1. IntroductionIncreased lifespans in the Western world have led to a rise in the frequencies of neurodegenerative diseases resulting from neuronal loss. Despite their increased prevalence, there are currently no effective preventive or therapeutic modalities for these deficits. Oxidative stress leading to neuronal cell death is a central process involved in the initiation and progression of neurodegenerative diseases and aging and, as such, is considered a potential target for the treatment of these diseases [1,2]. Reactive oxygen species (ROS)-elicited oxidative stress is one of the major factors involved in the aging process and may serve as one of the mechanisms underlying age-related degenerative diseases [3]. Indeed, increased ROS production damages almost every intracellular macromolecule in neurons, including proteins, lipids, and DNA, resulting in cellular dysfunction, mutations, and/or cell death. Therefore, the regulation of ROS levels is important to prevent or treat neurodegenerative diseases and aging. In recent years, natural dietary components with antioxidant activity have attracted much attention due to their role in modulating oxidative stress associated with brain aging and chronic conditions [4,5]. H2O2 is a key metabolite in oxidative stress which occurs in normal metabolism in mammalian cells at concentrations ~10 nM [6]. Higher concentrations lead to adaptive stress responses via nuclear factor erythroid 2–related factor 2 (Nrf2)/Keap1, while supraphysiological concentrations of H2O2 (>100 nM) lead to damage of biomolecules [6]. H2O2 was also shown to modulate the activity of the transcription factor cyclic AMP response element-binding protein (CREB) [7].In a previous study, we showed that an infusion prepared from the plant Pulicaria incisa (Lam.) DC. (Pi) protected astrocytes from oxidative-stress-induced cell death, attenuated the induced ROS levels, demonstrated antioxidant properties in cell-free assays, and induced the transcription of glial-derived neurotrophic factor (GDNF) [8,9]. The infusion also triggered anti-inflammatory activity and inhibited the activation of microglial cells which secrete ROS [9]. Pulicaria incisa (Pi) is a desert plant that is used in folk medicine to treat various diseases [10,11,12,13,14,15,16,17]. Interestingly, Bedouins in Israel, northern Sinai, Egypt, and Jordan, drink infusions prepared from the wild-growing Pi plant in place of tea and to treat various illnesses [11,12,18]. Since Pi infusion possessed properties of antioxidants and protective effects in glial cells [8,9], we hypothesized that Pi infusion might protect neuronal cells against oxidative stress. The present study aimed to examine the antioxidative and neuroprotective effects of Pi infusion on H2O2-mediated oxidative stress in cultured neuronal cells and on neuronal survival in the hippocampus of aging mice. The effects of Pi infusion on CREB and Nrf2 activation in neuronal cells was also assessed. The presented results support the potential of Pi infusion to be developed as a neuroprotective antioxidant beverage. 2. Materials and Methods 2.1. Plant Material and Preparation of Pi Infusion

The Pi plants were grown in Southern Arava Research and Development, Hevel Eilot, Israel. Aerial parts of Pi plants collected in January were air-dried. The Pi infusion was prepared by soaking dried aerial Pi parts in a beaker containing boiling autoclaved double distilled water (DDW) in a ratio of 1 g plant:20 mL DDW. The beaker was allowed to cool at room temperature (RT) for 45 min, and the infusion was transferred into tubes. The tubes were centrifuged (4000 RPM, 10 min, RT), the supernatant was collected, filtered through 0.2 µm filters, and aliquots were frozen and stored at −20 °C, until use. To determine the concentration of Pi infusion, a 40 mL sample of the filtered supernatant was lyophilized to obtain a powder that was weighed. The average concentration of the various preparations of Pi infusion was ~7 mg/mL. For in vivo experiments in mice, Pi infusions were prepared similarly, except that sterile autoclaved tap water was used instead of DDW.

2.2. Chemical Characterization of Pi Infusion 2.2.1. Sample PreparationThe infusion was lyophilized (Alpha-Christ, Christ, Osterode am Harz, Germany) and stored at −20 °C. Samples (30 mg) and 50 µL of 0.2 mg/mL ribitol (Sigma Aldrich, St. Louis, MO, USA) in methanol (Acros organics, Morris, NJ, USA) as internal standard, were dissolved in 0.2 mL DDW (Synergy® UV Water Purification System, Merck, Darmstadt, Germany). A SEP-PAK C18 cartridge (Waters, Milford, MS, USA) was pre-washed with 1 mL methanol, followed by 1 mL H2O. The sample was loaded on the cartridge and eluted with 1.5 mL DDW. The aqueous phase was collected. Next, the cartridge was washed with 1.5 mL methanol, and the methanolic phase was collected. A 0.5 mL sample of each phase was lyophilized and then derivatized, according to an established protocol [19]. 2.2.2. Silylation Derivatization

Briefly, 70 µL methoxyamine hydrochloride (Sigma, Aldrich, St. Louis, MO, USA) solution (40 mg/mL in pyridine) was added to each lyophilized sample in an Eppendorf tube. Samples were then incubated in a thermoshaker for 90 min, after which, 100 µL N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA; Sigma, Aldrich, St. Louis, MO, USA) were added. Samples were mixed in a thermoshaker at 30 °C for 30 min, and 70 µL were used for injection into GC/MS.

2.2.3. Gas Chromatography–Mass Spectrometry AnalysisGC/MS analysis was performed with an Agilent 6850 GC (Agilent, Santa Clara, CA, USA), equipped with Agilent 5975C single quadrupole MS, CTC-PAL RSI 85 auto-sampler, and HP-5MS capillary column (0.25 µm × 30 m × 0.25 mm). The following conditions were applied: injector temperature 250 °C, initial temperature 50 °C for 5 min, a gradient of 5 °C/min until 180 °C, a gradient of 10 °C/min until 270 °C and a hold time of 10 min, and increasing to 320 °C. The MS parameters were set as follows: source temperature 230 °C, transfer line 325 °C, quadrupole: 150 °C, detector 325, positive ion monitoring and electron ionization (EI)-MS measurement at 70 eV [19]. Helium was used as a carrier gas, infused at 0.6 mL/min. 2.2.4. Data Processing and Compound Annotation

Data were analyzed by the Unknown Analysis software (Agilent, Santa Clara, CA, USA). The percentage composition of the samples was computed from the GC peak areas. Library searches were conducted using the National Institute of Standards and Technology (NIST) 14 GC/MS Library and mass spectra from the literature. Component-relative percentages were calculated based on GC peak areas without using correction factors. Commercially available standards were used for 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, genistic acid, protocatechuic acid, quininic acid, gallic acid, ferulic acid, caffeic acid, (2R-E) catechin, and quercetin (Sigma, Aldrich, St. Louis, MO, USA).

2.3. Cell Growth

SH-SY5Y (ATCC CRL-2266) were grown in a 1:1 mixture of Eagle’s minimum essential medium (EMEM) (ATCC, Manassas, VA USA) and F12 medium (Biological Industries, Israel), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Biological Industries, Beit Haemek, Israel). Passages 12–20 were used in this study. All the experiments included a vehicle control group containing double distilled water (DDW) instead of the Pi infusion.

2.4. Measurement of Cell Viability

SH-SY5Y cells were seeded (30,000/well) in a 96-well plate and were grown for 24 h in a 1:1 mixture of EMEM: F12 medium, containing 2% FBS and 1% penicillin–streptomycin. The medium was replaced the next day with fresh medium containing different concentrations of Pi infusion, and cells were incubated for 2 h. Then, cells were treated with H2O2 (200 µM), and cytotoxicity was measured 20 h later. Cytotoxicity was measured using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, as per the manufacturer’s instructions (Sartorius AG, Goettingen, Germany). Absorbance was measured at 490 nm in a Synergy2 multi-detection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The absorbance of the plate before the addition of XTT served as the background absorbance.

2.5. Quantitation of Nrf2 Levels in Nuclear Extracts

SH-SY5Y cells were seeded (10 × 106/252 cm flasks) and cultured overnight in a 1:1 mixture of EMEM: F12 medium, containing 2% FBS and 1% penicillin–streptomycin. The medium was replaced the next day with a fresh medium. The cells were pretreated with Pi infusion, after which, 200 µM H2O2 was added for an additional 2 h or 18 h. Nuclear proteins were extracted using a nuclear extraction kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s instructions, and protein concentrations were determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, lL, USA). Equal amounts (20 µg protein) from each sample were tested (27 °C in a heated block) for Nrf2 levels using the Nrf2 Transcription Factor Assay kit (Cayman Chemical, Ann Arbor, MI, USA). This assay kit detects specific transcription factor DNA-binding activity in nuclear extracts. A specific dsDNA sequence containing the Nrf2 response element is immobilized onto a 96-well plate. Nrf2 from nuclear extracts specifically binds to the Nrf2 response element and is detected by the addition of a specific primary anti-Nrf2 antibody. A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm.

2.6. Immunocytochemistry

SH-SY5Y cells were replated (150,000 cells/well) into 24-well plates containing glass coverslips, in a 1:1 mixture of EMEM:F12 medium, containing 2% FBS and 1% penicillin–streptomycin. The medium was replaced the next day with fresh medium, and cells were then pretreated with Pi infusion for either 1 h or 2 h. Then, 200 µM H2O2 was added for either 2 h or 18 h. Following treatment, the cells were washed with warm Phosphate buffered saline (PBS) and then fixed with 4% formaldehyde for 15 min at room temperature, washed 3 times with PBS, and permeabilized for 3 min with 0.01% Triton-x in PBS. The cells were washed again and incubated with 10% normal mouse serum in PBS, for 30 min, at room temperature, then rinsed with PBS and incubated overnight, at 4 °C, with Alexa Fluor 488-conjugated mouse monoclonal anti-human Nrf2 antibody (sc-518033, Santa Cruz Biotechnology, Dallas, TX, USA) in PBS containing 1.5% normal mouse serum. To visualize the nuclei, the cells were washed again, stained with 4’,6-diamidino-2-phenylindole (DAPI) 1:10,000 in PBS (15 min, at room temperature), and rinsed 4 times with PBS. Images were acquired by a Leica SP8 laser scanning microscope (Leica, Wetzlar, Germany), equipped with solid-state lasers with 405 and 488 nm light, HC PL APO CS 63x/1.2 water immersion objective (Leica, Wetzlar, Germany), and Leica Application Suite X software (LASX, Leica, Wetzlar, Germany). DAPI and Alexa Fluor 488 emission signals were detected with PMT and HyD (hybrid) detectors in ranges of 415–490 and 500–550 nm, respectively. An average of 11 images/coverslip were captured, with the same exposure time for all samples. The mean fluorescence values in regions of interest were quantified with ImageJ software (NIH).

2.7. Determination of Caspase-3 Activity

SH-SY5Y cells were re-plated into 6-well plastic plates at a density of 2 × 106 cells/2.5 mL/well in 2.5 mL of a 1:1 mixture of EMEM:F12 medium, containing 2% FBS and 1% penicillin–streptomycin. The medium was replaced the next day with fresh medium and cells were treated with Pi infusion, for 2 h. Then, 200 µM H2O2 was added to each well, and cells were incubated for 20 h before being scraped into tubes and washed twice with 1 mL of cold PBS (125 g, 5 min). The supernatant was discarded, and pellets were incubated in a cold lysis buffer comprised 10 mM TRIS pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.01% Triton X-100, for 30 min, on ice. After freezing in liquid nitrogen and thawing, the supernatant was centrifuged (10,000× g, 15 min) and the lysates were frozen at −80 °C. Protein concentration was determined using a BCA kit. Equal amounts of protein (20 µg) were tested for caspase-3 activity using the EnzChek Caspase-3 assay kit (Molecular Probes, Eugene, OR, USA). Fluorescence was measured using the SynergyTM Neo2, Multi-Mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

2.8. Determination of Phospho-CREB Levels

SH-SY5Y cells were re-plated into 6-well plates at a density of 2 × 106 cells/2.5 mL/well in a 1:1 mixture of EMEM:F12 medium, containing 2% FBS and 1% penicillin–streptomycin. Medium was replaced the next day with fresh medium, and cells were treated with Pi infusions at the indicated concentrations, for 2 h. Thereafter, 200 µM H2O2 was added for 30 min. Medium was collected into tubes and cells were washed with cold PBS and then lysed on ice in 0.5 mL/well lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton x-100, 20 mM sodium pyrophosphate, 25 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin and 1 mM PMSF. After 5 min, cells were scraped and collected into tubes, frozen in liquid nitrogen, thawed, and centrifuged for 10 min at 13,000× g, 4 °C. The supernatant was collected and stored at −80 °C. Protein concentration was determined using the BCA kit protein assay kit (Pierce Biotechnology, Rockford, lL, USA). Equal amounts of protein were analyzed using the PathScan Phospho-CREB sandwich ELISA kit (Cell Signaling Technology, Danvers, MA, USA), according to the manufacturer’s instructions.

2.9. Evaluation of Intracellular ROS Levels

Intracellular ROS levels were determined using the non-fluorescent cell-permeable compound, 2’7’-dichlorofluorescein diacetate (DCF-DA; Sigma, Aldrich, St. Louis MO, USA). SH-SY5Y cells were plated onto 24-well plates (30,000 cells/0.5 mL/well) and incubated for 24 h in a 1:1 mixture of EMEM:F12 medium, containing 2% FBS and 1% penicillin–streptomycin. Cells were then labeled with DCF-DA (20 µM) for 30 min, at 37 °C. Following incubation with DCF-DA, cultures were rinsed twice with PBS which was then replaced with fresh medium. ROS levels (fluorescence) at time zero were measured in a plate reader with excitation at 485 nm and emission at 520 nm. SH-SY5Y cells were then treated with Pi infusions at the indicated concentrations, for 2 h, before H2O2 was added. Fluorescence intensities (indicative of ROS levels) were measured 1 h and 18 h later, by the SynergyTM Neo2, Multi-Mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The background fluorescence intensities at time zero were subtracted from the intensities at 1 h and 18 h.

2.10. In Vivo Establishment of the Aging Mouse Model

The research was conducted following The National Institute of Health (NIH) guidelines for the care and use of laboratory animals and was approved by The National Permit Committee for animal science (IL-20-2-60). Twenty-seven female C57BL mice (5-week-old, weighing ~15.5 g) were purchased from Envigo, Israel. The mice were maintained under a controlled 12 h light/12 h dark cycle, with free access to food and water. The mice were randomly allocated into three groups (9 mice/group). Control mice drank only water, without Pi infusion. Nine mice received a daily Pi infusion (200 mg/kg or 600 mg/kg body weight) in the drinking water for 18 months, starting from ~1.5 months of age. The Pi infusions were prepared as described above, except that instead of DDW, they were prepared in sterile tap water. Each day, a new frozen tube of Pi infusion was thawed and then diluted into autoclaved tap water, which was added to the drinking autoclaved tap water bottles to a final volume of 60 mL. Each bottle was covered with aluminum foil to minimize exposure to light.

2.11. Hematoxylin and Eosin Staining AssayAt 19 months of age, mice were sacrificed, and brains were harvested and stored for 48 h in 4% formaldehyde. The tissues were processed for paraffin embedding, and serial sections (4 µM thick) were cut off with a microtome. Paraffin-embedded sections were deparaffinized and rehydrated and then stained with hematoxylin and eosin (H&E) following a previously published method [20]. After H&E staining, histopathological changes were then assessed as described in Section 2.12. 2.12. Digital Morphometry and Histopathological Evaluation of Pyknosis in the HippocampusThe number of hippocampal neurons at the cornu Ammonis 3 (CA3) and dentate gyrus (DG) regions was quantified at a magnification of X20, using MATLAB software by brightness, color, and morphological-based segmentation. The image analysis system used was Image Pro Ver. 10.0.11 (Media Cybernetics, Rockwille, MD, USA). Identification of pyknosis in the hippocampus was based on the color and brightness of pixels, as measured using the ‘Smart Segmentation’ option (Machine Learning option) combined with the “Classification learning” process, which involves teaching the system morphometric and density parameters indicative of vital versus pyknotic cells [21]. This selection is saved and applied to each new image and can be changed if needed (the software keeps learning the changes). The percentage of the pyknotic neurons was calculated as the number of pyknotic neurons out of the total number of neurons (vital + pyknotic). 2.13. Statistical Analyses

Statistical analyses were performed with one-way ANOVA followed by Tukey–Kramer multiple comparison tests, using the JMP 7 statistical analysis software program (SAS Institute, Inc., Duxbury, MA, USA). In this analysis, columns with different letters (i.e., A, B, C) are significantly different (p < 0.05), and columns with the same letter are not significantly different (p > 0.05).

4. Discussion

The increasing life span and number of patients with neurodegenerative diseases have brought to a growing interest in the development of nutritional supplements and functional foods and beverages that can support neuronal health. This study examined the effects of an infusion prepared from the Pulicaria incisa plant on cultured neuronal cells under oxidative stress and in vivo in aging mice. Pi infusions successfully protected neurons against oxidative stress, reduced intracellular ROS levels to the levels observed in control samples, increases Nrf2 expression, and induced its translocation from the cytoplasm to the cell nucleus.

Long-term oxidative stress, ROS accumulation, and oxidative damage to macromolecules in the brain are strongly associated with neurodegenerative diseases and are critical contributors to aging and age-related pathologies through organelle dysfunction (e.g., mitochondrial dysfunction), DNA damage, and protein aggregation. Nrf-2 is a redox-sensitive transcription factor that activates the antioxidant system in the cell [38] and upregulates genes that combat these adverse effects and mitigates age-related pathologies [1,39,40]. Nrf2 target genes protect against the development of age-related pathologies, both by neutralizing free radicals and by preventing the damage caused by ROS. Under basal conditions, Nrf2 is constantly degraded in the cytoplasm, but under oxidative stress, degradation is halted, leaving it stabilized and free to translocate to the nucleus, and subsequently activate the transcription of antioxidant genes whose promoters contain the antioxidant response element (ARE) [41]. Nrf2 induction prevents senescence and promotes proteasome formation and cell survival by preventing an increase in ROS, controlling the expression of anti-apoptotic mitochondrial proteins, such as BCL-2 and BCL-xL, and by modulating apoptosis and autophagy-related signaling [42,43].The gradual reduction in Nrf2 expression with age resulting in increased oxidative stress appears to be a major risk factor for heightened susceptibility to neurodegenerative disorders and contributes to each of the hallmarks of aging, i.e., inhibited DNA repair, an overall increase in mutations and genomic instability, loss of proteostasis, telomere attrition, epigenetic alterations, mitochondrial dysfunction, deregulated nutrient metabolism and sensing, cellular senescence, stem cell exhaustion and altered intercellular communications [39,44,45,46,47].The hippocampus has been reported to be among the three most susceptible areas to oxidative stress and consequently is the first to undergo a functional decline with age [36,37,48]. Several studies have suggested that pyramidal cells of CA3 and granule cells of the DG are oxidative stress-prone areas, whereas others have suggested that pyramidal cells of CA1 are more susceptible to oxidative damage [2]. The present study demonstrated that long-term consumption of Pi infusion can prevent neuronal cell death in the hippocampus during brain aging in mice.Our observation that Pi infusion upregulates the levels of pCREB in neuronal cells can partially explain the neuroprotective effects of Pi infusion on neuronal cells under oxidative stress and in hippocampal neurons of aging mice. pCREB activates many genes, including genes that control brain development, function, and plasticity [33]. CREB is expressed in different regions of the brain, including the hippocampus [49]. Its levels decrease in aged rodent brains and dementia patients, with alterations and dysfunction in CREB signaling associated with cognitive deficits, as observed in normal aging and neurodegenerative diseases [50]. Therefore, CREB was suggested to be a therapeutic target for the treatment of age-related cognitive deficits [51].Our in vivo results support the neuroprotective activities of the bioactive phytochemicals present in Pi infusion. These effects can be attributed to the presence of the central low-molecular-weight constituents of Pi infusion, i.e., aucubin (MW 346.33 g/mol), chlorogenic acid (354.31 g/mol) and quercetin (302.236 g/mol). Chlorogenic acid (~17.8% of the total constituents of Pi infusion) is a phenolic acid, which is an ester of caffeic acid and quinic acid [24]. It has been ascribed neuroprotective, anti-inflammatory, and antioxidant effects [24,52,53] and reported to prevent cognitive decline and neuronal damage in the hippocampus, and to protect against cognitive impairment in aged mice [54,55]. Chlorogenic acid was shown to regulate the oxidative stress-related Nrf2 pathway [56] and to stimulate the CREB pathway [53]. Quercetin is a flavonoid comprising ~8% of the total constituents of Pi infusion. Flavonoids have been implicated in neuronal proliferation and survival, by reducing oxidative stress and modulating various cellular signaling cascades, including CREB [57]. In vivo administration of purified flavonoids led to the phosphorylation of CREB and long-term potentiation in the hippocampus and had neuroprotective effects [57,58,59]. Aucubin is an iridoid glycoside (a class of monoterpenes) comprising ~2.8% of the Pi infusion. It has a wide range of pharmacological properties, including neuroprotective and anti-aging effects [60]. Aucubin provides a neuroprotective effect in primary cortical neurons and the hippocampus of experimental traumatic brain injury by inhibiting ROS generation, oxidative stress, and inflammatory responses [61]. The mechanism involves the activation of the Nrf2-induced antioxidant system [61,62,63]. It is still to be determined whether the effects of Pi infusion are elicited by a single constituent or if they are the output of the synergistic activities of several components.