Multi-parameter magnetic resonance imaging of zebularine in liver fibrosis treatment and calcineurin/NFAT3 mechanism

Figure 1 Example of region of interest placement in Balb/C mice. A: Three regions of interest (ROIs) were allocated to the same liver-parenchyma region in the plain stage, and T1 relaxation time was measured; B: Three ROIs were allocated to the same liver-parenchyma region in the hepatobiliary stage, and T1 relaxation time was measured.

Figure 2 Toxicity of zebularine in LX-2 cells. A: Histogram showing zebularine concentration-cell viability; B: Nonlinear regression curves [log (zebularine) - relative cell viability]. ^cP < 0.001. ^dP < 0.0001.

Figure 3 Expression of regulator of calcineurin1. 4, calcineurin, and NFAT3 pairs in transforming growth factor-β1-activated LX-2 by zebularine action. A: Expression of regulator of calcineurin1.4 and calcineurin proteins; B: Intranuclear protein expression of NFAT3; C: Cytoplasmic protein expression of NFAT3. ^aP < 0.05. ^bP < 0.01. RCAN: Regulator of calcineurin; CaN: Calcineurin; TGF: Transforming growth factor.

Figure 4 Microscopic changes of LX-2 cells activated by transforming growth factor-β1 by zebularine action. A: Control group; B: Transforming growth factor-β1 group; C: Zebularine group; D: Transforming growth factor-β1 + zebularine group.

Figure 5 Effect of zebularine on anti-fibrotic and fibrotic gene expression in transforming growth factor-β1-activated LX-2. The message RNA levels of the following genes in LX-2 cells were measured before and after transforming growth factor-β1 activation and after zebularine treatment. A: α-smooth muscle actin (α-SMA); B: Type I collagen; C: SEPTIN9; D: PTEN; E: PPAR-γ; F: PTCH1; G: Protein expression of α-SMA in LX-2 cells following zebularine intervention at varying concentrations. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. ¹P vs control. ²P vs transforming growth factor-β1. ³P vs transforming growth factor-β1 + zebularine 70 mmol/L. ⁴P vs zebularine 70 mM. α-SMA: α-smooth muscle actin; TGF: Transforming growth factor; CoL1a1: Type I collagen; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 6 Receiver operating characteristic curves for T1pre, DT1, apparent diffusion coefficient, T2, and the combined diagnostic approach for liver fibrosis. AUC: Area under the receiver operating characteristic curve; ADC: Apparent diffusion coefficient; Combination: Combined diagnosis.

Figure 7 Spearman correlation analysis of magnetic resonance parameters with fibrosis and inflammation. A-D: Fibrosis; E-H: Inflammation. ADC: Apparent diffusion coefficient.

Figure 8 Changes in magnetic resonance parameters in the control, carbon tetrachloride, and carbon tetrachloride + zebularine groups. Red dotted lines indicate the entire liver border in: A: T2-weighted images; B: Pre-contrast T1 maps; C: Post-contrast T1 maps; D: T2 maps; E: Apparent diffusion coefficient maps. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. ¹P vs control. ²P vs carbon tetrachloride. T2WI: T2-weighted imaging; CCl₄: Carbon tetrachloride; ADC: Apparent diffusion coefficient.

Figure 9 Representative images of mouse livers after carbon tetrachloride and zebularine treatment. A: Carbon tetrachloride group; B: Carbon tetrachloride + zebularine group; C: Control group.

Figure 10  Changes in body weight, liver weight, and hepatosomatic index in mice treated with carbon tetrachloride and zebularine. A: Body weight at 2, 4, and 6 weeks; B: Mouse liver mass-timed histograms for each group; C: Hepatosomatic index-timed histograms for each group (n = 12). ^aP < 0.05. ^bP < 0.01. ^dP < 0.0001. ¹P vs control. ²P vs carbon tetrachloride. CCl₄: Carbon tetrachloride.

Figure 11  Effects of carbon tetrachloride and zebularine treatments on serum aspartate aminotransferase and aspartate aminotransferase levels in each experimental group. A: Alanine aminotransferase (U/L) time histogram; B: Aspartate aminotransferase (U/L) time histogram (n = 12). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. ¹P vs control. ²P vs carbon tetrachloride. CCl₄: Carbon tetrachloride; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

Figure 12  Masson’s staining showing the effect of carbon tetrachloride and zebularine treatment on collagen fibers in mouse liver tissue. A: Representative images of liver sections stained with Masson’s trichrome; B: Collagen fiber percentage at 2, 4, and 6 weeks; C: METAVIR fibrosis grading at 2, 4, and 6 weeks (n = 12 mice/group). Original magnification: × 200. ^aP < 0.05. ^bP < 0.01. ^dP < 0.0001. ¹P vs control. ²P vs carbon tetrachloride. CCl₄: Carbon tetrachloride.

Figure 13  Hematoxylin and eosin staining showing the effect of carbon tetrachloride and zebularine on inflammation in mouse liver tissue. A: Hematoxylin and eosin staining of liver sections from each experimental group; B: Liver inflammation grading (METAVIR) at 2, 4, and 6 weeks (n = 12 mice/group). Original magnification: × 200. ^cP < 0.001. ^dP < 0.0001. P vs control. HE: Hematoxylin and eosin; CCl₄: Carbon tetrachloride.