Neuroprotective effects of resveratrol against beta-amyloid-induced neurotoxicity in rat hippocampal neurons: involvement of protein kinase C

Abstract

1. Resveratrol, an active ingredient of red wine extracts, has been shown to exhibit neuroprotective effects in several experimental models. 2. The present study evaluated the neuroprotective effects of resveratrol against amyloid beta(Abeta)-induced toxicity in cultured rat hippocampal cells and examined the role of the protein kinase C (PKC) pathway in this effect. 3. Pre-, co- and post-treatment with resveratrol significantly attenuated Abeta-induced cell death in a concentration-dependent manner, with a concentration of 25 microm being maximally effective. 4. Pretreatment (1 h) of hippocampal cells with phorbol-12-myristate-13-acetate, a PKC activator, at increasing concentrations (1-100 ng x ml(-1)), resulted in a dose-dependent reduction in Abeta-induced toxicity, whereas the inactive 4alpha-phorbol had no effect. 5. Pretreatment (30 min) of hippocampal cells with GF 109203X (1 microm), a general PKC inhibitor, significantly attenuated the neuroprotective effect of resveratrol against Abeta-induced cell death. 6. Treatment of hippocampal cells with resveratrol (20 microm) also induced the phosphorylation of various isoforms of PKC leading to activation. 7. Taken together, the present results indicate that PKC is involved in the neuroprotective action of resveratrol against Abeta-induced toxicity.

Figure 1

Protective effects of resveratrol against Aβ_25–35-induced cell death in hippocampal neurons. Cells were pretreated with resveratrol at various concentrations, 2 h before the addition of Aβ_25–35 (20 μM) for 24 h. Cell viability was assayed with MTT (a) and LDH (b). Cells were also cotreated (c) or post (2 h)-treated (d) with resveratrol. Percentage of cell viability was relative to vehicle-treated controls (white bars). Values represent mean±s.e.m of three independent experiments, each performed in triplicates. ^*P<0.05, ^**P<0.01 by Student's t-test, compared to group that was treated with Aβ alone.

Figure 2

Protective effects of resveratrol against Aβ_1–40- and Aβ_1–42-induced toxicity and its rescuing effect in hippocampal neurons. Cells were cotreated with resveratrol at various concentrations in the presence of Aβ_1–40 (20 μM) (a) or Aβ_1–42 (20 μM) (b) for 24 h. (c) Cells were also post-treated with resveratrol at the indicated time after addition of Aβ_25–35 (20 μM). Cell viability was assayed with MTT. Percentage of cell viability was relative to vehicle-treated controls (white bars). Values represent mean±s.e.m of three independent experiments, each performed in triplicates. ^*P<0.05, ^**P<0.01 by Student's t-test, compared to group that was treated with Aβ alone.

Figure 3

Microscopic analysis of resveratrol against Aβ_25–35-induced cell death. Representative phase-contrast photomicrographs of primary cultured rat hippocampal neurons exposure for 24 h to (a) vehicle (0.1% DMSO); (b) Aβ_25–35 (20 μM); (c) resveratrol (20 μM); and (d) cells pretreated with 20 μM resveratrol for 30 min prior to exposure to Aβ_25–35 (20 μM). Scale bar=500 μm.

Figure 4

PMA reduces Aβ_25–35-induced toxicity in dose-dependent manner. Cultures were treated with PMA (solid bars) at the indicated concentrations and 100 ng ml⁻¹ 4α-phorbol (open bar) 1 h before the addition of Aβ_25–35. Cell viability was assessed 24 h later using the MTT assay. Data represent the mean (±s.e.m.) from a representative experiment (n=3). ^*P<0.05 relative to Aβ_25–35 alone.

Figure 5

Blockade by a PKC inhibitor of the protective effects of resveratrol against Aβ_25–35-induced cell death in hippocampal neurons. Hippocampal neurons were pretreated with GF 109203X (a), PD98059 (b) and LY294002 (c) 30 min before adding resveratrol (20 μM) in the presence (shadow bars) or absence (white bars) of Aβ_25–35 (20 μM). Treatment with Aβ_25–35 (20 μM) alone (black bar). After 24 h, the viability of cells was measured using the MTT assay. Results are expressed as mean (%)±s.e.m. ^*P<0.01 by Student's t-test, compared to group that was treated with resveratrol (20 μM) in the presence of Aβ_25–35 (20 μM).

Figure 6

Effects of resveratrol and Aβ_25–35 on the phosphorylation of PKC in hippocampal cells. The phosphorylation of PKC was detected in cell lysates by Western blot using an anti-phospho-PKC (pan) antibody. (a) Hippocampal cells treated with resveratrol (20 μM) for the indicated times. (b) Hippocampal cells treated with resveratrol at various concentrations for 30 min. (c) Hippocampal cells treated with Aβ_25–35 (20 μM) for the indicated times. (d) Hippocampal cells treated without or with Aβ_25–35 (20 μM) in combinations with resveratrol at various concentrations for 30 min. Western blots (left) were probed with anti-phospho-PKC (pan), and bands from phospho-PKC immunoblots were quantified using an MCID program, normalized to β-actin, and represented graphically (right). Results are the mean±s.e. of three independent experiments. Student t-test: ^*P<0.05, ^**P<0.01 versus control.

Figure 7

Effects of resveratrol and Aβ_25–35 on the PKC isoforms and the phosphorylation of PKC isoforms in hippocampal cells. PKC isoforms and the phosphorylation of PKC isoforms were detected in cell lysates by Western blot using a PKC sampler kit and an anti-phospho-PKC antibody sampler kit, respectively. (a) Western blots of PKC isoforms in hippocampal cells treated with Aβ_25–35 (20 μM) and/or resveratrol at various concentrations for 30 min. (b) Western blots of phospho-PKC isoforms in hippocampal cells treated with Aβ_25–35 (20 μM) and/or resveratrol (20 μM) for 30 min. Control was treated only with vehicle DMSO at a final concentration of 0.1%. Results are the representative of three independent experiments.

Figure 8

Effects of resveratrol and Aβ_25–35 on the phosphorylation of Akt and ERK1/2 in hippocampal cells. (a) Western blots of phospho-ERK1/2 in hippocampal cells treated with Aβ_25–35 (20 μM) and/or resveratrol at various concentrations for 30 min. (b) Western blots of phospho-Akt in hippocampal cells treated with Aβ_25–35 (20 μM) and/or resveratrol at various concentrations for 30 min. Control was treated only with vehicle DMSO at a final concentration of 0.1%. Results are the representative of three independent experiments.