Vitis Vinifera Leaf Extract Protects Against Glutamate-Induced Oxidative Toxicity in HT22 Hippocampal Neuronal Cells and Increases Stress Resistance Properties in Caenorhabditis Elegans

Abstract

Vitis vinifea has been used for traditional medicines, food, beverages, and dietary antioxidant supplements. The chemical compositions and biological activities of the fruits and seeds have been extensively investigated. However, the biological effects of the leaves are limited, and its anti-neurodegeneration or antiaging activities are little known. The current work aims to study the beneficial effects of V. vinifera leaf extract on neuroprotective effects in HT22 cells, antiaging, and oxidative stress resistance properties in the Caenorhabditis elegans model. The ethanol extract was characterized by phytochemical composition using gas/liquid chromatography-mass spectrometry and reversed-phase high-performance liquid chromatography. The beneficial effects of V. vinifera ethanol (VVE) extract on antioxidant properties, neuroprotective effects, and the underlying mechanisms were studied by in vitro and in vivo studies. In HT22 cells, we found that VVE has a protective effect against glutamate-mediated oxidative stress-induced cell death. The gene expression of cellular antioxidant enzymes such as CAT, SODs, GSTs, and GPx was upregulated by VVE treatment. Moreover, VVE was also shown to alleviate oxidative stress and attenuate reactive oxygen species accumulation in C. elegans. We demonstrated that VVE could upregulate the expression of stress-response genes gst-4 and sod-3 and downregulate the expression of hsp-16.2. Our results suggest that the oxidative stress resistance properties of VVE are possibly involved in DAF-16/FoxO transcription factors. VVE reduced age-related markers (lipofuscin) while did not extend the life span of C. elegans under normal conditions. This study reports the neuroprotective effect and antioxidant activity of V. vinifera leaf extract and suggests its potential as a dietary or alternative supplement to defend against oxidative stress and age-related diseases.

Keywords: HT22; aging; caenorhabditis elegans; daf-16; glutamate toxicity; neuroprotection; oxidative stress resistanc; vitis vinifera.

Figure 1

Representative bioactive compounds in VVE extract. LC-MS-MS (A) and HPLC (B) profiles of major compounds in VVE extract.

Figure 2

Protective effects of VVE extracts against glutamate-induced toxicity in HT22 cells. Cell viability by treatment with different concentrations of VVE extracts for 48 h (a) and cell viability by treatment with different concentrations of glutamate for different times (b). Cells were treated with different concentrations of VVE extracts for 48 h and exposed to 5-mM glutamate for 18 h. Then, cell viability was measured by MTT (c) and LDH (d) assay. Cell morphology was observed under a microscope at 5 × magnification (e). Samples were exposed to 5-mM glutamate (g) to induce toxicity. All data are shown as mean ± SEM of at least three independent experiments. ^####p < 0.0001 vs. DMSO control; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with glutamate-treated cells by one-way ANOVA following Bonferroni's method (post hoc).

Figure 3

Protective effect of VVE extracts against glutamate-induced oxidative stress in HT22 cells. VVE extracts treatment reduced ROS levels in HT22 cells when compared with glutamate-treated cells (a). VVE extract treatment increased endogenous antioxidant gene expression in HT22 cells when compared with DMSO control (b). Samples were pretreatment with VVE extracts for 48 h and exposed to 5-mM glutamate (G5) for 12 h to induce oxidative stress. Representative fluorescence micrographs of cells stained with H₂DCFDA were observed under a fluorescence microscope (c). All data are shown as mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with glutamate-treated cells; ^###p < 0.001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).

Figure 4

Protective effect of VVE extracts against juglone-induced oxidative stress in C. elegans. VVE extracts protect against oxidative stress in wild-type C. elegans. Survival rate of wild-type (N2) worms was significantly enhanced after pretreatment with extracts (a). VVE extracts treatment reduced ROS levels in N2 worms when compared with DMSO control (b). Representative pictures of DCFDA fluorescence in wild-type (N2) worms treated with 25 μg/ml VVE (c1); 50 μg/ml VVE (c2); 100 μg/ml VVE (c3); and DMSO control (c4). In survival assay, samples were exposed to 80-μM juglone (J) to induce oxidative stress. All data are shown as mean ± SEM of at least three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).

Figure 5

Stress resistance properties of VVE extracts mediated DAF-16/FoxO pathway in C. elegans. VVE extracts failed to increase survival rate (a) and decrease ROS levels (b) in CF1038 worms. Moreover, VVE extracts induced a significant translocation of DAF-16::GFP in mutant TJ356 worms (daf-16p::daf-16a/b::GFP + rol-6) (c). Representative fluorescent images of subcellular location of DAF-16 in nucleus, intermediate, cytosolic regions, and TJ356 worms after treated with VVE extracts (d). All data are shown as mean ± SEM of at least three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).

Figure 6

Effect of VVE extracts on expression of stress resistance-related genes in C. elegans. VVE extracts decreased hsp-16.2 expression in mutant TJ375 worms (a), increased sod-3 expression in mutants CF1553 worms (b), and gst-4 expression in mutants CL2166 worms (c). a2–a5, b2–b5, c2–c5: Representative pictures of GFP fluorescence in worms treated with 25 μg/ml VVE (a2/b2/c2); 50 μg/ml VVE (a3/b3/c3); 100 μg/ml VVE (a4/b4/c4); and DMSO control (a5/b5/c5). TJ375 and CL2166 worms were exposed to 20-μM juglone to induce mild oxidative stress. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).

Figure 7

Effect of VVE extracts on aging in C. elegans. VVE extracts attenuated autofluorescent pigment in BA17 worms (a). Autofluorescent granules were measured under blue wavelength band. VVE extracts had no effect on life span of wild-type (N2) worms in normal conditions (b). Brood size (c) and body length (d) of wild-type (N2) worms after VVE extracts treatment. Treatment with VVE extracts had no effect on egg-laying activity and body length. Results are expressed as mean ± SEM from three independent experiments (n = 30 worms in each experiment). Treatment groups are compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).