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

Figure 1 Evaluation of nonstructural protein 3-transactivated protein 1 expression in carbon tetrachloride-induced hepatic fibrosis and Transforming growth factor 1 beta 1-stimulated LX-2 cells. A: Real-time quantitative polymerase chain reaction analysis of fibrosis-related genes in liver tissues (n = 6); B: Immunofluorescence staining for alpha smooth muscle actin (α-SMA); red: α-SMA (n = 3), scale bar = 20 μm; C: Immunohistochemical staining for α-SMA, scale bar = 100 μm; D: Image J analysis of immunohistochemical staining for α-SMA (n = 3); E: Immunofluorescence staining for nonstructural protein 3-transactivated protein 1 (NS3TP1); green: NS3TP1 (n = 3), scale bar = 20 μm; F: Western blot analysis of NS3TP1 in L02, LX-2, Huh7, and G2 cells; G and H: NS3TP1 was overexpressed in LX-2 cells treated with transforming growth factor 1 beta 1 (TGFβ1) for 24 h (n = 3). The data was presented as mean ± SE. ^aP < 0.05, ^bP < 0.01 vs TGFβ1 (0 ng/mL) group; ^cP < 0.01 vs corn oil control group. NS3TP1: Nonstructural protein 3-transactivated protein 1; TGFβ1: Transforming growth factor 1 beta 1; CCl₄: Carbon tetrachloride; RT-qPCR: Real-time quantitative polymerase chain reaction; α-SMA: Alpha smooth muscle actin.

Figure 2 Influence of nonstructural protein 3-transactivated protein 1 on liver fibrosis in vitro. A: Western blot analysis of fibrosis-related genes after Nonstructural protein 3-transactivated protein 1 (NS3TP1) overexpression (n = 3); B: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of fibrosis-related genes after NS3TP1 overexpression (n = 3); C: Western blot analysis of the fibrosis-related genes after NS3TP1 interference (n = 3); D: RT-qPCR analysis of the fibrosis-related genes after NS3TP1 interference (n = 3); E and F: Cell proliferation was measured by cell counting kit-8 assays after interference or overexpression of NS3TP1 (n = 3); G and H: Western blot analysis of Bcl-2 and Bax after interference or overexpression of NS3TP1 (n = 3). The data was represented as mean ± SE. ^aP < 0.05, ^bP < 0.01 vs pcDNA-NS3TP1 group; ^cP < 0.05, ^dP < 0.01, ^eP < 0.001 vs siRNA-NS3TP1 group. NS3TP1: Nonstructural protein 3-transactivated protein 1; CCK-8: Cell counting kit-8; COL1A1: Type 1 collagen alpha 1 chain; COL1A2: Type 1 collagen alpha 2 chain; COL3A1: Type 3 collagen alpha 1 chain; COL4A2: Type 4 collagen alpha 2 chain.

Figure 3 Promotion of nonstructural protein 3-transactivated protein 1 in hepatic fibrosis via transforming growth factor beta 1 receptor/Smad3 and NF-kB signaling pathways. A and B: The protein levels of molecules in the transforming growth factor beta 1 (TGFβ1)/Smad3 and NF-kB signaling pathways after nonstructural protein 3-transactivated protein 1 (NS3TP1) interference or overexpression in LX-2 cells were analyzed by Western blot (n = 3); C and D: Western blot analysis of the above signaling pathway molecules in LX-2 cells after Smad3-specific inhibitor or licochalcone D treatment (n = 3); E: Coimmunoprecipitation analysis between NS3TP1 and Smad3 or p65; F: Luciferase activity analysis between NS3TP1 and the TGFβ1 receptor 1 (TGFβRI) promoter (n = 3); G: Luciferase activity analysis between TGFβ1 and the NS3TP1 promoter; TGFβ1 (L: 2.5 ng/mL, M: 5 ng/mL, H: 10 ng/mL) (n = 3); H: Luciferase activity analysis between NS3TP1 and the p65 promoter (n = 3); I: Luciferase activity analysis between p65 and the NS3TP1 promoter (n = 3). The data was represented as mean ± SE. ^aP < 0.001 pcDNA3.1 + TGFβ1R promoter vs pGL4.10 + pcDNA3.1, ^bP < 0.0001 NS3TP1 + TGFβ1R promoter vs NS3TP1 + pGL4.10; ^cP < 0.001; NS3TP1 + TGFβ1R promoter vs pcDNA3.1 + TGFβ1R promoter; ^dP < 0.01 pGL4.10 + NS3TP1 promoter vs pGL4.10; ^eP < 0.001 pcDNA3.1 + p65 promoter vs pGL4.10 + pcDNA3.1, ^fP < 0.01 NS3TP1 + p65 promoter vs NS3TP1 + pGL4.10; ^gP < 0.01 pcDNA3.1 + NS3TP1 promoter vs pGL4.10 + pcDNA3.1, ^hP < 0.0001 p65 + NS3TP1 promoter vs p65 + pGL4.10; ⁱP < 0.001 p65 + NS3TP1 promoter vs pcDNA3.1 + NS3TP1 promoter. NS3TP1: Nonstructural protein 3-transactivated protein 1; TGFβ1R: Transforming growth factor beta 1 receptor; p-smad3: Phosphorylated sekelsky mothers against decapentaplegic homolog 3; SIS3: Smad3-specific inhibitor; LD: Licochalcone D; Co-IP: Coimmunoprecipitation.

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.

Figure 5 The relationship between calcitriol and nonstructural protein 3-transactivated protein 1. A: RT-qPCR analysis of nonstructural protein 3-transactivated protein 1 (NS3TP1) in mice treated with calcitriol (n = 6); B: Immunohistochemical staining for α-smooth muscle actin (α-SMA) in hepatic tissue, scale bar = 100 μm; C: Image J analysis of immunohistochemical staining for α-SMA, (n = 3); D: Immunofluorescence staining for NS3TP1 in liver tissue, green: NS3TP1 (n = 3), scale bar = 20 μm; E and F: Western blot analysis of NS3TP1 in LX-2 cells stimulated with various concentrations of calcitriol or with 16 μmol/L calcitriol for various durations (n = 3); G: Analysis of luciferase activity between the NS3TP1 promoter and calcitriol in HepG2 cells, calcitriol (L: 8 μmol/L, M: 16 μmol/L, H: 32 μmol/L); H: Western blot analysis of the activity of both above signaling pathways in LX-2 cells treated with different concentrations of calcitriol; I: Collagen I, α-SMA, NS3TP1, and p-p65 in calcitriol-treated LX-2 cells were measured by Western blot and compared to LPS-treated cells; J: Extracellular matrix accumulation was evaluated by Western blot in LX-2 cells stimulated with calcitriol after NS3TP1 overexpression. The data was represented as mean ± SE. ^aP < 0.01 CCl₄ group vs corn oil control group, ^bP < 0.01 calcitriol group vs CCl₄ group; ^cP < 0.0001 pGL4.10-NS3TP1 promoter vs pGL4.10, ^dP < 0.0001 pGL4.10-NS3TP1 promoter + calcitriol (32 μmol/L) vs pGL4.10-NS3TP1 promoter. ECM: Extracellular matrix; LPS: Lipopolysaccharide; NS3TP1: Nonstructural protein 3-transactivated protein 1; TGFβ1: Transforming growth factor 1 beta 1; CCl₄: Carbon tetrachloride.