Resveratrol amplifies profibrogenic effects of free fatty acids on human hepatic stellate cells

Abstract

Aim: To ascertain whether resveratrol affects the expression of free fatty acids (FFA)-induced profibrogenic genes, death receptors, and/or apoptosis-related molecules in human hepatic stellate cells, using the LX-2 cell line.

Methods: Cells were cultured in the presence of FFAs (2:1 oleate : palmitate) and subsequently treated with resveratrol. Gene expression rates were determined by quantitative real-time PCR. The 50% lethal dose (LD(50)) of resveratrol in the presence of FFAs was assessed with the MTT viability test.

Results: Compared to vehicle controls, incubation of LX-2 cells with 0.5 mM FFAs induced profibrogenic genes (alpha-SMA x 2.9; TGF-beta1 x 1.6; TIMP-1 x 1.4), death receptors (CD95/Fas x 3.8; TNFR-1 x 1.4), and anti-apoptotic molecules (Bcl-2 x 2.3; Mcl-1 x 1.3). Subsequent addition of 15 microM resveratrol (LD(50) = 23.2 microM) significantly (P < 0.05) upregulated further these genes (alpha-SMA x 6.5; TGF-beta1 x 1.9; TIMP-1 x 2.2; CD95/Fas x 13.1, TNFR-1 x 2.1; Bcl-2 x 3.6; Mcl-1 x 1.9). Importantly, this effect was only observed in the presence of FFAs.

Conclusion: Resveratrol amplifies the profibrogenic activation of human hepatic LX-2 stellate cells. This finding raises the possibility that in obese patients with elevated FFAs reserveratrol could provoke hepatic fibrogenesis. In-vivo studies are necessary to further validate this conclusion.

Figure 1

Incubation human LX-2 hepatic stellate cells in the absence or presence of free fatty acids (FFAs; 0.5 mM) with or without the principal concentration of resveratrol (Res.; 15 μM). Untreated negative controls (upper left-hand side) as well as treated cells and are shown as doublets, each, i.e. by phase-contrast microscopy (top), and by fluorescence microscopy showing Nile-Red lipid staining and blue nuclear counterstaining with 4′-6-diamidino-2-phenylindole (DAPI) (bottom). The morphology and viability of cells treated with 15 μM of resveratrol did not differ from cells maintained without this polyphenol. All photographs are representative of multiples of cultures kept under the respective condition (bar = 20 μm).

Figure 2

Expression of activation- and fibrosis-related genes in LX-2 cells. The expression of mRNAs in FFA-treated (left), resveratrol-treated (center), and co-treated cells was determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). Asterisks indicating statistical significance refer to the faintly shaded negative controls kept without FFAs or resveratrol. Results were reproducible in all of the cases. See main text for detailed information and interpretation.

Figure 3

Expression of death receptor genes (CD95/Fas, TNFR-1) and of genes encoding for anti-apoptotic receptors (Bcl-2, Mcl-1) in LX-2 cells. Results were reproducible in all of the cases. For further specifications, see caption to Fig. 2. Detailed information and interpretation is given in the main text.

Figure 4

Determination of unstimulated and stimulated apoptosis in LX-2 hepatic stellate cells. Apoptosis was provoked via the CD95/Fas-agonistic monoclonal antibody, CH11. Asterisks indicating statistical significance refer to connected columns. Results were reproducible in all of the cases. See main text for detailed information and interpretation.