Antioxidant and Astroprotective Effects of a Pulicaria incisa Infusion

Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, an infusion prepared from the desert plant Pulicaria incisa (Pi) was tested for its protective and antioxidant effects on astrocytes subjected to oxidative stress. The Pi infusion attenuated the intracellular accumulation of ROS following treatment with hydrogen peroxide and zinc and prevented the H2O2-induced death of astrocytes. The Pi infusion also exhibited an antioxidant effect in vitro and induced GDNF transcription in astrocytes. It is proposed that this Pi infusion be further evaluated for use as a functional beverage for the prevention and/or treatment of brain injuries and neurodegenerative diseases in which oxidative stress plays a role.


Introduction
Increased life spans in the Western world have led to an elevated frequency of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Neurodegenerative diseases are the result of gradual and progressive loss of neural cell function.
Oxidative damage plays a pivotal role in the initiation and progress of many human diseases and is considered to be a salient and early pathogenetic event in many neurodegenerative disorders that are characterized by selective neuronal death [1][2][3][4][5]. Compared with other tissues, the brain is particularly vulnerable to oxidative damage due to its high rate of oxygen utilization and the great deal of oxidizable polyunsaturated fatty acids it contains [6,7]. In addition, certain regions of the brain are rich in iron, a metal that is catalytically involved in the production of damaging reactive oxygen species (ROS) [8]. Although ROS are critical intracellular signaling messengers [9], an excess of free radicals may lead to peroxidative impairment of membrane lipids and, consequently, to the disruption of neuronal functions and apoptosis. The ROS known to be responsible for neurotoxicity are hydrogen peroxide (H 2 O 2 ), superoxide anions (O 2 − ), and hydroxyl radicals (OH − ). Brain cells have the capacity to produce large quantities of peroxides, particularly H 2 O 2 [10]. Excess, H 2 O 2 accumulates in response to brain injuries and during the course of neurodegenerative diseases and can cross cell membranes to elicit biological effects from inside the cells [11]. Although H 2 O 2 is generally not very reactive, it is thought to be the major precursor of highly reactive free radicals that may cause damage in the cell, through iron ion-or copper ionmediated oxidation of lipids, proteins, and nucleic acids. This capability can partially account for H 2 O 2 -mediated neuronal [12] and glial [13] death. H 2 O 2 also induces differential protein activation, which accounts for its varied biological effects. In the mammalian central nervous system (CNS), the transition metal zinc is found only in the synaptic vesicles of glutamatergic neurons and plays a special role in modulating synaptic transmission. Chelatable zinc is released 2 Oxidative Medicine and Cellular Longevity into the synaptic cleft with the neurotransmitter during neuronal execution [14]. Under normal circumstances, the robust release of zinc is transient and the zinc is efficiently cleared from the synaptic cleft to ensure the performance of successive stimuli. However, under pathological conditions, elevated levels of extracellular zinc have been recognized as an important factor in neuropathology [15][16][17]. In neurotransmission, the amount of zinc in the synaptic cleft is in the range of 10 to 30 μM, but, under pathological conditions that involve sustained neuronal depolarization (e.g., ischemia, stroke or traumatic brain injury), the concentration of extracellular zinc can increase to 100 to 400 μM, which can contribute to the resulting neuropathology [18,19]. In vivo and in vitro studies have shown that at concentrations that can be reached in the mammalian CNS during excitotoxic episodes, injuries or diseases, zinc induces oxidative stress and ROS production, which contribute to the death of both glial cells [20] and neurons [21,22]. Zinc has been shown to decrease the glutathione (GSH) content of primary cultures of astrocytes [20,23], increase their GSSG content [23], and inhibit glutathione reductase activity in these cells [24]. Furthermore, it slows the clearance of exogenous H 2 O 2 by astrocytes and promotes intracellular production of ROS [24]. Thus, ROS generation, glutathione depletion, and mitochondrial dysfunction may be key factors in ZnCl 2induced glial toxicity [20].
Astrocytes are the most abundant type of glial cell in the brain and play multiple roles in the protection of brain cells. Under ischemic conditions, astrocytes can remove excess glutamate and K + to protect neurons from glutamate-mediated cytotoxicity and depolarization [25,26]. Astrocytes can also supply energy and promote neurogenesis and synaptogenesis in response to ischemia-induced brain damage [27,28]. Astrocytes release multiple neurotrophic factors, such as GDNF, to protect themselves and neighboring cells from in vitro ischemia and there is evidence that dysfunctional astrocytes can enhance neuronal degeneration by decreasing the secretion of trophic factors [29]. GDNF can promote the survival of substantia nigra dopaminergic neurons, induce neurite outgrowth and sprouting [30][31][32], upregulate tyrosine hydroxylase expression, and enhance synaptic efficacy [33]. Moreover, GDNF has been shown to protect cultured astrocytes from apoptosis after ischemia by inhibiting the activation of caspase-3 [34].
The study of astrocytes is particularly important in light of the coexistence of apoptotic death of neurons and astrocytes in brains affected by ischemia and neurodegenerative diseases. Despite their high levels of antioxidative activity, astrocytes exhibit a high degree of vulnerability and are not resistant to the effects of ROS. They respond to substantial or sustained oxidative stress with increased intracellular Ca 2+ , loss of mitochondrial potential, and decreased oxidative phosphorylation [35]. Since astrocytes determine the brain's vulnerability to oxidative injury and form a tight functional unit with neurons, impaired astrocytic energy metabolism and antioxidant capacity and the death of astrocytes may critically impair neuronal survival [36,37]. Thus, protection of astrocytes from oxidative stress appears essential for the maintenance of brain function.
Based on the pathophysiological roles of astrocytes in ischemic brains, it appears that astrocyte damage causes impairment of brain function during cerebral ischemia. In recent years, there has been an increased interest in plants as a source of bioactive substances that might be developed as potential nutraceuticals and various activities of such substances have been demonstrated in in vitro and in vivo experimental systems [38]. Several studies have shown that some herbal medications and antioxidants show promise toward preventing Alzheimer's disease [39][40][41].
Pulicaria incisa (Pi) is a desert plant that belongs to the Asteraceae family and has been used for many years in traditional medicine for the treatment of heart disease and as a hypoglycemic agent [42][43][44]. Pi has been found to contain high amount of unsaturated fatty acids [45], to decrease total lipid, total cholesterol, and triglyceride levels, and has been proposed as a potential hypocholesterolemic agent [46]. A Pi infusion is consumed in place of tea by many Egyptian Bedouins [42,43,47]. To the best of our knowledge, its effects in the context of neurodegenerative diseases have not yet been studied.
In light of the fact that oxidative stress has become accepted as a target of therapeutic interventions for the treatment of brain injuries and neurodegenerative diseases and the critical role of astrocytes in neuronal survival, the present study examined the effects of an infusion prepared from Pi on the susceptibility of astrocytes to oxidative stress.

Experimental Animals.
Newborn Wistar rats (0-2 days old) were obtained from Harlan Laboratories. The experiments were performed in compliance with the appropriate laws and institutional guidelines and were approved by the Institutional Animal Care and Use committee of the Volcani Center, Agricultural Research Organization (number IL-135/07). The beaker was allowed to cool at RT for 30 min. The tube was centrifuged (4000 RPM, 10 min, RT), the supernatant was collected, filtered, and aliquots were stored at −20 • C until use. In order to determine the extract concentration, a sample of the filtered supernatant was lyophilized to obtain extract powder. The averaged concentration of the various preparations that were tested was 10 mg/mL.

Preparation of Primary Astrocytes Cultures.
Cultures of primary rat astrocytes were prepared from cerebral cortices of 1-to-2-day-old neonatal Wistar rats. Briefly, meninges and blood vessels were carefully removed from cerebral cortices kept in Leibovitz-15 medium; brain tissues were dissociated by trypsinization with 0.5% trypsin (10 min, 37 • C, 5% CO 2 ); cells were washed first with DMEM containing soybean trypsin inhibitor (100 μg/mL) and 10% FBS and then with DMEM containing 10% FBS. Cells were seeded in tissue culture flasks precoated with poly-D-lysine (PDL, 20 μg/mL in 0.1 M borate buffer pH 8.4) and incubated at 37 • C in humidified air with 5% CO 2 . The medium was changed on the second day and every second day thereafter. At the time of primary cell confluence (day 10), microglial and progenitor cells were discarded by shaking (180 RPM, 37 • C) for 24 h. Astrocytes were then replated on 24-well PDL-coated plastic plates, (a) for toxicity assays, at a concentration of 1 × 10 5 /well, in DMEM (without phenol red) containing 2% FBS, 2 mM glutamine, 100 U/mL pennicilin, and 100 μg/mL streptomycin. (b) For the cellular antioxidant activity assay at a concentration of 3 × 10 5 /well, in DMEM containing 10% FBS 8 mM HEPES, 2 mM glutamine, 100 U/mL pennicilin, and 100 μg/mL streptomycin.

Treatment of Astrocytes.
Twenty four hours after plating, the original medium in which the cells were grown was aspirated off, and fresh medium was added to the cells. Dilutions of Pi infusion, ABAP, DCF-DA, zinc, and of H 2 O 2 in the growth medium were made freshly from stock solutions prior to each experiment and were used immediately.

Evaluation of Intracellular ROS Production. Intracellular
ROS production was detected using the nonfluorescent cell permeating compound, 2 ,7 -dichlorofluorescein diacetate (DCF-DA). DCF-DA is hydrolyzed by intracellular esterases and then oxidized by ROS to a fluorescent compound 2 -7 -DCF. Astrocytes were plated onto 24-well plates (300,000 cells/well) and treated with DCF-DA (20 μM) for 30 min at 37 • C. Following incubation with DCF, cultures were rinsed twice with PBS and then resuspended.

Induction of GDNF in Astrocytes.
Astrocytes were replated at 6-well PDL-coated plastic plates at a density of 2 × 10 6 /well, in DMEM/F12 containing 5% FBS, 2 mM glutamine, 100 U/mL pennicilin, and 100 μg/mL streptomycin. Twenty-four hr after plating, the original medium of the cells was aspirated off and fresh medium was added to the cells. Cells were then lysed and collected using RLT buffer containing 1% βME for RNA extraction.

Quantitative Real-Time PCR Analysis.
RNA was extracted by the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA was removed from the RNA samples by using 50 units of RNase-free DNaseI at 37 • C for 1 h. RNA (20 μg) was converted to cDNA using the Thermo Scientific Verso cDNA kit (Thermo Fisher Scientific Inc.) following the manufacturer's protocol. The cDNA was used for quantitative real-time PCR amplification with TaqMan chemistry (Applied Biosystems) using Rat GDNF predesigned TaqMan Gene Expression Assay from Applied Biosystems (Assay ID Rn00569510). Values were normalized relative to rat glyceraldehyde-3phospahte dehydrogenase (GAPDH; Assay ID Rn00569510). All results from three technical replicates were normalized to GAPDH and expressed as relative expression ratios calculated (relative quantity, RQ) using the comparative method and based on the data that were created by the ABI PRISM 7700 Sequence Detection System (using version 1.6 software).

Determination of Cell Viability.
Cell viability was determined using a commercial colorimetric assay (Roche Applied Science, Germany) according to the manufacturer's instructions. This assay is based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the growth medium. All measurements were made with distilled water as blank.
The scavenging ability (%) of the samples was calculated as (A control − A sample )/A control × 100).

Data Analysis.
Statistical analyses were performed with one-way ANOVA followed by Tukey-Kramer multiple comparison tests using Graph Pad InStat 3 for windows (GraphPad Software, San Diego, CA, USA).    To determine the optimal timing of the application of the Pi infusion to ameliorate the effect of H 2 O 2 , the cells were pre-incubated with Pi infusion for 2 h or 1 h, cotreated or posttreated with H 2 O 2 . Our results demonstrate that the Pi infusion is more effective when applied to the cells before H 2 O 2 and that the addition of Pi infusion to the cells after the application of H 2 O 2 did not provide significant protection (Figure 3).

The Pi Infusion Inhibited the H 2 O 2 -and ZnCl 2 -Induced Generation of ROS. H 2 O 2 -induced cell death is accompanied
by an increase in ROS levels. In order to determine whether our Pi infusion could inhibit the production of ROS that is Oxidative Medicine and Cellular Longevity 5 induced by H 2 O 2 , we assessed the intracellular generation of ROS and tested whether treatment of astrocytes with the Pi infusion affected intracellular ROS levels. For the study of the preventive effects against intracellular ROS formation, the cells were preloaded with the ROS indicator DCF-DA and were treated with various concentrations of Pi infusionbefore the application of H 2 O 2 . ROS formation was determined by examining fluorescence every hour for 4 h. As shown in Figure 4(a), H 2 O 2 induced the production of ROS in astrocytes, with the maximum levels of ROS produced after 1 h. Pretreatment of astrocytes with the Pi infusion inhibited the H 2 O 2 -induced elevation of the levels of intracellular ROS in a dose-dependent manner (Figure 4(b)). We also found that treatment with ZnCl 2 increased ROS generation in astrocytes and that, similar to the effect of the Pi infusion H 2 O 2 -induced generation of ROS, this infusion greatly attenuated ZnCl 2 -induced ROS generation ( Figure 5).
To determine the time at which the Pi infusion best ameliorates H 2 O 2 -induced ROS production, the cells were pre-incubated with Pi infusion for 2 h or 1 h, cotreated or posttreated (for 2 h or 1 h) with H 2 O 2 . Interestingly, in contrast to the results obtained in our cell protection assay (Figure 3), the Pi infusion was similarly effective in attenuating ROS production when it was pre-incubated with the cells and when it was applied after the H 2 O 2 ( Figure 6). This may indicate that Pi treatment sets the cells into a defensive state that helps them to contend with oxidative stress and that it takes about 1 h to get the cells into that defensive state. If this is the case, it indicates that the Pi infusion not only is an antioxidant cocktail, but also contains compounds that might bind to a cell surface or intracellular receptor(s) to conduct a protective signal.

The Pi Infusion Reduced 2,2 -Azobis(amidinopropane)-(ABAP-) Mediated Peroxyl Radicals Levels in Astrocytes.
In addition to H 2 O 2 , various other species, such as peroxynitrite (ONOO − ), nitric oxide (NO • ), and peroxyl radicals, have been found to oxidize DCFH to DCF in cell cultures [51]. Therefore, we used a cellular antioxidant activity assay to measure the ability of compounds present in the Pi infusion to enter the cells and prevent the formation of DCF by ABAP-generated peroxyl radicals [52]. The kinetics of DCFH oxidation in astrocytes by peroxyl radicals generated from ABAP is shown in Figure 7(a). As shown in that figure, ABAP generates radicals in a time-dependent manner and the treatment of cells with the Pi infusion moderated this induction. The increase in ROS-induced fluorescence was inhibited by the Pi infusion in a dose-dependent manner, as shown in Figure 7(b). This indicates that compounds present in the Pi infusion entered the cells and acted as efficient intracellular hydroperoxyl radical scavengers.

Free-Radical Scavenging Activity of Pi Infusion.
Since many low molecular weight antioxidants may contribute to cellular antioxidant defense properties, we analyzed radical scavenging activity rather than seeking specific antioxidants. The free-radical scavenging activity of the Pi infusion was determined by the 2,2-diphenyl-1-picryhydrazyl (DPPH) radical which is considered to be a model lipophilic radical. In this assay, the Pi infusion was found to be a very potent free-radical scavenger with an IC 50 value of 45 μg/mL and 80% inhibition of DPPH absorbance at 517 nm ( Figure 8). Astrocytes. The contrast between the need for a preincubation of the cells with the infusion (Figure 3) in order to gain the protective effect and the fact that the timing of the addition of the Pi infusion did not appear to affect the ability of the treatment to neutralize ROS levels ( Figure 6) led us to examine whether another factor aside from antioxidant activity might be involved in the beneficial effect of the Pi infusion. One possible factor could be GDNF secretion. It has been previously shown that GDNF protects astrocytes from ischemia-induced apoptosis [34]. Thus, we raised the possibility that the protective effect of Pi infusion on astrocytes suffering oxidative stress might be at least partially due to the induction of GDNF by the Pi infusion. This possibility might be supported by the need for preincubation of the Pi infusion with the cells in order to elicit the protective effect against H 2 O 2 -induced cell death. To test this possibility, we used real-time PCR and quantified GDNF mRNA. Primary astrocytes were treated with different concentrations of Pi infusion for several incubation periods and levels of GDNF transcripts were determined. The results of this work showed that incubation with Pi infusion at an optimal concentration of 250 μg/mL leads to a 5-fold increase in GDNF mRNA levels following 9 h of incubation and a 10-fold increase in GDNF mRNA levels following 24 h of incubation (Figure 9).

Discussion
In the present study, we evaluated the effectiveness of the Pi infusion for counteracting oxidative damage in cultured astrocytes treated with hydrogen peroxide, zinc, or ABAP. The main findings of this study are that the examined Pi infusion can protect primary cultures of rat brain astrocytes from H 2 O 2 -induced cell death and reduce the levels of intracellular ROS produced following treatment with H 2 O 2 , ZnCl 2 , or ABAP. The beneficial effects of the Pi infusion are also demonstrated by the induction of astrocyte GDNF expression. Substances that can protect brain cells from oxidative stress are potential tools for the treatment of brain injuries and neurodegenerative diseases. Specifically, the neurotrophic and protective effects of GDNF on neuronal cells that have been demonstrated in previous studies have made GDNF a promising candidate for gene and cell therapy for various neurodegenerative diseases [53][54][55].
H 2 O 2 has been shown to induce the phosphorylation of ERK1/2, AKT/protein kinase B, and ATF-2 in C6 glioma cells [56]. The components of the Pi infusion might exert their protective effects by different mechanisms affecting one or more of these processes. Alternatively, or in addition, compounds present in the Pi infusion might act as signaltransduction agents to enhance the resistance of astrocytes to oxidative stress induced by ZnCl 2 and H 2 O 2 , for example, by inducing the expression of neurotrophic factors such as Pi infusion (12.5 µg/mL) + H 2 O 2 * * * * * * * * * * * * * * * * * * * * * * * * Figure 6: Pi infusion is similarly effective in attenuating ROS production when applied before or after H 2 O 2 . Pi infusion (12.5 μg/mL) was added to astrocytes before (−2 h, −1 h) or after (1 h, 2 h) the addition of H 2 O 2 . ROS levels in DCF-DA preloaded astrocytes were measured 1, 4, and 6 h after the application of H 2 O 2 . The results are mean ± SEM of two experiments (n = 6). * * P < 0.001.
Strong free-radical scavenging activity of a methanolic extract of Pi was also reported before [45,57].
In our model, there are two opportunities for compounds present in the Pi infusion to elicit antioxidant effects. They can act at the cell membrane to disrupt peroxyl radical chain reactions at the cell surface or they can be taken up Oxidative Medicine and Cellular Longevity by the cell and react with ROS inside the cell. The efficiency of cellular uptake and/or membrane binding combined with that of the radical-scavenging activity dictate the efficacy of the tested infusion. In order to discriminate between these possibilities, astrocytes were pre-incubated with ABAP, which generates ROS inside cells. According to our results, the Pi infusion inhibited intracellular ROS levels, suggesting that, in addition to other possible activities, compounds present in the Pi infusion may enter the cells and react with ROS inside those cells. The most popular traditional remedies are infusions or decoctions prepared from plants and herbs. These herbal teas are frequently used in the treatment of chronic diseases, such as cancer, gastrointestinal diseases, and type 2 diabetes. Scientific documentation is available regarding the functionality of herbal and tea extracts that contain different ingredients and have a wide variety of biological effects. For example, green tea (unfermented Camellia sinensis) contains large amounts of catechins and vitamin C, has anticancer effects [58], and protects against age-related neurodegeneration [59]. Oolong tea (semifermented Camellia sinensis) is known to be an effective treatment for allergic diseases [60] and the suppression of the accumulation of neutral fat [61]. Animal model studies with rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) extracts indicate that teas prepared from both of these plants possess potent immunemodulating and chemopreventive activity [62]. Several other studies have revealed that some herbal medications and antioxidants show promise for the prevention of neurodegenerative diseases [63].

Conclusions
Neurodegenerative diseases are multifactorial and strategies for treating these diseases need to include a variety of interventions directed at multiple targets. On the basis of the current results, we suggest that various compounds present in the Pi infusion might have beneficial effects and should be further evaluated for the development of a health-promoting functional beverage for the prevention or treatment of brain injuries and neurodegenerative diseases in which oxidative stress and astrocytic cell death play a role. To the best of our knowledge, the effects of Pi in the context of neurodegenerative diseases have not been studied previously  Figure 9: Treatment with the Pi infusion increases the levels of GDNF transcript in primary astrocytes. Rat primary astrocytes were exposed to 250 Mg/mL of Pi infusion for 9 or 24 h. Total RNA was then extracted. GDNF transcripts were measured using quantitative real-time PCR. The results of three technical replicates were normalized to glyceraldehyde-3-phosphate (GAPDH) and are expressed as relative quantities of GDNF transcripts. The results are means ± SD of one out of three experiments. Means-paired, one-tailed t-tests were used to evaluate the effects of the infusion on the expression of GDNF and the effect of the duration of the incubation. These tests yielded P(t) values of 0.029 and 0.105, respectively. Therefore, we can conclude that, in cases in which the effect of the duration is not statistically significant, the effect of the Pi infusion is significant at a 95% significance level. and this is the first study to characterize the astroprotective effects of this plant.