Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons

Abstract

Animal and epidemiological studies suggest that polyphenol constituents of red wine possess antioxidant activities that favour protection against cardiovascular disease - the so-called. 'French paradox' - and possibly, central nervous system disorders such as Alzheimer's disease (AD) and ischaemia. In the present study, the potential of three major red wine derived-polyphenols to protect against toxicity induced by the nitric oxide free radical donors sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1) was examined in cultured rat hippocampal cells. Both co- and post-treatments with either the stilbene resveratrol (5 - 25 microM) or the flavonoids quercetin (5 - 25 microM) and (+)-catechin (1 - 10 microM) were capable of attenuating hippocampal cell death and intracellular reactive oxygen species accumulation produced by SNP (100 microM and 1 mM, respectively). However, among the phenolic compounds tested, only the flavonoids afforded significant protection against 5 mM SIN-1-induced toxicity. The effects of phenolic constituents were shared by Trolox (100 microM), a vitamin E analogue, but not by selective inhibitors of cyclo-oxygenases (COX) and lipoxygenases (LOX). Among the phenolic compounds tested, only quercetin (10 microM) inhibited 100 microM SNP-stimulated protein kinase C (PKC) activation, whereas none of them were able to attenuate nitrite accumulation caused by SNP (100 microM). Taken together, these data suggest that the neuroprotective abilities of quercetin, resveratrol, and (+)-catechin result from their antioxidant properties rather than their purported inhibitory effects on intracellular enzymes such as COX, LOX, or nitric oxide synthase. Quercetin, however, may also act via PKC to produce its protective effects.

Figure 1

Effect of co-treatment with quercetin (5–25 μM) (A), resveratrol (5–25 μM) (B) and (+)-catechin (1–10 μM) (C) against toxicity induced by SNP (100 μM), as estimated by the MTT and NR assays in rat hippocampal cell cultures. Values represent mean±s.e.mean from 3–5 independent experiments. ^#P<0.01 compared to vehicle-treated groups, ^*P<0.01 compared to groups treated with SNP alone.

Figure 2

Effect of co-treatment with quercetin (5–25 μM) (A), resveratrol (5–25 μM) (B) and (+)-catechin (1–10 μM) (C) against toxicity induced by SIN-1 (5 mM), as estimated by the MTT and NR assays in rat hippocampal cell cultures. Values represent mean±s.e.mean from 3–4 independent experiments. ^*P<0.05, ^**P<0.01 compared to groups treated with SIN-1 alone.

Figure 3

Effect of co-treatment with quercetin (5–25 μM) (A), resveratrol (5–25 μM) (B) and (+)-catechin (1–10 μM) (C) on either 100 μM SNP-induced intracellular ROS in rat hippocampal cell cultures. Phenolic compounds were applied at the onset of SNP exposure. ROS were determined 20 h after by quantification of DCF fluorescence as described in Methods. Values represent mean±s.e.mean from 3–5 independent experiments. #P<0.05, ##P<0.01 compared to groups treated with vehicle, ^*P<0.05, ^**P<0.01 compared to groups treated SNP.

Figure 4

Effect of 2 h post-treatment with quercetin (5–10 μM) (A), resveratrol (5–10 μM) (B) and (+)-catechin (1–5 μM) (C) against 1 mM SNP-induced toxicity, as estimated by the MTT and NR assays in rat hippocampal cell cultures. Values represent mean±s.e.mean from 3–5 independent experiments. ^*P<0.05, ^**P<0.01 compared to groups treated with SNP alone.

Figure 5

Effect of 2 h post-treatment with quercetin (5–10 μM), resveratrol (5–10 μM) and (+)-catechin (1–5 μM) on against 1 mM SNP-induced intracellular ROS in rat hippocampal cell cultures. Phenolic compounds were applied 2 h after the onset of SNP exposure. ROS were determined 24 h after by quantification of DCF fluorescence as described in Methods. Values represent mean±s.e.mean from 3–5 independent experiments. ^*P<0.01 compared to groups treated with either SNP.

Figure 6

Effect of quercetin (Que, 10 μM), resveratrol (Res, 10 μM) and (+)-catechin (Cat, 10 μM) on (100 μM, 5 min) SNP-induced PKC activity in rat hippocampal mixed cell cultures. PKC activity was measured as described in Methods. Values represent mean±s.e.mean from five independent experiments. ^*P<0.01 compared to SNP-treated groups.

Figure 7

Effect of quercetin (Que, 10 μM), resveratrol (Res, 10 μM) and (+)-catechin (Cat, 10 μM) on (100 μM, 20 h) SNP-stimulated nitrite accumulation into the culture medium of rat hippocampal mixed cells. At the end of incubation time, nitrite accumulation (A) and cell survival (B) were estimated as described in Methods. Values represent mean±s.e.mean from four independent experiments. ^*P<0.01 compared to SNP-treated groups.