Human bone marrow mesenchymal stem cells-derived exosomes alleviate liver fibrosis through the Wnt/β-catenin pathway

Abstract

Background: Mesenchymal stem cells (MSCs) are increasingly being applied as a therapy for liver fibrosis. Exosomes possess similar functions to their parent cells; however, they are safe and effective cell-free reagents with controllable and predictable outcomes. In this study, we investigated the therapeutic potential and underlying molecular mechanism for human bone mesenchymal stem cells-derived exosomes (hBM-MSCs-Ex) in the treatment of liver fibrosis.

Methods: We established an 8-week CCl₄-induced rat liver fibrosis model, after which, we administered hBM-MSCs-Ex in vivo for 4 weeks. The resulting histopathology, liver function, and inflammatory cytokines were analyzed. In addition, we investigated the anti-fibrotic mechanism of hBM-MSCs-Ex in hepatic stellate cells (HSCs) and liver fibrosis tissue, by western blotting for the expression of Wnt/β-catenin signaling pathway-related genes.

Results: In vivo administration of hBM-MSCs-Ex effectively alleviated liver fibrosis, including a reduction in collagen accumulation, enhanced liver functionality, inhibition of inflammation, and increased hepatocyte regeneration. Moreover, based on measurement of the collagen area, Ishak fibrosis score, MDA levels, IL-1, and IL-6, the therapeutic effect of hBM-MSCs-Ex against liver fibrosis was significantly greater than that of hBM-MSCs. In addition, we found that hBM-MSCs-Ex inhibited the expression of Wnt/β-catenin pathway components (PPARγ, Wnt3a, Wnt10b, β-catenin, WISP1, Cyclin D1), α-SMA, and Collagen I, in both HSCs and liver fibrosis tissue.

Conclusions: These results suggest that hBM-MSCs-Ex treatment could ameliorate CCl₄-induced liver fibrosis via inhibition of HSC activation through the Wnt/β-catenin pathway.

Keywords: Exosomes; Liver fibrosis; Wnt/β-catenin; hBM-MSCs.

Fig. 1

Exosome characterization. a Morphological appearance of cultured hBM-MSCs (bar = 100 μm). b Morphological analysis of hBM-MSCs-Ex by transmission electron microscopy (bar = 100 nm and 200 nm). c Size distribution measurements under flow conditions by nanoparticle tracking with the corresponding video frame. d Western blot assay indicated the positive expression of CD9, CD63, CD81, TSG101 and Alix proteins in hBM-MSCs-Ex

Fig. 2

hBM-MSCs-Ex alleviates liver fibrosis in rats. a Study design. b Representative images showing the gross morphology and histological analysis of liver (scale bar = 1 mm), n = 12. c Collagen proportionate area quantification by computer-assisted image analysis at 4 weeks. d Ishak scoring criteria. e, f Quantitative analysis of hepatic Hyp and MDA content. **p < 0.01, ***p < 0.001. Data: n = 12; mean ± SD

Fig. 3

hBM-MSCs-Ex improves liver function and decreases inflammation. a ALT, alanine aminotransferase. b AST, aspartate aminotransferase. c TP, total protein. d TBIL, total bilirubin. e ALP, alkaline phosphatase. f γ-GT, gamma glutamyl transpeptidase. g–l The relative inflammatory gene expression for IL-1, IL-2, IL-6, IL-8, IL-10, and TNF-α. *p < 0.05, **p < 0.01, ***p < 0.001. Data: n = 12; mean ± SD

Fig. 4

hBM-MSCs-Ex increases Ki-67⁺ and decreases α-SMA⁺ in a rat model of liver fibrosis. a Photomicrographs of liver tissue sections showing IHC staining for α-SMA and Ki-67 (black arrows indicate brown-positive cells, bar = 1 mm). b–d Quantification of α-SMA and Ki-67-positive cells was performed by computer-assisted image analysis. **p < 0.01, ***p < 0.001. Data: n = 12; mean ± SD

Fig. 5

hBM-MSCs-Ex inhibits the Wnt/β-catenin signaling pathway. a, c Representative western blotting analysis measuring PPARγ, Wnt3a, Wnt10b, β-catenin, WISP1, Cyclin D1, α-SMA, and Collagen I in HSCs and liver fibrosis tissue. b, d The quantification of protein relative intensity. ***p < 0.001, ***p < 0.001. Data, n = 3; mean ± SD

Fig. 6

hBM-MSCs-Ex treatment alleviates liver fibrosis in both HSCs and liver fibrosis tissues through inhibition of Wnt/β-catenin signaling (PPARγ, Wnt3a, Wnt10b, β-catenin) and the downregulation of downstream gene expression (WISP1, Cyclin D1). This inhibits HSC activation to prevent further liver fibrosis