Human umbilical cord mesenchymal stem cells enhance liver regeneration and decrease collagen content in fibrosis mice after partial hepatectomy by activating Wnt/β-catenin signaling

Abstract

Liver fibrosis is a critical stage in the progression of various chronic liver diseases to cirrhosis and liver cancer. Early inhibition of liver fibrosis is crucial for the treatment of liver disease. Hepatectomy, a common treatment for liver-related diseases, promotes liver regeneration. However, in the context of liver fibrosis, liver regeneration is hindered. Many studies have shown that mesenchymal stem cells (MSCs) can promote liver regeneration after partial hepatectomy (PH). However, there are few reports on the impact of MSC therapy on liver regeneration post-PH in the context of hepatic fibrosis. The objective of this study is to examine the impact of MSCs on liver regeneration following PH in the fibrotic liver and uncover the related molecular mechanisms. This study reveals that MSC therapy significantly enhances liver function and mitigates liver inflammation after PH in the context of hepatic fibrosis. MSCs also significantly promote liver regeneration and alleviate liver fibrosis. In addition, this study identifies the role of MSCs in promoting liver regeneration and alleviating liver fibrosis via the activation of Wnt/β-catenin signaling. The combination of MSCs with hepatectomy may offer a novel approach for the treatment of liver fibrotic diseases.

Keywords: liver fibrosis; liver regeneration; mesenchymal stem cells; partial hepatectomy.

Figure 1

Characterization of H-uc MSCs (A) Study design. (B) Optical micrographs of the morphological characterization of MSCs at passage 4 (P4). Osteogenic differentiation of MSCs was assessed by alizarin red staining. Adipogenic differentiation of MSCs was assessed by Oil Red O staining. The magnification is 200× (scale bar: 100 μm). (C) Flow cytometry analysis of specific phenotype markers of MSCs at P4.

Figure 2

H-uc MSCs improve liver function and decrease inflammation (A) Representative images showing the gross liver morphology (scale bar: 5 mm). (B) Representative images of liver tissue sections subjected to H&E staining. The magnifications are 100× and 200× (scale bar: 100 μm). (C–F) Serum enzyme activities of ALT, AST, ALP and γ-GT. (G–J) Relative inflammatory gene expressions of IL-1, IL-6, IL-10, and TNF-α. Data are presented as the mean ± SD, n = 6. *P < 0.05, **P<0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

H-uc MSC treatment promotes liver regeneration (A) Photomicrographs of liver tissue sections showing IHC staining for Ki67 (black arrows indicate brown-positive cells). (B) Photomicrographs of liver tissue sections showing IHC staining for PCNA (black arrows indicate brown-positive cells). (C) Quantification of Ki67-positive cells. (D) Quantification of PCNA-positive cells. (E) Liver regeneration rates in the four groups of mice. (F) Liver weight/body weight ratio. (G) Western blot analysis of the PCNA protein. (H) Quantification of the relative intensity of PCNA protein. (I–L) The relative growth factor gene expressions of HGF, EGF, VEGF, and FGF. Data are presented as the mean ± SD, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 100 μm.

Figure 4

H-uc MSCs alleviate liver fibrosis in mice (A) Representative images of liver tissue sections subjected to Masson staining. The magnifications are 100× and 200×. (B) Representative images of liver tissue sections subjected to Sirius red staining. The magnifications are 100× and 200×. (C) Photomicrographs of liver tissue sections showing IHC staining for α-SMA. The magnifications are 100× and 200×. (D) Quantitative results of Masson staining. (E) Quantitative results of Sirius red staining. (F) Quantification of α-SMA-positive cells. (G) Western blot analysis of the α-SMA protein. (H) Quantification of the relative α-SMA protein intensity. (I) Quantitative analysis of hepatic Hyp content. Data are presented as the mean ± SD, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 100 μm.

Figure 5

H-uc MSCs activate Wnt/β-catenin signaling (A) Representative western blots of Wnt3a, β-catenin, Cyclin D1, and c-Myc in liver tissue. (B) Quantification of the relative intensity of the Wnt3a protein. (C) Quantification of the relative β-catenin protein intensity. (D) Quantification of the relative intensity of the Cyclin D1 protein. (E) Quantification of the relative intensity of the c-Myc protein. Data are presented as the mean ± SD, n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.