Evaluation of a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced mouse model in a comparative experimental study of portal hypertension

Figure 1 The 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet mouse model (4 weeks) exhibited portal pressure comparable to bile duct ligation and carbon tetrachloride mouse models. A-C: Schematics of the modeling procedures and timelines for 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet (A), bile duct ligation (BDL) (B), and carbon tetrachloride (CCl₄) mouse models (C), respectively; D and E: Portal pressure measurements and survival curve of sham (n = 8) and BDL (n = 7) groups; F and G: Portal pressure and survival curve in oil (n = 8), 8-week CCl₄ (n = 7), and 12-week CCl₄ (n = 8) groups; H-J: Longitudinal comparisons of body weight, portal pressure, and survival curve between normal chow diet and DDC groups at 2 (n = 5), 4 (n = 5), 6 (n = 5), and 8 (n = 4) weeks post-modeling. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NCD: Normal chow diet; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; CCl₄: Carbon tetrachloride; HR: Hazard ratio; CI: Confidence interval; NS: No significant.

Figure 2 The 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet mouse model (4 weeks) exhibited moderate biliary fibrosis in the portal vein region. A: Hematoxylin-eosin, Masson, and Sirius red staining of mouse livers; B-G: Statistical analysis of Masson and Sirius red staining results; H: Immunohistochemistry staining for collagen-1, α-smooth muscle actin (SMA), and desmin in mouse livers; I-Q: Statistical analysis of collagen-1, α-SMA, and desmin staining results. Sample sizes (n): 8:8 (normal chow diet vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine), 8:7 (sham vs bile duct ligation), and 8:7:8 (oil vs 8-week carbon tetrachloride vs 12-week carbon tetrachloride). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: No significant; NCD: Normal chow diet; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; CCl₄: Carbon tetrachloride; HE: Hematoxylin-eosin; COL1: Collagen-1; SMA: Smooth muscle actin.

Figure 3 The 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet mouse model (4 weeks) exhibited significant liver sinusoidal endothelial cell dysfunction. A: Scanning electron microscopy (SEM) of liver sinusoids in normal chow diet (NCD) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet mouse models; B: Statistical analysis of fenestrae number and porosity; C: SEM of liver sinusoids in sham and bile duct ligation (BDL) mouse models; D: Statistical analysis of fenestrae number and porosity; E: SEM of liver sinusoids in oil, 8-week carbon tetrachloride (CCl₄), and 12-week CCl₄ mouse models; F: Statistical analysis of fenestrae number and porosity; G and H: Protein expression and quantitative analysis of total endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) in NCD and DDC diet mouse livers; I and J: Protein expression and quantitative analysis of total eNOS and p-eNOS in sham and BDL mouse livers; K-M: Protein expression and quantitative analysis of total eNOS and p-eNOS in oil, 8-week CCl₄, and 12-week CCl₄ mouse livers; N: Immunohistochemistry staining for lymphatic vessel endothelial hyaluronan receptor 1 (LyVE-1), cluster of differentiation (CD) 34, and von Willebrand factor (vWF) in livers of the three model groups; O-W: Quantitative analysis of positive staining areas for LyVE-1, CD34, and vWF. Sample sizes (n): 8:8 (normal chow diet vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine), 8:7 (sham vs bile duct ligation), and 8:7:8 (oil vs 8-week carbon tetrachloride vs 12-week carbon tetrachloride). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: No significant; NCD: Normal chow diet; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; CCl₄: Carbon tetrachloride; eNOS: Endothelial nitric oxide synthase; p-eNOS: Phosphorylated endothelial nitric oxide synthase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LyVE-1: Lymphatic vessel endothelial hyaluronan receptor 1; vWF: Von Willebrand factor; CD: Cluster of differentiation.

Figure 4 The 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet induces abnormal proliferation of intrahepatic vascular structures and extrahepatic portosystemic shunting. A: Immunohistochemistry (IHC) staining for vascular endothelial growth factor receptor 2, cluster of differentiation 31, and vascular endothelial growth factor A in mouse livers; B-J: Statistical analysis of positive staining areas from IHC results; K-M: Hematoxylin-eosin staining of mesenteric tissues. Sample sizes (n): 8:8 (normal chow diet vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine), 8:7 (sham vs bile duct ligation), and 8:7:8 (oil vs 8-week carbon tetrachloride vs 12-week carbon tetrachloride). ^bP < 0.01. ^cP < 0.001. NS: No significant; NCD: Normal chow diet; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; CCl₄: Carbon tetrachloride; CD: Cluster of differentiation; VEGFR2: Vascular endothelial growth factor receptor 2; VEGF-A: Vascular endothelial growth factor A.

Figure 5 The 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced intrahepatic inflammatory responses contribute to elevated portal pressure. A: Immunohistochemistry staining for F4/80 in mouse livers; B: Statistical analysis of F4/80-positive staining areas; C and D: Detection of hepatic inflammatory index messenger RNA and liver enzymes in normal chow diet and 3,5-diethoxycarbonyl-1,4-dihydrocollidine groups; E and F: Detection of hepatic inflammatory index messenger RNA and liver enzymes in sham and bile duct ligation groups; G and H: Detection of hepatic inflammatory index messenger RNA and liver enzymes in oil, 8-week carbon tetrachloride (CCl₄), and 12-week CCl₄ mouse models. Sample sizes (n): 8:8 (normal chow diet vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine), 8:7 (sham vs bile duct ligation), and 8:7:8 (oil vs 8-week carbon tetrachloride vs 12-week carbon tetrachloride). ^bP < 0.01. ^cP < 0.001. NS: No significant; NCD: Normal chow diet; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; CCl₄: Carbon tetrachloride; IL: Interleukin; TNF: Tumor necrosis factor; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; T-Bil: Total bilirubin; D-Bil: Direct bilirubin; TBA: Total bile acid.

Figure 6 The 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet induces ductular reactions comparable to those in the bile duct ligation model. A: Immunohistochemistry staining for cytokeratin (CK) 7, CK19, and SOX9 in mouse livers; B-J: Statistical analysis of positive staining areas for CK7, CK19, and SOX9. Sample sizes (n): 8:8 (normal chow diet vs 3,5-diethoxycarbonyl-1,4-dihydrocollidine), 8:7 (sham vs bile duct ligation), and 8:7:8 (oil vs 8-week carbon tetrachloride vs 12-week carbon tetrachloride). ^bP < 0.01. ^cP < 0.001. NS: No significant; NCD: Normal chow diet; DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; CCl₄: Carbon tetrachloride; CK: Cytokeratin.