Down-regulation of transforming growth factor beta 1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

Abstract

Aim: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-beta1 (TGF beta 1)/activin receptor-like kinase 1 (ALK1, type I receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells.

Methods: LX-2 cells were treated with TGF beta 1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGF beta 1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGF beta 1/ALK1 pathway. Real-time PCR was performed to examine the expression of alpha-SMA (alpha-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of alpha-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of alpha-SMA.

Results: In LX-2 cells, TGF beta 1/ALK1-pathway-related gene expression could be stimulated by TGF beta 1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of alpha-SMA mRNA and protein expression. This effect was related to inhibition of the above TGF beta 1/ALK1-pathway-related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGF beta 1 co-treatment for 24 h.

Conclusion: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGF beta 1/ALK1/Smad1 pathway.

Figure 1

Effect of different treatment on α-SMA, Id1, ALK1 expression after 24 h. Cpd861 could decrease the level of all these genes compared with untreated LX-2 cells (^aP < 0.05 vs control LX-2 cell group). Even with TGFβ1, Cpd861 could also exert its inhibitory roles in these genes expression (^cP < 0.05 vs TGFβ1 treated group). Samples were obtained from 6 wells of cells for examining the relative fold change of α-SMA (A), Id1 (B), ALK1 (C) expression .Three replicate reactions for per sample. Error bars, SD.

Figure 2

The level of α-SMA protein expression with different treatment after 24 h. Western blot was used as described in Materials and Methods. A: Representative Western blot results of α-SMA. The positions of protein size markers were given; B: Densitometry of Western blot analyzed by Gel-pro software. The levels of α-SMA were normalized to the level of β-actin protein. Six independent experiments were performed. ^aP < 0.05 vs control LX-2 cell group. Error bars, SD.

Figure 3

Expression of α-SMA in different treatment groups. Qualitative expression of α-SMA in control LX-2 cells (A), treated with TGFβ1 (B), Cpd861 (C) and Cpd861 together with TGFβ1 (D) for 24 h using immunohistochemical staining. α-SMA presented brown color in cytoplasm (× 200).

Figure 4

The level of Phosphorylated Smad1 with different treatment after 24 h. Western blot was used as described. A: Representative Western blot results of Phosphorylated Smad1. The positions of protein size markers were given; B: Densitometry of Western-blot analyzed by Gel-pro software. The levels of Phospho-Smad1 were normalized to the level of β-actin protein. Six independent experiments were performed. ^aP < 0.05 vs untreated LX-2 cell group, ^cP < 0.05 vs TGFβ1 treated group. Error bars, SD.