Dimerumic Acid and Deferricoprogen Activate Ak Mouse Strain

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Dimerumic Acid and Deferricoprogen Activate the Akt /HO-1 Pathway and Prevent Apoptotic Cell Death in an SH-SY5Y Cell Model of Parkinson’s Disease Wei-Ting Tseng, Ya-Wen Hsu, and Tzu-Ming Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01551 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 21, 2016

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Journal of Agricultural and Food Chemistry

Dimerumic Acid and Deferricoprogen Activate the Akt/HO-1 Pathway and Prevent Apoptotic Cell Death in 6-OHDA-induced SH-SY5Y Cells

Wei-Ting Tseng,a Ya-Wen Hsu,b and Tzu-Ming Panab *

a

Department of Biochemical Science and Technology, College of Life Science,

National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan b

SunWay Biotechnology Company, No. 139, Xing’ai Road, Taipei 11494, Taiwan

First Author: Wei-Ting Tseng *Corresponding Author: Tzu-Ming Pan, Professor; Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan Tel: +886-2-3366-4519 ext 10; Fax: +886-2-3366-3838; E-mail: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative disorder, which can be

2

modeled using the neurotoxin 6-hydroxydopamine (6-OHDA) to generate oxidative

3

stress. Here, we studied the effects of the antioxidants deferricoprogen (DFC) and

4

dimerumic acid (DMA), produced by rice fermented with Monascus purpureus NTU

5

568, on 6-OHDA-induced apoptosis in SH-SY5Y cells and their potential protective

6

mechanisms. DMA and DFC inhibited 6-OHDA-induced apoptosis and cellular

7

reactive oxygen species (ROS) in SH-SY5Y human neuroblastoma cells. Molecular

8

analysis demonstrated associated upregulation of the Ak mouse strain thymoma (Akt),

9

heme oxygenase-1 (HO-1), and signal-regulated kinase (ERK) pathways along with

10

inhibited phosphorylation of c-Jun N-terminal kinase (JNK) and p38 pathways and

11

altered homodimeric glycoprotein, N-methyl-D-aspartate (NMDA) receptor, and

12

immunoglobulin Fc receptor gene expression. These results suggested that the

13

neuroprotection elicited by DMA and DFC against 6-OHDA-induced neurotoxicity

14

was associated with the Akt, MAPK, and HO-1 pathways via regulating the gene

15

expression of NMDA receptor, homodimeric glycoprotein, and immunoglobulin Fc

16

receptor.

17 18

KEYWORDS: Monascus purpureus-fermented rice, Parkinson’s disease, apoptosis,

19

dimerumic acid, deferricoprogen 2


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20

INTRODUCTION

21

Parkinson’s disease (PD) is a degenerative nerve diseases associated with the

22

progressive loss of dopamingic producing nerve cells in the substantia nigra pars

23

compacta (SNpc). Treatment of levodopa is the therapeutic criterion for PD but is

24

only effective for symptomatic relief in the past stages of the disease. Accordingly,

25

there is a specific medicinal need for new curative for PD. Though there are not

26

completely understood about the mechanisms accountable for the death of

27

dopaminergic cell, multiply evidence from human post-mortem investigations and

28

animal studies indicated that oxidative stress was an important role in initiating this

29

process.1–3 Therefore, inhibition of oxidative stress might be a feasible therapeutic

30

approach for PD.

31

A large number of PD models have been developed to assistant in the detection of

32

neuroprotective curatives for PD. Many of these models utilize experimental

33

neurotoxins

34

6-hydroxydopamine (6-OHDA). 6-OHDA is widely used to induced in vitro and in

35

vivo PD models and it could lead up oxidative stress.4 Previous reports have indicated

36

that auto-oxidation contributed to the formation of reactive oxygen species (ROS).5

37

Moreover, ROS were established to play a critical role in 6-OHDA-induced cell

38

apoptosis.6 In addition, mitogen-activated protein kinases (MAPKs) are usually

like

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

3


(MPTP)

and


39

related to nerve cell death and apoptosis,7,8 whereas the Ak mouse strain thymoma

40

(Akt) pathway is regarded to be essentiality for the protection of neurons from death

41

and cell survival.8,9 Accordingly, various studies have shown that MAPK pathway

42

activation and Akt inhibition mediate 6-OHDA-induced neuronal damage.8,10

43

Monascus purpureus-fermented rice (red mold rice, RMR) has been indicated to be

44

efficient for the administration of hypercholesterolemia, diabetes, hypertension,

45

obesity, and Alzheimer’s disease as well as for the prevention of cancer.11-17 Our

46

research group has been found that extracts of rice which was fermented with M.

47

purpureus NTU 568 suppressed amyloid β (Aβ)-induced neuron cytotoxicity via

48

combined anti-inflammatory and antioxidant mechanisms.18 RMR contains some

49

bioactive compounds which were pigments, polyketide monacolins, deferricoprogen

50

(DFC), and dimerumic acid (DMA). DMA and DFC potent radical scavenging

51

activity and exhibit the structural features of iron chelating (siderophore).19 DMA in

52

particular has been exhibited to be defendable versus cytotoxicity in rat hepatocytes

53

which were induced by oxidative stress.20 Previously, we demonstrated that DMA and

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DFC may protect against 6-OHDA toxicity by inhibiting the formation of ROS and

55

apoptosis in rodent cells of neural crest origin.21 Furthermore, we showed that DMA

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and DFC-containing extract of rice which was fermented with M. purpureus NTU 568

57

(extracted with 50% ethanol, called R50E) may prevent degenerative nerve via 4


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anti-inflammatory and anti-oxidative mechanisms, indicating its potential curative

59

efficacy for PD therapeutics.22

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In the present study, SH-SY5Y human neuroblastoma cells were exposed to

61

6-OHDA was used to be a model to assess the mechanisms fundamental the

62

cytoprotection generated by the secondary metabolites DMA and DFC extracted from

63

rice which was fermented with M. purpureus NTU 568. Cell viability, apoptosis, ROS

64

formation, proteins and their modification states, and gene expression were examined.

5



65

MATERIALS AND METHODS

66

Materials. Potato Dextrose Agar (PDA) was purchased from Becton, Dickinson

67

and Company (Sparks, MD, USA). N-acetyl cysteine (NAC) and 6-OHDA were

68

purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Cell culture reagents

69

were purchased from Invitrogen Corp. (Carlsbad, CA, USA). All other assay kits were

70

purchased from Cayman Chemical Co., Ltd. (Ann Arbor, MI, USA). The antibodies

71

were purchased from Abcam Inc. (Cambridge, UK).

72

Preparation of Red Mold Rice (RMR). Long-grain rice was fermented with M.

73

purpureus NTU 568 as described previously23 to obtain RMR. The RMR was further

74

dried by freeze dryer and extracted with 50% ethanol.

75

Identification of Deferricoprogen (DFC) and Dimerumic Acid (DMA). The

76

50% ethanol extract of RMR, R50E, was separated and purified by MCI gel (CHP

77

20P, 75−150 µm; Mitsubishi Chemical Industries, Tokyo, Japan) to produce five

78

fractions tagged R50E-1 through R50E-5 after filtration and concentration. The

79

anti-oxidative activities of the five fractions were determined by using

80

2,2-diphenyl-1-picrylhydrazyl (DPPH) method. R50E-3 had the highest DPPH

81

scavenging activity, further separated and purified by Sephadex LH-20 gel

82

(Pharmacia Fine Chemicals AB, Uppsala, Sweden) to produce six fractions.

83

Ultimately, the highest DPPH scavenging activities fractions were accumulated and 6


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purified by preparative high performance liquid chromatography to acquire purified

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DMA and DFC.30

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SH-SY5Y Cells Culture and Drug Treatment. Ham’s F12 and MEM medium

87

(1:1), contained 10 mM sodium pyruvate, 2 mM glutamine, and 15% fetal calf serum

88

was used to culture SH-SY5Y human neuroblastma cells at 37 °C with 95% air and

89

5% CO2. SH-SY5Y cells were plated onto 96-well plates at a density of 2 × 104 cells

90

each well. After 24 h of incubation, the growth media were attended with various

91

concentrations of 6-OHDA. 6-OHDA was dissolved in 0.02% ascorbic acid saline as

92

a stock solution (the final concentration was 25 mM). Cells were exposed to R50E,

93

DMA, or DFC at different concentrations in the absence or presence of 6-OHDA for

94

24 h.

95

Cell Viability Assay. SH-SY5Y cells viability was assessed by the MTT method.

96

Solution of 0.5 mg/mL MTT (final concentration) was added to each well after

97

treatment with extract samples or 6-OHDA. After 4 h incubation, the MTT solution

98

was threw out and added 200 µL DMSO to solubilize the formazan in each well. The

99

absorbance was estimated with ELISA universal microplate reader (EXL 800,

100

BIO-TEK Instrument Inc., Winooski, VT, USA) at 570 nm.

101

ROS Assessment. The level of cellular oxidative stress was established by the

102

estimation of ROS. After drugs treatment, the collected cells were washed and 7



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suspended in 1 mL PBS which containing 15 µM dichlorodihydrofluorescein

104

diacetate (DCFH-DA), and incubated at 37 °C for 30 min. The cells were removed the

105

excess DCFH-DA with PBS. The cell pellets were suspended with 200 µL PBS and

106

the level of ROS was analyzed by flow cytometry (BD FACSCantoTM, BD

107

Biosciences, San Jose, CA, USA).

108

Apoptosis

Analysis

via

Annexin

V-FITC/Propidium

Iodide

(PI)

109

Double-Labeled by Flow Cytometry. The level of cell apoptosis was analyzed by

110

the Annexin V-FITC Apoptosis Detection Kit (Becton Dickinson). SH-SY5Y cells

111

were treated with 6-OHDA in the presence or absence of DMA and DFC for 12 h.

112

After incubation, the cells were washed with PBS, and then suspended in 1X binding

113

buffer. The density of cell was adapted to 1 × 106 cells/mL. The cell suspension were

114

mixed with Annexin V-FITC in 1.5 mL centrifuge tube, incubated in the dark for 30

115

min at 37 °C, and then washed with 1× binding buffer. PI (5 µL) was added to the

116

mixtures, which were then analyzed using a flow cytometry (BD FACSCantoTM).

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Cells negative for PI and positive for Annexin V were established as early apoptotic

118

cells; cells positive for PI and Annexin V were established as late apoptotic cells.

119

Western Immunoblotting. Cells were lysed and the cell lysates were centrifuged

120

(10,000 × g for 10 min) and the supernatant were assembled as the cell extract. The

121

concentrations of protein in the cell extracts were established by Bio-Rad protein 8


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122

assay

kit.

The

123

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a

124

polyvinylidene fluoride (PVDF) membrane. The 50 g/L nonfat dry milk solution used

125

to block the PVDF membranes for 1 h and then the PVDF membranes were incubated

126

with primary antibodies for 2 h. The membranes were washed with PBST and shaken

127

in a solution of horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary

128

antibody.

129

chemiluminescence reagent (Millipore, Billerica, MA, USA).

Protein

cell

extracts

expression

were

resolved

levels

were

by

10%

recognized

sodium

by

an

dodecyl

enhanced

130

Microarray Analysis. Total RNA was amplified using an Agilent Low Input

131

Quick-Amp Labeling kit (Agilent Technologies, Santa Clara, CA, USA) and labeled

132

with Cy3 (CyDye, Agilent) during the transcription process. Cy3-labeled cRNA was

133

fragmented to a general size of approximately 50–100 nucleotides by incubation with

134

fragmentation buffer for 30 min at 60 °C. The correspondingly fragmented labeled

135

cRNA was then combined and hybridized to an Agilent SurePrint G3 Human Gene

136

Exp V3 Array for 17 h at 65 °C. The microarrays were washed and blow-dried with a

137

nitrogen gun, and then examined with an Agilent microarray scanner at 535 nm to

138

detect Cy3. The images of scanner were assayed by Feature extraction10.5.1.1

139

software (Agilent), an image analysis and normalization software used to quantify

140

signal and background intensities for each feature. 9



141

Statistical Analysis. For SPSS statistical analysis (Chicago, IL, USA), all data

142

were analyzed using one-way analysis of variance followed by Dunnett’s test or the

143

Mann-Whitney test. Significant differences were exhibited as P < 0.05.

10


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RESULTS

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Identification of DMA and DFC from RMR. We purified DFC and DMA

146

from R50E by preparative HPLC and characterized them using nuclear magnetic

147

resonance (NMR) and mass spectrometry. The structures of DFC and DMA are

148

presented in Figure 1.

149

DMA and DFC Protect SH-SY5Y Cells against 6-OHDA-Induced Cell

150

Death. SH-SY5Y cells were treated with different concentrations of DMA or DFC (0,

151

5, and 10 µM) in the presence or absence or of 6-OHDA (100 µM). These results

152

indicated that DMA (5 and 10 µM) significantly increased cell survival rate to 61 and

153

91% (Figure 2) and that DFC (5 and 10 µM) significantly increased cell survival rate

154

to 80 and 86% (Figure 2), suggesting that DMA and DFC decreased cytotoxicity in

155

6-OHDA-induced SH-SY5Y cells.

156

DMA and DFC Reduce the Formation of 6-OHDA-Induced ROS in

157

SH-SY5Y Cells. Accumulation of intracellular and extracellular ROS is reflected a

158

key mediator of cytotoxicity and a premier trigger of apoptotic signaling ensuing

159

6-OHDA treatment. To establish whether the protective effects of DMA and DFC

160

were associated with inhibition of 6-OHDA-induced ROS formation in SH-SY5Y

161

cells. DCFH-DA, an oxidation-sensitive fluorescent dye, was used to determine the

162

intracellular ROS by flow cytometry. DFC or DMA (10 µM) significantly decreased 11



163

the mean fluorescence intensity in 6-OHDA-induced SH-SY5Y cells compared with

164

6-OHDA alone group (Figure 3).

165

DMA and DFC Diminish 6-OHDA-Induced SH-SY5Y Cell Apoptosis. To

166

establish whether treatment with DMA and DFC inhibited 6-OHDA-induced

167

apoptosis in neurons, the number of fluorescent apoptotic cells was quantified by

168

annexin V-FITC/PI staining and assessed by flow cytometry. The percentage of

169

apoptotic cells was significantly raised after a 12-h incubation with 6-OHDA at 100

170

µM (Figure 4). DMA and DFC treatment significantly reduced the percentage of

171

apoptotic cells in 6-OHDA-induced SH-SY5Y cells compared with 6-OHDA alone

172

group.

173

Reversal of Akt Pathway- and Heme Oxygenase-1 (HO-1)-Mediated

174

SH-SY5Y Cells Damage through the Protective Effects of DMA and DFC.

175

Neuronal damage from 6-OHDA primarily occurs through the Akt pathway and via a

176

reduce in detoxifying enzymes such as HO-1,24,25 we assessed the protein levels of

177

Akt and HO-1 in SH-SY5Y cells exposed to 6-OHDA and in the presence or absence

178

of DMA or DFC. The results of protein immunoblot are shown in Figure 5. 6-OHDA

179

reduced the ratio of phosphorylated Akt (p-Akt)/Akt and also reduced the levels of

180

HO-1 in SH-SY5Y cells. Both DMA and DFC at 10 µM restored the levels of HO-1

181

and p-Akt/Akt ratio in 6-OHDA-treated SH-SY5Y cells. In addition, DMA and DFC 12


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raised the p-Akt/Akt ratio compared with non-treated group in SH-SY5Y cells (Figure

183

5A). Data in Figure 5B showed that there were no significant differences in HO-1

184

levels between DMA alone group and non-treated group.

185

Reversal of Mitogen-Activated Protein Kinase (MAPK)-Mediated

186

SH-SY5Y Cells Damage through the Protective Effects of DMA and DFC.

187

MAPKs were containing c-Jun N-terminal kinase (JNK), p38, and extracellular

188

signal-regulated kinases (ERK) have been studied to be associated with neuronal

189

apoptosis and cell death.26,27 We therefore assessed the levels of these proteins in

190

SH-SY5Y cells exposed to 6-OHDA and in the presence or absence of DMA or DFC.

191

The results of protein immunoblot are shown in Figure 6. 6-OHDA raised the ratios of

192

p-JNK/JNK and p-p38/p38 in SH-SY5Y cells, whereas both DMA and DFC at 10 µM

193

restored these ratios. In addition, both DMA and DFC at 10 µM raised the ratio of

194

p-ERK/ERK in SH-SY5Y cells.

195

Microarray Analysis Identifies Distinct Changes in Gene Expression

196

Following 6-OHDA and DMA or DFC Treatment. To investigate the molecular

197

differences consequent to 6-OHDA, DMA, or DFC treatment, we performed

198

microarray analysis to compare the global gene expression profiles of SH-SY5Y cells

199

following treatment to 6-OHDA and DMA or DFC. Gene expression analysis was

200

achieved using one-color hybridization to Agilent SurePrint G3 Human Gene Exp V3 13



201

microarrays. The difference between the normalized log ratio values for each gene on

202

the array was estimated for the various treatment conditions in SH-SY5Y cells and is

203

displayed as a comparison between untreated and 6-OHDA groups, 6-OHDA and

204

6-OHDA+DMA groups, and 6-OHDA and 6-OHDA+DFC groups.

205

In the 6-OHDA treatment group, 2.6-fold up-regulated glutamate receptor,

206

ionotropic, N-methyl D-aspartate (NMDA) 2C (grin2c) gene expression (Figure 7A)

207

and 2.2 fold down-regulated stanniocalcin 2 (stc2) gene expression (Figure 7B) were

208

observed. In contrast, following co-treatment with DMA, grin2c was down-regulated

209

2.5 fold (Figure 7A) and stc2 was up-regulated 2.1 fold (Figure 7B) in

210

6-OHDA-induced SH-SY5Y cells. In turn, DFC might exert its protective function

211

through Fc gamma R-mediated phagocytosis regulation, as the expression of the

212

associated fcgr2a gene, which demonstrated 40.4 fold up-regulation in the 6-OHDA

213

treatment group, was down-regulated 28.3 fold in 6-OHDA-treated SH-SY5Y cells

214

co-treated with DFC (Figure 8).

14


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215

DISCUSSION

216

In this study, we showed that DMA and DFC from M. purpureus-fermented rice

217

reduced neurotoxicity in 6-OHDA-induced SH-SY5Y cells. These results indicate that

218

the neuroprotective effect might be mediated by the reduction of intracellular ROS

219

formation and the activation of HO-1, Akt, and ERK pathways. Furthermore, DMA

220

and DFC inhibited the phosphorylation of the p38 and JNK pathway in SH-SY5Y

221

cells. In addition, DMA and DFC might regulate the grin2c, stc2, and fcgr2a genes,

222

respectively.

223

RMR has been reported that containing several bioactive components associating

224

with chemoprevention. For example, extracts of rice which fermented by M.

225

purpureus NTU 568 fermented repressed amyloid β (Aβ)-induced neurocytotoxicity

226

via combined anti-inflammatory and antioxidant mechanisms.18 As R50E may

227

prohibit neurodegeneration through anti-oxidative and anti-inflammatory mechanisms,

228

it might thus have potential therapeutic value in PD.22

229

In the present study, we investigated the neuroprotective effects of the

230

R50E-extracted antioxidants DMA and DFC using human neuroblastoma-derived

231

SH-SY5Y cells. DMA has been found anti-oxidant effects in primary hepatocytes, and

232

ensured against hepatotoxicity via inhibiting oxidative stress.20 DMA also has been

233

reported to be efficient in diminishing in

a mouse model of carbon

15



234

tetrachloride-treated liver injury.28 Moreover, DMA inhibited the cell invasion and the

235

activation of H2O2-mediated MAPKs pathways in SW620 human colon cancer cells.29

236

In comparison, DFC was found to directly remove DPPH free radicals at lesser doses

237

(IC50 = 15.37 µg/mL) and exhibited protective effects against the cell death and

238

cytotoxicity in citrinin-induced HEK-293 human embryonic kidney cells.30

239

Furthermore, DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS

240

formation and apoptosis.21 Together, such evidence suggested that the secondary

241

metabolites of M. purpureus-fermented rice such as DFC and DMA exhibit

242

distinguished protective effects versus chemical and oxidative stresses.

243

The generation of ROS has been presented to being an important role in

244

neurodegenerative diseases such as PD.31 6-OHDA, one of the most commonly

245

applied catecholaminergic neurotoxins, is known to generate ROS32 via intracellular

246

hydrogen peroxide production,2 leading to cell death. Moreover, the system of

247

nitrogen oxide (NOX) was established as the most significant intracellular source of

248

ROS other than mitochondria.33 Notably, our research group has previously

249

demonstrated that DMA and DFC treatment reduced the 6-OHDA-induced formation

250

of extracellular and intercellular ROS and decreased NADPH oxidase-2 expression in

251

differentiated PC-12 cells.21

252

The SH-SY5Y human neuroblastoma cell line possesses many properties of 16


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substantia nigra nerve cells and is widely applied to research the death of

254

dopamine-producing nerve cells.34 In the present study, we utilized SH-SY5Y cells

255

induced with 6-OHDA as a model system to examine the neuroprotective capacity of

256

DMA and DFC and investigate the role of inhibition of the ROS-generating system in

257

their neuroprotection. We established that expose to 6-OHDA decreased SH-SY5Y

258

cell survival rate and increased the formation of intracellular ROS in SH-SY5Y cell

259

culture, inducing a combination of both necrotic and apoptotic cell death.4 Similarly,

260

this toxin was found to induce morphological changes in rat PC-12 cells that are

261

typical of apoptosis containing membrane blebbing, DNA fragmentation, and cell

262

shrinkage.35 In these cells, we demonstrated that DMA and DFC could reverse that

263

6-OHDA induced DNA fragmentation, activate caspase-3, down-regulate Bcl-2, and

264

up-regulate Bax.21 In the current study, we also found that DMA and DFC could

265

reduce and partially reverse the early apoptotic events of SH-SY5Y cells, suggesting

266

DMA and DFC could interrupt the 6-OHDA-induced apoptosis and oxidative stress in

267

these cells.

268

Akt is a critical role in essential cellular functions including cell survival and

269

proliferation via phosphorylating a type of enzymes such as detoxifying,

270

pro-apoptotic regulators, transcription factors, and antioxidant proteins.36 Certain

271

studies found that Akt plays an principal therapeutic role for curing stroke, 17



272

neurodegenerative diseases, and diabetes.36,37 In the present study, we established that

273

SH-SY5Y cells exposed to 6-OHDA for 9 h reduced the phosphorylation of Akt,

274

whereas DMA and DFC repaired Akt phosphorylation. Several cytoprotective

275

enzymes regulated by the Akt cascade such as HO-1 has been shown to be a critical

276

target in neuroprotective functions.38 Accelerating attestation established that a

277

pharmacological inducer, HO-1, expressing may maximize the internal antioxidant

278

potential of cells.39 Our data indicated that 6-OHDA reduced the levels of HO-1 in

279

SH-SY5Y cells and that DMA and DFC were also able to reverse this decrease.

280

Therefore, we propose that DMA and DFC induced Akt phosphorylation and further

281

activated the expression of HO-1 to neuralize the neurotoxicity and reduced the

282

neuronal injury in 6-OHDA-induced SH-SY5Y cells.

283

MAPK pathway plays critical roles in promoting neuronal cell survival and

284

suppressing apoptosis via the inhibition and phosphorylation of several downstream

285

substrates.40 A succession of studies have reported that MAPK signaling pathways

286

represent anti-apoptotic and survival factors in multiple examples including the

287

resistance against 6-OHDA-induced neurotoxicity.41 In the present study, we found

288

increased phosphorylation of JNK and p-38 after exposed with 6-OHDA, whereas

289

treated with DMA and DFC restored the phosphorylation of MAPK proteins in

290

6-OHDA-induced neurons. These results indicate that maintained MAPK activity 18


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291

might be adverse for SH-SY5Y cells survival and that the MAPKs pathways play a

292

important role in the neuroprotective effects of DMA and DFC in SH-SY5Y cells.

293

grin2c encodes a subunit of the NMDA, ion type glutamate (ionotropic glutamate)

294

receptor, is found in the central nervous system (CNS) and which regulates the flow

295

of primarily calcium, potassium, and sodium ions in and out of the cell. Accordingly,

296

an abnormal increase of grin2c gene expression leads to the death of nerve cells.42 In

297

this study, we found that DMA restored the elevated grin2c gene expression found in

298

6-OHDA-induced SH-SY5Y cells. stc2 encodes a secreted homodimer glycoprotein

299

and its expression has been associated with nerve cell growth and differentiation.43

300

Our data indicated that DMA also increased the reduced stc2 gene expression

301

observed in 6-OHDA-induced SH-SY5Y cells. These results indicate that DMA might

302

function through the glutamate signaling pathway and cell surface receptor linked

303

signal transduction regulation to protect against the 6-OHDA-induced apoptosis in

304

SH-SY5Y cells, as the grin2c and stc2 genes associated with these two pathways

305

(Figure 7). In turn, fcgr2a encodes an immunoglobulin Fc receptor on the cell

306

membrane, which has been shown to be associated with diseases of the central

307

nervous system.44 In this study, we found that DFC restored the elevated fcgr2a gene

308

expression seen in 6-OHDA-treated SH-SY5Y cells (Figure 8). These results indicate

309

that DFC might exert its protective function through Fc gamma R-mediated 19



310

phagocytosis regulation to protect against the 6-OHDA-induced apoptosis in

311

SH-SY5Y cells, as the fcgr2a gene associated pathway. Therefore, we suggest that

312

DMA regulation of grin2c and stc2 and DFC regulation of fcgr2a gene expression

313

served to reduce the neuronal damage in 6-OHDA-induced SH-SY5Y cells.

314

In conclusion, our findings indicated that DMA and DFC were able to interrupt

315

6-OHDA-induced apoptosis and oxidative stress in SH-SY5Y cells. Moreover, DMA

316

and DFC activated Akt phosphorylation, induced HO-1 expression, and reduced the

317

phosphorylation of JNK and p-38 to counteract the neurotoxicity in 6-OHDA-induced

318

SH-SY5Y cells. Furthermore, DMA and DFC regulated grin2c, stc2, and fcgr2a gene

319

expression to ameliorate the neuronal damage (Figure 9). These results supplied

320

critical implications for the development of mechanism-based therapies for PD. In

321

addition, we anticipate that the DMA and DFC derived from M. purpureus

322

fermentation might supply as a functional food for the prevention or treatment of PD.

20


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ACKNOWLEDGEMENTS This work was supported by the research grant (MOST 104-2320-B-002-056) from Ministry of Science and Technology, Taiwan.

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FIGURE CAPTIONS

474

Figure 1. Structures of dimerumic acid (DMA) (A) and deferricoprogen (DFC) (B).

475 476

Figure 2. SH-SY5Y cell survival following DMA or DFC co-treatment with 6-OHDA.

477

SH-SY5Y cells were exposed to 6-OHDA (100 µM) in the presence of DMA or DFC

478

for 24 h, and cell viability was measured using the MTT assay. Data represent the

479

means ± SD of survival as measured by cell viability of three independent

480

experiments. *P < 0.05, compared with the control group, #P < 0.05,

481

compared with the 6-OHDA group only.

##

P < 0.01,

482 483

Figure 3. ROS level of SH-SY5Y cells by DMA or DFC co-treated with 6-OHDA for

484

4 h. SH-SY5Y cells were exposed to 6-OHDA (100 µM) in the absence or presence of

485

10 µM DMA or DFC for 4 h. Intracellular ROS were stained with

486

dichlorodihydrofluorescein diacetate (DCFH-DA) for 30 min and analyzed by flow

487

cytometry. Data represent the means ± SD of survival as measured by cell viability of

488

three independent experiments. *P < 0.05, compared with the control group, #p < 0.05,

489

compared with the 6-OHDA group.

490

30


Page 31 of 43


491

Figure 4. DMA or DFC decreased apoptotic cells in 6-OHDA-treated differentiated

492

SH-SY5Y cells. SH-SY5Y cells were exposed to 6-OHDA (100 µM) in the absence or

493

presence of 10 µM DMA or DFC for 12 h. Apoptotic cells were stained with Annexin

494

V-FITC/PI stain and analyzed by flow cytometry. Data represent the means ± SD of

495

survival as measured by cell viability of three independent experiments. *P < 0.05,

496

compared with the control group, #P < 0.05, compared with the 6-OHDA group only.

497 498

Figure 5. DFC or DMA increased 6-OHDA-mediated reduction of the p-Akt/Akt

499

ratio (A) and HO-1 expression (B) in SH-SY5Y cells. SH-SY5Y cells were exposed

500

to 6-OHDA (100 µM) in the absence or presence of 10 µM DMA or DFC for 9 and 1

501

h, respectively and the protein extracts were assayed by immunoblot to measure p-Akt,

502

Akt, HO-1, and GAPDH expression. Data represent the means ± SD of survival as

503

measured by cell viability of three independent experiments.

504

with the control group, #P < 0.05, compared with the 6-OHDA group.

**

P < 0.01, compared

505 506

Figure 6. Both DFC and DMA decreased 6-OHDA-mediated enhancement of the

507

MAPK ratio in SH-SY5Y cells. SH-SY5Y cells were exposed to 6-OHDA (100 µM)

508

in the absence or presence of 10 µM DMA or DFC for 6 h. Protein extracts were

509

assessed for p-p38, p38, p-JNK, JNK, p-ERK, and ERK expression by Western blot 31



510

analysis. Data represent the means ± SD of survival as measured by cell viability of

511

three independent experiments.

512

0.05, ## P < 0.01, compared with the 6-OHDA group.

**

P < 0.01, compared with the control group, #P

Dimerumic Acid and Deferricoprogen Activate Ak Mouse Strain

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