Neuroprotective Effects Of Glochidion Zeylanicum Leaf Extract Against H2O2/Glutamate-Induced Toxicity in Cultured Neuronal Cells And Aβ-Induced Toxicity in Caenorhabditis Elegans

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Simple Summary: Antioxidants that are interrelated in the process of overcoming oxidative-stress-induced toxicity and neurite-outgrowth-inducing activity have become the main targets of neuroprotective therapy. The methanol extract of Gloclidion zeylanicum (GZM) exhibits neuroprotective properties that are not only limited against H2O2/glutamate/Aβinsults but also promote neurite outgrowth activity. The neuroprotective effects ofGZM extract were confirmed in cultured neuronal (HT-22 and Neuro-2a) cells and C.elegans models. To the best of our knowledge, this study is the first to report for the neuroprotective effects of GZ M extract, suggesting that G.zeylanicum may be a neuroprotectant applicant for the prevention anc alleviation of oxidative stress-induced neurodegenerative disorders, including Alzheimer's disease. However, additional studies are required to identify the mechanistic pathways involved in neuroprotection and to confirm the efficacy of the extract in more complex model organisms.

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Abstract: Oxidative stress plays a crucial role in the development of age-related neurodegenerative diseases. Previously, Glochidion zeylanicum methanol (GZM) extract has been reported to have antioxidant and anti-aging properties. However, the effect of GZM on neuroprotection has not been reported yet; furthermore, the mechanism involved in its antioxidant properties remains unresolved. The study is aimed to demonstrate the neuroprotective properties of GZM extract and their underlying mechanisms in cultured neuronal (HT-22 and Neuro-2a) cells and Caenorhabditis elegans models. GZM extract exhibited protective eeffects against glutamate/H2O2-induced toxicity in cultured neuronal cells by suppressing the intracellular reactive oxygen species (ROS) generation and enhancing the expression of endogenous antioxidant enzymes (SODs, GPx, and GSTs). GZM extract also triggered the expression of SIRT1/Nrf2 proteins and mRNA transcription of antioxidant genes (NQO1,GCLM,and EAAT3)which are the master regulators of cellular defense against oxidative stress. cistanche tubulosa benefits and side effects Additionally, GZM extract exhibited protective effects to counteract β-amyloid (Aβ)-induced toxicity in C. elegans and promoted neuritogenesis properties in Neuro-2a cells. cistanche stem Our observations suggest that GZM leaf extract has interesting neuritogenesis and neuroprotective potential and can possibly act as potential contender for the treatment of oxidative stress-induced Alzheimer's disease (AD) and related neurodegenerative conditions; however, this needs to be studied further in other in vivo systems.

Keywords: Glochidion zeylanicum; glutamate; anyloid-β;H2O2; neurite outgrowth; Nrf2/SIRT1;antioxidant; Caenorhabditis elegans;HT22;Neuro-2a

1. Introduction

Alzheimer's disease(AD) is a progressive neurologic ailment, which causes cognitive impairments and memory problems in patients [1]. The typical markers of AD histopathol-ogy are the accumulation of neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein and β-amyloid (Aβ) plaques in the brain tissue, which lead to neuronal dysfunction and cell death [1]. The abnormal accumulations of Aβ and tau protein affect neuroplas-ticity and neurodegeneration, which correlate well with the cognitive symptoms of AD patients [1,2]. The evidence supports that oxidative stress-associated neuronal cell damage plays a crucial role in the pathogenesis of AD [3]. Oxidative stress is closely associated with neurotoxicity, particularly by promoting Aβ aggregation and mitochondrial damage, which triggers neuronal cell death [1]. Glutamate is the main excitatory neurotransmitter that has been considered as an initiating factor of neuronal death in several neurodegenerative disorders[4,5]. Additionally, the high level of reactive oxygen species (ROS)accumulation is closely related to neuronal damage by glutamate [4,6,7], which occurs via receptor-mediated excitotoxicity and non-receptor-mediated oxidative toxicity [4,6].

Cistanche can anti-aging

The prevalence of AD is increasing among aging populations[1]. Currently, there are few effective drugs available for AD treatment. However, the drugs used to treat AD exert various adverse effects [1,8]. Recently, antioxidant compounds have been regarded as essential supplements for the alternative treatment and prevention of AD [9]Prevention against oxidative-stress-induced neuronal toxicity is a key parameter in aiding neuroprotection [10]. Therefore, natural bioactive compounds found in herbs or plants with potent antioxidant and neuroprotective effects may provide complementary and alternative approaches for the treatment or prevention of AD and other neurodegenerative disorders.

Glochidion zeylanicum(Gaertn.) A.Juss.(family Phyllanthaceae)(GZ), native to Eastern Asia including Thailand, is a rich source of antioxidant compounds. cistanche tubulosa extract In our previous study, the leaf extract of GZ was shown to promote oxidative stress resistance and exhibit anti-aging effects in the nematode Caenorhabditis elegans through the DAF-16/FoxO and SKN-1/Nrf-2 signaling pathways. [11,12]. Nevertheless, the neuroprotective and neuritogenesis properties of GZ extract have not been reported.

The current study explored the neuroprotective effects of GZ extract and its underlying mechanisms on neurodegenerative events using cultured neuronal (HT-22 and Neuro-2a)cells and C.elegans models. We also investigated the neuritogenesis properties of GZ extract in the context of neurite outgrowth. The study provides experimental evidence of GZ extract and its applications in the prevention or treatment for neurodegenerative conditions involving oxidative stress. 2.Materials and

Methods

2.1.Chemicals and Reagents

All the chemicals and reagents used in the study were purchased from Invitrogen (Carlsbad, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA). Antibodies (both pri-mary and secondary) were procured from Cell Signaling Technology (Danvers,MA, USA)(Supplementary Materials).

2.2.Plant Extraction

Glochidion zeylanicum (GZ) leaves were collected by Mrs. Laong Kwunpet and Mrs. Korakod Choosri from Jana district, Songkhla Province, in southern Thailand (7.205278°N, 100.596944°E).The plant samples were deposited for identification at the Kasin Suvatab-handhu herbarium, Department of Botany, Faculty of Science, Chulalongkorn University, Thailand (Voucher specimen No. BCU-016061). Dried and ground leaves (40 g) were extracted with methanol (400mL) using a Soxhlet apparatus as previously described [11]. The extracts were filtrated and evaporated at 35-45°C. The GZ methanol GZM) extract was prepared in DMSO and stored at -20℃ as a stock.

2.3. Qualitative Phytochemical Screening

High-performance liquid chromatography (HPLC) of GZM extract was performed at RSU Science and Technology Research Equipment Center, Rangsit University, Thailand for the analysis of the chemical constituents (Supplementary Materials).

2.4.Cell Culture

Cell cultures were maintained as previously described [13]. HT-22 cells (Salk Institute, CA, USA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and Neuro-2a cells (The JCRB Cell Bank, Osaka,Japan) were maintained in Ham's Nutrient Mixture F12, supplemented with 10% fetal bovine serum and 1% streptomycin. Both cells were grown at 37°C with 5% CO2 atmosphere.

2.5.Cell Treatment

Pre-treatment of HT-22 and Neuro-2a cells was performed with various concentrations (0.5-10 ug/mL) of GZM extract for 48 h. Glutamate or H2O2 was then mixed into the culture medium for inducing cell toxicity. For protective assays, the extract was co-treated with glutamate (HT-22:18h, Neuro-2a:24h) or H2O2(15min). DMSO (0.1% v) served as the control group. There was no significance between DMSO treatment and untreated control (Figure S3, Supplementary Materials).

2.6.Determination of Cell Viability

Cell viability was determined using MTT and LDH assays. The details are provided in the Supplementary Materials. 2.7.Measurement of Intracellular ROS

The measurement of intracellular ROS generated upon treatment was assayed with DCFH-DA dye as previously described [11]. The details are provided in the Supplementary Materials.

2.8.RNA Isolation and Quantitative RT-PCR

RNA extraction was performed with TRIzol reagent. Quantitative real-time PCR was performed using standard procedures[13]. The primer sequences are listed in the Supple-mentary Materials. β-actin was used as the normalization control [7,13,14] (Supplementary Materials).

2.9.Western Blot Analysis

Proteins were obtained from Neuro-2a cells by lysis 1X using RIPA buffer containing protease inhibitor cocktail (PMSF) and quantified using Bradford assay. Briefly, protein was electrophoresed through 10% SDS polyacrylamide gel, followed by transferring to PVDF membranes and blocking (5% skim milk).cistanche tubulosa reviews The membranes were subjected to immunoblot analysis using respective antibodies (SIRT1, fNrf2, and β-actin (Supplementary Materials). Full images of the blots are provided in the supplementary materials (Figure S2).

2.10.Neurite Outgrowth Analysis

Neurite outgrowth upon GZM extract was performed according to Eik et al. in Neuro-2a cells and number of neurite-bearing cells and neurite length were measured [15](Supplementary Materials).

2.11. C.elegans Strains and Culture Conditions

All of the C. elegans strains used in the study were procured from Caenorhabditis Genetics Center (University of Minnesota, U.S.A.) and maintained with Escherichia coli OP50 as the food source[16]. The nematodes were cultured in NGM agar plates at 16 ℃and synchronized populations were developed as described previously [7,12]. Strains used in this study include CL4176(smg-1(cc546) I;dvls27[(myo-3p:A-Beta(1-42):let-8513'UTR)+ rol-6(su1006)]X),CL2006 (dvls2 [pCL12(unc-54/human Abeta peptide 1-42minigene)+pRF4),CL2355(smg-1(cc546)dvls50[pCL45(snb-1:Abeta1-42:3'UTR(long)+mtl-2:GFP]I),and CL2122(dvls15[(pPD30.38)unc-54(vector)+(pCL26)mtl-2:GFP.The nematodes were treated with various concentrations (1.25,2.5, and 5 ug/mL) of GZM extract with DMSO(1% v/v) as the control.

2.12.Paralysis Assay

Transgenic worms (CL4176 and CL2006) expressing human Aβ1-42 were used to study the effect of GZM against Aβ toxicity[17]. The transgenic worm CL4176 expressed and aggregated human Aβ1-42 peptides in the muscle cells after a temperature shift to 25 ℃, leading to oxidative stress and paralysis [17]. The transgenic worm CL2006 constitutively express Aβ along the body-wall muscles leading to progressive paralysis in adulthood [17]. For Aβ-independent effects, CL802 strain was used as control. The worms were synchronized and treated with GZM extract at the L4 stage.

The CL4176 worms were maintained at 16 ℃ for 48 h and shifted to 25℃ to induce Aβ expression. The number of paralyzed worms was measured at 20,22,26,28,and 30 h after the shift in temperature.

The CL2006 worms were maintained at 16 ℃ and classified as paralyzed when they did not respond to touch or showed halo appearance around the worms' heads. Paralyzed worms were classified and excluded from the plates every second day.

2.13. Chemotaxis Assay

The transgenic strains CL2122 and CL2355 were used for the identification of Aβ-induced defects in chemotaxis behavior. The transgenic worms (L4 stage) were treated with GZM extract at 16 ℃ for 36 h, and then shifted to 23℃ for 36 h to induce Aβ1-42 expression. After the treatment period, plates were washed, and the nematodes were placed in the center of the plate. The attractant side contained a mixture of diacetyl (0.1% in absolute ethanol) and sodium azide(1 M) on the plate. The opposite side of the plate (control)contained a mixture of absolute ethanol and sodium azide(1M). Worms drawn toward the attractant and repellant sides were counted, and the chemotaxis index was calculated (the number of worms at attractant location—the number of worms at the control location)/the total number of worms).

2.14.Statistical Analysis

The data are shown as the mean ± SEM. Data handling and statistical processing were completed using GraphPad Prism 8.0 and analyzed by one-way ANOVA, followed by Bonferroni's test. and p ≤0.05 was considered to be significant.

3. Results

3.1.Measurement of Optimum Glutamate and HzO2 Conditions in Cultured Neuronal (HT-22 and Neuro-2a)Cells

Neuronal cell damage induced by oxidative stress is a major phenomenon in neurodegenerative conditions [3]. cistanche UK The optimum conditions at which H2O2/glutamate induces neurotoxicity in HT22 and Neuro-2a cells were investigated. Exposure of HT-22 and Neuro-2a cells to different doses of glutamate (2.5-10 mM) and HzO2 (100 to 400 uM) for 1-24h and 5-90 min, respectively. For H2O2 treatment, we found that cell viability of both cells reduced by 50% when compared with the untreated control after treatment with 20 and 400 uMH2O2 for 15 min in HT22 and Neuro-2a cells, respectively (Figure la,b).In the case of glutamate treatment, a reduction in cell viability by 50% was obtained at 5 and 10 mM glutamate in HT22(18h) and Neuro-2a cells (24 h), respectively (Figure 1c,d). Therefore, these optimum conditions were used in the following experiments.

3.2.Neuroprotective Effects of GZM Extract against H2O2/Glutamate-Induced Neuronal Death in Cultured Neuronal (HT-22 and Neuro-2a) Cells

The cytotoxicity of GZM extract in both cells was investigated to examine the non-lethal concentration. GZM extract (0.5-10 ug/mL) treatment was performed for 48h in HT22 and Neuro-2a cells. When compared with the DMSO control, GZM extract treatment （0.5-10 μg/mL） did not affect the cell viability of both cells （Figure le，f）. The results indicated that GZMextract is non-toxic at the tested doses (0.5-10 ug/mL). Therefore, these concentrations were used in subsequent experiments.

The time- and dose-dependent responses of H4O2/glutamate supported and confirmed the experimental model of neurotoxicity in cultured neuronal (HT22 and Neuro-2a)cells (Figure la-d). Cell survival, when treated with H2Oz alone, was significantly lower (approximately 50%) than the DMSO control cells. However, co-treatment with GZM

3.2.Neuroprotective Effects of GZM Extract against H2O2/Glutamate-Induced Neuronal Death in Cultured Neuronal (HT-22 and Neuro-2a) Cells

The cytotoxicity of GZM extract in both cells was investigated to examine the non-lethal concentration. GZM extract (0.5-10 ug/mL) treatment was performed for 48h in HT22 and Neuro-2a cells. When compared with the DMSO control, GZM extract treatment （0.5-10 μg/mL） did not affect the cell viability of both cells （Figure le，f）. The results indicated that GZMextract is non-toxic at the tested doses (0.5-10 ug/mL). Therefore, these concentrations were used in subsequent experiments.

The time- and dose-dependent responses of H4O2/glutamate supported and confirmed the experimental model of neurotoxicity in cultured neuronal (HT22 and Neuro-2a)cells (Figure land). Cell survival, when treated with H2Oz alone, was significantly lower (approximately 50%) than the DMSO control cells. However, co-treatment with GZM Similarly,co-treatment with GZM extract significantly improved the cell viability compared with that of glutamate-treated cells (Figure 3a-c,f). Furthermore,glutamate-induced LDH release was also attenuated by GZM treatment (Figure 3c,d). The results suggest that GZM extract exerted an influential neuroprotective effect against H2O2/glutamate-induced neurotoxicity.

This article is extracted from Biology 2021, 10, 800. https://doi.org/10.3390/biology10080800 https://www.mdpi.com/journal/biology