New role and molecular mechanism of Gadd45a in hepatic fibrosis

Figure 1 Expression of fibrogenic proteins and extracellular matrix deposition in the liver of mice exposed to CCl₄. A: Relative mRNA expression of collagen I, fibronectin, and α-SMA after 1, 2, 3, and 4 wk of CCl₄ treatment, as determined by quantitative RT-PCR; B: Protein expression of collagen I, fibronectin, and α-SMA after 1, 2, and 4 wk of CCl₄ treatment, as determined by Western blot; C: Picrosirius red staining shows hepatic collagen deposition in mice treated with CCl₄ for 4 wk; saline treatment served as the control group. SMA: Smooth muscle actin.

Figure 2 Chronic CCl₄ injury suppresses Gadd45a expression and activates TGF-β/Smad signaling. A: Relative mRNA expression levels of Gadd45a, Gadd45b, and Gadd45g in the livers of mice treated with CCl₄ for 1, 2, and 4 wk; the saline-treated group served as the control group; B: Relative protein expression levels of Gadd45a, Gadd45b, and Gadd45g in the livers of mice treated with CCl₄ for 1, 2, and 4 wk; C: Immunofluorescence staining of Gadd45a, Gadd45b, and Gadd45g (in green) in HSCs treated with CCl₄ for 4 wk (nuclei are stained blue with DAPI; scale bars = 10 μm); D: Expression levels of p-Smad2, p-Smad3, Smad2, Smad3, and Smad4 were analyzed in HSCs treated with CCl₄ for 1, 2, and 4 wk. HSC: Hepatic stellate cells; TGF: Transforming growth factor.

Figure 3 Gadd45a inhibits the expression of extracellular matrix proteins and α-smooth muscle actin in CCl₄-treated hepatic stellate cells. A: Expression of type I collagen, fibronectin, and α-SMA in HSCs treated with CCl₄vs saline for 24 h; B: Expression of Gadd45a, Gadd45b, and Gadd45g in HSCs treated with CCl₄vs saline for 24 h; C: Expression of type I collagen, fibronectin, α-SMA, Gadd45a, Gadd45b, and Gadd45g in HSCs transfected with pcDNA3.1-Gadd45a or siRNA-Gadd45a. HSC: Hepatic stellate cell; SMA: Smooth muscle actin.

Figure 4 Gadd45a inhibits hepatic fibrosis through the suppression of transforming growth factor-β/Smad signaling. A: Effects of Gadd45 on PAI-1-Luc activity in HSCs treated with pcDNA3.1, Gadd45a, and Gadd45a siRNA in both the CCl₄ and saline groups; B: Effects of Gadd45 on (CAGA)9 MLP-Luc activity in HSCs treated with pcDNA3.1, Gadd45a, and Gadd45a siRNA in both the CCl₄ and saline groups. HSC: Hepatic stellate cell; TGF: Transforming growth factor.

Figure 5 Gadd45a exerts reactive oxygen species scavenging effects by upregulating the expression of antioxidant enzymes. A: The effects of Gadd45a on ROS generation in CCl₄-pretreated HSCs treated with pcDNA3.1, PCDNA3.1-Gadd45a, scrambled siRNA, and Gadd45a siRNA were detected by flow cytometry; B: Effects of Gadd45a on HO-1 and NQO1 RNA expression in CCl₄-pretreated HSCs treated with pcDNA3.1, PCDNA3.1-Gadd45a, scrambled siRNA, and Gadd45a siRNA; C: Effects of Gadd45a on HO-1 and NQO1 protein expression in CCl₄-pretreated HSCs treated with pcDNA3.1, PCDNA3.1-Gadd45a, scrambled siRNA, and Gadd45a siRNA. HSC: Hepatic stellate cells; ROS: Reactive oxygen species.

Figure 6 Gadd45a inhibits the transforming growth factor-β/Smad signaling pathways partly due to its antioxidant properties. A: Relative mRNA expression of type I collagen and α-SMA as determined by quantitative RT-PCR. Hepatic stellate cells were stimulated with CCl₄ in the presence or absence of glutathione for 24 h; B: Western blot analyses of type I collagen and α-SMA expression; C: Activation of the TGF-β/Smad signaling pathways was measured by Western blot analyses of p-Smad2, p-Smad3, Smd2, Smad3, and Smad4. GSH: Glutathione; SMA: Smooth muscle actin; TGF: Transforming growth factor.