Original research paper
Protective effects of Drynaria fortunei against 6-hydroxydopamine-induced oxidative damage in B35 cells via PI3K/AKT pathway
Hui-Chun Kuo a,†, Hung-Chi Chang b,†, Wan-Cheng Lan a, Fan-Hsuan Tsai c, Jung-Chun Liao d,* and Chi-Rei Wu a,*
Affiliation
a The Department of Chinese Pharmaceutical Sciences and Chinese Medicine
Resources, College of Pharmacy, China Medical University, No.91, Hsueh Shih Road, Taichung, 40402, Taiwan, R.O.C.
b Department of Golden-Ager Industry Management, College of Management,
Chaoyang University of Technology
c The School of Chinese Medicines for Post-Baccalaureate, I-Shou University, No.8,
Yida Rd., Yanchao Township, Kaohsiung County 82445, Taiwan, R.O.C.
d The School of Pharmacy, College of Pharmacy, China Medical University, No.91,
Hsueh Shih Road, Taichung, 40402, Taiwan, R.O.C.
Correspondence
Dr. Chi-Rei Wu, The School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, 91, Hsueh Shih Road, Taichung, 40402, Taiwan, R.O.C. E-mail: [email protected] Phone: + 886 4 22053366 5506
† Both authors contribute equally 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Abstract
In this study, we demonstrated the antioxidant and protective properties of aqueous extract of two commercial Polydiaceae plants - Drynaria fortunei (DF) and
Pseudodrynaria coronans (PC) against 6-hydroxydopamine (6-OHDA)-induced oxidative damage in B35 neuroblastoma cells. The contents of their phytochemical profiles were determined by spectrophotometric methods and high performance liquid chromatography using a photodiode array detector. DF extract has better effects than PC extract in scavenging ROS and inhibiting 6-OHDA autoxidation. Following exposure of B35 cells to 6-OHDA, there was a marked decrease in cell survival and the activation of intracellular antioxidant enzymes and PI3K/AKT pathway, and then the level of lipid peroxidation was increased. Pretreatment with DF extract blocked these 6-OHDA-induced cellular events. Naringin and epicatechin are major
components of DF extract. These results show that DF extract exerts the protective effects against 6-OHDA toxicity via radical scavenging activity and the increase in the activation of PI3K/AKT pathway to elevate the levels of intracellular antioxidant enzymes including HO-1, NQO-1 and glutathione-related enzymes.
Keywords: Drynaria fortunei; naringin; antioxidant activity; 6-hydroxydopamine; PI3K/AKT; HO-1 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Introduction
The rhizome of Drynaria fortunei (Kunze) J. Smith (Polydiaceae) (DF) is a major resource for the traditional Chinese medicine “Gu-Sui-Bu” in China, which is used to prevent osteoporosis and aging-associated symptoms. Accumulating evidence shows that DF has osteoprotective effects through its osteogenic differentiating and
proliferating activities in cell culture and animal studies.1-4 Flavonoids and phenylpropanoids, such as epicatechin, naringin and neoeriocitrin, are the active constituents of DF extract for its osteoprotective activities.1-4 Other pharmacological studies indicate that DF has antioxidant and anti-apoptotic activities.5, 6 The above phytoconstituents also have neuroprotective and anti-apoptotic activities through their antioxidant mechanisms in vitro and in vivo.7-10 The rhizome of Pseudodrynaria coronans (Wall.) Ching (Polydiaceae) (PC) is an alternative medicinal resource for “Gu-Sui-Bu” in Taiwan. There are no phytochemical and pharmacological reports on PC. Therefore, this investigation is first that is aimed at comparing the
phytoconstituents and reactive oxygen species (ROS) scavenging activities of two commercial “Gu-Sui-Bu” resource plants, DF and PC, because ROS are major
intermediate neurotoxins and play an important role in intracellular oxidative damage due to aging-associated disorders.
Natural antioxidants, including flavonoids and phenylpropanoids, scavenge free radicals, which initiate and propagate oxidative chain reactions, thus, prevent intracellular oxidative damage.11 Intracellular oxidative stress cascades, including biomolecules, act through ROS and deficient intracellular antioxidant defenses, which might induce the aging process and aging-associated neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).12, 13 One of major
pathological mechanisms in PD is autoxidation and enzymatic oxidation of dopamine 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
in the substantia nigra, which causes selective apoptosis and a loss of dopaminergic neurons.14 6-hydroxydopamine (6-OHDA) is a toxic oxidative dopamine metabolite that is rapidly and non-enzymatically oxidized by molecular oxygen to form p-quinone and ROS, such as superoxide anion and hydrogen peroxide, under physiological conditions.15 Thus, 6-OHDA is a widely used compound for
investigating pathogenesis and progression of as well as drug development for PD. Therefore, we further attempted to demonstrate the role of intracellular antioxidants and protective enzymes on the protective effects of DF extract against 6-OHDA-induced neuronal damage in B35 neuroblastoma cells because neuronal cells have several antioxidants and protective enzymes to prevent ROS formation or detoxify ROS.16
Materials and methods
Preparation of the herb extracts
Two Polydiaceae plants (DF and PC) were identified and provided by Hung-Chi Chang. DF or PC was extracted with distilled water, and the resulting extract was concentrated under reduced pressure to obtain DF or PC extract.6 To assess
phytoconstituents and ROS-scavenging activities, the DF or PC extract was dissolved in distilled water. To clarify the protective effects from and mechanism for 6-OHDA-induced neuronal damage, the DF or PC extract stock solutions were prepared using sterile distilled water, filtered using a 0.22 M sterile filter and then diluted with DMEM without phenol red.
Chemicals 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
2-deoxyribose, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4”-disulfonic acid (ferrozine), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), 5,5’-dithio-bis(2-nitrobenzoic) acid (DTNB),
6-hydroxy-2,5,7,8-tetramethy-chroman-2-carboxylic acid (trolox), 6-OHDA, ascorbic acid, (+)-catechin, cyanidin, epicatechin, ferrous sulfate heptahydrate, Folin-Ciocalteu’s reagent, reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), homovanillic acid (HVA), horseradish peroxidase (HRPase), malodialdehyde (MDA), naringin, nitroblue tetrazolium chloride (NBT), quercetin, sodium carbonate, sodium molybdate, sodium nitrate, superoxide dismutase (SOD), thiobarbituric acid (TBA), trichloroacetic acid (TCA), verbascoside, xanthine and xanthine oxidase were purchased from Sigma-Aldrich Chem. Corp. (St. Louis, MO, USA). Hydrogen peroxide (H2O2) and all HPLC-grade solvents were purchased from Merck (Darmstadt, Germany).
Determination of phytoconstituents using a spectrophotometric reader The levels of all phytochemicals, including total phenol, phenylpropanoid, flavonols and anthocyanidins, were assayed using a 96-well microtiter
spectrophotometric method. The method used to determine the total phenolic levels is based on forming blue-colored products through a redox reaction with
Folin-Ciocalteu’s reagent and measuring its absorbance at 725 nm. The total phenolic concentration of the samples was expressed as mg of catechin equivalents per gram of sample 17. The method used to determine total phenylpropanoid levels is based on forming colored products using phenylpropanoid with the Arnow reagent (containing 5% (w/v) sodium nitrate and 5% sodium molybdate) and measuring its absorbance at 525 nm. The total phenylpropanoid concentration of the samples was expressed as mg of verbascoside equivalents per g of sample.17 The method used to determine
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flavonols and anthocyanidin levels is based on switching the absorbance wavelength through different hydrogen chloride concentration and measuring its absorbance at 360 and 520 nm.18
Determination of phytoconstituents using high performance liquid chromatography (HPLC)
Aqueous DF or PC extract was dissolved in distilled water and then filtered using a 0.22 m filter. Stock solutions of the standards were prepared in methanol to the final concentration 10 mg/mL. All standard and sample solutions were injected into 10 L in triplicate. The Shimadzu VP series HPLC and Shimadzu Class-VPTM chromatography data systems were used. All chromatographic operations were performed at 25 C. The epicatechin and naringin chromatographic peaks were confirmed by comparing their retention times and UV spectra. A LiChrospher® RP-18e (250 × 4 mm, 5 m) column (Merck KGaA, Darmstadt, Germany) was used. Certain separating conditions including the mobile phases and gradient program conditions, followed the description by Liu et al.19
Determination of ROS using scavenging activity assay in vitro
The superoxide anion, H2O2 and hydroxyl radical scavenging activities were determined as described previously.17 The superoxide anion scavenging activity is based on the reaction between NBT and the superoxide anion produced from xanthine and xanthine oxidase and was determined at 560 nm for 5-min kinetics using a microplate reader (PowerWaveX, Bio-Tek instruments, Inc., Winooski, VT, USA). The results were expressed as the U of SOD equivalents per milligram of sample. The H2O2 scavenging activity is based on HVA dimer formation through the reaction 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143
between H2O2 and HVA catalyzed by HRPase, which was measured by the fluorescence intensity at the excitation 315 nm and emission 425 nm using a
fluorescence microplate reader (FLX800, Bio-Tek instruments, Inc., Winooski, VT, USA). The H2O2 scavenging activity results were expressed as mol of trolox equivalents per gram of sample. The hydroxyl radical scavenging activity was monitored at 532 nm using the 2-deoxyribose-TBARS method. The hydroxyl radical scavenging activity results were expressed as mg of quercetin equivalents per gram of sample.
In vitro lipid peroxidation inhibition assay
The whole rat brain was homogenized (100 mg/mL) in ice-cold 0.1 M phosphate buffer (pH 7.4), and then brain homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C. The reaction mixture consisted of brain homogenate, 1 mM ferrous sulfate, 5 mM ascorbic acid and sample solution. The reaction solution was incubated at 37 °C for 30 min, and the thiobarbituric acid reactive substance (TBARS) test was
performed by rapidly adding 1.2% (w/v) TBA and 10% TCA. The TBARS test tubes were incubated at 90 °C for 60 min, cooled, and centrifuged at 3,000 rpm for 10 min; the absorbance of the supernatant was then determined at 532 nm.17
In vitro ferric ion reducing antioxidant power (FRAP) assay
Briefly, 25 L of sample solution or trolox standards were mixed with 25 L of freshly prepared FRAP reagent, which consisted of 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) solution in 40 mM HCl, 20 mM FeCl3, and 50 mM acetate buffer (pH 3.6). The reaction mixture absorption was measured at 595 nm. The results were 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
calculated based on a standard curve obtained using trolox and expressed as the relative trolox equivalent per gram of sample.17
In vitro iron-chelating activity (ICA) assay
This method is based on ferrozine-Fe+2 colored complex formation, and the absorbance is measured at 562 nm. The percentage of inhibition of ferronize-Fe+2 complex formation was calculated as we previously reported.17
Inhibition of 6-OHDA autoxidation in vitro
Autoxidation of 6-OHDA was followed spectrophotometrically by monitoring p-quinone formation at 490 nm.20 The assay was conducted in a cell free system under conditions that correspond to cellular OHDA treatments. Stock solutions of 6-OHDA (100 mM) were prepared in phosphate-buffered saline solution. The experiment was initiated by adding 6-OHDA to yield the final concentration 50 μM. The absorbance at 490 nm was monitored for 3 min at 30-sec intervals at 37℃
The protective effects of 6-OHDA-induced neuronal damage in B35 cells Rat B35 neuroblastoma cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 g/mL streptomycin in a water-saturated atmosphere with 5% CO2 at 37 C. The experiments were performed 24 h after the cells were seeded in 96-well sterile clear-bottom plates or a 90-mm dish. The cells were plated at an appropriate density according to the scale of each experiment. The DF or PC extract was treated 1 h before 6-OHDA (50 M) was added. 6-OHDA (50 M) was used after a 24-h exposure, as described below. B35 neuroblastoma cells were seeded in a 90-mm dish and incubated overnight in a water-saturated atmosphere 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192
with 5% CO2 at 37C. The cell morphology was observed 24 h after 6-OHDA exposure using a phase-contrast microscope (Nikon, Tokyo, Japan).
The MTT assay is based on the ability of living cells to reduce MTT to insoluble formazan, which is measured at 570 nm to study cell survival as we previously reported.21 Briefly, 24 h after 6-OHDA exposure, the medium was replaced and MTT was added to each well. After incubating for 2 h at 37C, the cells were washed with PBS, and DMSO was added. The experiments were performed in triplicate over four independent experiments. Cell viability was expressed as the percentage relative to untreated cells, which served as the control group (designated 100% viable).
Intracellular antioxidant enzyme and GSH level measurements
Following incubation for 24 h with 6-OHDA, B35 neuroblastoma cells were collected from culture dishes and sonicated on ice. The solution was centrifuged for 15 min at 4°C to eliminate cell debris, and the supernatant was used in enzyme activity assays. The antioxidant enzyme activities, including for GPx and GR, were measured as we previously reported.21 The GPx and GR activities were expressed as mU/mg of protein. The GSH levels were determined described previously.21 Briefly, the lysates (20 μg/50 μL) or GSH standard was pipetted into each well of a 96-well plate. The reaction solution, included DTNB, NADPH and GR, was added to each well and was recorded at 405 nm for 5 min in a microplate reader. The GSH levels were expressed as pmol/mg of protein.
The lipid peroxidation assay in B35 cells
Lipid peroxidation was measured using the TBARS assay in cell cultures. Briefly the lysates (200 g/100 L) or MDA standard was pipetted into 1.5 mL tubes, and a 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217
TBA test was performed. Next, the supernatant absorbance at 532 nm was
determined.21 The experiments were performed in triplicate over three independent trials. The MDA levels were expressed as nmol/mg of protein.
Western blot analysis
Twenty-four hours after 6-OHDA exposure, the cells were subjected to western blot analyses to determinate of the levels of phosphoinositide 3-kinase (PI3K)/AKT pathway, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO-1). Briefly, the protein samples were extracted from the cell and quantified using a Bradford protein assay kit (Bio-Rad), followed by electrophoretic separation through SDS-PAGE. After transferring the protein samples to PVDF membranes, the samples were incubated with primary antibodies against PI3K, AKT, phospho-AKT (serine 473) (p-AKT (ser)), phospho-AKT (threonine 308) (p-AKT (thr)), HO-1 or NQO-1, overnight at 4 °C and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG. The images were scanned using an LAS-4000 mini imaging system (Fujifilm, Kanagawa, Japan), and the optical density data were analyzed using MultiGauge v3.0 software (Fujifilm, Kanagawa, Japan). For the western blot analyses, β-actin served as an internal control.
Statistical Analyses
All results were expressed as the mean ± standard deviation (SD). The significant differences were calculated using SPSS software with a one-way ANOVA followed by Scheffe’s test, and P values < 0.05 were considered significant.
Results
218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242DF or PC extract phytoconstituents
The levels of total phenols, flavonoids, phenylpropanoids and anthocyanidins in the DF or PC extract were measured using 96-well microtiter spectrophotometric methods and are shown in Table 1. The levels of total phenols, flavonoids, phenylpropanoids and anthocyanidins in each gram of DF extract were equivalent to 268.41 mg catechin, 13.67 mg quercetin, 21.05 mg verbascoside and 2.07 mg cyanidin. However, the levels of total phenols, flavonoids, phenylpropanoids and anthocyanidins in each gram of PC extract were equivalent to 130.10 mg catechin, 7.42 mg quercetin, 4.50 mg verbascoside and 1.22 mg cyanidin.
The phytoconstituents of the DF or PC extract were further assayed using high performance liquid chromatography. Their chromatographs are shown in Figure 1; the certain phytoconstituent peak zones differ between the DF and PC extracts. Each gram of DF extract contained 26.23 mg of naringin and 1.00 mg of epicatechin. Each gram of PC extract contained 10.27 mg of naringin and 1.46 mg of epicatechin (Table 1).
The ROS-scavenging and lipid peroxidation inhibiting activities of DF or PC extract in vitro
The scavenging activity of DF or PC extract against ROS was also investigated using 96-well microtiter spectrophotometric methods. The scavenging activity of each gram of DF extract against superoxide anion, H2O2 and hydroxyl radical was equivalent to 11.46 U of SOD, 927.57 μmol of trolox and 14.95 mg of quercetin. The scavenging activity of each gram of PC extract was equivalent to 9.43 U of SOD, 582.60 μmol of trolox and 4.40 mg of quercetin (Table 2). Next, their lipid peroxidation-inhibiting effects were evaluated using the Fe2+/ascorbate method, for 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267
which rat brain homogenate was used as the oxidizable biomolecule target. The IC50
of DF extract against lipid peroxidation is 32.43 mg/mL and the IC50 of PC extract is
117.99 mg/mL (Table 2). Furthermore, their iron-chelating and reducing power activities were further investigated using 96-well microtiter spectrophotometric methods (ICA and FRAP tests). The reducing power of each gram of DF extract is equivalent to 1034.65 μmol of trolox and better than that of PC extract (each gram is equivalent to 622.64 μmol trolox) (Table 2). However, the above extracts did not have iron-chelating capacities at the concentrations used in the hydroxyl radical scavenging and lipid peroxidation-inhibiting assays (data not shown).
DF or PC extract inhibits 6-OHDA autoxidation in vitro
The DF or PC extract inhibiting activity against p-quinone production from 6-OHDA autoxidation under cell-free physiological conditions was also investigated through 9well microtiter spectrophotometric methods. The reaction mixture (only OHDA) absorbance at 490 nm was considered 100% of p-quinone produced from 6-OHDA. The DF extract at 25 - 250 μg/mL inhibited absorbance at 490 nm in a concentrationdependent manner (P < 0.05, P < 0.001). The PC extract at only 50 -250 μg/mL inhibited absorbance at 490 nm (P < 0.05, P < 0.01) (Figure 2)
The protective effects of the DF extract against 6-OHDA-induced neuronal damage in B35 neuroblastoma cells
We further evaluated the protective effects of DF extract at 10 - 250 g/mL against 6-OHDA-induced neuronal damage in B35 neuroblastoma cells using the MTT assay. Incubation with 50 M 6-OHDA to B35 neuroblastoma cells for 24 h, cell viability was decreased to 46.3% compared with the control (P < 0.001) (Fig. 3(A)). The DF 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292
extract at 50 - 250 g/mL increased the cell viability against 6-OHDA in a concentration-dependent manner (P <0.01, P < 0.001) (Fig. 3(A)). Furthermore, we observed morphological alterations of B35 neuroblastoma cells through phase-contrast microscopy. Incubation with 50 M 6-OHDA for 24 h decreased the cell number and cell shrinkage (Figure 3(B)-a and 3(B)-b). The DF extract (50 - 100 g/mL) attenuated the morphological changes (Figure 3(B)-c and 3(B)-d).
GSH cycle involvement in the protective effects of DF extract
To clarify the protective mechanism of the DF extract against 6-OHDA-induced neuronal damage in B35 neuroblastoma cells, we measured the intracellular antioxidant defenses including the levels of GSH and related antioxidant enzymes activities. The intracellular GSH levels as well as GPx and GR activities in B35 neuroblastoma cells treated with 50 M 6-OHDA for 24 h were lower (P < 0.01) (Table 3). The level of MDA in B35 neuroblastoma cells treated with 50 M 6-OHDA for 24 h was greater (P < 0.01) (Table 3). The DF extract at 100 g/mL partially restored the intracellular GSH levels, and partially reversed the decrease in activities of GPx and GR by 50 M 6-OHDA in B35 neuroblastoma cells (P < 0.05) (Table 3). The DF extract at 100 g/mL also decreased the level of MDA, which was increased by 6-OHDA (P < 0.05) (Table 3).
Reversal of PI3K/AKT-, NQO-1- and HO-1-mediated damage through the protective effects of DF extract
Because neuronal damage due to 6-OHDA mainly occurs through the PI3K/AKT pathway and a decrease in detoxifying enzymes such as HO-1 and NQO-1,32, 33 we 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316
assayed the levels of PI3K/AKT pathway proteins, HO-1 and NQO-1 in B35 neuroblastoma cells treated with 6-OHDA and DF extract. The protein immunoblot assay is shown in Figure 4(A). 6-OHDA decreased the levels of PI3K (P < 0.01) but did not alter the level of AKT (P > 0.05) (Figure 4(B) and Figure 4(C)). However, 6-OHDA decreased the ratio of p-AKT (thr) to AKT and p-AKT (ser) to AKT (P < 0.001) (Figure 4(D), Figure 4(E) and Figure 4(F)). 6-OHDA further decreased the levels of HO-1 and NQO-1 in B35 neuroblastoma cells (P < 0.01 for HO-1, P < 0.05 for NQO-1) (Figure 4(G) and Figure 4(H)). The DF extract at 50 ~ 100 g/mL restored the levels of PI3K and NQO-1 as well as the ratio of p-AKT to AKT (especially p-AKT (ser) vs AKT) (P < 0.01, P < 0.001), but only the DF extract at 100 g/mL could restore the levels of HO-1 and ratio of p-AKT (thr) to AKT (P < 0.05, P < 0.01) (Figure 4(B-H)).
Discussion
Gu-Sui-Bu is a common traditional Chinese medicine used to prevent aging-associated disorders, including PD, for centuries. DF is a major resource for Gu-Sui-Bu, and PC is an alternative resource in Taiwan. According to phytochemical reports on DF,3, 4, 19 flavonoids and phenylpropanoids are major phytoconstituents of DF. Our data indicate that the DF extract had higher levels of total phenolics and flavonoids (approximately two times) compared with the PC extract. Next, we further compared the DF and PC extract chromatograms and matched them with other reports.19 The DF extract might contain phenolic acids, dihydroxychromone, epicatechin and naringin. The PC extract might contain epicatechin and naringin. Epicatechin and naringin are co-existing phytoconstituents in the DF and PC extracts; however, the certain
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phytoconstituent peak zones differ between the DF and PC extracts. The DF extract contained higher levels of naringin (approximately two times) than the PC extract. Hence, we suggest that the DF extract has higher phenolic levels, especially for naringin.
These phenolic compounds have been shown to correlate with the plant antioxidant activities.17, 22 Moreover, in the pathogenesis of PD, both neurotoxic ROS and highly reactive, redox-cycling DA-derived quinones are formed due to enzymatic breakdown by monoamine oxidase (MAO) or autoxidation of excess cytosolic dopamine.15 ROS mainly include superoxide anion, H2O2 and hydroxyl radical. Superoxide anion is produced in a cell’s mitochondrion and has been implicated in the pathophysiology of certain diseases such as PD. H2O2 yields the highly reactive hydroxyl radical, which is the most reactive and severely damages adjacent biomolecules, such as
polyunsaturated fatty acids.11 Our results also indicate that the DF extract had higher ROS scavenging and lipid peroxidation-inhibiting activities as well as reducing power capacity (approximately two times) compared with the PC extract. Moreover, at the concentrations used in the hydroxyl radical scavenging and lipid peroxidation-inhibiting assays, neither the DF nor the PC extract had the capacity to chelate iron. From the above results, we suggested that the DF or PC extract inhibited lipid peroxidation in brain homogenate systems mainly by terminating oxidative chain reactions through its radical scavenging capacity and reducing power; however, certain reports indicate that the activity of certain antioxidant compounds may correlate with iron-chelation.11 Furthermore, at the concentrations used in the ROS-scavenging activity assays, the DF extract also inhibited p-quinone production from 6-OHDA autoxidation in a concentration-dependent manner; this effect was greater than for the PC extract. Based on the above phytochemical and antioxidant results, we 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365
suggest that ROS-scavenging activities of the DF or PC extract positively correlate with the levels of all phytochemicals; this relationship is consistent with earlier reports.17, 22 The enhanced ROS-scavenging activity of the DF extract compared with the PC extract may be due to the DF extract enrichment phenolic compounds, such as naringin, because their antioxidant activities are consistent with the pharmacological activities of naringin and epicatechin.10, 23
Similar to PD pathogenesis, 6-OHDA is a selective dopaminergic neurotoxin that induces PD-like cell or animal models via excessive ROS and p-quinone generation during 6-OHDA autoxidation.15 We further found that, at 50 - 250 g/mL, DF extract increased cell viability against 6-OHDA in a concentration-dependent manner and reversed the 6-OHDA-induced morphological changes in B35 neuroblastoma cells. Naringin is an active DF compound identified in our HPLC results and other reports that also protect against rotenone-induced neuronal damage in SH-SY5Y cells.9 Therefore, we suggest that the DF extract protect against 6-OHDA-induced neuronal damage in B35 neuroblastoma cells partially via scavenging ROS and inhibiting p-quinone. However, intracellular redox imbalance is mainly due to an imbalance between generating and eliminating the free radicals, specifically caused by lower intracellular antioxidant defenses.11, 13 Neuronal intracellular antioxidant defenses mainly include several antioxidants and protective enzymes that prevent ROS
formation or detoxify ROS.16 GSH recycling includes GSH and related enzymes. such as glutathione peroxidase (GPx) and GR, and is a major intracellular antioxidant defense. In PD patients, the degree of in symptom severity correlates with
intracellular GSH loss in substantia nigra.24 Many oxidants, such as 6-OHDA and H2O2, could deplete the intracellular GSH levels and decrease GPx and GR activation in cell culture and animals.21, 25 Our results are consistent with the above reports;21, 25 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
incubation with 50 M 6-OHDA in B35 neuroblastoma cells for 24 h decreased the GSH cycle activities and increased the oxidative damage. The DF extract at 100 g/mL reversed the GSH cycle activities that were decreased by 50 M 6-OHDA in B35 neuroblastoma cells, which in turn, decreased the oxidative damage. Hence, we suggest that the DF extract attenuated the 6-OHDA-induced neuronal damage by upregulating the antioxidant status via intracellular GSH regeneration and its radical scavenging activity. In fact, naringin which is a major DF extract constituent also protected against the neuronal damage caused by 3-nitropropionic acid or kainic acid via antioxidant activity and intracellular GSH regeneration.7, 8 Certain reports further indicated that naringin and epicatechin have neuroprotective activities against rotenone, aluminum and amyloid β peptide in vitro and in vivo. 7-10, 26 Based on the above results, we suggest that DF is a potential medicinal plant that protects against PD in a comparison between two commercial Polydiaceae plants.
AKT plays a pivotal role in fundamental cellular functions, such as cell
proliferation and survival, by phosphorylating a variety of enzymes, including pro-apoptotic regulators, detoxifying and antioxidant proteins, and transcription factors.27
AKT is mainly activated through phosphorylation of T308 (AKT (thr)) and S473 (AKT (ser)) by receptor tyrosine kinases, G-protein-coupled receptors, mTOR complex 2 (mTORC2), DNA-dependent protein kinase (DNA-PK), and other stimuli that induce phosphatidylinositol 3,4,5 triphosphates (PtdIns(3,4,5)P3) production through PI3K.28 Hence, certain reports indicate that AKT is an important therapeutic
target for the treating diabetes, stroke, and neurodegenerative disease.27, 28 In this
study, we found that treating with 6-OHDA for 24 h decreased the levels of PI3K and AKT phosphorylation, especially at S473, in B35 neuroblastoma cells. Hence, 6-OHDA mainly inhibited full activation of AKT via obstructing AKT phosphorylation 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415
at S473, which causes neuronal damage. The DF extract increased the PI3K levels and restored AKT phosphorylation. Among the various cytoprotective enzymes modulated by the AKT cascade, HO-1 and NQO-1 play an important role in neuroprotective functions.29 Recent, mounting evidence indicates that a pharmacological inducer of
HO-1 expression may maximize the intrinsic antioxidant potential of cells.30 NQO-1 is
a key enzyme that detoxifies reactive quinones produced from autoxidation and enzymatic oxidation of dopamine.31 Our data also indicate that 6-OHDA decreased the HO-1 and NQO-1 levels in B35 neuroblastoma cells. Hence, our results are consistent with other reports that 6-OHDA decreases the HO-1 and NQO-1 expression via inhibiting phosphorylation of the PI3K/AKT pathway, causing neuronal damage.32, 33
DF also reversed the HO-1 and NQO-1 levels that were decreased by 6-OHDA. Therefore, we suggest that the DF extract induced AKT phosphorylation and further activated HO-1 and NQO-1 expression to counteract the neurotoxicity and decrease the neuronal damage caused by 6-OHDA in B35 cells.
Conclusion
In conclusion, DF extract had the higher phenolic levels, especially for
phenylpropanoids, and exhibited the higher radical scavenging potency compared to PC extract. Its antioxidant activity can be correlated with its reducing power, which is not due to iron chelation, and this antioxidant activity depends on the phenolic
antioxidants such as naringin and epicatechin.19 The DF extract has a protective effect against 6-OHDA-induced neuronal damage in vitro. Naringin and epicatechin are its major active compounds because naringin and epicatechin can protect against neuronal damage caused by rotenone and amyloidβ peptide.9, 26 This protective mechanism might be related to its radical scavenging capacity and its ability to 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440
activate intracellular antioxidant defenses, including GSH recycling as well as HO-1 and NQO-1 via the phosphorylation in the PI3K/AKT pathway (Figure 5). Hence, we suggest that DF extract has potential therapeutic benefits for treating aging-associated symptoms and neurodegenerative disorders. However, the expressions of detoxifying and antioxidant enzymes such as GSH-related enzymes, HO-1 and NQO-1 are
coordinated and induced via a nuclear factor-E2-related factor 2 (Nrf2)-dependent and antioxidant response element (ARE)-mediated mechanism.34 Recent studies have reported that Nrf2 is a master redox regulator that upregulates HO-1 to protect dopaminergic neurons against 6-OHDA-induced neurotoxicity.35, 36 Studies have also suggested that Nrf2 nuclear translocation requires the activation of several signal transduction pathways, such as PI3K/AKT or the mitogen-activated protein kinase (MAPK) pathways.32, 34, 36 In addition, naringin is a major active compound in DF extract and has also been shown to protect against 3-nitropropionic acid-induced apoptosis via downregulating the pro-apoptotic gene Bax and the upregulating the anti-apoptotic genes Bcl-2 and Bcl-X(L).7 The expression of pro-apoptotic genes is also modulated by AKT.27, 28 Therefore, the anti-apoptotic effects from DF extract and the role of pro-apoptotic genes and Nrf2 translocation mediated by PI3K/AKT in the neuroprotective effects from DF extract must be further investigated.
Acknowledgments
We thank the financial support of the National Sciences Council NSC98-2320-B-039-041 MY3, NSC101-2320-B-039-051, NSC 101-2120-M-009-008 and NSC102-2320-B-039-037. 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464
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