2.1 Animals
Male C57BL/6J mice, 8–12 weeks of age, were purchased from the Animal Center of Chongqing Medical University. All mice were housed in specific pathogen-free animal housing in the Animal Laboratory Center and were allowed free access to water and food in a climate-controlled environment (24°C, humidity 50–60%) with a 12-h/12-h light/dark cycle. The CLP and LPS models were used to establish sepsis-induced injuries.
The CLP model was established according to a previous study[6]. Briefly, the abdomen of anesthetized (avertin) mice was cut, and the cecum was ligated to the appropriate length and perforated with a 23G needle. The abdomen was sutured and 1 ml of physiological saline was administered via subcutaneous injection. The sham mice had abdominal incisions made with no cecum ligated or punctured. All the mice were sacrificed 24 h after CLP.
For the LPS model, LPS (10 mg/kg, Sigma) was intraperitoneally injected and 24 h later, the mice were sacrificed. The mice in the control group were administered an equivalent amount of physiological saline.
2.2 Cell isolation and culture
HSCs and macrophages were isolated as described previous studies[13, 14]. The HBSS and collagenase IV(Sigma-Aldrich) were successively infused into the liver tissues of normal mice in situ at 37°C for 30 min. The obtained cell suspension was centrifuged at 25×g three times, and the collected supernatant liquid was centrifuged at 400×g for 10 min to obtain NPCs. HSCs were isolated by centrifugation at 400×g for 20 min using a 17.6/11.5% two-step Percoll density gradient centrifugation method (Percoll, Sigma, USA). Isolated HSCs were cultured in DMEM supplemented with 10% FBS. For hepatic macrophage isolation, NPCs were centrifuged at 1800 g × 15 min at 4°C using a 50/25% two-step Percoll gradient. Hepatic macrophages aspirated from the middle layer were washed twice with cold PBS and plated onto cell culture dishes in RPMI 1640 medium containing 10% FBS for 2h, followed by DMEM containing 10% FBS. The morphological features of hepatic macrophages (Fig. 1A) and HSCs (Fig. 1C) were observed under a light microscope. The purity of hepatic macrophages was determined using flow cytometry (Fig. 1B). After LPS treatment, HSCs activity was detected using immunocytochemistry of α-SMA (Fig. 1D).
Mouse monocytic cell line (RAW264.7) were obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China), RAW264.7 monocytes were cultured in DMEM medium (Gibco), supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C and 5% CO2.
2.3 Exosome isolation and quantification
Exosomes released from HSCs were purified by sequential centrifugation as described previously[15]. Briefly, cell culture supernatant was centrifuged at 500 g for 10 min at 4°C to remove large cell fragments or debris. The supernatant was centrifuged at 12,000 g for 20 min at 4°C to eliminate small cellular debris. exosomes were pelleted by ultracentrifugation at 100,000 × g for 70 min at 4°C, washed with PBS, and collected by ultracentrifugation at 100,000 × g for 70 min. Exosomes were identified by western blotting, electron microscopy, and Nanoparticle Tracking Analysis (NTA). Exosomes concentration was measured by BCA.
Moreover, PKH67 Green Fluorescent Dye (D0031, Solarbio, China) and PKH26 Red Fluorescent Dye (D0030, Solarbio, China) were used to label exosomes. Briefly, Dye: Diluent C was configured as the working fluid in a ratio of 1:9, and the working fluid was then added to the exosomes at a ratio of 1:25. Blow The mixture with a pipette and incubated at 4°C for 20 min. The mixture was then centrifuged by ultracentrifugation to remove uncombined dye at 100,000 × g for 60 min at 4°C.
2.4 Co-culture of macrophages and HSCs
HSCs obtained from mouse livers were treated with or without LPS for 24 h and the conditioned medium was collected. Hepatic macrophages were seeded in 6-well plates at a density of 1.5×10^6 cells every well in DMEM containing 10% FBS and 100 U/ml penicillin/streptomycin. After overnight incubation, the macrophages were treated with conditioned medium for 24 h. In the other experiments, HSCs were co-cultured with macrophages in a trans-well system for 24h.
2.5 Enzyme-linked Immunosorbent Assay
The concentrations of IL-1β (QZ-10247, Quanzhou Jiubang Biotechnology Co., Ltd.) and TNF-α (QZ-10225, Quanzhou Jiubang Biotechnology Co., Ltd.) were measured using ELISA kits, according to the manufacturer’s instructions.
2.6 RNA extraction and qRT-PCR
Total RNA was extracted from the cells and exosomes using TRIzol reagent (R401, Vazyme, China). miRNA cDNA was synthesized using the miDETECT A Track miRNA qPCR Kit (RuiBo, China). The mRNA cDNA was reverse-transcribed using RT Master Mix for qPCR II (HY-K0511A, MCE). The qRT-PCR assay was subsequently performed using the SYBR Green qPCR Master Mix (HY-K0523, MCE). mRNA and miRNA expression levels were normalized to those of ACTIN and U6, respectively. Relative expression was calculated using the 2 − ΔΔCT method.
2.7 miRNAs sequencing
An miRNA microarray was used to investigate differentially expressed miRNAs between LPS-Exos and N-LPS-Exos. Briefly, total RNA was extracted and purified using a miRNeasy Mini Kit (Qiagen, Hilden, Germany). Exosomal miRNAs were subsequently analyzed using an Illumina Hiseq2000/2500 system (Illumina, USA). Raw reads were processed by trimming adaptor sequences and culling low-quality reads using ACGT101-miR (LC Sciences, Houston, Texas, USA). The remaining sequences were then compared with various RNA database sequences (excluding miRNA), such as the mRNA, RFam, and Repbase databases, filtered, and the final data obtained were used for subsequent small RNA data analysis. To comprehensively analyze the microarray data, we applied filtering criteria (≥ 2-fold change, p < 0.05).
2.8 Western blot
Cells and exosomes were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail for 30 min. Total protein was quantified using a BCA kit (Beyotime Biotechnology, China) and denatured by boiling in water. The protein suspension was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Proteins were separated and electrophoretically transferred onto polyvinylidene fluoride membranes using an electroblotting apparatus (Bio-Rad). The membranes were blocked with 5% bovine serum albumin for 60 min at room temperature, and the blots were incubated overnight with primary anti-iNOS (1:1000, Abcam, ab178945), anti-TNF-α (1:1000, Abcam, ab183218), anti-IL-1β (1:1000, Abcam, ab283818), anti-actin (1:1000, Proteintech, 81115-1-RR), anti-CD81 (1:1000, Abcam, ab79559), anti-TSG101 (1:1000, Abcam, ab125011), and anti-KLF4 (1:1000, Proteintech, 11880-1-AP) antibodies. After incubation with the secondary HRP-conjugated antibody for 1 h at room temperature, the protein signals on the membrane were detected using a gel imaging scanning system (Bio-Rad, USA) and quantified using ImageJ (National Institutes of Health, USA) using actin as a reference protein.
2.9 Luciferase assay
The wild-type or mutant 3’-UTR of KLF-4 was constructed into dual luciferase reporters using the pmirGLO vector (Hanyin Biotechnology, China), and 100 nM miR-146a-5p mimics or negative control (GenePharma, China) were co-transfected with 2 µg wild-type or mutant luciferase reporter plasmids in HEK293T cells. Relative luciferase activity was measured using a dual luciferase reporter assay kit according to the manufacturer's protocol (Promega, USA).
2.10 Immunofluorescence and Immunohistochemical staining
Liver sections were fixed with formaldehyde, permeabilized with Triton X-100, and blocked with BSA. They were then incubated with the primary antibody CD86 (ab239075, 1:100; Abcam), followed by incubation with fluorescently labeled secondary antibodies. The nuclei were stained with DAPI and the samples were observed under a fluorescence microscope.
Liver sections were stained with hematoxylin and eosin (HE). For immunohistochemical staining, liver sections were rehydrated, processed for an antigen-unmasking procedure, and then incubated with primary antibodies α-SMA antibody (Abcam, ab7817, 1:100) overnight at 4°C. Images were acquired using an optical microscope.
2.11 Biochemical measurement
Serum ALT and AST levels were measured using a Micro Alanine Aminotransferase (ALT) Assay Kit (Solarbio, BC1555) and Micro Aspartate Aminotransferase (AST) Assay Kit (Solarbio, BC1565). The lipopolysaccharide (LPS) concentration was detected using the Chromogenic LAL Endotoxin Assay Kit (Beyotime, C276S). Briefly, 10ul of N-LPS-Exo or LPS-Exo was pipetted into a centrifuge tube, then 10ul LAL liquid was added and incubated at 37°C for 9 min away from light. 10ul color developing agent were added and incubate at37°C for 6 min. Then 50ul of Buffer A, 50ul of Buffer B and 50ul of Buffer C were added to the mixture, and the mixture was thoroughly mixed. Absorbance at 545 nm was read after five minutes using an enzyme labeler.
2.12 Statistical analysis
Measurement data are presented as mean ± standard deviation (SD). Multiple samples were compared using one-way and two-way analysis of variance to determine whether there were significant differences. The comparison of each group was performed using the t-test, and statistical significance was set at p < 0.05. All data were analyzed using GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, CA, USA).