Mesenchymal stem cell-derived exosomes promote hepatic regeneration in drug-induced liver injury models

Abstract

Introduction: Mesenchymal stem cell-conditioned medium (MSC-CM) has been shown to have protective effects against various cellular-injury models. This mechanism of protection, however, has yet to be elucidated. Recently, exosomes were identified as the active component in MSC-CM. The aim of this study is to investigate the effect of MSC-derived exosomes in an established carbon tetrachloride (CCl4)-induced liver injury mouse model. This potential effect is then validated by using in vitro xenobiotic-induced liver-injury assays: (1) acetaminophen (APAP)- and (2) hydrogen peroxide (H2O2)-induced liver injury.

Methods: The exosomes were introduced concurrent with CCl4 into a mouse model through different routes of administration. Biochemical analysis was performed based on the blood and liver tissues. Subsequently the exosomes were treated in APAP and H2O2-toxicants with in vitro models. Cell viability was measured, and biomarkers indicative of regenerative and oxidative biochemical responses were determined to probe the mechanism of any hepatoprotective activity observed.

Results: In contrast to mice treated with phosphate-buffered saline, CCl4 injury in mice was attenuated by concurrent-treatment exosomes, and characterized by an increase in hepatocyte proliferation, as demonstrated with proliferating cell nuclear antigen (PCNA) elevation. Significantly higher cell viability was demonstrated in the exosomes-treated group compared with the non-exosome-treated group in both injury models. The higher survival rate was associated with upregulation of the priming-phase genes during liver regeneration, which subsequently led to higher expression of proliferation proteins (PCNA and cyclin D1) in the exosomes-treated group. Exosomes also inhibited the APAP- and H2O2-induced hepatocytes apoptosis through upregulation of Bcl-xL protein expression. However, exosomes do not mitigate hepatocyte injury via modulation of oxidative stress.

Conclusions: In summary, these results suggest that MSC-derived exosomes can elicit hepatoprotective effects against toxicants-induced injury, mainly through activation of proliferative and regenerative responses.

Figure 1

Effect of exosomes on biochemical parameters and hepatocyte injury after CCl₄ treatment in vivo. Serum (A) aspartate aminotransferase (AST) and (B) alanine aminotransferase (ALT) levels were measured after 24-hour dosing of CCl₄ with or without exosomes administration (n = 6 per group; *P < 0.05 versus of CCl₄ control for both AST and ALT). Standard hematoxylin and eosin (H&E) staining for liver tissue harvested after 24 hours of (C) CCl₄ administration and (D) CCl₄ with exosomes treatment. Obvious hepatic necrosis appeared in the section from a CCl₄-treated animal. Original magnification × 80; scale bar, 100 μm.

Figure 2

Effect of exosomes on hepatocyte proliferation after CCl₄-induced injury in mice. (A) Expression of cyclin E, HGF, pMet, NF-κB p65, cyclin D1, and phosphorylated STAT3 was determined by immunoblotting after 24 hours of CCl₄ administration with or without exosomes (n = 6 per group (three per group were shown); *P < 0.05 versus CCl₄ control). The relative densities of all the proteins bands were normalized to actin in the same samples. Livers were processed for immunohistochemistry on PCNA to quantify hepatocyte proliferation after 24 hours of (B) CCl₄ administration and (C) CCl₄ with exosomes treatment. Original magnification × 200; scale bar, 100 μm.

Figure 3

Effect of exosomes in cell viability after APAP- and H₂O₂-induced injury. Experiments were performed in TAMH (A-C), THLE-2 (D-F), and Huh-7 (G-I) hepatocytes. (A, D, G) Cytotoxicity of exosomes at different concentrations in respective cell lines. 0.05 μg/ml and 0.1 μg/ml of exosomes were added to respective concentration of APAP (B, E, H) and H₂O₂(C, F, I), and MTT were performed 24 or 72 hours later. Cell viability was normalized against vehicle control group and expressed in percentage (n = 6 per group; *P < 0.05 versus APAP or H₂O₂ non-exosomes-treatment group.

Figure 4

Effect of exosomes in hepatoregenerative-expression genes on APAP- and H₂O₂-induced injury in hepatocytes. Expression of regenerative-response genes after 24 hours of exosomes in (A) APAP and (B) H₂O₂ treatment was determined by quantitative real-time PCR and normalized against GAPDH expression of the same sample and presented as fold-increase over the controls. Bars represent means ± SEM. *P < 0.05 versus APAP or H₂O₂ nonexosomes treatment group.

Figure 5

Effect of exosomes in G₁phase of the cell cycle after APAP- or H₂O₂-induced injury in hepatocytes. Expression of NF-κB (p65 and p50) and phosphorylated STAT3 was determined by immunoblotting after 24-hour treatment in APAP- or H₂O₂-injury models. The relative densities of NF-κB and phosphorylated STAT3 bands were normalized to actin in the same samples.

Figure 6

Effect of exosomes in cell proliferation after APAP- or H₂O₂-induced injury in hepatocytes. Expression of cyclin D1 and PCNA was determined by immunoblotting after 24-hour treatment in the APAP- or H₂O₂-injury model. The relative densities of cyclin D1 and PCNA bands were normalized to actin in the same samples.

Figure 7

Effect of exosomes on antiapoptosis in APAP- or H₂O₂-induced injury in hepatocytes. (A) Caspase 3/7 was measured after 24-hour treatment of exosomes in APAP or H₂O₂ injury. (B) Expression of Bcl-_xL was determined by immunoblotting after 24-hour treatment in the APAP- or H₂O₂-injury model. *P < 0.05 versus APAP or H₂O₂ non-exosomes treatment group. The relative densities of Bcl-_xL bands were normalized to actin in the same samples.

Figure 8

Effect of exosomes on antioxidation in APAP- or H₂O₂-induced injury in hepatocytes. Liver stress/defense-related genes expression in TAMH cells treated with or without exosomes concurrently in (A) 2 mM APAP or (B) 350 μM H₂O₂ for 24 hours. All the expressions were determined by quantitative real-time PCR, normalized against GAPDH expression of the same sample and presented as fold-increase over the controls. Bars represent mean ± SEM.