Extracellular vesicles derived from live or apoptotic mesenchymal stem cells: comparison of the effects of two extracellular vesicles on liver fibrosis

Abstract

Background

Mesenchymal stem cells (MSCs) have become a promising treatment of liver fibrosis which is a key process in liver diseases. Recent studies have shown that transplanted MSCs undergo rapid apoptosis and the apoptotic extracellular vesicles (ApoEVs) derived from MSCs exhibited stronger immunosuppressive capability. However, the effect and the mechanisms of ApoEVs in liver fibrosis remain unclear. The functional differences between ApoEVs and extracellular vesicles (EVs) have yet to be elucidated. This study aims to compare their therapeutic effects on liver fibrosis in order to optimize existing treatment strategies.

Methods

ApoEVs and EVs were isolated by density gradient centrifugation and illustrated by TEM and NTA. A CCl4-induced liver fibrosis mouse model was treated with equal doses of ApoEVs and EVs. Histopathological analysis was performed on liver sections, serological indicators, fibrosis-related gene expression, macrophage polarization, and the activation status of hepatic stellate cells (HSCs) were analyzed. Subsequently, miRNA-sequencing and untargeted metabolomics analysis were conducted to identify potential pathway.

Results

Our results demonstrated that ApoEVs had fourfold higher protein yield than EVs, and ApoEVs exhibited a significant superior ability to improve liver fibrosis. In vitro, ApoEVs enhanced macrophage polarization and suppressed HSC activation more effectively, thereby reducing the degree of fibrosis. The underlying molecular mechanism likely due to the enrichment of more miRNAs targeting the PI3K-AKT pathway in ApoEVs and more metabolite molecules that mediate inflammatory metabolic processes.

Conclusion

These findings showed that ApoEVs exhibit better effects than EVs in alleviating liver fibrosis. Besides, the findings highlighted their therapeutic potential which suggested that ApoEVs could be a promising approach for the treatment of liver diseases and further lay a research foundation for clarifying the therapeutic mechanism of MSCs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12876-026-04762-0.

Background

Liver fibrosis or cirrhosis may result from the progression of chronic liver diseases caused by various etiologies, which is facing a significant health challenge all over the world [1]. Without effective intervention in time, the morbidity and mortality will be increased significantly because of symptoms such as portal hypertension and gastrointestinal bleeding. In China, about 300 million people suffer from these diseases. In the world, liver cirrhosis is the 11th most common cause of death and together with liver carcinoma, accounts for 3.5% of all deaths [2]. Orthotopic liver transplantation is the only curative treatment for end-stage liver diseases. But its widespread application has been limited by issues such as donor shortage and poor prognosis [3]. Therefore, many regenerative or alternative therapies are studied aiming to treat liver fibrosis or cirrhosis.

In recent years, stem cell-based therapies have provided new possibilities for the treatment of liver diseases. In chronic liver diseases, mesenchymal stem cells (MSCs) play a crucial role in regulating the immune microenvironment through direct differentiation or regeneration [4]. MSCs, sourced from bone marrow, adipose tissue, or umbilical cord, offer clinical advantages such as easy to clinical translation, low immunogenicity, and strong proliferative capacity et al. Currently, numerous clinical trials have been conducted in the field of MSC therapy for liver cirrhosis to demonstrate its effectiveness [5]. However, the exact mechanism of action is still unclear. Previous studies have indicated that MSCs can affect the inflammatory phenotype of macrophages through its paracrine function, or alleviates fibrosis by degrading extracellular matrix (ECM) or reducing the activation level of hepatic stellate cells (HSCs) [6–8]. Meanwhile, extracellular vesicles (EVs) especially exosomes, as mediators of intercellular communication, also play a significant role in the therapeutic effects of MSCs [9]. Exosomes can alleviate liver fibrosis by influencing the differentiation phenotype of immune cells or delivering microRNAs (miRNAs) to mediate YAP down-regulation [10, 11].

The rapid apoptosis of MSCs following infusion has been considered a major factor limiting their sustained therapeutic effects [12]. Therefore, many studies aimed at enhancing the homing ability of MSCs, improving their survival, and ensuring their retention in vivo to improving their therapeutic efficacy. However, recent studies have shown that the immunosuppressive capacity of MSCs is attenuated when their apoptosis is resistant [13]. In other words, MSCs apoptosis is a crucial component of their therapeutic function on tissue repair and inflammation regulation [14, 15].

Apoptosis is an important form of programmed cell death. Apoptotic extracellular vesicles (ApoEVs) are formed during the process of apoptosis, through budding and fragmentation of the cell membrane. ApoEVs are a unique subtype of EVs, with diameters ranging from 50 to 5000 nm [16]. They participate in intercellular communication, signaling, and immune regulation, which are similar to EVs produced by non-apoptotic MSCs. ApoEVs contain numerous bioactive molecules including proteins (such as CD81, CD9, etc.), lipids, RNA (mRNA, miRNA, etc.). After interacting with the recipient cells, they play indispensable roles in a variety of physiological and pathological environments [16, 17]. Current research indicates that ApoEVs can play functional roles in a variety of diseases. In the event of inflammatory injury, ApoEVs modulate the population of immune cells and macrophage inflammatory phenotypes in various diseases, including acute kidney injury, colitis, allergic pulmonary inflammation, and skin wound healing. In addition, they can be engulfed by endothelial cells or basal cells for tissue regeneration. Moreover, ApoEVs can interact directly with recipient cells, regulating activity through membrane surface ligands et al. [18–20]. However, no studies have yet investigated ApoEVs in liver diseases, particularly in liver fibrosis.

In terms of liver fibrosis treatment, previous research has mainly focused on the regulation of macrophage inflammatory phenotypes by EVs, particularly exosomes. Or focusing on the inhibition of HSCs activation, the degradation of ECM. However, the precise function of ApoEVs in liver fibrosis has not yet been explored. At the same time, there has been no comparative study on the effects of ApoEVs and EVs on liver fibrosis. Therefore, this study aims to dissect the differences between these two types of vesicles and explore their respective therapeutic efficacy.

In this study, we comparatively analyzed the biological roles of ApoEVs and EVs secreted by MSCs before and after apoptosis. We evaluated the therapeutic efficacy of both types of vesicles in improving fibrosis levels in a carbon tetrachloride (CCl4)-induced mouse model. Additionally, we compared their effects on macrophages and HSCs to demonstrate potential differences in functional properties. This study aims to identify more effective therapeutic approaches for liver fibrosis.

Methods

Cell culture

Human umbilical cord-derived MSCs (hUC-MSCs) were provided by the National Engineering Research Center (Tianjin AmCellGene Engineering Co., Ltd, China). MSCs were cultured in Mesenchymal Basal Medium (DAKEWE Biotech Co., Ltd. China) containing 5% EliteGro-Adv (Elitecell, DAKEWE) mixed with 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. Cells in generation 4–6 were used for subsequent experiments.

RAW264.7 cells, obtained from the American Type Culture Collection (ATCC), which were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, NY) and 1% penicillin–streptomycin.

Mouse bone morrow-derived macrophages (BMDMs) were obtained as previously described [21]. BMDMs were isolated from the femurs and tibias of 6-8-week-old C57BL/6J mice. Subsequently, these cells were plated at a density of 2 × 10⁶ cells/mL and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, plus 40 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech Inc. USA) for 7 days.

Human HSC lines LX-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, NY) and 1% penicillin–streptomycin.

Induction of MSCs apoptosis

HUC-MSCs in generation 4–6 were selected and cultured in MSCBM supplemented with 5% EliteGro-Adv and 1% penicillin–streptomycin until they reached 80–90% confluence. Then removing the medium and MSCs were washed three times with PBS. Subsequently, the complete medium containing 250 nM staurosporine (STS) (Cell Signaling Technology, USA) was added to MSCs [22]. As a control, MSCs that had not undergone apoptosis treatment were replaced with fresh complete medium at the same time points and cultured for the same period. After 12 h of treatment, the apoptosis of MSCs was detected by morphological observation and flow cytometry analysis.

Flow cytometric analysis of cell phenotypes

Apoptotic MSCs were detected using an apoptosis staining kit (BD Biosciences, USA). After discarding the supernatant, the cells were washed three times with PBS and resuspended in the buffer provided by the apoptosis staining kit at a density of 1 × 10⁷/mL. PE-Annexin V and 7-AAD were added according to the recommended ratio, followed by incubation at room temperature in the dark for 15 min. Then, the flow cytometric data was analyzed on a FACSVerse flow cytometer (BD Bioscience, CA, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, US).

Isolation and characterization of ApoEVs and EVs

According to the previously optimized protocol, ApoEVs were collected through differential centrifugation [19, 23, 24]. Briefly, collected the culture supernatant from apoptotic MSCs at the 12-hour mark of MSCs apoptosis induction. Then, centrifuged the supernatant at 800 g for 10 min at 4 ℃ to to remove some cell debris and intact cells. Following, the supernatant was further collected and centrifuged at 16,000 g for 30 min at 4℃, discarded the supernatant, and retained ApoEVs pellet. Subsequently, resuspended the pellet gently in pre-cooled PBS, centrifuged at 16,000 g for 30 min at 4 ℃. Washed twice in this method to obtain final ApoEVs. ApoEVs were quantified by measuring the protein concentration via a BCA Protein Assay Kit (GlpBio, USA).

EVs derived from MSCs were isolated by differential centrifugation as previously described [25]. Briefly, MSCs (hUC-MSCs) in generation 4–6 were cultured in complete medium (EV-depleted) until they reached 70–80% confluence and then collected the culture supernatant and centrifuged at 300 g for 10 min to remove some cell debris and intact cells. Further collected the supernatant and centrifuged at 2000 g for 20 min to remove larger cell debris and apoptotic bodies. Then, centrifuged the supernatant at 10,000 g for 30 min at 4 °C to remove micro-vesicles and other larger particles. Finally, the supernatant was ultracentrifuged at 100,000 g for 70 min at 4 °C, and washed the pellet with PBS at 100,000 g for 70 min to obtain EVs. EVs were quantified by measuring the protein concentration via a BCA Protein Assay Kit (GlpBio, USA).

The morphology of ApoEVs and EVs were characterized using transmission electron microscopy (TEM). Nanoparticle tracking analysis (NTA) was used to analyze the particle size distribution and concentration of both ApoEVs and EVs. For apoptotic marker detection, purified ApoEVs were characterized by Western blot using anti-Caspase-3 and anti-β-Actin antibodies. For specific EVs markers, CD9, CD81 were used as positive controls, and calnexin, GM130 were used as negative controls for Western blot analysis.

Cell treatment

For the phenotypic induction of macrophages, the M1 (pro-inflammatory) phenotypes were established by stimulating BMDMs or RAW 264.7 cells with 100 ng/mL LPS (Sigma, USA)/20 ng/mL IFN-γ (Peprotech, USA), which are represented by “B1” in the figure.

For the phenotypic induction of HSCs, LX-2 cells were cultured in a medium containing 10% FBS (Gibco, NY) and 1% penicillin–streptomycin to a fusion degree of 60–70%, then switched to a medium containing 2% FBS concentration, and 8 ng/mL TGF-β1 (novoprotein, China) was added for 48 h to establish an activated phenotype. After 48 h, we added 20 µg/mL of ApoEVs or EVs respectively, then 12 h more hours the cells were collected for subsequent analyses such as Western blot or qRT-PCR.

To investigate the effects of ApoEVs and EVs on the inflammatory phenotype of macrophages and the activation of HSCs, 20 µg/mL of ApoEVs or EVs were added to the culture medium of BMDMs, RAW 264.7 cells or LX-2 cells, respectively. After 12 h of treatment, the cells were collected for subsequent analyses such as Western blot or qRT-PCR.

Transfection

Cell transfection was performed using transfection reagent CALNP™ RNAi in vitro (D-Nano Therapeutics, China), according to the manufacturer’s protocol. The inhibitor and NC inhibitor of let-7f-5p, miR-20a-5p, and miR-411-5p were obtained from Sangon Biotech (Shanghai, China), and their sequences were shown in Table S2.

Experimental animal models

The male C57BL/6J mice (aged 6–8 weeks) were purchased from and housed in the Laboratory Animal Center of the Fourth Military Medical University. The mice were housed in a specific pathogen-free (SPF) conditions under controlled temperature (23 ± 1℃) and at 65–70% relative humidity, with 12-hour light/dark cycles, and food and water were provided ad libitum. The animal study protocols were approved by the Animal Welfare and Ethics Committee of the Fourth Military Medical University and performed according to the “Guidelines for the Care and Use of Laboratory Animals”.

The liver fibrosis mouse model was established by continuous intraperitoneal injection of 0.2 mL/20 g CCl4 [20% (v/v), dissolved in olive oil] twice a week for 8 weeks. Subsequently, the liver fibrosis mice were randomly divided into groups (n = 4–6) that received 200 µl phosphate buffered saline (PBS), 150 µg ApoEVs diluted in 200 µl PBS and 150 µg EVs diluted in 200 µl PBS via tail vein injection. The normal control group consisted of wild-type mice from the same batch that were raised under the same conditions. Alternatively, in another group, the mice received 200 µl PBS, 10¹⁰ particles ApoEVs suspended in 200 µl PBS and 10¹⁰ particles EVs suspended in 200 µl PBS via tail vein injection. After treatment, the mice were observed for an additional two weeks and four weeks and were continually injected with CCl4 once a week. Two and four weeks later, liver tissues and serum samples were collected for subsequent experiments. In this experiment, blinding was implemented for both the animal caretakers and the data collectors and statisticians. Until the final unblinding stage, all participants were unaware of the group information.

The mice metabolic dysfunction-associated steatohepatitis (MASH) model was established by 8-week feeding with a diet deficient in methionine and choline (MCD) (Dyets, USA). Then, MCD mice were randomly divided into groups (n = 4–6) that received PBS, 150 µg ApoEVs diluted in 200 µl PBS and 150 µg EVs diluted in 200 µl PBS via tail vein injection. The mice were continuously maintained on the MCD diet after treatment, and liver tissues and serum samples were collected two weeks later for subsequent experiments.

At the end of the above experiments, mice were anesthetized with a single intraperitoneal injection of 1.25% tribromoethanol (Avertin) at a dose of 0.2 mL/10 g body weight. This method ensures rapid induction of anesthesia with stable effects. Anesthetic depth was monitored by the toe-pinch reflex and respiratory rate. Blood samples were collected from the orbital sinus after anesthesia. Mice were then euthanized by exposure to a chamber gradually filled with 60% carbon dioxide (CO₂). The chamber was inflated gradually to minimize distress, and CO₂ exposure was maintained for at least 5 min after respiratory arrest. Death was confirmed by the cessation of breathing and dilated pupils. All anesthesia and euthanasia procedures were performed in accordance with the AVMA Guidelines for the Euthanasia of Rodents to ensure minimal pain and distress.

This study was designed and reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines to ensure rigorous, transparent, and comprehensive reporting of animal research.

Biochemical analysis and histological staining

The serum of mice was obtained at each time point. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using an Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) and measured with a Microplate Reader (Thermo Fisher Scientific, USA). The liver tissue samples were prepared as paraffin-embedded sections by Servicebio, China.

And then, sections were stained with hematoxylin and eosin (HE) for routine histological examination or with Sirius Red and Masson for fibrosis evaluation. Further quantification and analysis of collagen fiber were assessed using Image-J software (National Institutes of Health, USA).

Immunofluorescent staining

Liver tissue samples were fixed with 4% paraformaldehyde for 30 min and washed with PBS. After antigen retrieval with sodium citrate in microwave condition, the sections were blocked with goat serum for 30 min. Then, primary anti-CD206 antibody (AB64693, 1:200, Abcam), anti-CD86 antibody (AB239075, 1:200. Abcam), and anti-F4/80 antibody (AB6640,1:100, Abcam), were added and incubated with tissue sections at 4 °C overnight. On the next day, the sections were washed three times with PBS and then treated with HRP 488 or HRP cy3-labeled secondary antibodies (Servicebio) for one hour at room temperature. After nuclear staining with DAPI, sections were mounted using anti-fade solution. Subsequently, the images were visualized using a laser scanning confocal microscope.

RNA isolation and real‑time PCR analysis

Total RNA was extracted from liver tissues or cells using the RNAeasy Plus Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China), and reverse transcription was generated using PrimeScript™ RT Master Mix (RR036A, Takara, Tokyo), 37 °C for 15 min, followed by 85 °C for 5 s. Then, qRT-PCR was conducted by TB Green Premix Ex Taq II (DRR820A, Takara, Tokyo) and a CFX96 Touch™ real-time PCR System (Bio-Rad, CA), 95 °C for 30 s, 60 °C for 30 s, with a total of 40 cycles. β-actin (Actb or ACTB) was used as an internal control for quantification. PCR primers are shown in Table S1.

Using kits for the reverse transcription (MR201, Vazyme, China) and quantification of miRNAs (MQ102, Vazyme, China). The conditions for reverse transcription were as follows: 37 °C for 60 min, followed by 85 °C for 5 min. The reaction program for qPCR was as follows: 95 °C for 10 s, 60 °C for 30 s, with a total of 40 cycles.

Western blot analysis

Whole proteins were extracted from tissues, cells or ApoEVs using RIPA Lysis Buffer (Beyotime biotechnology, China) supplemented with proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentration was quantified using a BCA Protein Assay Kit (GlpBio, USA). Subsequently, equal amounts of protein samples (30 µg) were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad Biotechnology, USA). The membranes were blocked in TBST buffer containing 2.5% skim milk for 30 min. Then, the membranes were incubated overnight at 4 ℃ with the following primary antibodies: anti-Caspase-3 (9662, Cell Signaling Technology, USA; diluted at 1:1000), anti-GM130 (ab52649, Abcam, UK; diluted at 1:1000), anti-Calnexin (10427-2-AP, Proteintech, USA; diluted at 1:1000), anti-CD81 (ab79559, Abcam, UK; diluted at 1:1000), anti-CD9 (ab236630, Abcam, UK; diluted at 1:1000), anti-Collagen I (ab270993, Abcam, UK; diluted at 1:1000), anti-αSMA (19245, Cell Signaling Technology, USA; diluted at 1:1000), anti-NOS2 (ab15323, Abcam, UK; diluted at 1:1000), anti-ARG-1 (ab124917, Abcam, UK; diluted at 1:1000), anti-STAT3 (10253-2-AP, Proteintech, USA; diluted at 1:1000), anti-STAT6 (9362, Cell Signaling Technology, USA; diluted at 1:1000), anti- NF-κB (8242, Cell Signaling Technology, USA; diluted at 1:1000), anti-AKT (4691, Cell Signaling Technology, USA; diluted at 1:1000), and anti-β-Actin (66009-1-Ig, Proteintech, USA; diluted at 1:5000).

After washing with TBS containing 0.1% Tween-20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Proteintech, China) at room temperature for 1 h. And blots were visualized using an enhanced chemiluminescence kit and detected by a gel imaging system (Bio-Rad Biotechnology, USA). Further quantification and analysis of protein expression were assessed using Image-J software (National Institutes of Health, USA).

The miRNA‑sequencing and bioinformatics analysis

The miRNA-sequencing was provided by LCBio Co., Ltd (Hangzhou, China). Briefly, we collected three independent ApoEVs and EVs samples. RNA samples from both typed of vesicles were extracted, sequenced, and analysed by LCBio Co., Ltd (Hangzhou, China). The purity and quantity of RNA were checked using a Bioanalyzer 2100 (Agilent, CA, USA). Small-RNA libraries were sequenced on the illumina NovaseqTM 6000 platform by LC Bio Technology CO.,Ltd. Then we analysed the differential miRNAs between ApoEVs and EVs. P < 0.05 and |log₂(foldchange)|>=1 was set as the threshold for significantly differential expression. A total of 96 significantly differential miRNAs were identified, with 69 of them being significantly enriched in ApoEVs. Subsequently, more than 111 miRNAs associated with liver cirrhosis were identified from the Human microRNA Disease Database. Compared with the 69 differential miRNAs, resulting in 10 highly differential miRNAs that are enriched in ApoEVs and strongly associated with liver cirrhosis. To predict the genes targeted by most aboundant miRNAs, two computational target prediction algorithms (TargetScan, v5.0 and Miranda, v3.3a) were used to identify miRNA binding sites. Finally, the data predicted by both algorithms were combined and the overlaps were calculated. The gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of these most aboundant miRNAs, miRNA targets were also annotated.

Untargeted metabolomics analysis

The untargeted metabolomics was provided by LCBio Co., Ltd (Hangzhou, China). The vesicles were dissolved in PBS and mixed with ice-cold methanol/acetonitrile (v: v, 1:1). Proteins were precipitated at − 20 °C for 1 h, followed by centrifugation at 13,000 rpm and 4 °C for 15 min. The supernatant was collected, concentrated at 4 °C and reconstituted for subsequent analysis. Meanwhile, an equal volume of each sample was aliquoted and pooled to prepare the quality control sample.

Chromatographic conditions: Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters). Mobile phase A consisted of 5 mmol/L ammonium acetate and 5 mmol/L acetic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program was set as follows: 0–1 min, 1% B; 1–9 min, linear gradient from 1% to 99% B; 9–9.1 min, linear gradient from 99% to 1% B; 9.1–12 min, 1% B. The flow rate was maintained at 0.35 mL/min, the injection volume was 2 µL, and the column temperature was set at 40 °C.

Mass spectrometric conditions: An electrospray ionization (ESI) source was employed, and data acquisition was conducted separately in both positive and negative ionization modes. The ion source temperature was set at 350 °C; the capillary voltage was + 3.8 kV in positive mode and − 3.4 kV in negative mode. The purge gas flow rate was 0 Arb, the auxiliary gas flow rate was 15 Arb, and the sheath gas flow rate was 50 Arb. The data acquisition mode combined full scan and data-dependent acquisition (DDA). In one acquisition cycle, the full scan range was 70–1050 Da with a resolution of 70,000, the automatic gain control (AGC) target was 3E6, and the maximum ion injection time (Maximum IT) was 100 ms. The top 5 precursor ions with a response intensity higher than 100,000 in the full scan were selected for MS² analysis. The MS² resolution was 17,500, the maximum ion injection time (Maximum IT) was 50 ms, and the dynamic exclusion time was set at 6 s.

Statistical analysis

The data were expressed as the mean values ± standard deviation. Technical replicates were performed three times for each experiment to ensure the reliability of the data. The normality of data distribution was assessed by the Shapiro-Wilk test. Homogeneity of variance was assessed by the F-test for two-group comparisons and by the Brown-Forsythe test for multiple group comparisons. Significant differences were determined using two-tailed unpaired Student’s t-tests for two-group comparisons and one- or two-way analysis of variance (ANOVA) with the Sidak’s test for multiple group comparisons. A P value < 0.05 was considered significant. Statistical analysis was plotted by GraphPad Prism 8.0 (GraphPad Software, CA, USA).

Results

Isolation and characterization of MSC-Derived ApoEVs and EVs

MSCs utilized in this study had been previously characterized and identified in our earlier literature [26]. For the induction of apoptosis, MSCs were stimulated with 250 nM STS for 12 h, which showed characteristic cell shrinkage morphological changes under microscope (Figure S2A). Additionally, flow cytometry analysis of dual markers 7-AAD and Annexin V-PE showed that the proportion of early apoptotic and late apoptotic cells reached up to 80% (Figure S2B). The supernatants were collected before or after apoptosis separately from the same number of MSCs, and ApoEVs and EVs were isolated following the optimized gradient centrifugation protocol (Fig. 1A).

Fig. 1.

Induction of apoptosis in MSCs and isolation and characterization of MSC-derived ApoEVs and EVs. A Schematic representation of the differential centrifugation method used to isolate ApoEVs and EVs from the same number of MSCs. B Representative TEM images showed the morphology of ApoEVs and EVs. Scale bar, 100 nm. C NTA showed the size distribution of ApoEVs and EVs. D The ratio of protein quantification in ApoEVs to that in EVs. E Western blot analysis the presence of Caspase3/Cleaved-caspase3, CD9, CD81, Calnexin and GM130 in MSCs, ApoEVs and EVs. The data in the figures represented the mean ± SD. Statistical analyses were performed by t-test. ***, p < 0.001

For the characterization of purified ApoEVs and EVs, TEM and NTA analyses were conducted. TEM analysis revealed that both ApoEVs and EVs displayed typical vesicular double-layered spherical structure (Fig. 1B). NTA provided information on the particle size distribution of vesicles, and ApoEVs exhibited multiple peaks ranging from 100 nm to 500 nm. In contrast, EVs were smaller with a size distribution around 100 nm (Fig. 1C). Strikingly, when comparing the protein content in ApoEVs and EVs produced by the same number of MSCs, we found that ApoEVs contained 4-fold higher protein yields than EVs per cell. Specifically, an equal number of MSCs produced approximately four times more protein in ApoEVs than in EVs (Fig. 1D). Furthermore, in protein level, Western blot analysis was utilized to detect the presence of apoptosis-specific marker Cleaved-caspase 3 in ApoEVs but not in EVS, at the same time identifying exosome-specific markers CD9 and CD81 in ApoEVs and EVs, whereas the non-exosomal markers Calnexin and GM130 were not detected in these vesicles (Fig. 1E). These results indicate that ApoEVs are a type of extracellular vesicles with specific apoptotic markers, and the changes in Caspase3/Cleaved-caspase3 are not observed in ordinary MSCs or EVs.

Comparison of the therapeutic effects between ApoEVs and EVs on CCl4-induced liver fibrosis mouse model

To assess the effects of ApoEVs and EVs on liver fibrosis progression, after 8 weeks of intraperitoneal CCl4 injection, we grouped the model mice as described in methods. We dissolved 150 µg ApoEVs or EVs in 200 µl PBS via tail vein injection into model mice (Fig. 2A). Two and four weeks later, histopathological samples and serum were collected to assess the therapeutic effects.

Fig. 2.

ApoEVs and EVs treatment could alleviate liver fibrosis in CCl4-induced model mice. A Schematic diagram illustrated the establishment of an in vivo liver fibrosis model and the treatment of ApoEVs or EVs. ApoEVs or EVs were transplanted after 8 weeks of CCl4 injury, and samples were collected at 2 weeks and 4 weeks post transplantation for subsequent analysis. B Histological analysis of health comparison, PBS, ApoEVs and EVs treatment after 2 and 4 weeks by HE, Masson and Sirius Red staining in liver sections. Representative images of the staining were shown (n = 4). Scale bar, 100 μm. C Liver fibrosis score analysis of Masson and Sirius Red-stained liver sections. The fibrotic area was presented as a percentage (n = 4). D Changes of serum parameters including ALT and AST in different groups (n = 4). The data in the figures represented the mean ± SD. Statistical analyses were performed by One-way analysis of variance (ANOVA) with the Sidak’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p > 0.05

Histological analyses of liver tissues revealed that compared with PBS group, the ApoEVs and EVs groups exhibited remarkably reduced fibrosis two and four weeks after treatment, and ApoEVs showed a lower degree of fibrosis (Fig. 2B, C). Specifically, ApoEVs treatment group showed reduction in inflammatory cell infiltration in HE staining. Masson and Sirius Red staining revealed that after ApoEV treatment, the fibrous septa became narrower and collagen deposition decreased obviously. Meanwhile, the serum parameters ALT and AST which related to liver function showed consistent findings with those of the pathological sections (Fig. 2D), suggesting ApoEVs had a superior capacity in reducing liver fibrosis in mice both at 2 weeks and 4 weeks after treatment. Subsequently, we set two weeks as the observation time point, Western blot analysis also confirmed that the expression levels of fibrosis-related markers Collagen I and α-SMA were lower in ApoEVs treatment group compared to EVs group (Figure S3A, B). In addition, when detecting the mRNA levels of inflammatory factors in liver tissue, the pro-inflammatory factors Tnf-α and Nos2 were significantly decreased, while the anti-inflammatory factors Arg-1 and Il-10 were significantly increased in the ApoEVs treatment group (Figure S3C). In conclusion, ApoEVs exhibited a superior efficacy in alleviating liver fibrosis and inflammation.

To more accurately compare the therapeutic efficacies of ApoEVs and EVs, this study continued to use an equal particle number as the dose standard. Specifically, both ApoEVs and EVs were administered to liver fibrosis mice via tail vein injection at a dose of 10¹⁰ particles/200 µl PBS, and samples were collected after two weeks (Figure S4A). Histological staining and serum biochemical assay results indicated that ApoEVs exert superior therapeutic efficacy, regardless of whether the dose is standardized by equal protein content or equal particle number (Figure S3B, C, D).

Furthermore, we evaluated the therapeutic efficacy using metabolic dysfunction-associated steatohepatitis (MASH) which is another classic model of liver fibrosis. We treated mice fed with a methionine-choline-deficient (MCD) diet for 8 weeks with ApoEVs and EVs, respectively (Figure S5A). Oil Red O and HE staining assays demonstrated that two weeks after treatment with ApoEVs or EVs, the severity of hepatic steatosis was significantly attenuated in MASH mice. Meanwhile, Masson staining revealed a reduction in fibrotic areas (Figure S5B, C). In addition, liver function parameters were partially restored in all treatment groups (Figure S5D). Notably, ApoEVs exerted more favorable therapeutic effects than EVs. These results suggested that ApoEVs possess remarkable advantages in ameliorating hepatic steatosis and fibrosis.

Modulatory effects of ApoEVs versus EVs on macrophage inflammatory phenotype transition

To explore the specific mechanisms by which ApoEVs and EVs ameliorate the inflammatory response in fibrosis, we investigated the regulatory effects of these two vesicles on the phenotype switching of macrophage in vivo. Immunofluorescence staining was performed on CCl4-induced mouse liver sections then indicated that pro-inflammatory macrophages (F4/80⁺CD86⁺) significantly reduced in ApoEVs treatment group compared with EVs (Fig. 3A). And a notable increase of anti-inflammatory macrophages (F4/80⁺CD206⁺) showed in ApoEVs group (Fig. 3B).

Fig. 3.

ApoEVs and EVs promote the transition to anti-inflammatory macrophages to inhibit the inflammatory response. A, B Immunofluorescence staining of liver sections showed the pro-inflammatory macrophages marker CD86+ (green) and the anti-inflammatory macrophages marker CD206+ (green) in each group. The nuclei were stained with DAPI (blue), and macrophages were stained with F4/80 (red). Representative images of the staining were shown. Scale bar, 20 μm. C The protein expression levels of NOS2 and ARG-1 using Western blot. D Quantitative analysis of protein expression in Western blot. E Western blot was used to detect the phosphorylation levels of STAT6, STAT3, NF-κB, and AKT. F Quantitative analysis of protein expression in Western blot. G qRT-PCR analyses of the mRNA levels of pro-inflammatory macrophage markers (Tnf-α, Nos2, Il-23) and anti-inflammatory macrophage markers (Arg-1, Cd206, Cd163). The data in the figures represented the mean ± SD. Statistical analyses were performed by One-way analysis of variance (ANOVA) with the Sidak’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p > 0.05

Subsequently, we isolated primary BMDMs to further assess the regulatory effects of ApoEVs and EVs on macrophages in vitro. A pro-inflammatory state in macrophages was induced using LPS/IFN-γ, and then co-cultured with ApoEVs at concentrations of 10 µg/mL, 20 µg/mL, and 30 µg/mL, respectively, for determining the optimal dose of the vesicles. The qRT-PCR results indicated that the inflammatory level of macrophages was negatively correlated with the dose of ApoEVs; however, there was no significant difference in efficacy between the 20 µg/mL and 30 µg/mL groups (Figure S6A, B). Therefore, pro-inflammatory macrophages were co-cultured with 20 µg/mL of ApoEVs or EVs, respectively. Then protein and RNA samples were collected, and the expression levels of pro-inflammatory or anti-inflammatory macrophage-associated genes was analyzed using Western blot and qRT-PCR techniques. At the protein level, NOS2 expression was significantly decreased in the ApoEVs and EVs groups, while the expression of ARG-1 was increased, and quantitative analysis further confirmed these results (Fig. 3C, D). Similarly, the mRNA expression levels of pro-inflammatory macrophage-related genes including Tnf-α, Nos2, and Il-23 were significantly downregulated in ApoEVs and EVs-treated groups, while regarding anti-inflammatory macrophages, the expression levels of marker genes Arg-1, Cd206, and Cd163 were markedly upregulated (Fig. 3G). The same experiments were repeated on the Raw264.7 cell line, and the results were consistent with those described above (Figure S7A, B). To further elucidate the mechanisms by which ApoEVs and EVs promote macrophage inflammatory phenotype switching, the activation states of canonical inflammatory pathways (JAK/STAT, PI3K-AKT, and NF-κB) were systematically analyzed. Pathway analysis revealed that both types of vesicles also inhibited the phosphorylation of STAT3, NF-κB, and AKT, while the phosphorylation of STAT6 was activated (Fig. 3E, F). This further suggested that ApoEVs and EVs may participate in inhibiting the pro-inflammatory effects of macrophages by modulating the activation status of transcription factors during the inflammatory process.

Investigating the effects of ApoEVs and EVs on the activation level of HSCs

HSCs are key effector cells in liver fibrogenesis [27]. But when liver injury occurs, inflammatory factors in liver can active HSCs, causing excessive ECM deposition as a wound-healing or scarring response [28]. Therefore, the regulation of HSCs activation has been recognized as a central target for the treatment of liver fibrosis [29]. Signals from pro-fibrotic macrophages can enhance the production of TGF-β, thereby stimulating the activation of HSCs [30–32]. In consequence, based on the preliminary findings we considered that ApoEVs and EVs may ameliorate liver fibrosis by either promoting the secretion of anti-inflammatory factors from macrophages or directly inhibiting the activation of HSCs (Fig. 4A).

Fig. 4.

ApoEVs and EVs inhibited the activation of HSCs. A Schematic illustration of ApoEVs and EVs promote the transition to anti-inflammatory macrophages and inhibit HSCs activation to alleviate liver fibrosis. B The protein expression levels of fibrosis-related markers Collagen I and α-SMA using Western blot. C Quantitative analysis of protein expression in Western blot. D qRT-PCR analyses of the mRNA levels of Col1a1 and α-SMA. The data in the figures represented the mean ± SD. Statistical analyses were performed by One-way analysis of variance (ANOVA) with the Sidak’s test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, p > 0.05

To examine the regulatory effects of ApoEVs and EVs on HSCs, the expression of activation-related marker genes Col1a1 and α-SMA were detected at both protein and mRNA levels. Western blot analysis showed Collagen I and α-SMA were downregulated in treated groups compared to activated HSCs (Fig. 4B, C). The same trend was observed through qRT-PCR analysis (Fig. 4D). These results suggested that ApoEVs and EVs could directly inhibit the activation of HSCs. But they may also indirectly act on HSCs while promoting the release of anti-inflammatory cytokines from macrophages. In summary, ApoEVs and EVs could treat liver fibrosis by altering the activation state of HSCs, and ApoEVs could reduce Col1a1 and α-SMA expression levels significantly.

Analyzed the potential mechanisms of ApoEVs’ superior therapeutic functions in liver fibrosis

Our study demonstrated that, ApoEVs showed significant anti-inflammatory and fibrosis-reversing capabilities in CCl4-induced liver fibrosis mouse model, as well as in vitro macrophage and HSCs. To futher elucidate the therapeutic mechanism of ApoEVs and to understand the reasons behind the differences in the efficacy of ApoEVs and EVs in reversing liver fibrosis, we examined the miRNAs contained within ApoEVs and EVs. Quality control results of the data are presented in Figure S8. We screened miRNAs with significant differential expression between ApoEVs and EVs, and focused on the group of miRNAs that are highly expressed in ApoEVs (Fig. 5A, B). Compared to the EVs group, ApoEVs exhibited a significant enrichment of 69 miRNAs. Through querying the HuDD database, 10 cirrhosis-associated miRNAs were identified from these 69 miRNAs (Fig. 5C). Subsequently, the target genes of the 10 miRNAs (including let-7a-5p, let-7f-5p, miR-101-3p, miR-197-3p, miR-20a-5p, miR-381-3p, miR-411-5p, miR-455-3p, miR-92a-3p, miR-98-5p) that were significantly upregulated in ApoEVs were predicted using TargetScan and Miranda, and these genes were then further bioinformatically analyzed using GO and KEGG. In the GO analysis, the top 20 pathways of GO biological process (BP), cellular component (CC), and molecular function (MF) were displayed. These target genes could influence various biological processes to affect cellular behavior, including “protein transport”, “intracellular signal transduction”, “cell cycle”, “intracellular protein transport” et al. which were related to cell communication, cellular transport and so on (Fig. 5D). In addition, the target genes of these 10 miRNAs were also associated with cellular components such as cytoplasm, nucleus, cytosol, and cytoskeleton et al. (Fig. 5E). Analysis of molecular functions revealed that the 10 miRNAs primarily target genes related to “ATP binding”, “hydrolase activity”, “kinase activity”, and “ubiquitin−protein transferase activity” which influenced the activity of some protein to regulate cell function (Fig. 5F). Furthermore, KEGG pathway analysis revealed that the target genes were closely associated with MAPK signaling pathway, PI3K − AKT signaling pathway, Hippo signaling pathway, and Ras signaling pathway, which play crucial roles in regulating inflammatory responses and fibrosis (Fig. 5G). These highly expressed miRNAs in ApoEVs and their functional analysis also explained why ApoEVs exhibit superior anti-inflammatory effects. The chord diagram illustrating the interaction analysis between miRNAs enriched in the classic inflammatory pathway PI3K-AKT and their corresponding target genes further revealed the potential characteristics of ApoEVs related to inflammation regulation, particularly in the context of liver cirrhosis-associated diseases (Fig. 5H).

Fig. 5.

Bioinformatics analysis of miRNA profiles and target genes involved in ApoEVs and EVs. A Heatmap showed the top 100 miRNAs in ApoEVs versus EVs difference analysis. B Volcano Plot showed some miRNAs with significant differences in ApoEVs versus EVs. C Venn diagram illustrated the intersection of miRNAs differential expression between highly expressed miRNAs in ApoEVs and liver cirrhosis-associated miRNAs. D-F GO analysis of the target genes of the 10 significantly upregulated miRNAs in ApoEVs versus EVs: D Biological Process (BP), E Cellular Component (CC), F Molecular Function (MF). G KEGG pathway enrichment analysis for the target genes of the 10 significantly upregulated miRNAs in ApoEVs versus EVs. H Chordal maps of miRNAs and target mRNAs of the PI3K-AKT signaling pathway

To further identify the core miRNAs that play a pivotal role among these 10 differentially expressed miRNAs, we used the miRPath v3.0 software which is a tool that can display the relationship between enriched pathways and miRNAs, to analyze KEGG enrichment on the 10 significantly upregulated miRNAs in ApoEVs. The results indicated that, among all upregulated miRNAs, let-7f-5p (targeting 62 genes), miR-20a-5p (targeting 50 genes), and miR-411-5p (targeting 8 genes) were significantly enriched in the PI3K-AKT pathway (Fig. 6A). To further validate these results, we detected the expression levels of let-7f-5p, miR-20a-5p, and miR-411-5p in both ApoEVs and EVs using qPCR. The results showed that the expression of these three miRNAs was significantly upregulated in ApoEVs (Fig. 6B), with a similar degree of upregulation. Therefore, to further verify the functions of these miRNAs, we transfected inhibitors of let-7f-5p, miR-20a-5p, and miR-411-5p in BMDM respectively, collected cellular proteins, and detected the phosphorylation level of the PI3K-AKT pathway as well as the impact on inflammatory markers. The results showed that after inhibitor transfection, the inhibitory effects of the three miRNAs on the PI3K-AKT pathway and inflammation were significantly attenuated (Fig. 6C). Quantitative analysis of NOS2, ARG-1, and phosphorylated AKT pathway were presented in Fig. 6D. In conclusion, let-7f-5p, miR-20a-5p, and miR-411-5p are indeed specific and effective components in ApoEVs, and play a key role in inhibiting the PI3K-AKT pathway and improving the inflammatory response of macrophages.

Fig. 6.

ApoEVs ameliorate the inflammatory state of macrophages by targeting the PI3K-AKT pathway through three specific miRNAs they contain. A KEGG enrichment analysis of up-regulated miRNAs of ApoEVs by miRPath v3.0. B qPCR analysis showed the relative expression levels of let-7f-5p, miR-20a-5p, and miR-411-5p in ApoEVs and EVs. C Western Blot analysis showed the protein levels of NOS2, ARG-1, p-AKT and AKT after transfection in BMDM with let-7f-5p, miR-20a-5p, and miR-411-5p inhibitors, respectively. D Quantitative analysis of protein expression in Western blot. The data in the figures represented the mean ± SD. Statistical analyses were performed by One-way analysis of variance (ANOVA) with the Sidak’s test. ***, p < 0.001; ns, p > 0.05

In summary, the bioinformatics analysis of miRNAs in ApoEVs and EVs suggested that ApoEVs outperformed EVs in reducing fibrosis, likely due to their enriched miRNA, which mediated the modulation and activation of inflammatory pathways such as the PI3K-AKT signaling pathway.

Comparative analysis of metabolite sequencing between ApoEVs and EVs revealed significant differences

To comprehensively investigate the metabolite differences between the two types of vesicles, we performed untargeted metabolomic sequencing analysis on ApoEVs and EVs. Metabolites in the ApoEVs and EVs groups were identified using LC-MS/MS. The overall distribution and analytical stability of the samples from the two groups were evaluated. Principal Component Analysis revealed significant differences in metabolite profiles between the ApoEVs and EVs groups, which accounted for 47.33% and 34.71% of the total variance, respectively, with good intra-group reproducibility (Fig. 7A). The Variable Importance in Projection (VIP) values and p-values of each metabolite were combined to screen for differentially abundant metabolites, with the thresholds set as p < 0.05 and VIP > 1. Volcano plots and bar charts indicated that a total of 179 differential metabolites were identified between the ApoEVs and EVs groups, among which 98 were upregulated and 81 were downregulated in ApoEVs (Fig. 7B, C). Based on the magnitude of p-values, the top 30 differential metabolites were visualized in a heatmap, which mainly included lipid metabolism intermediates (e.g., α-ketoisovaleric acid, 2-ketocaproic acid) and amino acids (e.g., valine, leucine, arginine) (Fig. 7D).

Fig. 7.

Comparative analysis of metabolite sequencing between ApoEVs and EVs. A Principal Component Analysis of the Metabolomes Between ApoEVs and EVs. B Volcano plot showed the differentially expressed metabolites between ApoEVs and EVs. The red dots represent significantly upregulated metabolites, the blue dots represent significantly downregulated metabolites, and the gray dots represent metabolites with no significant differential expression. C The bar chart showed a total of 179 significantly differentially expressed metabolites between ApoEVs and EVs, of which 98 were upregulated and 81 were downregulated. D Heatmap displayed the top 30 metabolites identified in the differential analysis between ApoEVs and EVs. E-G KEGG pathway enrichment analysis for the differential metabolites between ApoEVs and EVs to visualize the significantly enriched pathways. H HMDB superclass analysis was performed on the differential metabolites between ApoEVs and EVs. The ordinate represents the number of metabolites and the abscissa represents the superclass classifications annotated in HMDB. I Regulatory relationship network diagram between differential metabolites and pathways

Then KEGG and Human Metabolome Database (HMDB) were used to conduct functional annotation analysis to further elucidate the biological functions of these differential metabolites. HMDB analysis of all identified metabolites demonstrated that the differential metabolites were predominantly categorized into lipid and lipid-like molecules, along with nucleosides and organic compounds (Fig. 7H). The KEGG results showed that pathways related to inflammatory responses, such as efferocytosis, arachidonic acid metabolism, mTOR signaling pathway, and FoxO signaling pathway, were significantly enriched. Additionally, pathways associated with metabolic processes (e.g., linoleic acid metabolism, amino acid metabolism, nucleotide metabolism) exhibited substantial differences between the two groups (Fig. 7E, F, G, I). Notably, efferocytosis, arachidonic acid metabolism, mTOR signaling pathway, and FoxO signaling pathway were closely involved in macrophage polarization, inflammatory cytokine secretion, and oxidative stress regulation, suggesting that these pathways may underlie the divergent effects of ApoEVs and EVs on modulating the inflammatory phenotype of macrophages. Collectively, these findings suggested that the vesicles may exert anti-inflammatory and anti-fibrotic effects by regulating lipid metabolism and inflammatory cytokine secretion through inflammation-related signaling pathways.

Discussion

Chronic liver diseases, such as fatty liver disease and viral hepatitis, are often associated with the persistent activation of inflammatory responses and sustained progression of liver fibrogenesis [33]. In recent years, results from clinical trials have suggested that stem cell therapy may hold considerable potential for addressing these conditions [34]. But a large proportion of infused stem cells undergo apoptosis, and these apoptotic cells still exhibit certain immunomodulatory functions—though the exact mechanisms underlying this phenomenon have yet to be fully elucidated [18]. ApoEVs were emerging as bioactive substances secreted during the process of apoptosis, and they have shown promising therapeutic prospects in the management of several diseases. In our previous research, we found that stem cells can exert potent therapeutic effects through their paracrine functions, with exosomes serving as key mediators [25]. This study aimed to comprehensively evaluate the immunomodulatory effects of two important secretory vectors from stem cells — ApoEVs and EVs — using two liver fibrosis models, with the expectation of providing a reference for optimizing strategies for the clinical application of stem cell therapy. Our study conducted a functional comparison of ApoEVs and EVs from the perspectives of anti-fibrosis and anti-inflammation. Furthermore, we analyzed the differences in the loaded functional molecules including miRNAs and metabolites to explore the molecular mechanisms underlying their functional disparities. Thus, our study confirmed that cellular apoptosis holds the potential to enhance the immunomodulatory and antifibrotic efficacy of MSCs and their derivatives, thereby providing a novel therapeutic perspective for liver diseases.

Over the past decades, MSCs have been extensively investigated for their clinical translation, with promising preclinical results in various animal disease models [9]. In chronic liver diseases, MSCs contribute to tissue homeostasis maintenance, regeneration, and immune microenvironment modulation, but their in vivo therapeutic mechanisms remain poorly defined. Notably, transplanted MSCs undergo rapid apoptosis in adverse disease microenvironments, such as hypoxia and inflammation [35]; this process does not impair their immunosuppressive effects, but instead enhances therapeutic efficacy in certain settings [36]. Accumulating evidence indicates that apoptotic MSCs exert comparable therapeutic effects to live MSCs [37], prompting intense research interest in ApoEVs which are secreted by apoptotic MSCs as key mediators of these effects. Inherited with parental MSCs’ molecular traits, ApoEVs play pivotal roles in immunomodulation, angiogenesis, and homeostasis regulation [35]. Most previous studies have focused on EVs secreted during the normal culture of MSCs [38], while esearch on functional differences between ApoEVs and EVs remains limited. In this study, two secretory products of MSCs under distinct states — ApoEVs and EVs — were isolated and characterized. Intrinsic differences were identified between the two in terms of surface markers, particle size, protein content, and encapsulated cargos. Overall, ApoEVs exhibits higher yields and more abundant protein content than EVs. Researches have shown that apoptotic cells release a greater number of microparticles than viable cells [39]. Apoptosis induction may also alter the membrane and cargo composition of vesicles; these changes in physicochemical properties may affect their biodistribution, immunomodulatory capacity, and therapeutic efficacy [40, 41].

The functional differences between ApoEVs and EVs are verified in subsequent in vitro and in vivo experiments. The secretion of pro-inflammatory cytokines by macrophages and the activation of HSCs are widely considered key events that drive the progression of liver fibrosis [42]. The polarization of macrophages is believed to be crucial for tissue repair and the resolution of inflammatory responses [43]. Our study confirms that ApoEVs exhibit a more potent capacity than EVs in promoting macrophage polarization and inhibiting HSCs activation, which suggests that, ApoEVs hold distinct advantages in liver fibrosis therapy and highlights their pivotal role in immunomodulation that over EVs.

The observed differences in functionality between ApoEVs and EVs can be attributed to several key factors. Among these, the two types of vesicles may differ in the types and quantities of encapsulated bioactive molecules, including proteins, nucleic acids, and lipids. These differential molecules exert profound impacts on their functional properties. MiRNA sequencing and metabolomics analyses provide important insights into the molecular mechanisms underlying these functional differences.

MiRNAs, which possess specific biological functions, represent the most extensively studied category of RNAs in extracellular vesicles. They are a class of small non-coding endogenous RNA molecules that regulate gene expression through translational repression or mRNA degradation. They are thought to serve as important mediators of intercellular communication via extracellular vesicles and may play a role in modulating the activation, infiltration, and resolution of inflammatory responses in macrophages and HSCs [44, 45]. In this study, we systematically analyzed the miRNAs encapsulated in ApoEVs and EVs, and identified 10 miRNAs with significant differences, including let-7a-5p, let-7f-5p, miR-101-3p, miR-197-3p, miR-20a-5p, miR-381-3p, miR-411-5p, miR-455-3p, miR-92a-3p, and miR-98-5p. We found that the target genes of these differential miRNAs were significantly associated with inflammatory signaling pathways, including the MAPK and PI3K-AKT pathways, providing evidence for the anti-inflammatory capacity of ApoEVs. Notably, three specific miRNAs were screened out, which were proven to significantly downregulate NOS2 expression and upregulate ARG-1 expression—key contributors to the superior anti-inflammatory efficacy of ApoEVs. Furthermore, these miRNAs were involved in intracellular signal transduction, protein transport, and the regulation of enzyme activity in various biological processes, all of which were closely linked to immunomodulatory functions. This set of miRNAs was identified and validated for their diverse functional roles in ApoEVs for the first time, which provided robust evidence for clarifying the effector molecules responsible for the functional performance of ApoEVs.

Untargeted metabolomics analysis further indicated that ApoEVs contained a greater variety of metabolites than EVs. Most of these differential metabolites exhibited lipid-molecule characteristics, which enhanced the tissue uptake and biological barrier penetration abilities of the vesicles and facilitated more efficient delivery of functional molecules (e.g., miRNAs). Moreover, these differential metabolites were mostly enriched in signaling pathways such as arachidonic acid metabolism, nucleoside metabolism, ABC transporters, mTOR, and FoxO pathways. Moreover, most of these differential metabolites exhibited lipid-molecule characteristics, which enhanced the tissue uptake and biological barrier penetration abilities of the vesicles and facilitated more efficient delivery of functional molecules (e.g., miRNAs).

Comparative studies on the efficacy of ApoEVs and EVs are conducive to the further optimization of MSC applications. However, it should be noted that this study has several limitations. First, both ApoEVs and EVs used in this study were heterogeneous mixtures obtained via centrifugation. Although their relatively stable anti-inflammatory efficacy was verified by technical replicates, batch-to-batch variations are inevitable. The long-term stability of the vesicles was not investigated, nor were variables such as storage methods and storage duration included in the study design. Second, the relatively small sample size of this study may limit the statistical power of the results and reduce the generalizability of our findings to larger populations. Future research will focus on developing standardized isolation protocols to improve reproducibility and optimizing storage conditions to enhance stability. Meanwhile, larger-scale studies involving a broader range of donors will strengthen the general applicability of the research outcomes.

In addition, although our current findings demonstrate that ApoEVs exert a more pronounced effect on alleviating liver fibrosis, there is a lack of observations on the long-term therapeutic efficacy in model mice, such as efficacy assessments at three or six months post-treatment. Moreover, the precise molecular mechanisms underlying this therapeutic effect have not been fully elucidated. Future studies should extend the treatment duration, further identify the key factors mediating the antifibrotic effects of ApoEVs, and clarify the important role of apoptosis in the clinical application of MSCs.

In conclusion, ApoEVs are a unique class of extracellular vesicles secreted during cellular apoptosis, featuring convenient isolation procedures and high yields. Enriched with molecules such as miRNAs and metabolites, ApoEVs exert potent antifibrotic effects, suppress the inflammatory phenotype of macrophages, and inhibit the activation of HSCs. These findings provide novel insights into the role of apoptotic events in the practical application of MSCs.

Conclusions

In summary, our results highlight the distinct characteristics of vesicles secreted by MSCS in different apoptotic states and explore the great potential of ApoEVs in the treatment of liver fibrosis. Notably, in the inflammatory model established in this study, ApoEVs exhibited more pronounced effects than EVs in alleviating the inflammatory response of macrophages, and this advantage is at least partially mediated by their enriched let-7f-5p, miR-20a-5p, and miR-411-5p through targeting the PI3K-AKT pathway. In addition, the regulation of inflammation-related pathways and metabolic processes involving differential metabolites may also contribute partially to this effect. Overall, our results suggested that ApoEVs could be a promising approach for the treatment of liver diseases and further lay a research foundation for clarifying the therapeutic mechanism of MSCs.

Abbreviations

MSCs

mesenchymal stem cells

MMPs

matrix metalloproteinases

ECM

extracellular matrix

HSCs

hepatic stellate cells

EVs

extracellular vesicles

miRNA

microRNA

ApoEVs

apoptotic extracellular vesicles

CCl4

carbon tetrachloride

SPF

specific pathogen-free

PBS

phosphate buffered saline

MASH

metabolic dysfunction-associated steatohepatitis

MCD

methionine and choline

hUC-MSCs

human umbilical cord-derived MSCs

STS

staurosporine

TEM

transmission electron microscopy

NTA

nanoparticle tracking analysis

BMDMs

bone morrow-derived macrophages

ALT

alanine aminotransferase

AST

aspartate aminotransferase

GO

gene ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

BP

biological process

CC

cellular component

MF

molecular function

Authors’ contributions

Ruobing Ju, Siyuan Tian and Bo Li performed the most of experiments, analyzed data and wrote the manuscript. Bo Li, Xia Zhou isolated and collected exosomes. Yinan Hu and Erzhuo Xia built animal models. Bo Li and Fangfang Yang contributed to flow cytometry analysis. Miao Zhang, Shuoyi Ma and Rui Su conducted the statistical analysis. Ying Han, Yulong Shang and Xia Zhou designed the study and revised the manuscript. All authors read and approved the current version of the manuscript. All the above-mentioned authors were involved in the writing and reviewing of the manuscript.

Funding

This study was funded by National Natural Science Foundation of China (No. 82270551, No. 82200680, No.82300672), Key Research and Development Program of Shaanxi province, China (No. 2024SF-GJHX-16), National Key Research and Development Program of China (No. 2020YFA0710803), Science and Technology Association youth talent lifting program of Shaanxi province, China (No.20230324) and Xijing Hospital Project (No. XJZT25ZH08).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The animal study protocol was approved by the Animal Welfare and Ethics Committee of the Fourth Military Medical University and performed according to the “Guidelines for the Care and Use of Laboratory Animals” (No. 20230205).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.