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

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.


INTRODUCTION
Neurogenesis describes the process of growth, survival, proliferation, differentiation and regeneration of neurons (Lam et al., 2016). Impairment of neurogenesis affects neuronal differentiation and neuronal cell loss in various neurodegenerative disorders (Lam et al., 2016). During neuronal differentiation, neurite outgrowth is an essential step for functional networks (connectome) of neurons. Regulation of neurite outgrowth can promote neuronal regeneration from nerve injury or neurological disorders, which plays an important role in development of therapies for neurodegenerative diseases (Bonaterra et al., 2018). Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system and plays a role in neurogenesis. Ten-4 expression mediates neurite outgrowth of the Neuro-2a cells (Suzuki et al., 2014).
Glutamate, the main excitatory neurotransmitter in the brain, has been recognized as one initiating factor for several neurodegenerative disorders (Jin et al., 2014;Sukprasansap et al., 2017). High levels of glutamate activate structural degradation, ROS/RNS production, mitochondrial and DNA damage, which further lead to neurotoxicity and neuronal cell damage (Jin et al., 2014;Hirata et al., 2018). Glutamate oxidative stress and neurotoxicity play a major role in a variety of neurodegenerative diseases, especially Alzheimer's disease (AD) (Jin et al., 2014;Mittal et al., 2014). Sirtuin 1 (SIRT1) is a Class III histone deacetylases that plays an important role in physiological and biochemical cell processes, including aging, inflammation and neuroprotection (Xue et al., 2016). SIRT1 regulates transcription factors, including nuclear factor-E2-related factor 2 (Nrf2) that is a major regulator in antioxidant defenses. Evidence suggests that SIRT1 and Nrf2 are involved in the CNS redox balance of neurodegenerative disorders by promoting antioxidant responses (Peng et al., 2017;Bi et al., 2018). In addition, enhancing SIRT1 and Nrf-2/HO-1 expression can protect neurons against oxidative injury in neuronal cells (Peng et al., 2017;Bi et al., 2018).
Reduction of oxidative stress and induction of neuronal differentiation are key parameters for neuroprotective effects. Thus, natural products from herbs or plant extracts with antioxidative and neuroprotective properties could provide an alternative approach to treat neurodegenerative diseases.
Anacardium occidentale L (AO) originates from Brazil, but is presently cultivated in many tropical countries around the globe. Leaf extracts from A. occidentale have been widely used as food and medicine in Thailand. The secondary metabolites which are presented in Anacardium plants exhibit substantial anti-oxidant (Salehi et al., 2019;Fusco et al., 2020;Siracusa et al., 2020), anti-inflammatory Siracusa et al., 2020) and anti-microbial (Muroi and Kubo, 1996;Salehi et al., 2019) effects. A recent study reported high content of antioxidant bioactive secondary metabolites from A. occidentale leaf extracts, including quercetin 3-O-glucoside, quercetin 3-(2galloylglucoside), quercetin 3-arabinoside, quercitrin/kaempferol-7-O-glucoside, α-tocopherols, and salicylic acid (Duangjan et al., 2019a). Moreover, the leaf extracts exerted anti-aging and oxidative-stress resistance in the Caenorhabditis elegans model (Duangjan et al., 2019a). However, neuritogenesis and neuroprotective effects of A. occidentale leaf extracts against oxidative stress in neuronal cell models have not been reported.

Plant Extraction
The leaves of Anacardium occidentale L (AO) were collected from Jana district, Songkhla Province, in southern Thailand (7. 205278°N , 100.596944°E) by Mrs. Laong Kwunpet and Mrs. Korakod Choosri. The leaves were stored as a voucher specimen (No. BCU-015863) at the herbarium of Kasin Suvatabhandhu (Department of Botany, Faculty of Science, Chulalongkorn University, Thailand). The leaves were sequentially extracted with hexane, dichloromethane and methanol using a Soxhlet apparatus as described before (Duangjan et al., 2019a).

Qualitative Phytochemical Screening
The secondary metabolites of the hexane and methanol extract were submitted to characterize and quantify the bioactive compounds by Gas/Liquid Chromatography-Mass Spectrometry (GLC-MS) and High-Performance Liquid Chromatography (HPLC) (Duangjan et al., 2019b)

Cell Culture
Mouse hippocampal neuronal HT22 cells have been used to study the neuroprotective properties. These cells lack ionotropic glutamate receptors and are resistant to excitotoxicity as a cause for glutamate-stimulated neuronal death (Sukprasansap et al., 2017). Mouse neuroblastoma Neuro-2a cells have been extensively used to study neuronal differentiation and neurite growth (Park et al., 2015). Thus, HT22 cells and Neuro-2a cells were used for the neuronal cell models in this study.

Cell Treatment
HT22 and Neuro-2a cells were pretreated with different concentrations of A. occidentale hexane extract (AOH) (10-50 μg/ml) and A. occidentale methanol extract (AOM) (0.5-10 μg/ml) for 48 h. To induce cell toxicity, glutamate or H 2 O 2 were added to the culture medium. For protective assays, the extracts were co-treatment with glutamate or H 2 O 2 for 18-24 h or 15 min respectively. Stock solutions of glutamate and H 2 O 2 were prepared in DMEM. Stock solutions of the AO extracts were prepared in DMSO. For the untreated control group, cells were treated with 0.1% (v/v) DMSO. For the positive control group, cells were treated with 4 μM quercetin.

Determination of Cell Viability
Cell viability was evaluated by using MTT and LDH assays (Supplementary Materials).

Measurement of Intracellular Reactive Oxygen Species
ROS production was quantified by the DCFH-DA method. After treatment, 10 μM H2DCFDA was added to the culture medium and incubated for 30 min at 37°C, followed by washing with Hank's balanced salt solution (HBSS). Fluorescence intensity (excitation 485 nm; emission 535 nm) was measured using an EnSpire ® Multimode Plate Reader (Perkin-Elmer). Data were expressed as the percentage of fluorescence intensity of treated cells relative to the untreated control.

Western Blot Analysis
Whole-cell lysates were prepared in 1× RIPA buffer for 45 min on ice according to the manufacturer's protocol. The cell lysates were collected and the protein concentration was determined using the Bradford protein assay. An equal amount of protein (20 μg) was denatured by heating in Laemmli loading buffer at 95°C for 10 min, subsequently separated on 6-10% SDS polyacrylamide gel and then transferred to PVDF membranes.

Measurement of Neurite Outgrowth and Neurite-Bearing Cells
Neuro-2a cells were performed in neurite outgrowth stimulation assay according to Eik et al. (2012)

Knockdown of Teneurin-4 Expression
For Teneurin-4 (Ten-4) knockdown, On-Target Plus small interfering RNA (siRNA), which contains the targeting sequence of Ten-4 (Thermo Fisher Scientific), was used. Specific siRNAs of the Ten-4 gene were designed according to Suzuki et al.

Statistical Analysis
All experiments were performed at least in triplicate. The data are shown as the mean ± SEM and were analyzed with GraphPad Prism 6. A comparison between the control and treatments was analyzed by one-way ANOVA following Bonferroni's method (post hoc). Differences were considered significant at the p ≤ 0.001 level.

RESULTS AND DISCUSSION
Phytochemical Constituents of Anacardium Occidentale L. Extracts In our previous study, AO hexane and methanol extracts demonstrated oxidative stress resistance properties in a C.
elegans model (Duangjan et al., 2019a). Thus, hexane and methanol extracts were explored in the present study. GLC-MS profiles represented the main compounds in the AO hexane extract, which were identified as palmitic acid (8495.95 mg/100 g of crude extract) and α-linolenic acid (4073.13 mg/100 g of crude extract) ( Figure 1A). Moreover, HPLC showed the presence of bioactive compounds in AO methanol extract that were identified as gallic acid (305.92 mg/100 g of crude extract), catechin (1924.13 mg/100 g of crude extract) and quercetin (707.10 mg/100 g of crude extract) ( Figure 1B). These results were consistent with our previous study (Duangjan et al., 2019a).

Cytotoxicity of Anacardium Occidentale L. Extracts on HT22 and Neuro-2a Cells
Cytotoxic activity of the extracts were tested in HT22 and Neuro-2a cells to show that the extracts were innocuous to a Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 627738 5 FIGURE 3 | Protective effects of AO extracts on H 2 O 2 -induced toxicity in HT22 and Neuro-2a cells. Cells were exposed to various concentrations of H 2 O 2 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 H 2 O 2 (H200: 200 µM H 2 O 2 , H400: 400 µM H 2 O 2 ) 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 H 2 O 2 only.
Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 627738 6 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.
Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 627738 7 normal cells line. We found that the treatment with different concentrations of AO extracts for 48 h did not change the cell viability of HT22 and Neuro-2a cells compared to the untreated control (Figures 2A,B). Results indicated that the AO extracts were relatively non-cytotoxic at the tested doses in HT22 and Neuro-2a cells.  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.  Figures 3A,B). However, the viability of both cells pretreated with AO extracts had significantly lower H 2 O 2 -induced cell death compared to that of the cells exposed to H 2 O 2 alone ( Figures 3C,E). FIGURE 6 | Effect of AO extracts on SIRT1/Nrf2 signaling pathway. AO methanol extract treatment increased the SIRT1 (A), Nrf2 expression (B) and antioxidantrelated 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.
Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 627738 9 In a complementary set of experiments, the optimal condition for glutamate-induced toxicity was elucidated. The cells were exposed to various concentrations of glutamate for 1-24 h. Cell viability of HT22 and Neuro-2a exposure to glutamate (5 mM for 18 h, HT22 cells; 10 mM for 24 h, Neuro-2a cells) decreased by 50% when compared to the untreated control ( Figures 4A,B). Surprisingly, the viability of the both cells pretreated with AO extracts had significantly lower glutamate-induced cell death compared to that of the cells exposed to glutamate alone ( Figures 4C,E).
Results were in a similar range as the quercetin positive control which is a well-known neuroprotective compound (Park et al., 2019), and were confirmed by LDH assay (Figures 3D,F, 4D,F) as well as morphological examination ( Figures 3G,H, 4G,H). Results suggest that AO extracts exert a potent neuroprotective effect against cytotoxicity induced by H 2 O 2 /glutamate in neuronal cells.

Effect of Anacardium Occidentale L. Extracts on Glutamate-Induced Oxidative Stress in HT22 and Neuro-2a Cells
In our previous study, AO extracts exhibited powerful antioxidant activity in vitro and in vivo (Duangjan et al., 2019a). To investigate whether AO extracts could suppress glutamate-induced oxidative stress, the antioxidant properties of AO extracts in neuronal (HT22 and Neuro-2a) cells were explored. Intracellular ROS level was significantly elevated in HT22 (approximately 1.7 fold) and Neuro-2a (approximately 1.9 fold) cells after exposure to glutamate, compared to the untreated control. Therefore, glutamate-induced cytotoxicity in neuronal (HT22 and Neuro-2a) cells was indeed associated with intracellular ROS increase. However, HT22 and Neuro-2a cells pretreated with AO extracts significantly reduced the elevated levels of ROS in the same range as the quercetin positive control (Figures 5A-D, Supplementary Material Figure S5, Supplementary Materials). Results suggest that AO extracts protect against glutamate-induced cytotoxicity by suppressing intracellular ROS production.

Effect of Anacardium Occidentale L. Extracts on Gene Expression of Antioxidant Enzymes in HT22 and Neuro-2a Cells
It is well known that glutamate-induced oxidative stress result in neuronal cell death (Fukui et al., 2009). To investigate the protective effects of AO extracts on glutamate-induced oxidative stress through endogenous antioxidant enzymes, we investigated the expression of antioxidant and phase II enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) which, have a major role in preventing ROS-mediated cellular damage (Sukprasansap et al., 2017).
Previous results showed that 25 μg/ml AO hexane extract and 10 μg/ml AO methanol extract exhibited a powerful neuroprotective effect in HT22 and Neuro-2a cells (Figures 3-5). Thus, these concentrations were used for the following experiments. HT22 and Neuro-2a cells, pretreated with 25 μg/ml AO hexane extract and 10 μg/ml AO methanol extract, showed significantly increased expression of endogenous antioxidant enzymes including SOD1, GPx, GSTo1, and GSTa2 ( Figures 5E,F). These results agree with our previous study that AO extracts also stimulated the expression of stress-response genes including, SOD-3 and GST-4 in C. elegans (Duangjan et al., 2019a). However, the expression of the CAT gene was not significantly changed in either HT22 or Neuro-2a ( Figures  5E,F). Results demonstrated that the protective effect of AO extracts against glutamate/H 2 O 2 -induced cytotoxicity was achieved not only by suppressing intracellular ROS production, but also through enhancing the expression of endogenous antioxidant enzymes. The neuroprotective effects of AO extracts may occur because of antioxidant activity from bioactive compounds such as palmitic acid (Ghareghani et al., 2017), α-linolenic acid (Lee et al., 2018), gallic acid (Oiram Filho et al., 2018), catechin (Herges et al., 2011;Zhang et al., 2020), and quercetin (Yang et al., 2014;Khan et al., 2018;Park et al., 2019), which were found in several studies to have neuroprotective properties.

Effect of Anacardium Occidentale L. Extracts on Cellular Antioxidant Defense Via SIRT1/Nrf2-Dependent Response
To investigate the underlying neuroprotective mechanism of AO extracts, we studied the SIRT1/Nrf2 signaling pathway. Pretreatment with 10 μg/ml AO methanol extract significantly increased the expressions of SIRT1 and Nrf2 (mRNA and protein levels) ( Figures 6A,B). However, 1 μg/ml AO hexane extract did not cause a significant change in SIRT1 and Nrf2 expression compared to the untreated control ( Figures 6A,B). To further extend our study, the effects of AO methanol extract on antioxidant-related target genes that are regulated by the SIRT1/ Nrf2 signaling pathway were elucidated. In addition, 10 μg/ml AO methanol extract also induced antioxidant-related target genes including NQO1, GCLM, and EAAT3 in Neuro-2a cells ( Figure 6C). Collectively, the findings demonstrate that AO methanol extract promotes antioxidant defense in Neuro-2a cells may be involved in the SIRT1/Nrf2 signaling pathway.
Recent studies demonstrated that phenolic antioxidants and aromatic compounds can activate ARE and induce the Nrf2/ARE signaling pathway (Jung and Kwak, 2010;Khan et al., 2018;Duangjan et al., 2019b;Yang et al., 2020), AO methanol extract contains phenolic (flavonoid glycoside) compounds including gallic acid, catechin and quercetin. Thus protective effects mediated by SIRT1/Nrf2 signaling pathway may be due to phenolic compounds in AO methanol extract. Moreover, a previous study also reported the oxidative stress resistance properties of AO methanol extract via the SKN-1/Nrf-2 signaling pathways in C. elegans (Duangjan et al., 2019a).

Effect of Anacardium Occidentale L. Extracts on Neurite Outgrowth Activity in Neuro-2a Cells
Neuritogenesis or neurite outgrowth is a process in the differentiation of neurons that plays a central role in neuronal development and the Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 627738 formation of synapses (Lam et al., 2016). The induction of neuronal differentiation is one of the neuroprotective factors. The effects of AO extracts on neurite outgrowth activity were explored in this study. Optimal concentrations of AO extracts (1 μg/ml AO hexane and 10 μg/ml AO methanol) were used for neurite outgrowth activity (Supplementary Material Figure S2, Supplementary Materials).
To investigate the effect of AO extracts on neurite outgrowth activity in Neuro-2a cells, the cells were maintained in a lowserum medium (DMEM supplemented with 1% FBS). Neuro-2a cells, that were treated with 1 μg/ml AO hexane extract, exhibited significantly increased neurite lengths (23.36 µm) and neurite bearing cells (43.25%) when compared to the 1% FBS control (neurite length, 17.68 µm; neurite bearing cells, 22.06%) ( Figures  7A,B). In addition, Neuro-2a cells that were treated with 10 μg/ml AO methanol extract, showed significantly increased neurite length (30.38 µm) and neurite bearing cells (54.06%) when compared to the 1% FBS control (Figures 7A,B). The neurite outgrowth inducing effects were similar to those of retinoic acid, which is a well-known inducer of neuronal differentiation (Kumar and Katyal, 2018).
To further confirm neurite outgrowth activities, GAP-43 expression, a marker of neurite outgrowth, was measured. Neuro-2a cells treated with AO extracts had significantly increased GAP-43 expression (mRNA and protein levels) when compared to 1% FBS control ( Figures 7C,D). Results suggest that AO extracts have an effect on neuritogenesis in Neuro-2a cells. These results agree with several recent studies regarding the neurodegeneration properties of α-linolenic acid, gallic acid (Siddiqui et al., 2019), catechin (Herges et al., 2011) and quercetin (Chan et al., 2018;Katebi et al., 2019).

Involvement of Signaling Pathway in Anacardium Occidentale L. Extracts Induced Neurite Outgrowth in Neuro-2a Cells
To investigate whether Ten-4 expression is involved in AO extracts-induced neurite growth in Neuro-2a cells, mRNA and protein expression levels of Ten-4 were examined. Neuro-2a cells treated with 10 μg/ml AO methanol extract exhibited significantly increased expression of Ten-4 mRNA and corresponding protein levels ( Figures 8A,B). However, 1 μg/ml AO hexane extract was inactive ( Figures 8A,B).
To confirm the role of Ten-4 in AO methanol extract-induced neurite outgrowth in Neuro-2a cells, Ten-4 siRNA (siTen-4) were used. When Ten-4 expression was knocked down by siTen-4, AO methanol extract failed to induce neurite length and neurite bearing cells in Neuro-2a cells agreeing with GAP-43 expression (Figures 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.
The available evidences suggest that SIRT1 and Nrf2 are also involved in neuroprotection and neurogenesis (Sugino et al., 2010;Liang et al., 2019). Thus, the neuroprotective and neurogenesis effects of AO methanol extract may be mediated by the SIRT1/Nrf2 signaling pathway. Nevertheless, further studies are needed to investigate the effects of AO methanol extract on neurite outgrowth via the SIRT1/Nrf2 signaling pathway.
AO contains a number of total phenolic compounds such as flavonoids, anthocyanins, and tannins which are therapeutically recognized in the treatment of several age-related diseases (Salehi et al., 2019). Moreover, AO contains phenolic lipids and anacardic acids, which have been reported in antimicrobial, antitumoral and antioxidant activities (Salehi et al., 2019;Siracusa et al., 2020).
The high concentrations of anacardic acids exhibited toxicity effects in bacteria and melanoma cells by inhibiting bacterial cell growth (Muroi and Kubo, 1996) and cancer cell proliferation (Radde et al., 2016;Tan et al., 2017). In contrast, the proper concentrations appeared antioxidant activities to modulate the immune responses and angiogenesis (Gomes Júnior et al., 2020).
Our previous study observed that AO extracts can effectively protect C. elegans against severe oxidative stress and attenuate intracellular ROS levels at moderate concentrations (Duangjan et al., 2019a). Higher concentrations of AO extracts cannot attenuate ROS levels in the worms, possibly due to a pro-oxidant activity of the plant extracts according to a high level of anacardic acids concentration (Duangjan et al., 2019a). 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. Wholecell 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.
Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 627738 In this study, we observed that AO extracts can effectively protect neuronal cells against severe oxidative stress and attenuate intracellular ROS levels. We suggested that the optimal concentration of AO extract is need for antioxidant and protective properties (Supplementary Material Figure S4). The neuroprotective effects of AO extracts are consistent with antioxidant properties against neurotoxicity of anacardic acids (Salehi et al., 2019), gallic acid (Oiram Filho et al., 2018), catechin (Herges et al., 2011;Zhang et al., 2020) and quercetin (Yang et al., 2014;Khan et al., 2018;Park et al., 2019).
Phytotherapy has many potentially significant advantages associated with synergistic interactions such as increased efficiency and reduced undesirable effects (Haroun and Al-Kayali, 2016). The synergistic interactions of bioactive compounds in AO extracts may involve neuroprotective effects and neurite outgrowth properties. There is the imitation for using the crude extracts in this study. It cannot conclude that the therapeutic effects are from a single compound or the synergistic interactions of bioactive compounds. However, there are several strengths for using the crude extracts such as the synergistic effect of the compounds and using the plants as dietary supplements. Further experiments of isolated bioactive components from AO extracts need to be done to confirm our interpretation. Moreover, the investigation focusing on the active components of AO extracts e.g., anacardic acids, gallic acid, catechin and quercetin, are interesting topics to clarify the neuroprotective properties of AO extracts.

CONCLUSION
In conclusion, these findings demonstrate the neuroprotective effects and neurite outgrowth properties of AO extracts in cultured neuronal (HT22 and Neuro-2a) cells. AO extracts exhibit neuroprotective effects against glutamate/H 2 O 2 -induced oxidative toxicity in neuronal cells which are mediated via inhibition of ROS accumulation, up-regulation of endogenous antioxidant enzymes, and the increase of the SIRT1/Nrf2 signaling. Significantly, AO extracts promoted neurite outgrowth via the up-regulation of Ten-4 expression. These results suggest that the leaf extracts have an interesting neuritogenesis and neuroprotective potential against glutamate/H 2 O 2 -mediated toxicity and could be a potential therapeutic candidate for neurodegenerative diseases. However, further studies focusing on the active components of AO extracts are crucial to verify the exact mechanisms involved in order to support the therapeutic potential of the plant extracts for alternative or adjunct treatment of neurodegenerative diseases.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
CD performed the experiments, analyzed data, and was a major contributor in writing the manuscript. PR performed the gene expression assay by RT-PCR. CD, PR, and SZ and designed the study and prepared media and reagents. MW review and editing the manuscript. TT provided materials for the study, conceived and supervised research. MW, TT, PR, and SZ corrected the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

FUNDING
This work was supported by the 90th Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) funding code GCUGR1125603032D No. 29. This work was supported by a scholarship from the "72nd Birthday Anniversary of His Majesty the King's for Doctoral Scholarship".