Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

Figure 4 The role of calcitriol in hepatic fibrosis. A: Western blot analysis of collagen I and α-smooth muscle actin (α-SMA) in LX-2 cells treated with different concentrations of calcitriol; B: The protein levels of collagen I and α-SMA after calcitriol treatment at various time points were analyzed by Western blot; C: Calcitriol reduced the transforming growth factor beta 1 (TGFβ1)-induced elevation of collagen I and α-SMA at the protein level (n = 3); D: Western blot analysis of the calcitriol-induced level of Bcl-2 and Bax; E and F: An Annexin V-FITC/7-AAD kit was utilized to measure cellular apoptosis by flow cytometry; G and H: LX-2 cell migration was measured using the wound-healing test at 0 h, 24 h, 48 h, and 72 h (× 100) (n = 3); I: Hematoxylin-eosin, Masson staining, Sirius red staining, and immunohistochemical staining for α-SMA in hepatic tissue, scale bar = 100 μm; J: Immunofluorescence staining for α-SMA in hepatic tissue, red: α-SMA (n = 3), scale bar = 20 μm; K and L: Protein expression of α-SMA in the hepatic tissues was evaluated by Western blot (n = 3); M: The fibrosis score was analyzed according to the Ishak scoring system (n = 6); N: Levels of alanine aminotransferase and aspartate aminotransferase in plasma for the three groups. The carbon tetrachloride (CCl₄) group was contrasted with the corn oil group, while the calcitriol group was contrasted with the CCl₄ group (n = 6). The data was presented as mean ± SE. ^aP < 0.05, ^bP < 0.01, ^cP < 0.0001 vs without calcitriol control group; ^dP < 0.001 CCl₄ group vs corn oil control group, ^eP < 0.001 calcitriol group vs CCl₄ group; ^fP < 0.01 CCl₄ group vs corn oil control group, ^gP < 0.01 calcitriol group vs CCl₄ group; ^hP < 0.0001 CCl₄ group vs corn oil control group, ⁱP < 0.0001 calcitriol group vs CCl₄ group. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CCl₄: Carbon tetrachloride; TGFβ1: Transforming growth factor beta 1.