Polysaccharides from wolfberry antagonizes glutamate excitotoxicity in rat cortical neurons

Abstract

Glutamate excitotoxicity is involved in many neurodegenerative diseases including Alzheimer's disease (AD). Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. Wolfberry (Lycium barbarum) is a common ingredient in oriental cuisines. A number of studies suggest that wolfberry has anti-aging properties. In recent years, there is a trend of using dried Wolfberry as food supplement and health product in UK and North America. Previously, we have demonstrated that a fraction of polysaccharide from Wolfberry (LBA) provided remarkable neuroprotective effects against beta-amyloid peptide-induced cytotoxicity in primary cultures of rat cortical neurons. To investigate whether LBA can protect neurons from other pathological factors such as glutamate found in Alzheimer brain, we examined whether it can prevent neurotoxicity elicited by glutamate in primary cultured neurons. The glutamate-induced cell death as detected by lactate dehydrogenase assay and caspase-3-like activity assay was significantly reduced by LBA at concentrations ranging from 10 to 500 microg/ml. Protective effects of LBA were comparable to memantine, a non-competitive NMDA receptor antagonist. LBA provided neuroprotection even 1 h after exposure to glutamate. In addition to glutamate, LBA attenuated N-methyl-D-aspartate (NMDA)-induced neuronal damage. To further explore whether LBA might function as antioxidant, we used hydrogen peroxide (H(2)O(2)) as oxidative stress inducer in this study. LBA could not attenuate the toxicity of H(2)O(2). Furthermore, LBA did not attenuate glutamate-induced oxidation by using NBT assay. Western blot analysis indicated that glutamate-induced phosphorylation of c-jun N-terminal kinase (JNK) was reduced by treatment with LBA. Taken together, LBA exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate.

Fig. 1

Pre-treatment of LBA against glutamate neurotoxicity. Rat primary cortical neurons were treated with different dosages of LBA for 1 h and then co-incubated with 30 or 60 μM glutamate for 24 h. The level of neuronal cell death in culture treated with a LBA only c LBA and 30 μM glutamate and e LBA and 60 μM glutamate was accessed by LDH assay. Cultured neurons have a normal turnover with baseline of LDH of 12.5 ± 0.5% of total lysis. The level of apoptosis in culture treated with b LBA only d LBA and 30 μM glutamate and f LBA and 60 μM glutamate was accessed by colorimetric caspase-3 like activity assay. The specific activity of caspase-3 was 0.042 ± 0.007 pmol/min/μg of protein in the control group. Data represent mean ± SE from at least 3 independent experiments. The significance of differences among treatment groups was determined by one-way ANOVA, followed by Student Newman-Keuls as post hoc test. ^#  P < 0.001 compared with control. ^Δ  P < 0.05 compared with cultures treated with 30 μM glutamate. ** P < 0.001 compared with cultures treated with 60 μM glutamate. * P < 0.05 compared with cultures treated with 60 μM glutamate

Fig. 2

Morphological changes in LBA- or glutamate-treated groups. Neurons were incubated with or without glutamate for 24 h. LBA was added 1 h before the addition of glutamate. Photographs shows phase-contrast images of representative fields of cells after various treatments. a Control, b 30 μM glutamate, c 60 μM glutamate, circled areas indicate fragmented neuritis, d 500 μg/ml LBA, e 500 μg/ml LBA + 30 μM glutamate and f 500 μg/ml LBA + 60 μM glutamate

Fig. 3

Neuroprotective effects of LBA against glutamate toxicity were comparable to that of memantine. Neurons were treated with LBA or memantine for 1 h prior to the addition of glutamate for 24 h. The protective effects of LBA and memantine against glutamate-induced cell death and apoptosis were accessed by a LDH assay and b caspase-3 like activity assay. The effects of memantine at 0.8 and 1 μM were 1.02 ± 0.01 fold of control and 1.07 ± 0.02 fold of control, respectively. Data represent mean ± SE from at least three independent experiments. The significance of differences among treatment groups was determined by one-way ANOVA, followed by Student Newman-Keuls as post hoc test. ^#  P < 0.001 compared with control. ** P < 0.001 compared with cultures treated with 60 μM glutamate. * P < 0.05 compared with cultures treated with 60 μM glutamate. ^ΔΔ  P < 0.001 compared with cultures treated with 30 μM glutamate. ^Δ  P < 0.05 compared with cultures treated with 30 μM glutamate

Fig. 4

Effects of post-treatment of LBA on glutamate neurotoxicity. Neurons were exposed to glutamate for 1 h prior to LBA for another 23 h. Release of LDH was measured in a neurons treated with LBA and 30 μM glutamate c LBA and 60 μM glutamate. Level of apoptosis in b neurons treated with LBA and 30 μM glutamate d LBA and 60 μM glutamate was accessed by measuring the caspase-3 like activity. Data were analyzed by one-way ANOVA for multiple comparisons, followed by Student Newman-Keuls as post hoc test. ^#  P < 0.001 compared with control. ^ΔΔ  P < 0.001 compared with cultures treated with 30 μM glutamate. ^Δ  P < 0.05 compared with cultures treated with 30 μM glutamate. * P < 0.05 compared with cultures treated with 60 μM glutamate

Fig. 5

Effects of LBA on NMDA neurotoxicity. LBA was added into the culture for 1 h and then co-incubated with 40 or 60 μM NMDA for 24 h. a LDH assay and b caspase-3 like activity assay were performed. Data represent mean ± SE from at least 3 independent experiments. The significance of differences among treatment groups was determined by one-way ANOVA, followed by Student Newman-Keuls as post hoc test. ^#  P < 0.001 compared with control. ^##  P < 0.05 compared with control. ^ΔΔ  P < 0.001 compared with cultures treated with 40 μM NMDA. ** P < 0.001 compared with cultures treated with 60 μM NMDA

Fig. 6

Effects of LBA on H₂O₂ toxicity. Neurons were treated with LBA for 1 h prior to the exposure to H₂O₂ for 24 h. a LDH assay and b caspase-3 like assay were carried out to examine its protective effect. Data represent mean ± SE from at least 3 independent experiments. The significance of differences among treatment groups was determined by one-way ANOVA, followed by Student Newman-Keuls as post hoc test. ^#  P < 0.001 compared with control. * P < 0.05 compared with cultures treated with H₂O₂

Fig. 7

Effects of LBA on glutamate-induced production of ROS. Neurons were treated with LBA for 1 h prior to the exposure to 60 μM glutamate for 2 h. NBT reduction assay were performed to access the level of intracellular ROS. Data represent mean ± SE from at least 3 independent experiments. The significance of differences among treatment groups was determined by one-way ANOVA, followed by Student Newman-Keuls as post hoc test. ^#  P < 0.001 compared with control. * P < 0.05 compared with cultures treated with glutamate only

Fig. 8

Effects of LBA on phosphorylation of JNK by glutamate. Neurons were treated with indicated dosages of LBA for 1 h prior to the exposure to 60 μM glutamate for 2 h. Neurons were harvested for measurement of phosphorylated JNK and non-phosphorylated JNK by western blotting. β-Actin was used as internal control. The images of western blots films were scanned and quantified with Image J. Data showed the ratio of p-JNK to JNK. Data represent mean ± SE from at least three independent experiments. Statistical analysis was performed with one-way ANOVA, followed by Student Newman-Keuls as post hoc test. ^# P < 0.05 compared with control. * P < 0.05 compared with cultures treated with 60 μM glutamate