Grape powder supplementation prevents oxidative stress-induced anxiety-like behavior, memory impairment, and high blood pressure in rats

Abstract

We examined whether or not grape powder treatment ameliorates oxidative stress-induced anxiety-like behavior, memory impairment, and hypertension in rats. Oxidative stress in Sprague-Dawley rats was produced by using L-buthionine-(S,R)-sulfoximine (BSO). Four groups of rats were used: 1) control (C; injected with vehicle and provided with tap water), 2) grape powder-treated (GP; injected with vehicle and provided for 3 wk with 15 g/L grape powder dissolved in tap water), 3) BSO-treated [injected with BSO (300 mg/kg body weight), i.p. for 7 d and provided with tap water], and 4) BSO plus grape powder-treated (GP+BSO; injected with BSO and provided with grape powder-treated tap water). Anxiety-like behavior was significantly greater in BSO rats compared with C or GP rats (P < 0.05). Grape powder attenuated BSO-induced anxiety-like behavior in GP+BSO rats. BSO rats made significantly more errors in both short- and long-term memory tests compared with C or GP rats (P < 0.05), which was prevented in GP+BSO rats. Systolic and diastolic blood pressure was significantly greater in BSO rats compared with C or GP rats (P < 0.05), whereas grape powder prevented high blood pressure in GP+BSO rats. Furthermore, brain extracellular signal-regulated kinase-1/2 (ERK-1/2) was activated (P < 0.05), whereas levels of glyoxalase-1 (GLO-1), glutathione reductase-1 (GSR-1), calcium/calmodulin-dependent protein kinase type IV (CAMK-IV), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) were significantly less (P < 0.05) in BSO but not in GP+BSO rats compared with C or GP rats. We suggest that by regulating brain ERK-1/2, GLO-1, GSR-1, CAMK-IV, CREB, and BDNF levels, grape powder prevents oxidative stress-induced anxiety, memory impairment, and hypertension in rats.

FIGURE 1

Analysis of markers of oxidative stress 8-isoprostane and MDA in rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine–treated (BSO), and grape powder plus BSO. The 8-soprostane concentrations in urine (A) and serum (B) were measured by using an Enzyme Immuno Assay kit (Cayman). (C) MDA was measured as previously described by us (7, 16) and quantified by using the molar extinction coefficient 1.56 × 10⁵ M⁻¹ cm⁻¹. (A, B) Means without a common letter differ, P < 0.05. (C) Means without a common letter differ within each brain region, P < 0.05. Bars represent means ± SEMs, n = 6–10 rats/group. BSO, BSO-treated rats; C, vehicle-treated control rats; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats; MDA, malondialdehyde.

FIGURE 2

Examination of anxiety-like behavior tests including light-dark (A) and open-field (B–D) tests in rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine (BSO), and grape powder plus BSO. The open-field test determined center time (B), rearing (C), and fecal boli (D). Means without a common letter differ, P < 0.05. Bars represent means ± SEMs, n = 10 rats/group. BSO, BSO-treated rats; C, vehicle-treated control rats; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats.

FIGURE 3

Examination of radial arm water maze (RAWM) memory tests in rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine–treated (BSO), and grape powder plus BSO. Short-term (A) and long-term (B) memory was assessed by using a series of 12 RAWM trials. The RAWM apparatus is shown as an insert containing a circular water pool with 6 swim paths. Means without a common letter differ, P < 0.05. Bars represent means ± SEMs, n = 10 rats/group. BSO, BSO-treated rats; C, vehicle-treated control rats; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats.

FIGURE 4

Blood pressure measurement in rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine (BSO), and grape powder plus BSO. Means (in left and right panels) without a common letter differ, P < 0.05. Bars represent means ± SEMs, n = 8–10 rats/group. BSO, BSO-treated rats; C, vehicle-treated control rats; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats.

FIGURE 5

Analysis of ERK-1/2 activation in the hippocampus, amygdala, and cortex of rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine (BSO), and grape powder plus BSO. ERK-1/2 activity was determined as previously published (16) (details described in the Methods section). Representative blots for phospho-ERK-1/2 (upper panel) and total ERK-1/2 (lower panel). Density ratios of phospho-ERK-1/2 and total ERK-1/2 are represented as bars. Means without a common letter differ within the hippocampus, amygdala, and cortex, P < 0.05. Bars represent means ± SEMs, n = 6–8 rats/group. BSO, BSO-treated rats; C/CON, vehicle-treated control rats; ERK-1/2, extracellular signal-regulated kinase-1/2; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats.

FIGURE 6

Examination of GLO-1 and GSR-1 protein levels in the hippocampus, amygdala, and cortex of rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine (BSO), and grape powder plus BSO. Protein levels of GLO-1 (A) and GSR-1 (B) were determined by Western blotting. The upper panels shown in (A) and (B) are representative blots for GLO-1 (A), GSR-1 (B), and the protein loading control β-actin (A, B). Bar graphs in (A) and (B) are ratios of GLO-1 to β-actin and GSR-1 to β-actin, respectively. Means without a common letter differ within the hippocampus, amygdala, and cortex, P < 0.05. Bars represent means ± SEMs, n = 6–8 rats/group. BSO, BSO-treated rats; C/CON, vehicle-treated control rats; GLO-1, glyoxalase-1; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats; GSR-1, glutathione reductase-1.

FIGURE 7

Examination of CAMK-IV, CREB, and BDNF protein levels in the hippocampus, amygdala, and cortex of rats treated with vehicle, grape powder, l-buthionine-(S,R)-sulfoximine (BSO), and grape powder plus BSO. Protein expression levels of CAMK-IV (A), CREB [phospho (P)/total] (B), and BDNF (C) in the hippocampus, amygdala, and cortex were determined by using Western blotting. Upper panels are representative blots for CAMK-IV/β-actin (A), phospho-CREB/total CREB (B), and BDNF/β-actin (C). Bar graphs are ratios of CAMK-IV to β-actin, phospho to total CREB, and BDNF to β-actin, respectively. Means without a common letter differ within the hippocampus, amygdala, and cortex, P < 0.05. Bars represent means ± SEMs, n = 6–8 rats/group. BDNF, brain-derived neurotrophic factor; BSO, BSO-treated rats; CON, vehicle-treated control rats; CAMKIV, calcium/calmodulin-dependent protein kinase type IV; CREB, cAMP response element–binding protein; GP, grape powder–treated rats; GP+BSO, grape powder plus BSO–treated rats; P-CREB, phosphorylated cAMP response element–binding protein.