Anacardium Occidentale L. Leaf Extracts Protect Against Glutamate/H2O2-Induced Oxidative Toxicity and Induce Neurite Outgrowth: The Involvement of SIRT1/Nrf2 Signaling Pathway and Teneurin 4 Transmembrane Protein

Abstract

Neurodegenerative diseases are linked to neuronal cell death and neurite outgrowth impairment that are often caused by oxidative stress. Natural products, which have neuroprotective against oxidative stress and neurite outgrowth inducing activity, could be potential candidates for alternative treatment of neurodegenerative diseases. This study aims to investigate the neuroprotective effects and neuritogenesis properties of Anacardium occidentale leaf extracts in cultured neuronal (HT22 and Neuro-2a) cells. We found gallic acid, catechin and quercetin as the main compounds in A. occidentale extracts. The extracts have a protective effect against glutamate/H₂O₂-mediated oxidative stress-induced cell toxicity. The gene expression of cellular antioxidant enzymes (SODs, GPx and, GSTs) were up-regulated by this treatment. The treatment also triggered SIRT, Nrf2 proteins as well as the mRNA transcriptions of relevant anti-oxidation genes (NQO1, GCLM, and EAAT3). We demonstrated that the extracts promote antioxidant defense in neuronal cells via the SIRT1/Nrf2 signaling pathway. Moreover, the extracts increase neurite outgrowth and Ten-4 expression in Neuro-2a cells. However, the neuritogenesis properties did not occur, when Ten-4 expression was knocked down by corresponding siRNA. These results suggest that the leaf extracts have an interesting neuritogenesis and neuroprotective potential against glutamate/H₂O₂-mediated toxicity and could be a potential therapeutic candidate for neurodegenerative diseases.

Keywords: H2O2; Nrf2/SIRT1; anacardium occidentale; glutamate; neurite outgrowth; teneurin-4.

GRAPHICAL ABSTRACT

FIGURE 1

Representative bioactive compounds in AO extracts. (A) GLC-MS profile of the AO hexane extract and (B) HPLC chromatograms of the AO methanol extract with the chemical structures of their main constituents.

FIGURE 2

Cytotoxicity effect of AO extracts in neuronal (HT22 and Neuro-2a) cells. The effects of AO extracts on cell viability of HT22 (A) and Neuro-2a (B) cells. The effects of quercetin on cell viability of HT22 (C) and Neuro-2a (D) cells. Cells were treated with different concentrations of the extracts or compounds for 48 h following MTT assay. The results are expressed as the means ± SEM of independent experiments (n = 3); p ≤ 0.001 compared to the untreated control.

FIGURE 3

Protective effects of AO extracts on H₂O₂-induced toxicity in HT22 and Neuro-2a cells. Cells were exposed to various concentrations of H₂O₂ for different times in HT22 (A) and Neuro-2a cells, cell viability was measured by MTT assay (B). Cell viability of HT22 (C,D) and Neuro-2a cells (E,F), cell morphology of HT22 (G) and Neuro-2a (H) after treatment with different concentrations of AO extracts. Cell morphology was observed under microscope at 5× magnification. Samples were treated with AO extracts for 48 h and exposed to H₂O₂ (H200: 200 µM H₂O₂, H400: 400 µM H₂O₂) for 15 min to induce toxicity. The results are expressed as the means ± SEM of independent experiments (n = 3). ****p < 0.0001 compared to the untreated control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 and ^#### p < 0.0001, compared to the group exposed to H₂O₂ only.

FIGURE 4

Protective effects of AO extracts on glutamate-induced toxicity in HT22 and Neuro-2a cells. Cells were exposed to various concentrations of glutamate for different times in HT22 (A) and Neuro-2a cells, cell viability was measured by MTT assay (B). Cell viability of HT22 (C,D) and Neuro-2a cells (E,F), cell morphology of HT22 (G) and Neuro-2a (H) after treatment with different concentrations of AO extracts. Cell morphology was observed under microscope at 5× magnification. Samples were treated with AO extracts for 48 h and exposed to glutamate (G5: 5 mM glutamate, G10: 10 mM glutamate) for 18 h (HT22 cells) or 24 h (Neuro-2a cells) to induce toxicity. The results are expressed as the means ± SEM of independent experiments (n = 3). ****p < 0.0001 compared to the untreated control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 and ^#### p < 0.0001, compared to the group exposed to glutamate only.

FIGURE 5

Protective effect of AO extracts on glutamate-induced oxidative stress. AO extract treatment reduced ROS levels in HT22 (A) and Neuro-2a (B) cells when compared to glutamate-treated cells. Representative fluorescence micrographs of HT22 (C) and Neuro-2a (D) cells stained with DCFH-DA were observed under a fluorescence microscope (10×) (Representative microscopy images from DCFH-DA, phase contrast and nuclear staining can be found in the Supplementary Material Figure S5. Samples were treated with AO extracts for 48 h and exposed to glutamate (G5: 5 mM glutamate, G10: 10 mM glutamate) for 12 h (HT22 cells) or 18 h (Neuro-2a cells) to induce oxidative stress. AO extract treatment increased endogenous antioxidant gene expression in HT22 (E) and Neuro-2a (F) cells when compared to untreated control. β-actin was used as the internal control for RT-PCR assay. The results are expressed as the means ± SEM of independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, compared to the untreated control; #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001, compared to the group exposed to glutamate only.

FIGURE 6

Effect of AO extracts on SIRT1/Nrf2 signaling pathway. AO methanol extract treatment increased the SIRT1 (A), Nrf2 expression (B) and antioxidant-related target genes (C) in Neuro-2a cells when compared to the untreated control. Samples were treated with AO extracts for 48 h. Whole-cell lysates were subjected to western blot analysis of the SIRT1 and Nrf2 levels after AO extract treatment. β-actin was used as an endogenous loading control for western blot assay and internal control for RT-PCR assay. All data were normalized to endogenous control levels and the results are expressed as the means ± SEM of independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, compared to the untreated control.

FIGURE 7

Effect of AO extracts on neurite outgrowth. AO extract treatment increased the average of neurite lengths (A) and the percentage of neurite-bearing cells (B) in Neuro-2a cells. Cell morphology of Neuro-2a cells was observed under a microscope at 10× magnification. Relative expression levels of mRNA (C) and protein (D) GAP-43 in Neuro-2a cells. Samples were treated with AO extracts for 48 h. Whole-cell lysates were subjected to western blot analysis of the GAP43 level after AO extract treatment. β-actin was used as an endogenous loading control for western blot assay and internal control for RT-PCR assay. All data were normalized to 10% FBS control level and the results are expressed as the means ± SEM of independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to the 1% FBS control; ^### p < 0.001 and ^#### p < 0.0001 compared to the 10% FBS control.

FIGURE 8

Effect of AO extracts on Ten-4-mediated neurite outgrowth. AO methanol extract treatment increased expression level of Ten-4 mRNA (A) and protein (B). AO methanol extract failed to induced neurite length (C) and neurite-bearing cells (D) in siTen-4-Neuro-2a cells. Results were confirmed by GAP43 mRNA expression (E). Cell morphology of Neuro-2a cells was observed under a microscope at 10× magnification (F). Samples were treated with AO extracts for 48 h. Whole-cell lysates were subjected to western blot analysis at the Ten-4 level after AO extract treatment. β-actin was used as endogenous loading control for western blot assay and internal control for RT-PCR assay. All data were normalized to 10% FBS control levels in siCont-Neuro-2a cells and the results are expressed as the means ± SEM of independent experiments (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to the 1% FBS control.