DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl4-induced liver fibrosis by suppressing Wnt/β-catenin signaling

Abstract

Liver fibrosis, as a consequence of chronic liver disease, involves the activation of hepatic stellate cell (HSC) caused by various chronic liver injuries. Emerging evidence suggests that activation of HSC during an inflammatory state can lead to abnormal accumulation of extracellular matrix (ECM). Investigating novel strategies to inhibit HSC activation and proliferation holds significant importance for the treatment of liver fibrosis. As a member of the doublecortin domain-containing family, doublecortin domain containing 2 (DCDC2) mutations can lead to neonatal sclerosing cholangitis, but its involvement in liver fibrosis remains unclear. Therefore, this study aims to elucidate the role of DCDC2 in liver fibrosis. Our findings revealed a reduction in DCDC2 expression in both human fibrotic liver tissues and carbon tetrachloride (CCl₄)-induced mouse liver fibrotic tissues. Furthermore, exposure to transforming growth factor beta-1(TGF-β1) stimulation resulted in a dose- and time-dependent decrease in DCDC2 expression. The overexpression of DCDC2 inhibited the expression of α-smooth muscle actin (α-SMA) and type I collagen alpha 1 (Col1α1), and reduced the activation of HSC stimulated with TGF-β1. Additionally, we provided evidence that the Wnt/β-catenin signaling pathway was involved in this process, wherein DCDC2 was observed to inhibit β-catenin activation, thereby preventing its nuclear translocation. Furthermore, our findings demonstrated that DCDC2 could attenuate the proliferation and epithelial-mesenchymal transition (EMT)-like processes of HSC. In vivo, exogenous DCDC2 could ameliorate CCl₄-induced liver fibrosis. In summary, DCDC2 was remarkably downregulated in liver fibrotic tissues of both humans and mice, as well as in TGF-β1-activated HSC. DCDC2 inhibited the activation of HSC induced by TGF-β1 in vitro and fibrogenic changes in vivo, suggesting that it is a promising therapeutic target for liver fibrosis and warrants further investigation in clinical practice.

Keywords: DCDC2; Hepatic stellate cell activation; Liver fibrosis; Wnt/β-catenin signaling.

Figure 1

Low expression of DCDC2 in human and mouse liver fibrotic tissues. (A) Hematoxylin and eosin (H&E), Sirius red staining and immunohistochemistry of DCDC2 were performed in tissues from human healthy controls and liver fibrosis samples. (B) The mRNA expression level of DCDC2 was measured using qPCR in human healthy control and liver fibrosis samples. (C) The protein expression levels of DCDC2, α-smooth muscle actin (α-SMA), and type I collagen alpha 1 (Col1α1) were assessed via western blot analysis in human healthy control and liver fibrosis samples. (D) H&E, Sirius red staining, Masson trichrome staining and immunohistochemistry analyses were conducted on liver tissues obtained from mice. (E) The protein expression levels of DCDC2, α-SMA, and Col1α1 were assessed via western blot analysis in liver tissues obtained from mice. (F) Immunofluorescence staining of DCDC2 (green) and α-SMA (red) in liver tissues from mice. Scale bar, 50 µM. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with Control, * P < 0.05.

Figure 2

Downregulation of DCDC2 in HSC activated by TGF-β1. (A) LX-2 cells were treated with different concentrations of TGF-β1 for 24 h. The protein expression levels of α-SMA and Col1α1 were detected using western blot. (B) LX-2 cells were treated with 10 ng/mL TGF-β1 at different time points. The protein expression levels of DCDC2, α-SMA and Col1α1 were assessed using western blot analysis. (C) The mRNA levels of DCDC2, α-SMA and Col1α1 were measured using qPCR. (D) Immunofluorescence staining of DCDC2 (green) and α-SMA (red) in LX-2 cells at 0 and 24 h following treatment with 10 ng/mL TGF-β1. (E) The efficiency of DCDC2 overexpression or silencing was measured by western blot. Scale bar, 50 µM. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with TGF-β1 0 ng/mL group (A) or TGF-β1 10 ng/mL 0 h group (D), * P < 0.05.

Figure 3

DCDC2 inhibits the activation of HSC induced by TGF-β1 by targeting the Wnt/β-catenin pathway. (A) The protein expression levels of β-catenin, α-SMA, and Col1α1 were assessed via western blot analysis subsequently to transfecting LX-2 cells with DCDC2 overexpressing lentiviral vector and negative control vector, followed by stimulation with TGF-β1. (B) The protein expression levels of β-catenin, α-SMA, and Col1α1 were evaluated through western blot analysis after transfecting LX-2 cells with DCDC2-sgRNA and negative control sgRNA, followed by stimulation by TGF-β1. (C) The protein expression levels of β-catenin, cyclinD1, and c-myc were detected by western blot after LX-2 cells were transfected with DCDC2 overexpressing lentiviral vector and/or then stimulated by SKL2001. (D) The protein expression levels of β-catenin, cyclinD1, and c-myc were assessed via western blot analysis after transfecting LX-2 cells with DCDC2-sgRNA and/or then stimulated by ICG001. (E) The protein expression levels of β-catenin in the cytoplasm or nuclear after DCDC2 was overexpressed or knockdown was measured using western blot. (F) Immunofluorescence staining of β-catenin nuclear translocation in LX-2 cells following DCDC2 was overexpressed. Scale bar, 50 µM. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with Con, # compare with TGF-β1 +VE group (A, B), OE group (C) or sgRNA group (D), *, # P < 0.05.

Figure 4

DCDC2 suppresses HSC proliferation and attenuates EMT-like transitions in vitro. (A,B) The cell cycle phase distribution was measured using flow cytometry analyses after LX-2 cells were transfected with DCDC2 overexpressing lentiviral vector or DCDC2-sgRNA. (C,D) EdU assay was performed to examine the proliferation of LX-2 cells following DCDC2 overexpression or knockdown. (E) The protein expression of E-cadherin, N-cadherin, vimentin, and Snail1 was detected using western blot after LX-2 cells were transfected with DCDC2 overexpressing lentiviral vector or DCDC2-sgRNA. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with VE group or NC group, *P < 0.05.

Figure 5

Overexpression of DCDC2 improves liver fibrosis in CCl₄-induced mouse model. (A) The timeline of the animal experiment. Two weeks following the initial CCl₄ injection, a single dose of lentivirus carrying DCDC2 or the control was administered via the tail vein. After 5 weeks of CCl₄ treatment, the mice were sacrificed. (B) The protein expression levels of β-catenin, α-SMA, and Col1α1 were detected by western blot after overexpression of DCDC2 in mice. (C) Immunohistochemistry was performed to detect the expression of β-catenin and α-SMA in tissues from mice treated with either control virus or LV-DCDC2. (D) H&E, Sirius red staining and Masson trichrome staining in CCl₄ mice following LV-DCDC2 treatment. (E) Serum ALT and AST levels were determined in CCl₄ mice after LV-DCDC2 treatment. The experiment was repeated at least three times and statistical data were presented as mean ± SD. * compare with Con group, # compare with CCl₄ group, *, # P < 0.05.