Erigeron annuus Protects PC12 Neuronal Cells from Oxidative Stress Induced by ROS-Mediated Apoptosis

Reactive oxygen species (ROS), associated with oxidative stress, are involved in many biological processes such as apoptosis, necrosis, and autophagy. Oxidative stress might induce neuronal damage via ROS generation, causing neurodegenerative diseases. Erigeron annuus (EA) has antioxidant properties and could protect neurons from oxidative stress. In this study, we investigated the protective effect of the aerial parts (EAA) and flowers (EAF) from EA on ROS-mediated apoptosis in pheochromocytoma 12 cells. We quantified 18 types of phenolic compounds using high-performance liquid chromatography. Pretreatment of the cells with EAA and EAF attenuated ROS generation and induced the expression of antioxidant enzymes such as superoxide dismutase 2, catalase, and glutathione peroxidase. In addition, EAF reduced the expression of apoptotic proteins such as Bax/Bcl-xL, caspase-3, and caspase-8 to a greater extent than that with EAA. These results suggested that the protective effect of EAF against oxidative stress-induced apoptosis might be due to the prevention of ROS generation mediated by oxidative enzymes.


Introduction
Reactive oxygen species (ROS) play an important role in regulating normal physiological and developmental functions such as cell cycle progression, proliferation, differentiation, migration, and cell death. ROS are generated in the mitochondria as byproducts of cellular metabolism [1]. Oxidative stress induced by ROS, such as superoxide (O 2 ) or hydrogen peroxide (H 2 O 2 ), has been associated with several pathologies and diseases such as diabetes, arthrosis, and Alzheimer's and Parkinson's diseases [2]. When the production of ROS surpasses the cellular antioxidant capacity, damage to macromolecules such as protein and DNA contributes to cell toxicity or apoptosis directly or indirectly [1][2][3]. Among the enzymes that are involved in ROS generation, catalase (CAT) and glutathione peroxidase (GPx) convert H 2 O 2 to H 2 O; meanwhile, superoxide dismutase (SOD) converts O 2 to H 2 O 2 [4,5]. Additionally, the SOS response has the effect of eliminating the ROS reaction, only if its enzymatic activity interacts with that of CAT and/or CAT.
Apoptosis is controlled by extrinsic and intrinsic pathways (mitochondrial pathway) [6]. ROS-mediated mechanisms drive apoptosis through intrinsic pathways to regulate cell death [3]. e intrinsic apoptosis pathway consists of intracellular signaling between proapoptotic proteins. For example, the Bcl-2 family includes proteins such as the antiapoptotic activator Bcl-xL and the proapoptotic effector Bax, which interacts with other proteins [7]. Additionally, overexpression of the antiapoptotic protein Bcl-xL, which appears to be bound to the mitochondrial membrane, can block apoptosis [8]. Conversely, Bax causes apoptosis by inducing the release of cytochrome-c, a key component of the mitochondria electron transport chain, into the cytoplasm [8,9].
is intrinsic pathway induces caspase-independent cell death, as it is controlled by Bcl-2 family proteins such as Bax and Bcl-xL [7]. In contrast, procaspase-8 is the apical caspase in the extrinsic pathway, for which activation occurs within the DISC (death-inducing signaling complex), and proceeds by directly activating procaspase-3 [6]. Procaspase-3 exists in the cytosol as an inactivated dimer, and the activation of procaspase-3 takes places after the cleavage of caspase-8 [10]. Cleaved caspase-3 acts as a direct executioner of apoptosis [10].
Erigeron annuus (EA), which belongs to the Asteraceae family, has white flowers and is commonly found in grasslands and roadsides. In addition, EA has traditionally been used as a medicinal plant for dyspepsia, abdominal pain, urine bleeding, and hypoglycemic effects [11]. Many compounds such as flavanone, erigeroflavanone, sesquiterpenoids, ergosterol peroxide, caffeic acid, and pyromeconic acid can be derived from the aerial part (EAA) and flowers (EAF) of EA [12][13][14][15][16][17]. It has been reported that these compounds have several activities such as reductase inhibitory in aldose, antiatherosclerotic, neuroprotective, antioxidant, and cytoprotective effects [12,[14][15][16][17]. Although several studies have demonstrated the effect of EA as an antioxidant and neuroprotective agent, studies on its effect against damage to neuronal cells due to oxidative stress are scarce. In this study, we demonstrated that EAA and EAF can effectively block the intrinsic and extrinsic apoptosis pathways via ROS-mediated signaling. Our data suggest that EAA and EAF could inhibit ROS mediated-apoptosis in PC12 cells under oxidative stress by upregulating the expression of antioxidant enzymes and downregulating apoptotic proteins.

Materials and Methods
2.1. Chemicals, Antibodies, and Apparatus. All reagents were purchased from Sigma Aldrich (Saint Louis, MO, USA), unless otherwise indicated. CellTiter 96 ® AQ ueous One Solution (MTS) was obtained from Promega (Madison, WI, USA). Pheochromocytoma (PC12) cells were purchased from the ATCC (Manassas, VA, USA). All cell culture reagents were obtained from Gibco (Burlington, ON, Canada). Radio immunoprecipitation (RIPA) cell lysis buffer was purchased from GenDepot (Katy, TX, USA). Bradford and enhanced chemiluminescence (ECL) reagents for protein assays were from Bio-Rad (CA, USA). All antibodies were from Abcam (Cambridge, UK), unless otherwise stated. Antibodies against β-actin, cleaved caspase-8, and caspase-8 were purchased from Santa Cruz (Dallas, TX, USA). Meanwhile, anticleaved caspase-3 was purchased from Cell Signaling (Danvers, MA, USA). Evaporation was conducted using an evaporator system under reflux in vacuo from BÜCHI (Sankt Gallen, Swiss). Multimode-plate reading was performed with a Synergy H1 Hybrid Reader from BioTek Instruments (Winooski, VT, USA). e confocal microscope for fluorescent imaging was purchased from Zeiss (Oberkochen, German). Protein expression levels were assessed with a chemiluminator from Davinch-K (Seoul, Korea). Analysis was performed using a high-performance liquid chromatography (HPLC) 2790/5 system equipped with a photodiode array (PDA) 2996 from Waters (Milford, MA, USA). e INNO column was obtained from Young Jin Biochrom Co., Ltd., (Seoul, Korea). Acetonitrile and water labeled as HPLC grade solvents were purchased from Fisher Scientific Ltd., (Sunnyvale, CA, USA).

Plant Materials, Extraction, and
Yields. EA samples were collected from Eumsung (Chungcheongbuk-do, Korea) in 2018. e plant material was identified by Jeong Hoon Lee, PhD. (Department of Herbal Crop Research), and the voucher specimen (NIBRVP0000456433) was deposited at National Institute of Biological Resources. ey were then divided into two groups, the aerial part (stem and leaves, EAA) and flower part (EAF). For the preparation of extracts, EAA and EAF (100 g, each) were grinded, sifted through a testing sieve (aperture 1.40 mm, wire 0.71 mm), and extracted three times with 70% ethanol at a 1 : 10 (v: v) ratio for 24 h, at room temperature. After filtration, all extracts were evaporated in vacuo, freeze-dried (20 mTorr, − 40°C, 1 week), and stored at − 80°C. Extraction yields were calculated as previously described [18] as follows: yields (%) � total extract dried weight/raw material weight × 100.

Determination of Total Phenolic Contents (TPC).
TPCs of extracts were determined by the Folin-Denis's phenol method, with slight modifications [19]. Specifically, 500 μL of each extract (2 mg/mL) was mixed with 50 μL of 1 N Folin-Ciocalteu reagent for 3 min. en, 100 μL of 20% sodium carbonate solution was added. After 1 h, the absorbance was measured at 725 nm using a multimode-plate reader. TPC was calculated from the calibration curve using gallic acid, and the results were expressed as gallic acid equivalents (y � 0.064x + 0.024).

Determination of Total Flavonoid Contents (TFC).
TFCs of each extract were determined by the aluminum chloride colorimetric method, with slight modifications [20]. Briefly, 150 μL of each extract (2 mg/mL) was mixed with 10 μL of 10% aluminum chloride solution, 10 μL of 1 M potassium acetate, and 280 μL of distilled water. e mixture was kept at room temperature for 30 min and then measured at 415 nm using a multimode-plate reader. TFC was expressed as quercetin equivalents (y � 0.001x + 0.015), which reflected the amount of quercetin (μg/mL).

Antioxidant Activity Assay
2.4.1. ABTS + Radical Scavenging Assay. ABTS + (2,2′-azinobis 3-ethylbenzothiazolin-6-sulfonic acid) radical scavenging activity was measured as described by Van den Berg, with some modifications [21]. ABTS + solutions containing 7.4 mM 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt and 2.6 mM potassium persulfate were prepared in distilled water for 24 h. e absorbance of the solution was adjusted to 0.70 ± 0.05 at 732 nm. Next, 20 μL samples were mixed with 180 μL of ABTS + solution and incubated for 30 min in the dark at room temperature. Absorbance values of ABTS radicals were measured for the different samples using a multimode-plate reader at 732 nm. e following samples were tested: Treatment, Blank1, Blank2, and Control corresponded to the sample, H 2 O, H 2 O + sample, respectively. An ABTS radical scavenging assay (RSA) was performed for each concentration according to the following equation: Additionally, the half-maximal inhibitory concentration (IC 50 ) was calculated for each extract.

DPPH Radical Scavenging
Assay. DPPH (2,2diphenyl-1-picryllydrazyl) radical scavenging activity was measured according to the Bondet method, with some modifications [22]. DPPH solution containing 300 μM of DPPH in 95% ethanol was prepared. e absorbance of the solution was adjusted to 1.00 ± 0.05 at 515 nm. Next, 25 μL of samples was mixed with 225 μL of DPPH solution and incubated for 30 min in the dark covered with aluminum foil at room temperature. Absorbance was measured using a multimode-plate reader at 515 nm. Among the samples tested were the following: Treatment, Blank1, Blank2, and Control corresponding to the sample, H 2 O, and H 2 O + sample, respectively. DPPH radical scavenging activity (RSA) was calculated for each concentration using the following equation: e half-maximal inhibitory concentration (IC 50 ) was also calculated for each extract.

Sample Preparation for HPLC.
To investigate phenolic compounds in EAA and EAF (5 g), the modified method of Ahn et al. was applied [23]. Each extract was redissolved in water and fractionated with ethyl acetate/ether (1 : 1 � v/v) to obtain the phenol-rich fraction. Each fraction was concentrated under reduced pressure, dissolved in methanol (10 mg/mL each), filtered through a 0.22 μM polyvinylidine difluoride (PVDF) membrane, and analyzed by HPLC.

Cell
Culture. PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO 2 incubator. e medium was replaced every 2 days for subculture. Cells from passages 5-10 were used in all experiments.

Intracellular ROS Generation.
Intracellular ROS generation was measured by a modified dichloro-dihydrofluorescein diacetate (DCFH-DA) method [24]. PC12 cells were cultured with EAA and EAF in black 96-well plates at a density of 1.0 × 10 4 cells/well. After 24 h, the culture cells were treated with 50 μM H 2 O 2 in SFM for 20 min and then with 20 μM DCF-DA in serum-free medium for 30 min; samples were then washed, and 100 μL Dulbecco's phosphate buffered saline (DPBS) was added to each well. Vitamin C (10 μg/mL) was used as a positive control. Vitamin C is well known as an antioxidant and is widely used as a positive control for antioxidant studies. e fluorescence was measured with a multiplate reader at 485 nm/535 nm (excitation/emission). In addition, fluorescent micrographs were obtained with a confocal microscope.

Preparation of Protein
Samples. PC12 cells were pretreated with EAA and EAF (50, 100, and 200 μg/mL) for 24 h and with H 2 O 2 (50 μM) for 20 min. en, cells were rinsed, scraped off, and collected with DPBS on ice. After centrifugation at 3000 rpm, the DPBS was removed completely, and cells were lysed with RIPA buffer for total protein extraction, following the manufacturer's instructions. Protein amounts were determined using the Bradford assay. Protein samples were mixed with 5x loading buffer, boiled for 5-10 min, and stored at − 80°C.
2.9. Western Blotting. Western blotting was performed with 10-15% tris-HCl gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). e proteins were transferred onto PVDF membranes, washed three times, and subsequently incubated with primary antibodies (at a 1 : 1000 dilution) at 4°C overnight. e membranes were washed three times again and incubated with horseradish peroxidase-conjugated secondary antibody (at a 1 : 2000 dilution) for 1 h at room temperature [25]. Protein expression was visualized using the ECL reagent, following the manufacturer's instructions.

Statistical Analysis.
All experimental data were expressed as means ± standard deviations (SDs) of three independent experiments. e statistical analyses in this study were performed using a one-way analysis of variance (ANOVA) with a Duncan's test, t-tests and correlation analysis based on Pearson's correlation coefficient (Statistical Package for the Social Sciences, ver. 21.0 for Window ver. 10).

Antioxidant Components and Activities of EAA and EAF.
It has been reported that 70% ethanol is the most effective solution to extract triterpenoids from Jatropha curcas leaves and phenols from Moringa oleifera and Curcuma longa extractions [26]. erefore, the extraction of EAA and EAF rendered yields of 25.16% and 25.84%, respectively (Table 1). e compound contents were analyzed by measuring TPC and TFC. e concentrations of the phenolic compounds, namely, TPC and TFC, with antioxidant activity [27] were determined for each EA part, specifically for EAA (9.8 and 20.8 mg/g) and EAF (8.9 and 10.1 mg/g) ( Table 1). Moreover, we calculated the levels of DPPH and ABTS + , indicative of antioxidant activity, by simple colorimetric methods [28]. e antioxidant activities of EAA and EAF were examined using ascorbic acid (AA) as the positive control [29]. IC 50 values of EAA and EAF were 11.0 and 17.5 μg/mL for ABTS + and 114.0 and 112.2 5 μg/mL for the DPPH-scavenging effect, respectively (Table 1).

Quantification of Phenolic
Compounds from EAA and EAF by HPLC. Phenolic compounds, which are known to be responsible for the beneficial effects of EAA and EAF, were analyzed by HPLC using a PDA detector. For the determination of phenolic compounds, EAA and EAF were reextracted from phenol-rich fractions. e chromatograms of phenolic compounds and phenol-rich fractions of EAA and EAF are shown in Figure 1. e homogentisic acid peak was the highest for both EAA and EAF and the peak of kaempferol was higher with EAF than with EAA. In addition, gallic, phloretic, and ferulic acids were not detected, but veratric and cinnamic acids were detected specifically in EAF. Phenolic compound contents in EAA and EAF were identified based on the calibration curve (Tables 2 and 3). e total phenolic compound (TP) contents of EAF (97.32 mg/g, 1.43-fold), as analyzed by HPLC, were higher than those of EAA, and these results were similar for TPC and TFC. e contents of homogentisic acid (1.11-fold), protocatechuic acid (1.18-fold), (+)-catechin (3.03-fold), naringin (2.36-fold), and naringenin (1.79-folds) were higher in EAA than in EAF. Conversely, the contents of chlorogenic acid (2.06-fold), p-coumaric acid (1.47-fold), salicylic acid (5.89-fold), hesperidin (2.68-fold), quercetin (3.60-fold), and kaempferol (21.31-fold) were higher in EAF than in EAA. As a result, EAA and EAF might have higher scavenging effects because of these phenolic compounds.

EAA and EAF Reduce Intracellular ROS Generation in PC12
Cells. ROS production was analyzed by staining with DCFH-DA and detecting and quantitatively measuring the fluorescence by confocal microscopy (Figure 2(a)). Incubation of PC12 cells with H 2 O 2 led to an increase in DCF fluorescence intensity, which was proportional to the amount of ROS generated. However, after treatment with EAA and EAF, the green fluorescence signal of H 2 O 2 -treated cells decreased compared with that observed for the controls. e addition of vitamin C (50 μg/mL) served as a positive control with an inhibition rate of approximately 60%. EAF (200 μg/mL) resulted in a more pronounced reduction than the positive control. ese results showed that ROS generation was significantly elevated in a dose-dependent manner (Figure 2(b)

Inhibitory Effects of EAA and EAF on Apoptosis in H 2 O 2 -
Treated PC12 Cells. We finally investigated whether ROS affects EAA-and EAF-induced apoptosis based on the two major pathways (intrinsic and extrinsic). To determine the effect of EAA and EAF on apoptosis in H 2 O 2 -treated PC12 cells, protein levels of components of the intrinsic and extrinsic pathways were evaluated by western blotting. Results from the intrinsic pathway are shown in Figure 3(a). Specifically, treatment with H 2 O 2 only upregulated the expression of Bax/Bcl-xL compared with that in untreated cells. Exposure of H 2 O 2 -treated PC12 cells to EAA and EAF reduced the expression of Bax/Bcl-xL (0.09-0.67-fold) in a dose-dependent manner. Results of the extrinsic pathway are shown in Figure 3(b). In this investigation, the cells treated with H 2 O 2 only showed increased levels of cleaved-caspase8 and cleaved-caspase 3 (1.20-and 1.31-fold, respectively) compared with those in untreated cells. However, our results showed that EAA and EAF reduced pro-and cleaved-caspase8 and cleaved-caspase 3 levels in a dosedependent manner.

Discussion
Air pollution caused by ambient air particulate matter such as diesel exhausts particles, and nitrate induces oxidative stress that can trigger ROS [31,32]. ROS generates toxic byproducts that cause damage to the cell by producing oxidative stress; however, the cell expresses several antioxidant enzymes to protect against this effect [1]. e overexpression of ROS causes an imbalance between oxidant 4 Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine production and antioxidant capacity and leads to the onset of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases [2]. ROS can decrease through nonenzymatic and enzymatic reactions [30]. Nonenzymatic antioxidants such as phenolic acids, flavonoids, and other compounds increase the activity of glutathione peroxidase GSH (which reduces H 2 O 2 to H 2 O) [30]. Phenolic compounds are present in various medicinal plants [33]. e phenolic hydroxyl groups in phenolic compounds play an important role in antioxidant properties by donating hydrogens and scavenging radicals [34]. erefore, we identified antioxidants in the phenol-rich fractions of EAA and EAF by HPLC analysis. Linearity of the calibration curve with a correlation coefficient >0.99 was obtained from the plot of a minimum of five standard concentrations (10,25,50, 100, and 200 μg/mL) by using the least square method (Table 3) [35]. Homogentisic and salicylic acids, which were abundant in both EAA and EAF, confer beneficial effects such as the inhibition of oxidation, anti-inflammation [36][37][38], and disease tolerance in plants [39]. Kaempferol, which was significantly present in EAF, exerts other beneficial effects such as anticancer cell   Evidence-Based Complementary and Alternative Medicine proliferation, in vitro antioxidation, and the inhibition of autophagy [40][41][42][43]. ese phenolic and flavonoid compounds produced by photosynthesis are stored in plant leaves and accumulate in the vacuoles of flowers [44]. Indeed, phenols and flavonoids in plants have antioxidant effects by transferring hydroxyl groups to the radicals in ABTS + and DPPH and participating in electron delocalization, thus providing stability [45]. Our results suggested that the antioxidative effects of EAA and EAF might be due to their TPC and TFC contents. Importantly, antioxidant contents and activities were not significantly different between EA parts. However, EAA and EAF have stronger antioxidant activities than AA in ABTS + scavenging (3.1and 1.9-fold, respectively). e correlation between antioxidant activities (ABTS + and DPPH) and the content of TPs and phenolic compounds (homogentisic acid, salicylic acid, and kaempferol) in EAA and EAF was also investigated (  . ese results showed that the antioxidant effects on ABTS + and DPPH were due to TPs, salicylic acid, and kaempferol contained in EAA and EAF. Enzymatic antioxidants, which decrease ROS generation (such as SOD2 (which reduces O 2 − to H 2 O 2 ) and CAT and GPx (which reduce H 2 O 2 to H 2 O)), are usually secreted into the cytosol to protect tissue damage due to oxidative stress [3,30]. In addition, H 2 O 2 generates intracellular ROS which regulate apoptosis [3]. To measure the inhibition of intracellular ROS generation in the presence of H 2 O 2 , we tested the application of various concentrations of EAA and EAF (50, 100, and 200 μg/mL) to PC12 cells after treating the cells with 50 μM of H 2 O 2 for 20 min, as those conditions resulted in the highest ROS generation (data not shown). Our results showed that intracellular ROS generation was reduced in a dose-dependent manner after pretreatment with EAA and EAF, compared with that in PC12 cells only treated with H 2 O 2 (Figures 2(a) and 2(b)). e maintenance of homeostasis in multicellular organisms depends on complex networks of intracellular signals [1]. Cell organelles generate intracellular ROS via oxidative stress injury [2]. When ROS are overproduced in cells, they cause various diseases in humans, including neurological diseases [1,2]. However, ROS can be inhibited by antioxidant components [2] [15,42,46]. Our results showed higher levels of TPC, TPF, and phenolic compounds, which have antioxidant effects, following the treatment of PC12 cells with EAF, compared with those observed with EAA treatment. Moreover, among TPs, salicylic acid and kaempferol were significantly correlated with ROS generation as shown in Table 4 (− 0.965 and − 0.976). Our study indicated that EAF contained more antioxidant compounds than EAA, and thus EAF further prevented oxidation in H 2 O 2 -treated PC12 cells. Apoptosis-induced ROS comprises two major pathways, the intrinsic and extrinsic [6]. e proapoptotic members (Bcl-xL and Bax) release cytochrome c to the mitochondrial membrane through the intrinsic pathway [3]. In the extrinsic pathway, cleaved-caspase-8 cleaves and activates procaspase-3 [10]. rough these two pathways, caspase 3 directs cell death directly [10]. In our investigation, EAA and EAF reduced the levels of apoptosis-activating factors such as caspase-3 and caspase-8, as well as the Bax/Bcl-xL ratio. Specifically, these apoptotic factors were further decreased with EAF treatment compared with that with EAA. Antioxidant enzymes are known to prevent apoptosis via ROS and directly reduce ROS generation [3,30]. EAF reduced apoptotic protein levels, and its antioxidant effect was higher than that of EAA, even in the presence of nonenzymatic antioxidants such as phenolic acid and flavonoid. Further, EAF downregulated the intracellular apoptosis pathways induced by ROS generation (Figures 2(c)-2(e) and 3).

Conclusions
e present study demonstrates that EAA and EAF have neuroprotective effects on PC12 cells. EAA and EAF exerted protective effects against apoptosis by targeting oxidant and antioxidant agents such as SOD2, CAT, and the GPx system. Specifically, EAF contained abundant phenolic compounds and had a higher antioxidant effect than EAA. EAF could exert its antiapoptotic effect by reducing the number of ROS and enzymatic factors. erefore, it could be a good source of natural antioxidants to prevent neurodegenerative diseases arising from oxidative stress-induced neuronal damage by regulating the expression of apoptotic proteins.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.