Figure 1 Induction of IL-8 and AMCase expression by IL-13 and TNF-a. Cultured HPAEC were treated with different concentrations of IL-13 and TNF-a, individually or combined. (A) Total RNA was isolated from control and treated HPAEC at 6 h following stimulation and IL-8 and AMCase mRNA was assayed by RT-PCR. The PCR products were separated on agarose gels according to size and visualized by ethidium bromide staining (top panel). IL-8 or AMCase mRNA levels following treatments were quantified and normalized against GAPDH shown in the bottom panel. Statistically significant difference between cytokine-treated groups compared with vehicle controls was indicated by asterisks (*). (B) The expression of transcription factors, NF-?B subunits and STAT3 and STAT6 mRNA in control and treated HPAEC using the indicated cytokines, for 3 h was also assayed by RT-PCR. The respective PCR signals were quantified and normalized against the value of GAPDH for each treatment condition. Asterisks (*) indicated statistically significant difference between cytokine-treated groups compared with vehicle controls at 0 h.

Figure 2 Modulation of IL-13 and TNF-a induced IL-8 or AMCase expression by resveratrol and/or its metabolites. (A) HPAEC was pretreated by 5 or 25 然 resveratrol or piceatannol for 1 h and stimulated with IL-13 (50 ng/ml), TNF-a (10 ng/ml), alone or in combination, for 3 h. IL-8 mRNA levels were assayed by RT-PCR, quantified by densitometry, and normalization against GAPDH. (B) Effect of the metabolites of resveratrol on IL-13 and TNF-a induced IL-8 expression. HPAEC was pretreated with 25 然 piceid, 4'-O-D-glucuronide, or 3-O-D-glucuronide for 1 h and stimulated with IL-13 (50 ng/ml) and TNF-a (10 ng/ml) for 3 h. Changes in mRNA of IL-8 were assayed by RT-PCR (left panel) and quantified by ImageJ and normalized against GAPDH (right panel). Values are expressed as mean崆EM for three experiments. (C) Effects of resveratrol on AMCase expression. HPAEC was pretreated by 10 or 25 然 resveratrol for 1 h and stimulated with IL-13 (100 ng/ml), TNF-a (100 ng/ml), alone or in combination, for 6 h. AMCase mRNA levels were assayed by RT-PCR, quantified by densitometry, and normalization against GAPDH.

Figure 3 Effects of the resveratrol and its metabolites on IL-13 and TNF-a induced change of STAT3, STAT6, NF-kB p50 and NF-kB p65 expression. (A) HPAEC was pretreated with 50 然 resveratrol, piceatannol, piceid, 4'-O-D-glucuronide, or 3-O-D-glucuronide for 1 h and stimulated with IL-13 (50 ng/ml) and TNF-a (10 ng/ml) for 3 h. Changes in mRNA of STAT3/6 and NF-kB p50/p65 were assayed by RT-PCR and quantified and expressed as a fold difference against GAPDH. (B) HPAEC were pretreated with 50 然 resveratrol then stimulated with IL-13 (50 ng/ml) and TNF-a (10 ng/ml) for 3 h. Western blot analysis was performed to measure the subcellular changes in protein expression of JAK-1, p-STAT6, STAT6 and two subunit of NF-?B on cells treated with 50 ng/ml IL-13 and 10 ng/ml TNF-a. Intensity of specific bands from control and treated cells in panel B was each normalized to actin and expressed as a fold of control.

Figure 4 Effects of resveratrol and its metabolites on NQO1 mRNA and protein expression and cell morphology in HPAEC. (A) HPAEC was treated with 50 然 piceid, 4'-O-D-glucuronide, or 3-O-D-glucuronide for 4 h. Changes in mRNA of NQO1 were assayed by RT-PCR and quantified by ImageJ and expressed as a fold difference against GAPDH. (B) Effects of resveratrol and its metabolites on NQO1 protein expression. Western blot analysis were performed on the HPAEC cells treated with 50 然 resveratrol and/or its metabolites for 48 h. Changes in protein expression of NQO1 were further quantified the intensity of NQO1 bands from control and treated cells and each normalized against actin and expressed as a fold of control. (C) Cell morphological change of HPAEC induced by exposure to 50 然 resveratrol or its metabolites for 48 h.

Figure 5 A mechanism showing modulation by resveratrol on induction of IL-8 and AMCase gene transcription by IL-13 and TNF-a. Resveratrol is postulated to suppress IL-8 or AMCase transcription and expression induced by IL-13 and TNF-a, and that its effects are partially mediated through the inhibition of STAT3/6 and NF-?B p50/p65 expression using signaling events whose details remain to be elucidated. The scheme also shows effects of resveratrol and its metabolites on NQO1, in part contributing to cardioprotection.