Silibinin Inhibits Proliferation and Migration of Human Hepatic Stellate LX-2 Cells

Abstract

Background: Proliferation of hepatic stellate cells (HSCs) play pivotal role in the progression of hepatic fibrosis consequent to chronic liver injury. Silibinin (SBN), a flavonoid compound, has shown to possess cell cycle arresting potential against many actively proliferating cancers cell lines. The objective of this study was to evaluate the anti-proliferative and cell cycle arresting properties of SBN in rapidly proliferating human hepatic stellate LX-2 cell line.

Methods: LX-2 cells were fed with culture medium supplemented with different concentrations of SBN (10, 50 and 100 μM). After 24 and 96 h of treatment, total cell number was determined by counting. Cytotoxicity was evaluated by trypan blue dye exclusion test. The expression profile of cMyc and peroxisome proliferator-activated receptor-γ (PPAR-γ) protein expressions was evaluated by Western blotting. Oxidative stress marker genes profile was quantified using qPCR. The migratory response of HSCs was observed by scrape wound healing assay.

Results: SBN treatments significantly inhibit the LX-2 cell proliferation (without affecting its viability) in dose dependent manner. This treatment also retards the migration of LX-2 cells toward injured area. In Western blotting studies SBN treatment up regulated the protein expressions of PPAR-γ and inhibited cMyc.

Conclusion: The present study shows that SBN retards the proliferation, activation and migration of LX-2 cells without inducing cytotoxicity and oxidative stress. The profound effects could be due to cell cycle arresting potential of SBN.

Keywords: AKR1C1, aldo-keto reductase family 1, member C1; ARE, antioxidant responsive element; CDKI, cyclin dependent kinase inhibitor; CYP450, cytochrome P450; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethylsulphoxide; ECM, extracellular matrix; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMOX1, heme oxygenase (decycling) 1; HSCs, hepatic stellate cells; NQO1, NAD(P)H dehydrogenase, quinone 1; Nrf-2, nuclear respiratory factor; PPAR-γ, peroxisome proliferator-activated receptor-γ; PPIA, peptidylprolyl isomerase A; ROS, reactive oxygen species; SBN, silibinin; TXNRD1, thioredoxin reductase 1; cytotoxicity; hepatic stellate cells; oxidative stress; qPCR, quantitative polymerase chain reaction; wound healing.

Figure 1

Morphology of LX-2 cells treated with silibinin for 96 h. (A) Control, (B) DMSO, (C–E) SBN 10, 50 and 100 μM treatments respectively (200×).

Figure 2

Effect of SBN treatment on LX-2 cells (A) cytotoxicity, (B) cell proliferation, (C) oxidative stress. Values are presented as mean ± S.D. of 3 nos. of observations for 96 h of different concentrations of SBN exposure respectively. Multiple comparisons between treatment groups were performed by using Newman–Keul's test. *Control compared to DMSO and SBN treatment groups. ^#DMSO group compared to all the SBN treated groups. **P < 0.01; ***P < 0.001. ^##P < 0.01; ^###P < 0.001.

Figure 3

Effect of SBN (24 h) on the protein expression of cMyc and PPAR-γ on LX-2 cells. β-Actin used as an internal control for optimization (representative images of at least three experiments in triplicate).

Figure 4

Inhibitory effects of SBN on HSC motility in wound healing model. A scrape wound was generated in the cell layer to remove a linear area of cells. 10% FBS used as a positive control (representative images of at least three experiments in triplicate).