealed methodology , hepatocytes had been quickly disadvantaged of oxygen by immersing them in mineral oil. Briefly, IAR20 or LO2 cells had been washed twice with PBS, soaked utterly with mineral oil (Aladdin, Shanghai, China), and positioned at 37 °C in a 5% CO2 incubator for 4 h. After repeated washing with PBS, MEM containing 10% FBS or 1640 medium containing 10% FBS had been added for additional tradition for six h.
First, steatosis in IAR20 cells was induced after which the cells had been handled with H/R or Erastin (20 μM) (MCE), Fer-1 (1 μM), and the apoptosis inhibitor Emricasan (30 μM) (MCE) for twenty-four h. In one other experiment, each regular IAR20 cells and steatotic IAR20 cells had been handled with H/R on the similar time, and cells had been collected 6 h later for detection. All experiments had been repeated 3 times.
Mobile uptake of exosomes
Exosomes had been labeled with CM-DiI as described above, after which added to IAR20 and LO2 cells for six h. The cells had been mounted utilizing 4% paraformaldehyde for 30 min. The nuclei had been stained with DAPI after which noticed below a confocal microscope (FluoView™ -fv1000, Olympus, Tokyo, Japan).
Complete RNA was extracted from M-exos and HM-exos utilizing the Trizol reagent (Takara, Dalian, China), and RNA integrity was assessed utilizing a 2100 bioanalyzer (Agilent Applied sciences). An Illumina TruSeq Small RNA package (Illumina, San Diego, CA, USA) was used to assemble the library, and a high-throughput sequencing platform was used to sequence the enriched 18–32 nt Small RNA fragments. The differentially expressed miRNAs within the two teams of exosomes had been screened (Fold change > 1.5; p-value < 0.05), and miRDB  and miRWalk  databases had been used to foretell the goal genes of the miRNAs, and KEGG enrichment evaluation was carried out for the recognized goal genes. The miRNA sequencing knowledge had been deposited within the Sequence Learn Archive (PRJNA794949).
Cell viability assay
In response to the producer’s directions, cell viability was measured utilizing a Cell Counting Package 8 (CCK-8) (Solarbio). In brief, 5 × 103 cells had been inoculated right into a 96-well dish, and 10 μL of CCK-8 resolution was added and incubated at 37 ℃ for 3 h. The absorbance at 450 nm was then measured utilizing a microplate meter.
The cells had been digested with trypsin, the unique supernatant was added to cease digestion, and the cells had been collected by gently rubbing and shaking. After centrifugation, the cells had been resuspended in 1 mL of PBS, after which transferred to a 12-well plate. Propidium iodide (PI) and fluorescein diacetate (FDA) dye (Sigma-Aldrich) had been added (5 μL every), and incubated for 1 min at nighttime, adopted by statement below a fluorescence microscope.
Lipid-ROS and ROS assay
Lipid-reactive oxygen species (ROS) ranges had been measured utilizing BODIPY-C11 dye (Invitrogen). Briefly, the cells had been inoculated right into a 12-well plate. On the second day, the tradition medium was modified to recent tradition medium containing 5 µM BODIPY-C11, and incubated at 37 ℃ for 20 min, washed with PBS twice, after which used for evaluation by move cytometry. ROS detection was carried out utilizing a Reactive oxygen species assay package (Beyotime Biotechnology, Shanghai, China). Cells in 12-well plates had been digested with trypsin with out EDTA (Solarbio), after which the cells had been suspended in clean tradition medium containing 10 μmol/L Dichloro-dihydro-fluorescein diacetate (DCFH-DA). The cells had been incubated at 37 ℃ for 30 min, washed with PBS twice, and analyzed utilizing move cytometry.
Malondialdehyde (MDA) assay
In response to the producer’s directions, a lipid peroxidation assay package (Beyotime Biotechnology) was used to detect the MDA focus in cells and liver tissues. A microplate reader was used to measure the absorbance at 532 nm.
In response to the producer’s directions, intracellular and liver tissue Fe2+ ranges had been measured utilizing an iron assay package (Abcam). Samples had been homogenized in an iron buffer, and the supernatant was retained and incubated with an iron decreasing agent and an iron probe. The absorbance of the samples at 532 nm was measured utilizing a microplate reader.
Labile iron pool (LIP) assay
In response to a beforehand revealed methodology , the LIP was detected utilizing Calcein-acetoxymethyl (AM; Beyotime Biotechnology). The cells had been incubated with 2 μM Calcein-AM at 37 °C for 8 min, the cells had been then suspended in Hank’s equilibrium salt resolution, the focus was adjusted to 1 × 106, and the samples had been incubated with Trypan blue. The fluorescence depth was measured at 488 nm for excitation and 517 nm for emission, after which 100 μM 2, 2 bipyridine (Sigma-Aldrich) was added and incubated for 10 min. The fluorescence modifications in every pattern had been calculated after the addition of two, 2 bipyridine.
Transfection of miRNA mimics and inhibitors, siRNA and plasmids
miRNA mimics, inhibitors, and small interfering RNAs (siRNA) had been designed and synthesized by Genepharma (Jiangsu, China). The detailed sequences are proven in Further file 2: Desk S1. Lipofectamine 3000 (Invitrogen) was used to transfect the cells. Briefly, the cells had been inoculated into 6-well plates, and when the diploma of fusion was 60–70%, the cells had been transfected with 10 μL of detrimental management (NC)-mimic and miR-124-3p-mimic; 5 μL of NC-inhibitor and miR-124-3p-inhibitor; and 5 μg of Steap3-siRNA (an siRNA concentrating on the mRNA encoding prostate six transmembrane epithelial antigen 3 (STEAP3), respectively. A plasmid expressing the Steap3 gene was constructed (GeneChem) and a pair of μg of this plasmid was transfected into IAR20 cells utilizing Lipofectamine 3000 to overexpress Steap3.
Twin-luciferase reporter assay
The three’ UTR sequence of Steap3 was inserted into the pmirGLO vector because the wild-type (WT), and the anticipated binding goal of miR-124-3p within the Steap3 3′-UTR was mutated within the pmirGLO vector because the mutant (MT) (Genepharma). HEK-293T cells had been co-transfected with luciferase reporter plasmid and NC-mimic or miR-124-3p-mimic. At 48 h after transfection, the firefly luciferase exercise was decided and normalized by the Renilla luciferase exercise.
Immunohistochemistry and immunofluorescence staining
In response to a earlier revealed methodology , immunohistochemical staining was carried out to detect glutathione peroxidase 4 (GPX4) (ProteinTech), 4-hydroxynonenal (4-HNE) (Abcam), STEAP3 (Abcam), and Myeloperoxidase (MPO) (ProteinTech) expression in liver tissue. Immunofluorescence staining of hepatocytes had been additionally carried out in accordance with beforehand revealed strategies . Briefly, IAR20 or LO2 cells had been inoculated into 12-well plates, handled based on the designated teams, and stained with main and secondary antibodies based on the producer’s directions. DAPI was added to label the nuclei and the cells had been noticed below a fluorescence microscope.
Complete RNA was extracted from exosomes, cells, and tissues utilizing the Trizol reagent (Takara). TB Inexperienced® Premix Ex Taq™ and PrimeScript™ RT reagent Package with gDNA Eraser (Takara) had been used for reverse transcription and amplification. The mRNA and miRNA ranges had been normalized to the expression of Actb (encoding β-actin) or U6 and calculated by way of the usual 2 − ΔΔCt methodology . The sequences of the primers used on this examine are proven in Further file 2: Tables S2 and S3. All primers had been synthesized by Sangon Biotech (Shanghai, China).
Radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology) containing protease inhibitors was used to extract proteins from tissues and cells. Western blotting was carried out based on our earlier method , utilizing antibodies concentrating on HO-1, GPX4 (each ProteinTech), STEAP3 (Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Expertise, Danvers, MA, USA), and divalent steel transporter 1 (DMT1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Picture J 7.0 software program (NIH, Bethesda, MD, USA) was used to investigate the picture grayscale values and calculate relative protein expression.
The info had been analyzed utilizing GraphPad Prism 8.0 (GraphPad Software program, Inc., La Jolla, CA, USA). All experiments had been carried out independently at the very least 3 times. Knowledge are expressed because the imply ± commonplace deviation. The imply values of two teams had been in contrast utilizing an unpaired Scholar’s t-test. Comparability amongst teams was carried out utilizing one-way evaluation of variance (ANOVA). P < 0.05 was thought-about statistically important.