Gan Shen Fu Fang ameliorates liver fibrosis in vitro and in vivo by inhibiting the inflammatory response and extracellular signal-regulated kinase phosphorylation

Figure 1 Gan Shen Fu Fangdecreased serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin in common bile duct-ligated rats and the liver and spleen coefficients (n = 8). A: Alanine aminotransferase level; B: Aspartate aminotransferase level; C: Total bilirubin level; D:Liver coefficient; E: Spleen coefficient. The data are presented as the mean ± SD. ^bP < 0.01, ^eP < 0.001, compared with the sham group; ^cP < 0.05, compared with the 2W-CBDL rats; ^gP < 0.05, ^kP < 0.001, compared with the 4W-CBDL rat. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBIL: Total bilirubin; CBDL: Common bile duct-ligated; GSFF: Gan Shen Fu Fang.

Figure 2 Gan Shen Fu Fang inhibited liver fibrosis and alleviated the inflammatory response. A: Hematoxylin and eosin staining and Masson staining of the liver tissue; B: The collagen content was measured by quantitative histomorphometry; C: Hydroxyproline concentration in the liver; D and E: Tumour necrosis factor-α and IL-1β levels in the liver. The data are presented as the mean ± SD. ^bP < 0.01, ^eP < 0.001, compared with the sham group; ^cP < 0.05, ^dP < 0.01, compared with the 2W-CBDL rats; ^hP < 0.01, ^kP < 0.001, compared with the 4W-CBDL rats. HE: Hematoxylin and eosin; CBDL: Common bile duct-ligated; GSFF: Gan Shen Fu Fang; TNF-α: Tumor necrosis factor-α; IL-1β: Interleukin-1β; Hyp: Hydroxyproline.

Figure 3 Gan Shen Fu Fang inhibited HSC-T6 cell viability and collagen synthesis. A and B: GSFF inhibited HSC-T6 cell viability and Col. I release; C and D: GSFF inhibited the viability and Col. I release of HSC-T6 cells stimulated with transforming growth factor β1 (TGF-β1); E and F: TGF-β1 promoted α-SMA expression in HSC-T6 cells, which was decreased by GSFF. The relative α-SMA content was measured by quantitative histomorphometry, and the results are shown in panel E. ^aP < 0.05, ^bP < 0.01, compared with the control cells. ^cP < 0.05, ^dP < 0.01, ^fP < 0.001, compared with cells stimulated with TGF-β1 only. GSFF: Gan Shen Fu Fang; TGF-β1: Transforming growth factor β1.

Figure 4 Gan Shen Fu Fang reduced extracellular signal-regulated kinase phosphorylation and NF-κB expression in common bile duct-ligated rats and HSC-T6 cells. A-D: Liver tissue homogenates were subjected to immunoblotting as indicated. Representative bans and quantitative data are shown. N = 4; E-G: HSC-T6 cells were not stimulated, and whole-cell lysates were used. Representative images and quantitative data are shown; H: HSC-T6 cells were stimulated with platelet-derived growth factor-BB to further evaluate the effect of GSFF on extracellular signal-regulated kinase phosphorylation. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001, compared with control cells; ^cP < 0.05, ^dP < 0.01, ^fP < 0.001 compared with sham rats. ^gP < 0.05, ^hP < 0.01 compared with the 2W-CBDL rats; ⁱP < 0.05, ^lP < 0.001, compared with the 4W-CBDL rats; ⁿP <0.01, compared with cells stimulated with only platelet-derived growth factor-BB. PDGF: Platelet-derived growth factor; GSFF: Gan Shen Fu Fang; ERK: Extracellular signal-regulated kinase; CBDL: Common bile duct-ligated.