Cell culture

Human hepatic stellate cell line LX2 (#CC4023) was purchased from Saiku Culture Bank (Guangzhou, China) and cultured in H-DMEM (Cytiva, USA, AI30093259) with 10% FBS (ExCell, USA, FND50). Human hepatic cell line L02 (#CL0192) was purchased from Fenghui Biology Co., Ltd (Changsha, China) and cultured in RPMI 1640 (Gibco, USA, C11875500) containing 10% FBS. All cells were grown in the incubator at 37 °C with saturated humidity and 5% CO2.

Isolation and cultures of primary mouse hepatocyte

Primary mouse hepatocytes were isolated from the liver of 6-week-old C57BL/6 male mice. For hepatocyte isolation, primary mouse hepatocytes were isolated using a standard two-step collagenase perfusion method [16]. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg/kg). A catheter was then inserted into the exposed inferior vena cava and the portal vein drainage was incised. The liver was perfused with Hank’s Balanced Salt Solution (HBSS)-EGTA solution (BOSTER, Wuhan, China, PYG0079) for 10 min, followed by infusion with 100 U/mL collagenase IV (Biofroxx, Einhausen, Germany, 2275GR001). After 15 min of perfusion, the livers were collected and minced to release hepatocytes. The resulting cell suspension was filtered through a sterile 100 μm cell strainer Falcon (Corning, USA, 352,360) and centrifuged at 150 ×g for 5 min. These cells were cultured in H-DMEM medium (Cytiva, USA, AI30093259) containing 10% FBS (ExCell, USA, FND50), supplemented with 1 × penicillin–streptomycin solution (Biosharp, China, BL505A) at 37 °C in a humidified incubator containing 5% CO2.

High-fat Diet (HFD) induced mice model

Male C57BL/6 mice were maintained in a pathogen-free environment with a temperature of 21–23 °C, relative humidity of 50–60%, and a 12 h light and dark cycle. The mice model of HFD-induced NAFLD was generated as described in previous study [17]. The mice were randomly assigned to four groups (6 mice per group). The groups were as follows: the NCD group, which was fed a normal chow diet; the HFD 4w group, which was fed a high-fat diet for 4 weeks; the HFD 8w group, which was fed a high-fat diet for 8 weeks; and the HFD 12w group, which was fed a high-fat diet for 12 weeks. The normal chow feed, which contained 10% kcal from fat, and the HFD feed, which contained 45% kcal from fat, were purchased from Medicience (Jiangsu, China).

Fatty acid treatment

Combination of palmitic acid (Sigma, USA, P0500) and oleic acid (Sigma, USA, O7501) was performed to induce lipotoxic hepatocyte according to the standard protocol described previously [18]. Briefly, after reached 80% confluence, 660 μM oleic acid conjugated with BSA and 330 μM palmitic acid conjugated with BSA was added to the cultured medium for treating L02 cells for 8 h, 16 h, 24 h to develop lipotoxic L02 cells.

Immunohistochemistry

Following dewaxing and rehydration of the paraffin sections, antigen retrieval was performed using citric acid buffer (10 mM citric acid, 0.05% Tween 20, pH = 6.0). The endogenous peroxidase activity was inactivated by treating with 3% H2O2 for 30 min. To prevent non-specific binding of antibodies, a blocking solution of 5% BSA was applied to the slides. The liver tissue sections were then incubated overnight at 4 °C with primary antibodies against LIMA1 (1:100, Proteintech) and α-SMA (1:100, Proteintech). Subsequently, the sections were incubated with biotin-conjugated anti-rabbit IgG and streptavidin–biotin. Finally, the liver tissue sections were visualized using the DAB Horseradish Peroxidase Color Development Kit (BOSTER, Wuhan, China, 18E16B22) and counterstained with hematoxylin. Random fields were captured using a pathological section scanner (InteMedic, Guangzhou, China).

Western blot

RIPA buffer (Beyotime, Shanghai, China, P0013K) containing 1% protease inhibitor PMSF (Biosharp, Anhui, China, BL507A-S) was used to lyse samples from different groups at 4℃. After centrifugation, the supernatants were collected. The lysates were brought to a boil while adding loading buffer (0.25 M Tris–HCl pH 6.8, 1 M sucrose, 5 mM EDTA, 0.1% bromophenol blue, 10% SDS, and 5% β-mercaptoethanol). Protein (30 μg) was added in equal proportions to 8–12% Bis–Tris gels and then transferred to polyvinylidene difluoride membranes. After transfer, the membranes were treated with blocking buffer (PBS, 0.1% Tween20, and 5% BSA) for 2 h. The primary and secondary antibodies used in this study are listed in Additional file 2: Table S1. Protein bands were visualized using an enhanced chemiluminescence (ECL) solution (Beyotime, Shanghai, China, P0018M) according to the manufacturer’s instructions and quantified using Image J software.

Quantitative RT-PCR (qRT-PCR)

The total RNA of LX2 cells was extracted using Trizol (GIBCO, USA, 15,596–026) following the manufacturer’s instructions. For the reverse transcription of RNA into cDNA, 1 µg of total RNA was used in a reaction with the SuperScript Reverse Transcriptase Kit (Vazyme, Nanjing, China, Q511-02) according to the manufacturer’s instructions. qRT-PCR assays were performed using SYBR Green (CWBIO, Beijing, China, CW0659). The sequences of the qPCR primers used in this study can be found in Additional file 2: Table S2. The fluorescence signals were detected using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). All measurements were normalized using β-actin expression as an internal standard, and relative expression was calculated using the 2−ΔΔCt method.

Oil Red O staining

To detect lipid droplets, cells were stained with Oil Red O (Sigma, USA, O0625). A 0.5% Oil Red O Stock Solution (in isopropanol) was used. The staining solution consisted of 6 parts of stock Oil Red O and 4 parts of distilled water, which was filtered with filter paper before use. After fixing the cells in PFA, they were stained with 1 ml of the staining solution for 20 min. The background was then cleared using 60% isopropanol. Nuclei were stained with hematoxylin and the samples were sealed for observation.

Nile Red staining

The Nile red staining assay utilized a stock solution consisting of 1 mg of Nile red (Sigma, USA, 72,485) dissolved in 1 ml of 100% acetone. The OPA treated L02 cells were then stained with a 1 ml working solution of fluorescence Nile red (diluted at 1:5000) for 30 min at room temperature. The stained cells were subsequently analyzed using a fluorescence microscope (Olympus, Tokyo, Japan).

FDA fluorescence

The cells were removed from the culture medium and washed three times with PBS. The cells were then treated with 10 μg/mL FDA (BOSTER, Wuhan, China, AR1186), placed in a dark place at room temperature for 5 min for staining, and imaged by fluorescence microscope (Olympus, Tokyo, Japan).

Lentiviral knockdown of LIMA1 in L02 cells

L02 cells were infected with a Lentiviral vector (pLKO.2-U6-L02-hPGK-copGFP-Puro-shLIMA1) containing LIMA1 shRNA sequence (sequence: GTC TCT GAA TTG GTC GAG TTT) or a negative control vector (pLKO.1-Puro-shRNA) purchased from Vigen Biotech (Zhenjiang, China). The infection was done at a multiplicity of infection (MOI) of 20% and 50% confluency. The expression of green fluorescent protein (GFP) was observed using fluorescence microscopy. To remove untransfected cells, puromycin was added at a final concentration of 1 µg/ml within 96 h after infection.

Isolation and identification of sEV

The sEV were isolated from cell culture medium by differential centrifugation. Briefly, L02 cells and LIMA1 stable knockdown L02 cells were cultured in 100 mm dishes, treated with 660 μM oleic acid and 330 μM palmitic acid, and then replaced with FBS-free medium to continue culturing for 24 h. The conditioned medium was collected and centrifuged at 300g for 10 min, at 2000g for 10 min, and at 10,000g for 30 min to remove cells and cell debris. Supernatants were then concentrated using a 100 kDa molecular weight cut-off (MWCO) ultrafiltration filter according to the manufacturer’s instructions (Millipore, USA). The concentrated supernatant was ultracentrifuged at 100,000g for 70 min (Optima L-90 K; Beckman Coulter, Brea, CA, USA). The sEV-enriched pellet was collected from the bottom of the tube and resuspended in PBS, and the final sEV were passed through a 0.22 μm filter and stored at -80 °C. The obtained sEV were named LTH-sEV and LTH-sEVshLIMA1 respectively. The supernatant of normally cultured L02 cells was collected and the extracted sEV were named L02-sEV. In addition, the supernatant of OPA-treated primary mouse hepatocytes was collected, and the extracted sEV were named pLTH-sEV. The protein concentration of the extracted sEV was quantified by a BCA protein assay kit (Pierce, ThermoFisher). The morphology of sEV was observed by transmission electron microscopy (TEM). The amount and size distribution of sEV was measured by NanoSight tracking analysis (NTA) with NTA 3.1 Software (NanoSight, Malvern, UK). Expression of sEV markers TSG101, CD63, and CD9, and endoplasmic reticulum protein Calnexin was analyzed by western blot.

Co-culture of sEV with LX2

LX2 were cultured in 6-well plates until it reached approximately 50–60% confluence. LTH-sEV were then co-cultured with LX2 at different concentrations and divided into 4 groups: control group, LTH-sEV 50 μg/ml group, LTH-sEV 100 μg/ml group and LTH-sEV 200 μg/ml group. Similarly, L02-sEV were co-cultured with LX2 at different concentrations and divided into 4 groups: control group, L02-sEV 50 μg/ml group, L02-sEV 100 μg/ml group and L02-sEV 200 μg/ml group. These cells were treated for 48 h for further investigation. In addition, LX2 was co-cultured with pLTH-sEV (200 μg/ml) for 48 h. To verify the role of mitochondrial autophagy in LTH-sEV-treated LX2, the ctrl group, the LTH-sEV group, and the LTH-sEV (200 μg/ml) + Urolithin A (50 μM) group were set up.

Immunofluorescence

The cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization in PBS containing 0.1% Triton X-100 (Beyotime, Shanghai, China, P0096) for 10 min. After being washed twice with PBS, the cells were incubated with a 5% BSA solution for 30 min to block nonspecific binding sites. Liver tissue sections and cultured cells were then incubated with specific primary antibodies. Subsequently, the slides were incubated with the secondary antibody for 30 min at room temperature and in the dark. The primary and secondary antibodies used for immunofluorescence are provided in Additional file 2: Table S1. The nuclei were stained with Hoechst 33,342, and fluorescence images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan) in a random field of view.

Cell counting kit-8 (CCK-8) assay

Cell proliferation was assessed using the CCK-8 assay (Beyotime, Shanghai, China, BS350A) following the standard procedure recommended by the reagent manufacturer. LX2 were seeded onto 96-well plates at a density of 2 × 103 cells per well and subjected to different treatments. After incubation at 37 °C for approximately 2 h, 10 μl CCK-8 solution was added to each well. The absorbance values at 450 nm were measured using a multi-function microplate reader (ThermoScientific, Waltham, MA, USA).

sEV internalization assay

PKH26 dye (Solarbio, Beijing, China, D0030) was used to label sEV according to the manufacturer’s instructions. LX2 were incubated with PKH26-labelled sEV for 6 h, and then fixed with 4% paraformaldehyde. The actin cytoskeleton was visualized using phalloidin (Solarbio, Beijing, China, CA1610), and nuclei were detected using Hoechst 33,342 dye. Fluorescence microscopy (Olympus, Tokyo, Japan) was employed to evaluate the internalization of sEV.

Cell transfection of plasmid and siRNA

For overexpression of LIMA1, the LIMA1 expression vector (LIMA1-pcDNA3.1-3xFlag-C) and control vector were purchased from Fenghui Biology Co., Ltd. (Changsha, China) and transfected into LX2. For LIMA1-knockdown cell lines, control scrambled siRNA and Homo-LIMA1 siRNA (siLIMA1) sequences (Additional file 2: Table S3) were purchased from GenePharma Co., Ltd. (Shanghai, China). Cells were transiently transfected by use of Lipofectamine 2000 reagent (Invitrogen, USA, 11,668,500) according to the manufacturer’s instructions. At 48 h after transfection, cells were harvested and used for experiments.

Construction of mt-Keima-COX8 transfected LX2

For the detection of mitophagy, mt-Keima-COX8 lentivirus titration solution samples were purchased from Vigen Biotech (Zhenjiang, China). The cell fusion rate was about 70% when the virus was infected. Lentivirus stocks were carefully diluted in FBS-free culture medium. Add the calculated virus solution to LX2, mix well and place in a carbon dioxide incubator (37 °C, 5% CO2). Selecting infected cells with puromycin at an appropriate concentration after 48 h.

Transmission electronic microscopy

The cells were processed and collected following the provided instructions, and subsequently fixed in 2.5% glutaraldehyde at 4 °C for an overnight duration. Following this, the cells underwent three washes with PBS and were then fixed in 2% osmium tetroxide for a period of 1.5 h at room temperature. The cells were then subjected to dehydration through a series of ethanol concentrations, followed by embedding and fixation, and ultimately stained with uranyl acetate/lead citrate. Finally, the sections were examined using a transmission electron microscope.

ATP assay

To determine the ATP content, an ATP colorimetric assay kit (Beyotime, Shanghai, China, S0027) was used following the manufacturer’s instructions. LX2 were lysed with lysate after being rinsed with PBS, and then centrifuged at 12,000g for 5 min. All reactions were carried out in 96-well plates by mixing 20 μl of the lysis supernatant with 100 μl of the ATP assay working solution. The Luminance (RLU) was measured immediately using a fluorescent chemical analyzer (ThermoScientific, Waltham, MA, USA).

Measurement of mitochondrial membrane potential (ΔΨm)

Mitochondrial membrane potential (ΔΨm) was assessed using the JC-1 fluorescence probe (Beyotime, Shanghai, China, C2003S). The JC-1 probe was diluted in fresh medium at a ratio of 1:1000 and incubated with cells in the dark at 37 °C for 20 min. After fixation with 4% paraformaldehyde, LX2 were stained with Hoechst 33,342. Fluorescence images were captured using a fluorescence microscope (Olympus, Tokyo, Japan) in a randomly selected field of view.

Mitochondrial autophagy promoters and mitochondrial autophagy inhibitors

To investigate the role of mitophagy in HSCs activation, we conducted experiments using mitophagy inhibitors Liensinine (Solarbio, Beijing, China, IL0640, 50 μM) and mitophagy promoters Urolithin A (Solarbio, Beijing, China, IU0200, 50 μM) in co-cultivation with LX2 for 48 h. Subsequently, protein and RNA were collected for further analysis.

Protein–protein docking

The X-ray crystal structures of LIMA1 were retrieved from the Protein Data Bank. The predicted structures of PINK1 were generated by Alphafold. To ensure the accuracy of the docking results, the protein was prepared by the AutoDockTools-1.5.7, and the water molecules were manually eliminated from the protein and the polar hydrogen was added. Docking Web Server (GRAMM) was used for protein–protein docking. The resulting protein–protein complex was also manually optimized by removing water and adding polar hydrogen by the AutoDockTools-1.5.7. Finally, the protein–protein interactions were predicted and the protein–protein interaction figure was generated by PyMOL (https://pymol.org).

Co-immunoprecipitation (Co-IP))

Samples for Co-IP were suspended in lysis buffer (50 mM Tris–HCl, pH 7.4, 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 10% glycerophosphate) at 4 °C for 30 min. Cell lysates were incubated with the anti-LIMA1 antibodies for 2 h. Then protein A/G agarose beads (Beyotime, Shanghai, China, P2197S) were added and the mixtures allowed incubating at 4 °C with gentle rocking overnight. Beads were washed with an appropriate amount of lysis buffer and boiled with 2 × SDS loading buffer (0.25 M Tris–HCl pH 6.8, 1 M sucrose, 5 mM EDTA, 0.1% bromophenol blue, 10% SDS, and 5% β-mercaptoethanol) before analysis by Western blot.

Cycloheximide chase assay

The half-life of PINK1 protein in LX2 was determined using a cycloheximide (CHX)-chase assay. This assay allows for the examination of protein degradation over a specific time period. LX2 were seeded in six-well cell culture plates and transfected with LIMA1 overexpression plasmid and LIMA1 small interfering RNA. After 48 h of transfection, the cells were treated with 20 μg/ml CHX (Sigma-Aldrich, Shanghai, China, C104450) for different time-points. Subsequently, the cells were collected and analyzed using Western blot.

sEV injection in HFD mice

For the sEV-injected mice model, mice were randomly divided into four groups (6 mice per group): NCD group, mice fed with normal chow diet for 14 weeks; HFD group, mice fed with high-fat diet for 14 weeks; HFD + LTH-sEVshCtr group, HFD mice injected with LTH-sEVshCtr; HFD + LTH-sEVshLIMA1 group, HFD mice injected with LTH-sEVshLIMA1. LTH-sEVshCtr (10 mg/kg) and LTH-sEVshLIMA1 (10 mg/kg) were injected once a week for four consecutive weeks through the tail vein in HFD-fed mice starting at 10 weeks. All mice were euthanized to collect blood and liver samples for further analysis.

Masson staining and Sirius Red staining

The collected specimens were fixed in a 10% formaldehyde solution for 24 h. Sections of 4 μm thickness were prepared after paraffin embedding. To examine histopathological changes in the liver, sections from each group were deparaffinized with xylene, rehydrated with varying concentrations of ethanol, and then stained with the Masson trichromatic staining kit (Solarbio, Beijing, China, G1340) and Sirius red staining kit (Solarbio, Beijing, China, G1473) following the manufacturer's instructions. The samples were observed under a pathological section scanner (InteMedic, Guangzhou, China).

Statistical analysis

Results were presented as the mean ± SD from at least three independent experiments. Normality of data was tested with the Shapiro–Wilk test. Statistical analysis was conducted using GraphPad Prism 8.0 software (GraphPad, San Diego, CA) with Student’s t-test for comparisons between two groups or One-way analysis of variance (ANOVA) with Tukey’s post hoc test for comparisons among multiple groups. P value ≤ 0.05 was considered statistically significant.