Despite increasing knowledge of the cellular and molecular mechanisms underlying liver fibrosis, no Western medicine has been approved to treat the condition. Mesenchymal stem cells (MSCs), which are multipotent progenitor cells, offer an attractive therapeutic approach for treating tissue damage and inflammation. A recent study published in Hepatology International identified the protective effects and mechanisms of human umbilical cord blood-derived MSCs (UC-MSCs) on thioacetamide (TAA)-induced liver fibrosis.
The researchers induced liver fibrosis in mice via intraperitoneal injections of TAA, followed by the administration of either UC-MSCs or UC-MSC-derived exosomes (UC-MSCs-Exo) through tail vein injection in some mice. Histological analyses were then performed on liver tissue samples.Identification and Characterization of UC-MSCs and UC-MSCs-Exo
The researchers isolated UC-MSCs from human umbilical cord tissue. These cells were spindle-shaped, adherent, and capable of differentiating into adipocytes, osteoblasts, and chondrocytes. Flow cytometry analysis revealed high expression of CD90, CD105, and CD73 in UC-MSCs, with minimal expression of CD34, CD19, CD45, and HLA-DR. Transmission electron microscopy showed that purified UC-MSCs-Exo had a characteristic disc-shaped morphology, encased in a lipid bilayer, with an average diameter between 30 and 150 nm. The exosomes expressed Alix and CD9 but did not express Calnexin.
Reduction of TAA-Induced Liver Inflammation and Improved Liver Function
The effectiveness of UC-MSCs-Exo was evaluated in the TAA-induced mouse liver fibrosis model. Hematoxylin and eosin (H&E) staining showed significant increases in inflammatory cell infiltration in the liver tissues of TAA-treated mice compared to controls. However, mice treated with UC-MSCs or UC-MSCs-Exo exhibited a marked reduction in inflammatory cell infiltration. Biochemical analyses revealed that serum ALT and AST levels were significantly lower in the UC-MSCs and UC-MSCs-Exo treatment groups.
Additionally, the researchers measured the expression of pro-inflammatory mediators in liver tissue using qRT-PCR. UC-MSCs and UC-MSCs-Exo treatment reduced mRNA levels of pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, and Mcp-1, compared to the PBS-treated control group. Expression of Adgre1 was also significantly lower in the UC-MSCs and UC-MSCs-Exo groups. Thus, both treatments improved liver function and alleviated liver inflammation.
Alleviation of Liver Fibrosis
Sirius red staining and Masson’s trichrome staining showed significantly reduced collagen deposition in the livers of mice treated with UC-MSCs and UC-MSCs-Exo compared to controls. Immunofluorescence staining revealed decreased expression of α-SMA, a specific marker for liver fibrosis and hepatic stellate cell (HSC) activation, in the treatment groups. qRT-PCR analysis demonstrated significant reductions in the mRNA levels of fibrosis-related genes, including Acta2, Col1α1, Col3α1, and Timp1, in the UC-MSCs and UC-MSCs-Exo groups. These findings indicate that both UC-MSCs and UC-MSCs-Exo significantly alleviated TAA-induced liver fibrosis in mice.
Inhibition of SMO Expression in Fibrotic Liver
Analysis of the GEO database showed that SMO mRNA levels were significantly higher in liver tissues of TAA-induced fibrotic mice compared to controls, but lower in recovered liver tissues. Further analysis revealed a positive correlation between SMO mRNA levels and the mRNA levels of Col1α1 and Acta2.
Western blot analysis demonstrated increased SMO protein expression in the liver following TAA treatment, which was significantly reduced by UC-MSCs and UC-MSCs-Exo treatment. Immunofluorescence staining further confirmed that UC-MSCs and UC-MSCs-Exo treatment significantly inhibited SMO expression in the liver. These results suggest that UC-MSCs and UC-MSCs-Exo exert their therapeutic effects by inhibiting SMO expression.
UC-MSCs-Exo Inhibit HSC Activation by Suppressing the Hedgehog/SMO Pathway
The relationship between UC-MSCs-Exo’s anti-fibrotic effects and the Hedgehog/SMO pathway was further investigated in vitro. Western blot analysis revealed that α-SMA expression was significantly upregulated in TGF-β-treated LX2 cells but was suppressed by UC-MSCs-Exo treatment. Additionally, UC-MSCs-Exo reduced the expression of SMO and Gli1 in LX2 cells. Immunofluorescence staining corroborated these results, showing that UC-MSCs-Exo inhibited the expression of α-SMA, SMO, and Gli1 in LX2 cells. Furthermore, UC-MSCs-Exo treatment decreased mRNA levels of Hedgehog target genes, including Ptch1, Gli1, and Cyclin D1, in LX2 cells. Pre-treatment of LX2 cells with the SMO agonist SAG reversed the inhibitory effects of UC-MSCs-Exo on HSC activation.
UC-MSCs-Exo’s Therapeutic Effects Reversed by SAG Treatment In Vivo
To further confirm that UC-MSCs-Exo alleviated liver fibrosis by targeting the Hedgehog/SMO signaling pathway in vivo, SAG was injected into TAA-induced fibrotic mice. H&E staining and biochemical analysis of serum ALT/AST levels showed that SAG+UC-MSCs-Exo treatment increased liver injury compared to UC-MSCs-Exo treatment alone. This was accompanied by increased expression of pro-inflammatory mediators, including TNF-α, IL-6, Mcp-1, and Adgre1. Sirius red and Masson’s trichrome staining demonstrated that SAG treatment reversed the anti-fibrotic effects of UC-MSCs-Exo in TAA-induced fibrotic mice. Analysis of liver tissue gene expression showed that Acta2, Col1α1, Col3α1, and Timp1 mRNA levels were significantly upregulated in the SAG+UC-MSCs-Exo treatment group.
Overall, this study suggests that UC-MSCs-Exo exerts its therapeutic effects on liver fibrosis by inhibiting the Hedgehog/SMO signaling pathway.
