Salacia chinensis L. Stem Extract Exerts Antifibrotic Effects on Human Hepatic Stellate Cells Through the Inhibition of the TGF-β1-Induced SMAD2/3 Signaling Pathway

Abstract

: Salacia chinensis L. (SC) stems have been used as an ingredient in Thai traditional medicine for treating patients with hepatic fibrosis and liver cirrhosis. However, there is no scientific evidence supporting the antifibrotic effects of SC extract. Therefore, this study aimed to determine the antifibrotic activity of SC stem extract in human hepatic stellate cell-line called LX-2. We found that upon TGF-β1 stimulation, LX-2 cells transformed to a myofibroblast-like phenotype with a noticeable increase in α-SMA and collagen type I production. Interestingly, cells treated with SC extract significantly suppressed α-SMA and collagen type I production and reversed the myofibroblast-like characteristics back to normal. Additionally, TGF-β1 also influenced the development of fibrogenesis by upregulation of MMP-2, TIMP-1, and TIMP-2 and related cellular signaling, such as pSmad2/3, pErk1/2, and pJNK. Surprisingly, SC possesses antifibrotic activity through the suppression of TGF-β1-mediated production of collagen type 1, α-SMA, and the phosphorylation status of Smad2/3, Erk1/2, and JNK. Taken together, the present study provides accumulated information demonstrating the antifibrotic effects of SC stem extract and revealing its potential for development for hepatic fibrosis patients.

Keywords: LX-2 cells; Salacia chinensis L., hepatic fibrosis; hepatic stellate cells; transforming growth factor-beta 1.

Figure 1

Effects of SC stem extract on stimulated hepatic stellate cell (HSC) morphology. Cell viability of LX-2 cells after SC treatments at 0.01 to 0.1 mg/mL are shown in (A). A schematic diagram of the experimental design is shown in (B). LX-2 morphology observed under a phase contrast microscopy is shown in (C). UT: untreated; TGF-β: TGF-β1 2 ng/mL; SC: Salacia chinensis L. extract. Scale bar = 50 µm. * p < 0.05.

Figure 2

Effects of SC stem extract on HSC 2D migration. Cell migration was observed at 0 h, 24 h, and 48 h after SC treatments (A). The percentage of cell migration at 24 h and 48 h after SC treatments is quantified in (B). UT: untreated; SC: Salacia chinensis L. extract. Scale bar = 50 µm. * p < 0.05.

Figure 3

Effects of SC stem extract on mRNA expressions. The mRNA expression of (A) α-SMA, (B) COL1A1, (C) MMP-2, (D) MMP-9, (E) TIMP-1, and (F) TGF-β1 was measured by quantitative RT-PCR. GAPDH was used as an internal control. UT: untreated; TGF-β: TGF-β1 2 ng/mL; SC: Salacia chinensis L. extract. * p < 0.05 indicates statistical significance from TGF-β1-treated group.

Figure 4

Effects of SC stem extract on α-SMA and collagen type I production. Both α-SMA and COL1A1 protein expression (Green) were examined by immunofluorescent staining (A). The nuclei (Blue) were stained with Hoechst 33342. The quantitative protein production of α-SMA and collagen type I (COL1A1) (B) was measured by western blotting. The bar graphs represent the relative expression of these proteins after normalization to β-actin (C,D). UT: untreated; TGF-β: TGF-β1 2 ng/mL; SC: Salacia chinensis L. extract. * p < 0.05 indicates statistical significance from the TGF-β1-treated group. Scale bar = 200 um.

Figure 5

Effects of SC stem extract on secreted extracellular matrix components. Western blot analysis of MMP-9, MMP-2, TIMP-1, and TIMP-2 protein expressions in supernatant of LX-2 cells was shown (A). The bar graphs represent the relative protein expressions of MMP-9 (B), MMP-2 (C), TIMP-1 (D), and TIMP-2 (E) after normalization to β-actin. MMP-2 and MMP-9 activity was investigated by gelatin zymography (F) and the bar graphs represent the percentage of relative MMP-9 (G) and MMP-2 (H) activities. UT: untreated; TGF-β: TGF-β1 2 ng/mL; SC: Salacia chinensis L. extract. * p < 0.05 indicates statistical significance from TGF-β1-treated group.

Figure 6

Effects of SC stem extract on phosphorylation of SMAD2/3, ERK1/2, p38, and JNK kinase. Western blot analysis of phosphorylated SMAD2/3, phosphorylated ERK1/2, ERK1/2, phosphorylated p38, p38, phosphorylated JNK, and JNK proteins are shown in (A). The bar graphs represent the relative expression of phosphorylated SMAD2/3 to β-actin (B), phosphorylated ERK1/2 to ERK1/2 (C), phosphorylated p38 to p38 (D), and phosphorylated JNK to JNK (E). UT: untreated; TGF-β: TGF-β1 2 ng/mL; SC: Salacia chinensis L. extract. * p < 0.05 indicates statistical significance from TGF-β1-treated group.