Exosomes derived from human adipose mesenchymal stem cells ameliorate hepatic fibrosis by inhibiting PI3K/Akt/mTOR pathway and remodeling choline metabolism

Characterization of hADMSCs and hADMSCs‑Exo

In the beginning, primary cells isolated from human adipose tissues underwent identification of immunophenotype and multipotential differentiation ability, for the purpose of providing exosome constantly. The morphology of hADMSCs was monolayer adherent and fibroblast-like spindle-shaped (Fig. 1A). For detecting multilineage differentiation potential of hADMSCs, we evaluated osteogenic, adipogenic and chondrogenic differentiation of hADMSCs by using different inducing conditioned medium respectively. Morphological changes were shown in Additional file 1: Fig. S1A, compact cell layer and intense Alizarin red staining illustrated calcium deposition (bright red spots) of osteogenically differentiated tissues, supporting the osteogenic differentiation ability of hADMSCs. Lipid droplets (dark red spots) formed during adipogenic differentiation of hADMSCs were visualized by Oil Red O staining. After 1 month of chondrogenic differentiation in culture, we observed clear cartilage formation, and production of proteoglycan (blue circle) by Alcian blue staining, suggesting the multi-lineage differentiation ability of hADMSCs. In addition, to better understand the immunophenotype of hADMSCs, we examined expression of characteristic stem-cell surface markers (CD44, CD73, CD90, and CD105) by utilizing flow cytometry. The result showed positive rate for CD44, CD73, CD90 and CD105 of hADMSCs were 85.1%, 99.3%, 99.6% and 98.7% respectively (Additional file 1: Fig. S1B). Collectively, these data identified the osteogenic, chondrogenic, and adipogenic differentiation hADMSCs isolated from human adipose tissues, as well as their immune phenotypes.

Fig. 1figure 1

Characterization of and hADMSCs and hADMSCs-Exo. A Morphological appearance of cultured hADMSCs (bar = 100 μm). B Representative TEM images of hADMSCs-Exo (bar = 500 nm and 100 nm). C Flow cytometry for nanoparticle analysis of hADMSCs-Exo. D Size distribution measurements of hADMSCs-Exo by NTA under flow conditions. E Western blot analysis of TSG101, CD63, CD81 and GAPDH in hADMSCs-Exo and whole-cell lysis of hADMSCs. F CLSM images of hADMSCs-Exo labeled with PKH26. Red: PKH26-Exo. Blue: DAPI. Scale bar, 40 μm

Next, exosomes derived from hADMSCs underwent evaluation on morphology and content. The ultra-microstructure of purified hADMSCs-Exo was elliptical vesicle structures with the similar size of ~ 100 nm (Fig. 1B). Result of NTA-based characterization showed that the size of hADMSC-Exo was concentrated in the range of 50–100 nm (average size = 76.14 nm), while the particles of 30–150 nm accounted for 96.66% of the total particles with a concentration of 4.11E + 11 Particles/mL (Fig. 1C, D). Through detecting exosomal biomarkers by western blot assay, we observed strong expression of TSG101, CD63 and CD81 in hADMSCs-Exo rather than hADMSCs lysates (Fig. 1E). Furthermore, hADMSCs-Exo were labeled with PKH26 (red) and cocultured with LX-2 cells. Labeled hADMSCs-Exo were engulfed by LX-2 cells and were distributed around their nucleus (Fig. 1F).

hADMSCs-Exo inhibits hepatic stellate cell proliferation by arresting cell cycle and inducing apoptosis

To investigate the repairment function of hADMSCs-Exo on fibrosis, we designed both in vitro and in vivo experiments as illustrated in Fig. 2A. Considering that TGF-β1-regulated hepatic stellate cells (HSCs) are responsible for liver fibrosis, we constructed the fibrotic cell model by treating LX-2 cells with TGF-β1. Morphologic change and expression of both profibrotic and EMT-related proteins in LX-2 cells were examined at different time points (12 h, 24 h, 48 h and 72 h). As shown in Additional file 1: Fig. S2A, compared to that quiescent HSCs were long spindle shape, LX-2 cells at 12 h after TGF-β1 treatment formed pseudopodia with elongated synapses. At the timepoint of 24 h after activation, LX-2 cells exhibited clearer filopodia, lamellipodia, and polygonal shapes. The cells displayed disordered arrangment, tight junction, aggregative growth, and cellular protrusions obviously at 48 h or 72 h. Western blot results showed EMT appeared in LX-2 cells after 12 h, and profibrogenic markers (α-SMA) could be stably expressed at 24 h (Additional file 1: Fig. S2B). Based on these observations, the time point of 24 h after TGF-β1 stimulation was used for constructing fibrotic cell model in the subsequent experiments.

Fig. 2figure 2

hADMSCs-Exo inhibits hepatic stellate cell proliferation by impeding cell cycle progression and inducing apoptosis. A A schematic representation of the experimental design. B In vitro cell viabilities of activated LX-2 cells incubated with hADMSCs-Exo at the indicated concentration in the presence of TGF-β1 (10 ng/ml) for 24 h, 48 h or 72 h, (n = 5). C IC50 were analysed in activated LX-2 cells exposed to hADMSCs-Exo for 24 h. D Edu assay showed that HSCs proliferation was suppressed by hADMSCs-Exo in a concentration-dependent manner. EdU% is used as an approximation for proliferation rate. E Cell cycle analysis showed an increase in the sub-G1 subpopulation and cell cycle arrest after hADMSCs-Exo. F The quantitative analysis of the apoptosis was performed using the Annexin-FITC staining based flow cytometry. Exo, exosome. Data are presented as means with SEM (n = 3 independent experiments). ns, not significant, *p < 0.05, **p < 0.01 and ***p < 0.001

To determine the effect of hADMSCs-Exo on activated HSCs, we evaluated cell proliferation after being treated with hADMSCs-Exo. As showed in Fig. 2B, proliferation of pro-fibrotic HSCs was inhibited by hADMSCs-Exo in a concentration-dependent fashion. High concentration of hADMSCs-Exo (0.3 μg/μl) decreased cell survival rate by half, whereas low‐dose hADMSCs-Exo (range from 0 to 0.1 μg/μL) did not alter proliferation of activated HSCs by CCK8 assay (Fig. 2C). Dose-dependent inhibiting effect of hADMSCs-Exo on HSCs were confirmed by Edu proliferation assay, which showed an augmented HSCs proliferation in hADMSCs-Exo-treated groups (Fig. 2D). In addition, we observed that hADMSCs-Exo arrested cell cycle arrest in G1 phase of cell cycle, a small, non-significant increase in cells in S phase, and gradually declined at the G2 phase (Fig. 2E). Similarly, we also tested the impact of hADMSCs-Exo on apoptosis of activated HSCs. A concentration-dependent increase in apoptosis occurred in activated HSCs after 24 h of exposure to hADMSCs-Exo (Fig. 2F).

hADMSCs-Exo internalized by LX-2 cells exhibit antifibrotic and anti-EMT effect in vitro

Subsequently, we explored the mechanism involved in influence of hADMSCs-Exo on activated HSCs in a LX-2 and hADMSCs co-culture system. As shown in Additional file 1: Fig. S3, hADMSCs significantly inhibited proliferation of activated LX-2 cells and blocked expression of α-SMA. To investigate whether the inhibitory effect on LX-2 is mediated exosome, we treated activated LX-2 cells with purified hADMSCs-Exo. As showed in Fig. 3A, our results showed that compared to original LX-2 cells, TGF-β1 activated LX-2 cells formed filopodia- and lamellipodia-like extensions. These cell protrusions shrunk gradually when being incubated with hADMSCs-Exo, or even completely disappeared after being incubating with high concentration of hADMSCs-Exo. Concurrently, the immunofluorescence assay results revealed that when aHSCs were treated with hADMSCs-Exo, the fluorescence signals of α-SMA significantly diminished in a dose-dependent manner, indicating excellent antifibrotic effect of hADMSCs-Exo (Fig. 3B). In addition, antifibrotic and anti-EMT effect of hADMSCs-Exo were evaluated by western blotting. After being educated with hADMSCs-Exo, ECM of LX-2 cells was markedly degraded, performed with downregulated collagen I and α-SMA expression. Reduced TGF-β1, vimentin and β-catenin whereas increased E-catenin suggested that EMT process was substantially inhibited as well. These results suggested that hADMSCs-Exo could inactivate the activated HSCs through EMT inhibition and ECM degradation.

Fig. 3figure 3

hADMSCs-Exo internalized by LX2 cells exhibit antifibrotic and anti-EMT effect in vitro. A Representative phase-contrast images of quiescent HSCs and aHSCs were treated with hADMSCs-Exo at the indicated concentration in the presence of TGF-β1 (10 ng/ml). Analyses were conducted 24 h after the indicated treatments. Scale bars, 100 mm. B Representative immunofluorescence images of α-SMA (red) and DAPI (blue) of aHSCs were treated with hADMSCs-Exo. Scale bars, 100 mm. C Western blot analysis of LX-2 cell incubated with hADMSCs-Exo for 24 h in the presence of TGF-β1 (10 ng/ml). Data expressed as the mean ± SEM (n = 3 independent experiments). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

hADMSCs-Exo could alleviate CCl4-induced mice liver fibrosis in vivo

For further verification, therapeutic effect of hADMSCs-Exo and hADMSCs were examined in fibrotic mice, and the samples were analyzed by RNA-seq and LC–MS/MS. Before that, we used CCl4 to establish liver fibrosis mouse model, which is verified by gross inspection and histological analysis (Additional file 1: Fig. S4A, B). We investigated the organ distribution of hADMSCs-Exo in fibrotic mice especially in their liver tissue. PKH26-labeled hADMSCs-Exo were injected through tail vein into the models, internal distribution of them were traced using a NIRF fluorescence imaging system. As expected, higher fluorescence signals of hADMSCs-Exo in models were detected in fibrotic liver tissue earlier than in other organs (Additional file 1: Fig. S5A, B).

Based on the fibrotic mice model, we assessed the therapeutic potential of hADMSCs-Exo as depicted in Fig. 4A. SWE verified the success of modeling, and showed a significant increase in mice liver stiffness in the other groups compared with sham group (Additional file 1: Fig. S6). We next assess liver stiffness measurement (LSM) by SWE after 2 weeks and 4 weeks of treatment. Compared to normal liver tissue, liver stiffness value was significantly elevated in regression group (REG). Hepatic fibrosis wasn’t completely repaired after 2 weeks of hADMSCs-Exo treatment. Liver matrix stiffness significantly declined to normality after 4 weeks of treatments or hADMSCs or hADMSCs-Exo, demonstrating a repaired status compared to REG group (Fig. 4B, Additional file 1: Fig. S7). In addition, the body weight of hADMSCs group and hADMSCs-Exo group increased faster compared with sham group and REG, demonstrating recovered status under treatment with hADMSCs or hADMSCs-Exo (Fig. 4C).

Fig. 4figure 4

hADMSCs-Exo could alleviate CCl4-induced mice liver fibrosis in vivo A Diagram of experimental scheme. B Elastograms obtained using shear wave elastrography (reliable images were obtained when uniform colour filled > 90% of the sampling area) and liver stiffness quantification. C Body weight changes during treatment. D Representative photographs of lives from mice in each group and histopathological images of liver sections were evaluated using H&E, Masson trichrome staining and Sirius red staining in animals with CCl4-induced cirrhosis. Liver histopathology grading was evaluated by necro-inflammatory scoring and Ishak (modified Knodell) scoring system after treatment. E Images of immunohistochemistry staining of extracellular matrix (ECM) proteins (collagen I and α-SMA). F The protein levels of ECM and EMT-related proteins in the liver tissues of the mice with different treatments. SWE, shear wave elastrography. LFG, liver fibrosis group, REG, regression group. Data are expressed as mean ± SEM (n = 6), ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

At four weeks post to treatment, liver tissues were collected for evaluating effectiveness of hADMSCs-Exo in recovering liver injury, hepatic inflammation and fibrotic degree. As shown in Fig. 4D, fibrotic liver manifested with hard texture, rough surface with brownish color and nodule formation, indicating a serious injured condition. Intriguingly, liver in the hADMSCs-Exo group showed clear morphological restoration along with a smooth surface and bright red color in macroscopic images. The results showed that hADMSCs-Exo and hADMSCs groups showed a reduction of hepatocyte piecemeal, confluent necrosis, ductal proliferation and infiltration of immune cells in histological examinations and their staging score of liver fibrosis, which was dramatically lower than that in LFG and REG (1.5, 1.75, 3.25, 5.25, p < 0.05). Masson’s trichrome and Sirius Red staining were performed to indicate collagen deposition in liver sections. The collagen-stained area of the hADMSCs-Exo group was 2.6 times lower than that of the REG and 3.4 times lower than that of the LFG. These results were consistent with those of α-SMA and collagen I staining, showing that a remarkable reduction was found in the α-SMA and collagen I stained areas of the hADMSCs-Exo-treated group (Fig. 4E). Furthermore, Western blot experiments verified the above result and confirmed anti-EMT effect of hADMSCs-Exo (Fig. 4F). Thus, these data substantiated that systemic administration of hADMSCs-Exo could histologically and functionally alleviate liver fibrosis in CCl4-induced mice liver fibrosis.

hADMSCs-Exo treatment improves liver function and regeneration, reduces liver inflammation and apoptosis

Next, we examined levels of hydroxyproline (Hyp) and malondialdehyde (MDA) in liver tissues, which indicate the presence of liver collagen deposition and lipid peroxidation changes, reflecting the degree of hepatic oxidative stress, inflammation and hepatocellular injury, respectively. Treatment with hADMSCs reduced Hyp and MDA accumulation in comparison with the LFG and REG, which was even lower in hADMSCs-Exo-treated group (Fig. 5A), suggesting a superior therapeutic effect of hADMSCs-Exo than hADMSCs in regulating collagen deposition and oxidative stress. Similar results were obtained when performing serum biochemical tests and quantifying inflammatory cytokines. Serum levels of ALT, AST and ALP were significantly suppressed in the hADMSCs and hADMSCs-Exo-treated mice when compared with LFG and REG (Fig. 5B). Furthermore, expression of inflammatory cytokines including Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were significantly decreased in livers of hADMSCs-Exo group compared with those of the LFG and REG, demonstrating effective anti-inflammatory effects of hADMSCs-Exo in liver fibrosis mice.

Fig. 5figure 5

hADMSCs-Exo treatment improves liver function and regeneration, reduces liver inflammation and apoptosis. A The Hyp and MDA levels of normal mice (Sham), fbrotic mouse model and the mice injected with PBS, hADMSCs or hADMSCs-Exo were measured with corresponding test kit B Serum levels of AST, ALT, ALP in different groups. C The relative inflammatory gene expression for IL-1β, IL-6, IL-10 and TNF-α. D Immunohistochemical staining was performed to detect the protein expressions of Ki67, HNF-4α and caspase 3 in the injured liver of mice treated with hADMSCs and hADMSCs-Exo. Scale bar, 40 μm. AST, aspartate aminotransferase, ALT, alanine aminotransferase, ALP, alkaline phosphatease. LFG, liver fibrosis group, REG, regression group. Data are expressed as mean ± SEM (n = 4), ns, not significant, *p < 0.05, **p < 0.01 and ***p < 0.001

To evaluate whether hADMSCs-Exo treatment could reduce hepatocellular injury and apoptosis, improve liver regeneration, we performed IHC to quantify Ki-67, caspase 3 and HNF-4α. As shown in Fig. 5D, the percentage of Ki-67+ cells in hADMSCs (5.09%) and hADMSCs-Exo (7.35%) treated groups was decreased significantly when compared to the LFG (35.31%) and REG (28.64%). The percentage of HNF-4α+ cells in hADMSCs-Exo (64.27%) group increased significantly when compared with the LFG (38.50%) and REG (40.38%). hADMSCs-Exo exhibited decreased hepatocyte death characterized by remarkably decreased caspase-3 expression, indicating that hADMSCs-Exo reduced hepatocellular injury and apoptosis, and recovered from CCl4-induced liver damage.

hADMSCs-Exo inhibited HSCs activation and liver fibrosis through PI3K/AKT/mTOR signaling pathway

To unearth the molecular mechanism involved in regulation of hADMSCs-Exo in liver fibrosis, we compared transcriptomic profiles between the LFG, REG, hADMSCs and hADMSCs-Exo group as presented in Fig. 6A. Among these differentially expressed genes (DEGs), we observed a partial intersection of 264 unique DEGs between the hADMSCs-Exo group versus REG, and 350 unique DEGs between the hADMSCs-Exo group versus LFG (Fig. 6B). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using these unique DEGs (Fig. 6C and Additional file 1: Fig. S7A, B). In GO terms, the major differentially expressed pathways involved are ECM components and related pathways, which is consistent with our results showing that hADMSCs-Exo regulated ECM remodeling in HSCs. Phosphatidylinositol 3- kinase/protein kinase B (PI3K/AKT) was identified as the top-ranked signaling pathway (Fig. 6D, E, Additional file 1: Fig. S7C, D). A batch of factors in PI3K/AKT signal pathway was downregulated by hADMSCs-Exo, including Cdkn1a, down-regulated genes: Col6a1, Lamb1, Itga8, Tnc, Lama4, Col6a2, Thbs2, Col1a1, Col1a2 (Fig. 6F, Additional file 1: Table S3). Reduced expression of these genes by hADMSCs-Exo group was confirmed by qRT–PCR (Fig. 6G). Through immunofluorescence assay, we observed that AKT protein expression was remarkably reduced following hADMSCs-Exo treatment, while fibronectin protein expression was notably influenced (Fig. 6H). This result suggests that hADMSCs-Exo may downregulate nuclear expression of fibrosis-related genes by inhibiting PI3K-AKT signaling pathway, inhibiting the formation of ECM to ameliorate the progression of liver fibrosis.

Fig. 6figure 6

hADMSCs-Exo inhibited HSCs activation and liver fibrosis through PI3K/AKT/mTOR signaling pathway. A Heatmap of differential expression analysis of RNA-sequencing data from LFG, REG and hADMSCs-Exo. B Venn diagram of LFG vs REG, LFG vs hADMSCs-Exo and REG vs hADMSCs-Exo. C GO analysis results of the DEGs between REG and hADMSCs-Exo, including cell component (CC), molecular function (MF) and biological process (BP). Red represents CC, green represents MF, blue represents BP. D The top 10 significant GO terms of DEGs. E KEGG analysis of significant pathway of DEGs. F Volcano plot and significantly DEGs in PI3K/AKT pathway. Red represents up-regulated, blue represents down-regulated genes. G The mRNA levels of significantly DEGs in PI3K/AKT pathway were determined by qRT–PCR. H Fibronectin and AKT protein levels in LX-2 or treated with pbs, hADMSCs-Exo in the presence of TGF-β1 (10 ng/ml) for 24 h were measured by immunofluorescence staining. I Western blot assay for AKT, mTOR and phosphorylated and total PI3K p85 and PI3K p110 in mouse liver issue. J Quantification of AKT and mTOR by qRT–PCR. K The expression level of AKT, mTOR and phosphorylated and total PI3K p85 and PI3K p110 after HSCs were incubated with hADMSCs-Exo. LFG, liver fibrosis group, REG, regression group. Data are presented as means with SEM (n = 3 independent experiments). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

To determine whether PI3K/AKT signaling pathway mediates efficacy of hADMSCs-Exo, we analyzed activities of AKT and mTOR expression in activated HSCs and liver tissues. In consistent with transcriptomic result, western blot analysis showed that phosphorylation levels of PI3K were significantly inhibited, a statistically significant decrease was accordingly observed in the AKT and mTOR expressions in vivo (Fig. 6I). qRT–PCR also confirmed decreased mRNA expression of AKT, mTOR in hADMSCs-Exo group ((Fig. 6J, Additional file 1: Fig. S8A). Similarly, PI3K/AKT/mTOR signaling was also inhibited in HSCs treated with hADMSCs-Exo in a concentration-dependent manner (Fig. 6K, Additional file 1: Fig. S8B). These results confirmed that PI3K/AKT/mTOR signaling pathway is participated in function of hADMSCs-Exo.

hADMSCs-Exo inhibits liver fibrosis by regulating choline metabolism

To unearth mechanisms underlying hADMSCs-Exo-mediated metabolic activities in inhibiting liver fibrosis, we quantified and compared metabolites in liver with hADMSCs-Exo treatment by LC/MS technology. Through principal components analysis (PCA), metabolites of mouse liver tissue in each group were obviously discriminated to five different groups (Fig. 7A), and Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed a clear and statistically significant separation among each group (OPLS-DA model: R2X = 0.381, R2Y(cum) = 0.993, Q2(cum) = 0.849) (Fig. 7B). A total of 1452 changed metabolites were identified with the threshold of variable importance in the projection (VIP) ≥ 1 and p ≤ 0.05. In particular 162 differential metabolites were identified between hADMSCs-Exo-treated group and control group, while 267 differential metabolites were identified between hADMSCs-Exo-treated group and the regression group (Fig. 7C). Information of the top 20 most altered metabolites (VIP, p value and fold change) in hADMSCs-Exo-treated group compared to REG or LEG were listed in Additional file 1: Table S4 and Table S5, respectively. As presented in the clustered heatmap (Fig. 7D), Oleamide, sphingosine, FMN, sphinganine, tridemorph ranked by the front of significantly up-regulated metabolites, while the most significantly down-regulated metabolites were betaine, choline, D-Fructose, 2-Hydroxy-2-ethylsuccinic acid.

Fig. 7figure 7

hADMSCs-Exo inhibits liver fibrosis by regulating choline metabolism. A Score plots from the PCA model derived from the UPLC-MS profile of liver obtained from mice in different groups. B Score plots from the OPLS-DA model from metabolic profiles of different groups. C Venn diagram of differential metabolites between LFG, REG, hADMSCs and hADMSCs-Exo-treated group. D The hierarchical clustering heatmap of the top 30 metabolites between different groups. E Summary of pathway analysis of differential metabolites between REG and hADMSCs-Exo-treated group. F Summary of pathway analysis of differential metabolites between LFG and hADMSCs-Exo-treated group. G The quantitative analysis of the key metabolites in choline metabolism based on metabolic profiles. H Quantitative analyses of the key metabolites in choline metabolism by ELISA. LFG, liver fibrosis group, REG, regression group. Data expressed as the mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Through pathway analysis of those differential metabolites, we noticed that lipid and energy metabolic pathways were enriched in comparison between hADMSCs-Exo with REG group or hADMSCs-Exo with LFG group, such as PPAR signaling pathway, choline metabolism, and fatty acid metabolism (Fig. 7E, F). hADMSCs-Exo perturbated metabolomic profiling in fibrotic liver tissue. The increased liver levels of sphingolipid and sphinganine and decreased levels of betaine, glycerophosphocholine (GPC) were reversed in the hADMSCs-Exo-treated group compared with those in the LFG and REG (Fig. 7G). Through ELISA, levels of choline, phosphorylcholine, phosphatidylcholine and sphingomyelin were determined to be reduced in hADMSCs and hADMSCs-Exo groups (p < 0.05) compared to LFG and REG (Fig. 7H). These results suggested that the mechanisms of hADMSCs-Exo against liver fibrosis might involve in the regulation of choline metabolism. The discovery is worthy of further study, regarding the importance of choline metabolism and its regulation of PI3K/AKT signaling.

hADMSCs-Exo regulates the choline metabolism, which involved in PI3K/AKT/mTOR signaling pathway to anti-liver fibrosis

To illustrate the role of hADMSCs-Exo in choline metabolism, we counted changes of intracellular and hepatocyte cytoplasm metabolites as well as expression of metabolic genes following hADMSCs-Exo treatment. Secreted and intracellular levels of choline were detected by performing ELISA on cell supernatants and whole cell lysates respectively. We found that hADMSCs-Exo promoted uptake of the choline by the activated LX-2 cells (Fig. 8A). Meanwhile, intracellular betaine, phosphatidylcholine and GPC levels. The results showed that decreased total intracellular GPC and increased phosphatidylcholine content but no change in betaine content in hADMSCs-Exo-treated cells (Fig. 8B). Next, we examined expression of genes encoding key enzymes in choline metabolism by qRT–PCR. The results revealed that the expression of genes coding for the choline transporter CTL1 (Slc44a1, Slc44a2, Slc44a3, Slc44a4), choline kinase alpha (Chkα, Chkβ) and phosphocholine cytidylyltransferase (Pcyt1α, Pcyt1β) had no statistically significant differences between hADMSCs-Exo-treated group and LEG or REG. However, exposure to hADMSCs-Exo increased transcription of Chpt1a, which encodes the choline phosphotransferase 1 (CHPT1) (Fig. 8C). These results were consistent with the transcriptome results (Additional file 1: Fig. S9). Furthermore, CHPT1 was confirmed to be augmented by hADMSCs-Exo both in vivo (Fig. 8D) and in vitro (Fig. 8E) by western blot analysis, while CHPT1 induction correlated with phosphatidylcholine biosynthesis from CDP-choline, suggesting that hADMSCs-Exo might activate activity of CHPT1 and played a central role in the formation and maintenance of vesicular membranes.

Fig. 8figure 8

hADMSCs-Exo regulates the choline metabolism, which involved in PI3K/AKT/mTOR signaling pathway to anti-liver fibrosis. A Quantitative analyses of extracellular and intracellular choline content in activated LX-2 cells or incubated with hADMSCs-Exo. B Quantitative analyses of total intracellular betaine, phosphatidylcholine and glycerophosphocholine content. C The mRNA levels of choline metabolism related genes were determined by qRT–PCR in livers of mice. D, E The protein levels of CHPTI were determined by western blotting in the liver tissues of the mice with different treatments and HSCs incubated with hADMSCs-Exo. F, G The expression level of profibrogenic markers (Collagen I, Vimentin and α-SMA) and PI3K/AKT signalling pathway proteins were determined by western blotting in activated LX-2 cells incubated with hADMSCs-Exo and treated (or not treated) with choline or phosphorylcholine. GAPDH was used as a loading control. Exo, exosomes. LFG, liver fibrosis group, REG, regression group. Slc44a1-4, Solute Carrier Family 44 Member. Chka, Choline Kinase Alpha. Chkb, Choline Kinase beta. Pcyt1a, Phosphate Cytidylyltransferase 1A. Pcyt1a, Phosphate Cytidylyltransferase 1B. CHPT1, Choline Phosphotransferase 1. CHPT1, diacylglycerol cholinephosphotransferase 1. PC, phosphorylcholine. Data expressed as the mean ± SEM (n = 3 independent experiments). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

We further investigated whether supplementing choline or phosphorylcholine synergize therapeutic effect of hADMSCs-Exo in LX-2 cells. As shown in Fig. 8F and Additional file 1: Fig. S10A, following supplementation with hADMSCs-Exo and 10 mM choline or 10 mM phosphorylcholine simultaneously in vitro, pro-fibrogenic protein expression of α-SMA, collagen I and vimentin significantly decreased compared to either individually. This demonstrated that the hADMSCs-Exo and choline synergistically enhanced anti-hepatic fibrosis efficacy. To determine whether hADMSCs-Exo-mediated enhanced choline uptake affects PI3K/AKT/mTOR signaling pathway in LX2 cells, we further examined protein expression and activated status of key proteins related to the PI3K/AKT/mTOR signaling pathway by western blot. Compared to PBS control group, addition of choline or phosphorylcholine resulted in decreases in the p-PI3K/PI3K ratio, lower AKT and mTOR levels (Fig. 8G, Additional file 1: Fig. S10B). These data support a role for hADMSCs-Exo in choline-mediated inhibition of the PI3K/AKT/mTOR signaling. In conclusion, these aforementioned data strongly demonstrated that hADMSCs-Exo played a vital role in alleviating LX2 cell activation and suppressing the progression of liver fibrosis through regulating the choline metabolism and inhibiting the PI3K/AKT/mTOR signaling pathway.

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