Effect of Yiguanjian decoction on cell differentiation and proliferation in CCl4-treated mice

Figure 1 Serum alanine aminotransferase activity in mice. ALT activities in CCl₄ groups increased continuously but declined in YGJ + CCl₄ groups over the entire period. ^bP < 0.01 vs the week 0 control group; ^cP < 0.05, ^dP < 0.01 vs the same time-point CCl₄ group. Results are presented as mean ± SD. ALT: Alanine aminotransferase; CCl₄: Carbone tetrachloride; YGJ: Yiguanjian.

Figure 2 Collagen staining with Sirius red and Hyp content. A: Collagen staining. Week 0 control mice have limited collagen in the portal area, but fibrous septa bridging the portal tracts were found in the livers of CCl₄-treated mice, and this was significantly attenuated in YGJ-treated mice, × 200; B: Semi-quantification of collagen staining, the week 0 control group level was set as the basal level; C: Hyp content in the liver. Results of collagen staining and Hyp content show that they both continuously increased in the CCl₄-treated group but declined in the YGJ + CCl₄ group over the entire period. ^aP < 0.05, ^bP < 0.01 vs week 0 control group; ^cP < 0.05, ^dP < 0.01 vs the same time-point CCl₄ group. Results are presented as mean ± SD. CCl₄: Carbone tetrachloride; YGJ: Yiguanjian; Hyp: Hydroxyproline.

Figure 3 Immunostaining and Western blot analysis of α-smooth muscle actin in liver tissues. A: α-smooth muscle actin (α-SMA) immunostaining, × 200; B: Semi-quantification of α-SMA positive area, with the level in the week 0 control group set as the basal level; C: Western blotting of α-SMA; D: Semi-quantification of the α-SMA Western blot results, with the week 0 control group level set as the basal level. ^bP < 0.01 vs week 0 control group; ^dP < 0.01 vs the same time-point CCl₄ group. Results are mean ± SD. CCl₄: Carbone tetrachloride; YGJ: Yiguanjian; GAPDH: Glyceraldehydes-3-phosphate dehydrogenase.

Figure 4 Immunofluorescent colocalization of enhanced green fluorescent protein and α-smooth muscle actin in CCl₄-induced chronic liver injury in bone marrow chimera mice, × 200. EGFP is shown in green and α-smooth muscle actin (α-SMA) in red. A yellow color confirms colocalization in the merged images. The number of EGFP/α-SMA double positive cells was found to increase in all mice after receiving CCl₄ injection and they were mainly found in the areas of scarring. In contrast, the number of EGFP⁺/α-SMA⁺ cells decreased in the YGJ treatment group. EGFP: Enhanced green fluorescent protein; YGJ: Yiguanjian.

Figure 5 Immunofluorescent colocalization of enhanced green fluorescent protein and albumin in CCl₄-induced chronic liver injured bone marrow chimera mice, × 200. EGFP is shown in green and Alb in red. No yellow color is visible in the merged images in control, CCl₄ or CCl₄ + YGJ groups. EGFP: Enhanced green fluorescent protein; YGJ: Yiguanjian; Alb: Albumin.

Figure 6 Immunofluorescent colocalization of enhanced green fluorescent protein and F4/80 in CCl₄-induced chronic liver injured bone marrow chimera mice, × 200. EGFP is shown in green and F4/80 in red. A yellow color confirms colocalization in the merged images. The number of EGFP/F4/80 double positive cells was found to increase in all mice after receiving CCl₄ injection. In contrast, the number of EGFP⁺/F4/80⁺ cells decreased in the YGJ treatment group. EGFP: Enhanced green fluorescent protein; YGJ: Yiguanjian.

Figure 7 Gene expression analyzed by real-time polymerase chain reaction and protein expression analyzed by Western blotting of monocyte chemotaxis protein-1 and CC chemokine receptor 2 in CCl₄-induced chronic liver injured bone marrow chimera mice. A: MCP-1 gene and protein expression. For both genes the relative expression is quantified using the control group as the basal level; B: MCP-1 and CCR2 bands by Western blotting. Band 1, control group; band 2, CCl₄ 7 wk group; band 3, CCl₄ 13 wk group; and band 4, CCl₄ + Yiguanjian (YGJ) group; C: CCR2 gene and protein expression. For both genes the relative expression is quantified using the control group as the basal level. MCP-1: Monocyte chemotaxis protein-1; CCR2: CC chemokine receptor 2; GAPDH: Glyceraldehydes-3- phosphate dehydrogenase. ^aP < 0.05, ^bP < 0.01 vs week 0 control group; ^cP < 0.05 vs the same time-point CCl₄ group.

Figure 8 Hepatocyte function. A: Ki-67 immunostaining in liver tissues, × 200; B: Semi-quantification of Ki-67 staining, with the week 0 control group level as the basal level; C: Serum Alb content; D: Liver Alb Western blotting bands; E: Semi-quantification of Alb based on Western blotting results, with the week 0 control group level as the basal level. ^aP < 0.05, ^bP < 0.01 vs week 0 control group; ^cP < 0.05, ^dP < 0.01 vs the same time-point CCl₄ group. Results are presented as mean ± SD. GAPDH: Glyceraldehydes-3-phosphate dehydrogenase; YGJ: Yiguanjian; Alb: Albumin.

Figure 9 Expression of progenitor markers in liver tissues. A: PKM2 immunostaining, × 200; B: Semi-quantification of PKM2 staining, with the week 0 control group level as the basal level; C: PKM2 and AFP Western blotting bands; D: Semi-quantification of PKM2 in Western blotting results, with the week 0 control group level as the basal level; E: Semi-quantification of Western blotting results of PKM2, with the week 0 control group level as the basal level. ^aP < 0.05, ^bP < 0.01 vs week 0 control group; ^cP < 0.05, ^dP < 0.01 vs the same time-point CCl₄ group. Results are presented as mean ± SD. GAPDH: Glyceraldehydes-3-phosphate dehydrogenase; YGJ: Yiguanjian; PKM2: Mitogen-activated protein kinase-2; AFP: α fetoprotein.