Withagulatin A inhibits hepatic stellate cell viability and procollagen I production through Akt and Smad signaling pathways

Abstract

Aim: To investigate the effects of the natural product Withagulatin A on hepatic stellate cell (HSC) viability and type I procollagen production. The potential mechanism underlying the pharmacological actions was also explored.

Methods: The effect of Withagulatin A on cell viability was evaluated in HSC and LX-2 cells using a sulforhodamine B (SRB) assay. Cell cycle distribution was analyzed using flow cytometry. Type I procollagen gene expression was determined using real-time PCR. Regulation of signaling molecules by Withagulatin A was detected using Western blotting.

Results: Primary rat HSCs and the human hepatic stellate cell line LX-2 treated with Withagulatin A (0.625-20 micromol/L) underwent a dose-dependent decrease in cell viability, which was associated with S phase arrest and the induction of cell apoptosis. In addition, the natural product decreased phosphorylation of the Akt/mTOR/p70S6K pathway that controls cell proliferation and survival. Furthermore, Withagulatin A (1, 2 mumol/L) inhibited transforming growth factor-beta (TGF-beta) stimulated type I procollagen gene expression, which was attributable to the suppression of TGF-beta stimulated Smad2 and Smad3 phosphorylation.

Conclusion: Our results demonstrated that Withagulatin A potently inhibited HSC viability and type I procollagen production, thereby implying that this natural product has potential use in the development of anti-fibrogenic reagents for the treatment of hepatic fibrosis.

Figure 1

Withagulatin A suppresses HSC activation and proliferation. (A) Chemical structure of Withagulatin A. (B) Withagulatin A suppresses α-SMA expression. Primary rat HSCs and human hepatic stellate LX-2cells were incubated with Withagulatin A (0, 1, and 2 μmol/L) for 24 h and the expression of α-SMA was determined by Western blotting. GAPDH was used as a loading control. The results shown are from three representative independent experiments. (C) Bands were quantified and data are expressed as fold of control. (D) Withagulatin A inhibited HSC proliferation. Primary rat HSCs, LX-2 cells, and LO2 hepatocytes were treated with a series of concentrations (0–20 μmol/L) of Withagulatin A for 48 h and cell viability was determined using a SRB assay. These experiments were carried out in triplicate. ^bP<0.05, ^cP<0.01 vs control cells (0 μmol/L).

Figure 2

Withagulatin A induces cell cycle arrest. (A) Withagulatin A arrests cells at S phase. LX-2 cells were incubated with Withagulatin A (0, 1, and 2 μmol/L) for 24 h. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry as described in Materials and methods. The experiment was repeated three times and representative data are shown. (B) Withagulatin A affects cell cycle regulatory proteins. LX-2 cells were incubated with Withagulatin A (2 μmol/L) for the indicated time points (0–24 h). Cells were harvested and cell lysates were analyzed by Western blotting for cyclin D1, p-CDK2, CDK2, and cyclin B1 levels. GAPDH was used as a loading control. Cells treated with DMSO were used as controls for each time point. Results shown are representative of three independent experiments. (C) Bands were quantified and data are expressed as fold of control. ^bP<0.05, ^cP<0.01 vs control cells.

Figure 3

Withagulatin A induces cell apoptosis. (A) Induction of an apoptotic morphology by Withagulatin A. LX-2 cells were treated with Withagulatin A (0, 1, and 2 μmol/L) for 24 h. Cells were fixed, stained with Hoechst 33342, and visualized with a fluorescence microscope (scale bar=0 μm). Nuclear condensation and fragmentation are indicated by arrows. Representative views from five fields for each slide are shown and the treatments were performed in triplicate. (B) Withagulatin A affects apoptotic protein levels. LX-2 cells were treated with Withagulatin A (0, 1, and 2 μmol/L) for 24 h. Cells were harvested and lysates were analyzed by Western blot analysis to determine the levels of Bcl-2, Bax, cleaved-caspase3 (c-caspase3), and caspase3. GAPDH was used as a loading control. Results shown are representative of three independent experiments. (C) Bands were quantified and data are expressed as fold of control. ^bP<0.05, ^cP<0.01 vs control cells.

Figure 4

Withagulatin A inhibits the Akt pathway. (A, B) Primary rat HSCs were incubated with Withagulatin A (0, 1, and 2 μmol/L) for 24 h (C, D) or treated with 2 μmol/L Withagulatin A for 0 to 24 h. Cells were harvested and analyzed by Western blotting for phosphorylated Akt (Ser 473), Akt, phosphorylated mTOR (Ser 2448), phosphorylated p70S6K (Thr 421/Ser 424) and p70S6K. GAPDH was used as a loading control. Results shown are representative of three independent experiments. Bands were quantified and data are expressed as fold of control. ^bP<0.05, ^cP<0.01 vs control cells.

Figure 5

Withagulatin A inhibits TGF-β stimulated procollagen I mRNA expression and Smad2/3 phosphorylation. (A, B) Withagulatin A inhibits TGF-β stimulated procollagen I mRNA expression. (A) Primary rat HSCs and (B) LX-2 cells were treated with Withagulatin A (0, 1, and 2 μmol/L) in the presence of TGF-β (2 ng/mL) for 24 h in DMEM supplemented with 0.2% BSA. mRNA levels of α1 procollagen I and α2 procollagen I were analyzed by real-time PCR assays. Ribosomal 18s RNA was used as an internal control. ^bP<0.05, ^cP<0.01 compared with DMSO treated cells. (C) Withagulatin A suppresses TGF-β stimulated Smad2/3 phosphorylation. Primary rat HSCs were treated with Withagulatin A (0, 1, and 2 μmol/L) in the presence of TGF-β (2 ng/mL) for 24 h in DMEM supplemented with 0.2% BSA. Cells were harvested and subjected to Western blot analysis for phosphorylated Smad2 (Ser 465/467), Smad2, phosphorylated Smad3 (Ser 432/425), and Smad3. GAPDH was used as a loading control. Results shown are representative of three independent experiments. (D) Bands were quantified and data are expressed as fold of control. (E, F) Withagulatin A suppresses TGF-β induced Smad2 nuclear translocation. CHO/EGFP-Smad2 cells were pretreated with Withagulatin A (0, 1 and 2 μmol/L) for 10 h and then stimulated with TGF-β (2 ng/mL) for 2 h in serum free F-12 medium supplemented with 0.2% BSA. Finally, cells were stained with 2 μmol/L Hoechst 33342 for 15 min and fluorescent images were taken by an INCell Analyzer 1000. Each treatment was repeated in 3 wells and 5 fields were photographed for each well. (E) Representative views are presented (scale bar=50 μm) and (F) data were quantified using the INCell Analyzer analysis software. ^cP<0.01 vs TGF-β stimulated cells.

Figure 6

Proposed model illustrating the anti-fibrotic mechanism of Withagulatin A (With A). During liver fibrosis, stimulatory signals from the fibrogenic cytokines including PDGF and TGF-β are transduced into target cells through their corresponding receptors, which in turn activate Akt and Smad proteins by phosphorylation. Withagulatin A inhibited the phosphorylation of Akt and its downstream targets such as mTOR and p70S6K, which finally led to reduced HSC proliferation. Meanwhile, Withagulatin A suppressed Smad2/3 phosphorylation and consequently blocked translocation to the nucleus, thereby resulting in reduced transcription of ECM proteins.