Reperfusion-Evoked Oxidative

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Green tea catechin prevents hypoxia/reperfusion-evoked oxidative stressregulated autophagy-activated apoptosis and cell death in microglial cells Chang-Mu Chen, Cheng-Tien Wu, Ting Hua Yang, Ya-An Chang, Meei Ling Sheu, and Shing-Hwa Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01513 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 5, 2016

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Page 1 of 29

Journal of Agricultural and Food Chemistry

1

Green

tea

catechin

prevents

hypoxia/reperfusion-evoked

oxidative

2

stress-regulated autophagy-activated apoptosis and cell death in microglial

3

cells

4 5

Chang-Mu Chen†, Cheng-Tien Wu‡, Ting-Hua Yang#, Ya-An Chang‡, Meei-Ling Sheu§,ǁ,

6

Shing Hwa Liu‡,Δ,ǁ,*

7 8

†

9

National Taiwan University, College of Medicine, Taipei, Taiwan

Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital and

10

‡

Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan

11

#

Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan

12

§

13

University, Taichung, Taiwan

14

Δ

15

University, Taichung, Taiwan

Institute of Biomedical Sciences, College of Life Sciences, National Chung Hsing

Department of Medical Research, China Medical University Hospital, China Medical

16 17

*Address correspondence to:

18

Shing Hwa Liu, PhD, Institute of Toxicology, College of Medicine, National Taiwan

19

University, Taipei, Taiwan. E-mail: [email protected]. Tel.: +886-2-23123456 ext.

20

88605. Fax: +886-2-23410217

21

1

ACS Paragon Plus Environment


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22

Abstract

23

Defective activation and proliferation in microglial cells has been suggested to be associated

24

with the increase of cerebral ischemia/reperfusion injury. We investigated the protection and

25

molecular mechanism of green tea catechin on hypoxia/reperfusion-induced microglial cell

26

injury in vitro. Microglial cells were cultured in hypoxia condition (O2 < 1%) and then

27

re-incubated to the complete normal culture medium (reperfusion). Hypoxia/reperfusion

28

obviously decreased cell viability and induced apoptosis in microglial cells, but not in

29

neuronal cells. Catechin significantly inhibited the hypoxia/reperfusion-induced decreased

30

cell viability and increased reactive oxygen species (ROS) and apoptosis in microglia. The

31

administration of both PI3K/Akt inhibitor LY294002 and mTOR inhibitor rapamycin

32

demonstrated

33

hypoxia/reperfusion-induced microglia apoptosis/death. Catechin upregulated the Akt and

34

mTOR phosphorylation and inhibited the hypoxia/reperfusion-induced autophagy in

35

microglia. These results suggest that hypoxia/reperfusion can evoke autophagy-activated

36

microglia apoptosis/death via an ROS-regulated Akt/mTOR signaling pathway, which can be

37

reversed by catechin.

that

Akt/mTOR-regulated

autophagy

was

involved

38 39

Key Words: Catechin/ Autophagy/ Apoptosis/ Hypoxia reperfusion/ microglia

40 41 42 43 44 45 46 2


in

the

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47


INTRODUCTION

48

According to World Health Organization, ischemic stroke, a cerebrovascular insult,

49

is the second leading cause of death worldwide. It predominantly occurs in mid-age and

50

older adults and may cause paralysis, impaired speech or loss of vision due to interruption

51

of blood flow caused by thrombosis or embolism.1 At present, there are few available

52

therapeutic options for ischemic stroke. Clinically, when thrombosis is removed by surgery

53

or anticoagulants, brain cells will undergo reperfusion by blood flow again and leads to the

54

secondary injury known as ischemia/reperfusion injury. The continual communication

55

between neurons and vasculature, neurons and astrocytes, and neurons and microglia has

56

been suggested to be probably bidirectional for which is responsible for neuronal

57

homeostasis and injury.2 Microglial cells are resident immune cells in central nerve system

58

and named as “brain macrophages”.3 They can be triggered by brain insult and exert

59

phagocytosis or release diverse factors to eliminate invading microorganisms and dead

60

cells.4 Under brain ischemia, the dying cells release HMGB1 and ATP and activate

61

microglia to protect neurons via phagocytic removal of pathogens and dead cell debris as

62

well as secretion of neurotrophic factors.5,6 The defective activation and proliferation in

63

microglia has been suggested to be associated with increase in the ischemic lesion size after

64

cerebral ischemia/reperfusion, suggesting microglia might play a protective role in

65

ischemic stroke.7

66

Oxidative stress is known to play an important role in brain damage after cerebral

67

ischemia/reperfusion.8 The antioxidant system can prevent the neuronal apoptosis and

68

death in response to brain ischemia/reperfusion.9 Oxidative stress also acts to regulate

69

autophagy and its related neuronal death.10 Catechins are the type of plant flavonoids and

70

possess antioxidant activity. Epigallocatechin 3-gallate has been shown to inhibit

71

macrophage NO generation and autophagy inhibition.11 Green tea catechin administration 3



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has been found to improve the learning and memory deficits in a senescence mouse

73

model.12 Moreover, Ashafaq et al. have also shown that catechin can prevent the

74

ischemia/reperfusion-induced cerebral injury in rats via an amelioration of the redox

75

imbalance.13

76

ischemia/reperfusion-induced cerebral injury is still unclear.

However,

the

molecular

mechanism

of

catechin

on

77

In this study, we tried to investigate the protective effect and molecular mechanism

78

of catechin on microglial cell injury during hypoxia/reperfusion condition in vitro. We

79

utilized microglia cell line BV-2 cells cultured in a hypoxia/reperfusion condition to mimic

80

brain ischemia/reperfusion.

81 82

MATERIALS AND METHODS

83

Cell culture and chemicals

84

IMR-32 human neuronal cells were purchased from the Bioresource Collection and

85

Research Center (Hsinchu, Taiwan) and maintained in minimum essential medium

86

supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium

87

pyruvate, 0.1 mM non-essential amino acids and 1% antibiotics (100 IU/mL penicillin, 100

88

µg/mL streptomycin, 2.5 µg/ml Amphotericin B) at 37°C under 5% CO2. In addition, BV-2

89

immortalizing mouse microglial cell line, which was developed in the laboratory of Dr. E.

90

Blasi,14 was kindly provided by Dr. M. L. Sheu (National Chung Hsing University, Taichung,

91

Taiwan) and cultured in the Dulbecco’s Modified Eagle’s Medium contain with 10% FBS and

92

1% antibiotics at 37°C under 5% CO2. Cells grew to appropriate 80% adaptive condition and

93

then sub-cultured every 2-3 days. Chemicals: LY294002 (phosphoinositide 3-kinase

94

(PI3K)/Akt inhibitor, Sigma-Aldrich, St. Louis, MO, USA), rapamycin (mTOR inhibitor,

95

Calbiochem, Bad Soden, Germany), (+)-catechin from green tea [≥98% (HPLC),

96

Sigma-Aldrich]. 4


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97 98


Hypoxia/reperfusion treatment in cells The hypoxia/reperfusion-induced injury in BV-2 cells was performed as previously

99

described.15 Briefly, microglial cells were pre-incubated in deprivation medium (without FBS)

100

for 1 h. Following, microglia cells were transferred to hypoxia chamber (O2 < 1%) for 6 h,

101

and then re-incubated to the complete culture medium and cultured in normal condition for

102

0.5-24 h.

103

Cell viability assay

104

Cells were seeded in the 96 well plates for 24 h. After adaptation, cells were treated

105

with hypoxia/reperfusion procedure for 12 or 24 h. Following, the cells were translated to the

106

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-contained medium at 37°C

107

for 2 h, and then the formazan crystals were completely dissolved by DMSO. The absorbance

108

was detected at 540 nm.

109

Caspase-3 activity assay

110

After hypoxia/reperfusion treatment, cells extracts were collected for caspase 3 activity

111

assay according to the instruction of manufacturer (Promega, Madison, WI, USA). Briefly,

112

100µg of total cell protein in a 100 µl total volume was mixed with 100 µl of equilibrated

113

Caspase-GloTM reagent, and then incubated at less 1 h at room temperature. Afterwards,

114

luminescence was measured using a Luminometer reader system.

115

Western blotting

116

After treating with hypoxia/reperfusion procedure for 0.5-24 h, cells were harvested

117

and lysed by the RIPA buffer. Subsequently, the cell lysates were centrifuged at 10,000×g for

118

20 min at 4oC and proteins were determined by bicinchoninic acid assay. Equal proteins (40

119

µg) were added to 6-15% SDS–polyacrylamide gel electrophoresis and electrophoretically

120

transferred to a polyvinylidene difluoride membrane. Following to block with 5% fat-free

121

milk in Tris-buffered saline/Tween-20 buffer (20 mM Tris, 150 mM NaCl, 0.01% Tween-20, 5



122

pH 7.5) for 1 h. The primary antibodies caspase-3 (1:1000, Cell Signaling),

123

microtubule-associated protein 1 light chain 3 (LC3) (1:1000, Cell Signaling),

124

phosphorylated-mTOR (Serine 2448) (1:1000, Cell Signaling), phosphorylated-Akt (Serine

125

473) (1:1000, Abcam, Cambridge, MA, USA), poly (ADP-ribose) polymerase (PARP),

126

GAPDH, and β-actin (1:1000; Santa Cruz) were incubated for overnight at 4°C. After

127

washing for three times, membranes were reacted with secondary goat anti-rabbit or

128

anti-mouse horseradish peroxidase-conjugated antibodies (Santa Cruz). Finally, the signals

129

were detected by the GelDoc system machine (Bio-Rad, Espoo, Finland). The relative values

130

for each protein were normalized by the β-actin.

131

Apoptosis and necrosis dual staining

132

Cells were seeded in the 6-well plates for 24 h and then treated with

133

hypoxia/reperfusion procedure for 24 h. Subsequently, the cells were harvested and staining.

134

Briefly, the floating and adherent cells were collected and centrifuged with 160 x g for 10

135

min. The condense cells were then re-suspended in the PBS containing with acridine orange

136

(200 ng/ml; Sigma-Aldrich) and ethidium bromide (200 ng/ml; Sigma-Aldrich) for 10 min.

137

Two-parameter fluorescences in samples were analyzed using an EPICS XL/MCL flow

138

cytometer (Beckman Coulter, Brea, CA, USA) with a 488 nm laser detection machine. Live,

139

necrotic or apoptotic cell populations were analyzed using FlowJo 8.8.6 software (Ashland,

140

Oregon, USA).

141

Detection of reactive oxygen species (ROS) by 2'-7'-dichlorodihydrofluorescein diacetate

142

(DCFDA) staining

143

Cells were seeded in the 6-well plates for 24 h and then treated with

144

hypoxia/reperfusion procedure for 0.5-24 h. DCFDA was added to experimental medium and

145

incubated for 30 min at 37°C, and then collected the total cells to centrifuge with 160 x g for

146

5 min. The media were discarded and the cells were re-suspended in the PBS. Finally, the 6


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147

samples, a total of 10000 cells, were analysis by the EPICS XL/MCL flow cytometer

148

(Coulter Cytometry).

149

Statistical analysis

150

Results are presented as mean ± SEM for at least three independent experiments. The

151

significant difference from the respective controls for each experimental test condition was

152

assessed by one-way analysis of variance (ANOVA) and post-hoc test. A value of p < 0.05

153

was considered statistically significant.

154 155

RESULTS

156

Effects of hypoxia/reperfusion on cell viability and apoptosis-related molecules in neuronal

157

cells and microglial cells

158

To mimic brain ischemia/reperfusion, IMR-32 neuronal cells and BV-2 microglial cells

159

were cultured with serum deprivation condition in hypoxia chamber (O2 < 1%) for 6 hours

160

and then recovered with complete growth medium at different time courses. To evaluate the

161

viability of IMR-32 neuronal cells and BV-2 microglial cells after hypoxia/reperfusion

162

treatment, the cell viability was determined by MTT assay. As shown in Fig. 1,

163

hypoxia/reperfusion did not affect the viability of IMR-32 neuronal cells (Fig. 1A), but

164

significantly decreased BV-2 microglial cell viability after 24 h of reperfusion (Fig. 1B).

165

There was no caspase-3 activation (cleavage of caspase-3 protein) (Fig. 1C) and PARP

166

cleavage (Fig. 1E) in IMR-32 cells during hypoxia/reperfusion. The caspase-3 cleavage (Figs.

167

1D) and PARP cleavage (Fig. 1E) and caspase-3 activity (Fig. 1F) were significantly

168

increased

169

hypoxia/reperfusion induces cell apoptosis and death in microglia but not in neuron under

170

this in vitro condition.

171

Effects of catechin on cell viability, ROS, and apoptosis in microglial cells under

after

hypoxia/reperfusion

in

BV-2

cells.

These

7


results

indicate

that


172

hypoxia/reperfusion

173

Catechin (10-250 µM) did not affect the cell viability of BV-2 cells under normal

174

growth medium (Fig. 2A), but significantly reversed the hypoxia/reperfusion-decreased

175

microglia viability (Fig. 2B). Oxidative stress is known to be involved in the reperfusion

176

injury after cerebral ischemia.8,9 As shown in Fig. 3A, the production of ROS was

177

significantly elevated in BV-2 microglial cells during hypoxia and reperfusion periods.

178

Catechin is a type of flavonoids and antioxidant. Administration of catechin (100 µM)

179

significantly decreased hypoxia/reperfusion-increased ROS production (Fig. 3B). Moreover,

180

hypoxia/reperfusion induced the activation of caspase-3 (cleavage of caspase-3) and

181

apoptosis (acridine orange/ethidium bromide staining) in BV-2 cells, which could also be

182

significantly reversed by catechin (Fig. 4).

183

The role of Akt/mTOR signaling-related autophagy and apoptosis in microglia injury during

184

hypoxia/reperfusion

185

The PI3K/Akt/mTOR signaling pathway is known to play an important regulatory role

186

in autophagy in neuronal cells.16 The role of PI3K/Akt/mTOR in microglial cell injury during

187

hypoxia/reperfusion is unclear. As shown in Fig. 5A, the LC3-II protein level was increased

188

during hypoxia period and early reperfusion period (0.5 and 1 h), and then the level was

189

markedly attenuated in period after 3 and 6 h of reperfusion; however, the level was markedly

190

and significantly increased in period after 24 h of reperfusion. In addition, the

191

phosphorylation of mTOR and Akt was significantly increased during hypoxia period, but

192

significantly decreased in period after 0.5 h of reperfusion, and then gradually increased in

193

periods after 1-6 h of reperfusion (Fig. 5A). Moreover, administration of PI3K/Akt inhibitor

194

LY294002 significantly suppressed the phosphorylation of mTOR and increased

195

LC3-II/LC3-I protein ratio in BV-2 microglial cells during hypoxia and reperfusion periods

196

(Fig. 5B-a). Administration of mTOR inhibitor rapamycin also significantly suppressed the 8


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phosphorylation of mTOR and increased LC3-II/LC3-I protein ratio in BV-2 microglial cells

198

during hypoxia and reperfusion periods (Fig. 5B-b). LY294002 could also significantly

199

enhance hypoxia/reperfusion-increased apoptosis (Fig. 6B-a) and decreased cell viability (Fig.

200

6B-b)

201

hypoxia/reperfusion-increased apoptosis (Fig. 6B-a) and decreased cell viability (Fig. 6B-b)

202

in BV-2 microglial cells. These results indicate that the activation of autophagy by increasing

203

LC3-II was inversely correlated with the expression of mTOR and Akt/mTOR-regulated

204

autophagy is involved in the hypoxia/reperfusion-induced microglia cell apoptosis and death.

in

BV-2

microglial

cells.

Rapamycin

could

also

significantly

enhance

205

On the other hand, administration of catechin (100 µM) effectively activated the

206

phosphorylation of Akt and mTOR (Fig. 6A), decreased LC3-II/LC3-I protein ratio (Fig. 6A),

207

reduced apoptosis (Fig. 6B-a), and increased cell viability (Fig. 6B-b) in microglial cells

208

under hypoxia/reperfusion condition. These results suggest that catechin protects microglia

209

against hypoxia/reperfusion-induced cell injury via an inhibition of Akt/mTOR-regulated

210

autophagy-activated apoptosis.

211 212

DISCUSSION

213

During ischemic stroke condition, microglia plays a role of double-edged sword that

214

phagocytoses tissue debris and secrete both pro- and anti-inflammatory mediators, which may

215

exacerbate ischemic damage or induce repair depending on different molecular signaling

216

pathways.17,18 The activated microglia can play a neuroprotective role and trigger

217

regeneration via growth/neurotrophic factors secretion and immune response modulation.1819

218

Damage of microglial activation and proliferation has been demonstrated to increase the

219

infarction size and apoptotic neuronal cells in response to ischemic injury.7 These findings

220

suggested that microglial cells play a pivotal role during cerebral ischemia/reperfusion. The

221

hypoxia/reperfusion-induced neuronal cell apoptosis in vitro has been reported; the neuronal 9



222

cells were cultured in hypoxia condition (< 1% O2) with serum and glucose deprivation for

223

1.5-2 h and re-oxygenation for 24 h.15,19 In the present study, the neuronal and microglial

224

cells were cultured in hypoxia condition (< 1% O2) with only serum deprivation for 6 h and

225

re-oxygenation for 24 h. We found that cell viability inhibition, apoptosis, and autophagy

226

occurred in microglial cells during mimic cerebral ischemia/reperfusion in vitro. However, in

227

this hypoxia/reperfusion condition, the cell viability inhibition, apoptosis, and autophagy did

228

not occur in neuronal cells. Increase in autophagy by the inhibition of PI3K/Akt or mTOR

229

signaling enhanced microglia apoptosis, indicating that autophagy plays a regulatory role in

230

microglia apoptosis during hypoxia coupled to reperfusion. We further found that polyphenol

231

catechin

232

autophagy-activated apoptosis via the activation of Akt/mTOR signaling.

effectively

protected

microglia

against

hypoxia/reperfusion-induced

233

Accumulating evidence indicates that autophagy possesses multitiered immunological

234

functions, which regulate immunity, inflammation, and infection.20,21 Autophagy also plays a

235

role in the regulation of cell death.20 The impairment of autophagy has been suggested to

236

contribute to abnormal protein aggregation and organelle damages in the neurons after

237

ischemia.22 Autophagy has been demonstrated to be an essential cytoprotective response to

238

pathological stresses during several diseases including ischemia.23 On the contrary,

239

autophagy can result in neuronal damage or death, known as “autophagic cell death” during

240

cerebral ischemia.24,25 Yang et al. indicated that autophagy induction triggered microglial cell

241

death under hypoxia condition in vitro.26 Yang et al. further found that cerebral ischemia

242

induced microglia autophagy, which contributed to the ischemic neuronal injury in a

243

permanent middle cerebral artery occlusion mouse model.27 In addition, the present study

244

also found that hypoxia coupled to reperfusion effectively evoked autophagy-related

245

microglial cell apoptosis and death. These findings suggest that autophagy contributes to

246

maintenance of metabolic homeostasis in the cells; but in some situations, autophagy is 10


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247

associated with apoptosis and cell death such as microglia under exposure of hypoxia coupled

248

to reperfusion.

249

Autophagy can be induced in response to multiple cellular stress, including nutrient or

250

growth factor deprivation, hypoxia, ROS, DNA damage, protein aggregates, or intracellular

251

pathogens.28 Under normal nutrient condition, mTOR complex 1 (containing mTOR, Raptor,

252

mLST8/GßL, Deptor, and PRAS40) contains kinase activity and interacts with a complex

253

(containing ULK1, Atg13, FIP200, and Atg101) and subsequently inhibits autophagy.29

254

Moreover, upon growth factor action, the PI3K/Akt signaling pathway is activated and

255

subsequently triggers the activation of mTOR.28 In conditions such as starvation, amino acid

256

deprivation, or growth factor withdrawal, the activation of mTOR signaling is inhibited,

257

leading to autophagy induction.30 In the present study, we found that the phosphorylation of

258

mTOR and Akt was significantly decreased in period after 0.5 h of reperfusion and then

259

gradually increased in periods after 1-6 h of reperfusion, which was consistent with the

260

time-course changes of LC3-II level associated with autophagy during reperfusion. Moreover,

261

activation of autophagy by the inhibition of both PI3K/Akt and mTOR signals by LY294002

262

and rapamycin, respectively, markedly enhanced microglia apoptosis and cell death under

263

hypoxia/reperfusion. These results indicate that hypoxia/reperfusion induces microglia

264

apoptosis and cell death via a PI3K/Akt-mTOR-regulated autophagy pathway. In contrast,

265

green tea catechin effectively upregulated the Akt and mTOR activation and decreased the

266

microglia autophagy and apoptosis under hypoxia/reperfusion. However, we also found that

267

both Akt phosphorylation and LC3-II expression were relatively high at 24 h of reperfusion

268

(Figure 5A). It seems to suggest that PI3/Akt or even mTOR pathway was indirectly involved

269

in ROS-mediated microglial cell autophagy at 24 h of reperfusion. The detailed mechanism

270

needs to be clarified in the future.

271

The phytochemicals of phenolic derivatives, iridoid glycosides, terpenoids, alkaloids, 11



272

and steroidal saponins have been found to possess neurotrophin-mediated neuroprotection on

273

neurodegenerative disease.31 The unregulated activation of microglia, like as exposure to

274

environmental toxins, has been found to produce toxic factors and propagate neuronal injury;

275

anti-inflammatory herbal medicine and its constituents have been suggested to be potent

276

neuroprotective agents.32 Carey et al. have shown that blueberry and its active components

277

can reduce the inflammatory responses in lipopolysaccharide-stimulated BV2 microglia.33

278

However, defective activation and proliferation in microglial cells has been suggested to be

279

associated with the increase of cerebral ischemia/reperfusion injury.5-7 In the present study,

280

we found that green tea catechin can prevent hypoxia/reperfusion-evoked microglia apoptosis

281

and death, which may protect neuronal cells from injury induced by hypoxia/reperfusion.

282

The physiological levels of flavonoids in plasma have been shown to be micromolar

283

range,34,35 but in the gut it could be much higher (millimolar range).36 Sehm et al. found that

284

EGCG (0.1-10 µM) and (+)-catechin (30-100 µM) possessed the potential to influence the

285

mRNA expression of TNFα in conA-stimulated white blood cells.37 Catechin hydrate (150

286

µg/ml and 300 µg/ml, about 517 and 1034 µM, respectively) has also been shown to suppress

287

the cell proliferation in MCF-7 human breast cancer cells.38 EGCG at concentrations of 10-40

288

µg/ml (about 22-87 µM) has also been found to reduce the invasive potential of A375 and

289

Hs294t human melanoma cells.39 Chen et al. showed that tea catechins (50 and 100 µM)

290

protected against lead-induced injury in PC12 cells.40 In the present study, we found that

291

catechin (10-250 µM) did not affect the BV-2 cell viability under normal growth medium, but

292

significantly reversed the decreased cell viability and induced apoptosis in BV-2 cells under

293

hypoxia/reperfusion exposure. Therefore, the concentrations of catechin used in the present in

294

vitro study are reasonable.

295

The previous studies suggested that dietary polyphenols could penetrate the blood–

296

brain barrier (BBB) and exert the neuroprotective action.41,42 Ferruzzi et al. have shown that 12


Page 12 of 29

Page 13 of 29


297

catechin and epicatechin can be detectable in brain tissues of rats administered a grape seed

298

polyphenolic extract for 10 days.43 An in vivo study of brain pharmacokinetics and BBB

299

penetration has also demonstrated that (+)-catechin and (-)-epicatechin can pass through the

300

BBB.44 These findings support the therapeutic availability of (+)-catechin on cerebral

301

ischemia/reperfusion injury.

302

In conclusion, oxidative stress can act to regulate autophagy and its related neuronal

303

death.10 In the present study, we found that hypoxia/reperfusion induced the ROS production

304

in microglia, which could be reversed by catechin. Catechin also significantly increased the

305

Akt and mTOR phosphorylation and reversed the induction of autophagy and apoptosis in

306

microglia during hypoxia/reperfusion. The proposed molecular mechanism underlying the

307

action of catechin is illustrated in Figure 7. Taken together, these findings suggest that

308

hypoxia/reperfusion, which condition is unharmful to neuronal cells, evokes oxidative

309

stress-regulated autophagy-activated apoptosis in microglia. Catechin effectively inhibits

310

ROS production and autophagy-activated apoptosis through the upregulation of the

311

PI3K/Akt/mTOR axis that can further prevent microglia injury during hypoxia/reperfusion

312

condition.

313 314

AUTHOR INFORMATION

315

Corresponding Author

316

*E-mail: [email protected] (SHL)

317

Author Contributions

318

ǁ

319

Funding

320

This study was supported by a grant from the National Science Council of Taiwan (NSC

321

100-2314-B-002-084).

M.L.S., S.H.L. contributed equally

13



322

Notes

323

The authors declare no competing financial interest.

Page 14 of 29

324 325

Abbreviations Used:

326

DCFDA: 2'-7'-dichlorodihydrofluorescein diacetate; FBS, fetal bovine serum; LC3,

327

microtubule-associated

328

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose)

329

polymerase; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species

protein

1

light

chain

3;

MTT,

330 331

REFERENCES

332

1. Moskowitz, M. A.; Lo, E. H.; Iadecola, C. The science of stroke: mechanisms in search of

333

treatments. Neuron 2010, 67, 181-198.

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2. Turner, R. C.; Dodson, S. C.; Rosen, C. L.; Huber, J. D. The science of cerebral ischemia

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and the quest for neuroprotection: navigating past failure to future success. J. Neurosurg.

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2013, 118, 1072-1085.

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3. Guillemin, G. J.; Brew, B. J. Microglia, macrophages, perivascular macrophages, and

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4. Nakamura Y. Regulating factors for microglial activation. Biol. Pharm. Bull. 2002, 25,

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6. Iadecola, C.; Anrather, J. The immunology of stroke: from mechanisms to translation. Nat.

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Figure Legends

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Fig. 1. Effects of hypoxia/reperfusion on cell viability, caspase-3 cleavage, and PARP

474

cleavage in neuronal cells and microglial cells. IMR-32 neuronal cells (A, C, and E) and

475

BV2 microglial cells (B, D, E, and F) were incubated in hypoxia chamber (O2 < 1%) for 6 h

476

(hypoxia, H) and then recovered with the complete growth medium for 0.5-24 h (reperfusion).

477

Cell viability was assessed by MTT assay (A and B). The cleavage of caspase-3 (C and D)

478

and PARP proteins (E) was assessed by Western blotting. The caspase-3 activity was also

479

determined in microglia BV-2 cells (F). Data are presented as mean ± SEM (n=4). *:P < 0.05

480

vs control.

481 482

Fig. 2. Effect of catechin on microglial cell viability during hypoxia/reperfusion.

483

BV2 cells were cultured in normal growth medium (A) or treated with hypoxia/reperfusion

484

procedure (B) for 24 h in the presence or absence of catechin (10-500 µM). Cell viability was

485

determined by MTT assay. Data are presented as mean ± SEM (n=4). *P < 0.05 vs control.

486

#P < 0.05 vs hypoxia/reperfusion group.

487 488

Fig. 3. Effect of catechin on oxidative stress in microglia during hypoxia/reperfusion.

489

BV2 cells were treated with hypoxia/reperfusion procedure for 0.5-6 h in the presence or

490

absence of catechin (100 µM). The ROS production was determined by DCFDA fluorescence.

491

Data are presented as mean ± SEM (n=4). *P < 0.05 vs control. #P < 0.05 vs

492

hypoxia/reperfusion group.

493 494

Fig. 4. Effect of catechin on hypoxia/reperfusion-induced apoptosis in microglia. BV2

495

microglial cells were incubated in hypoxia chamber (O2 < 1%) for 6 h (hypoxia, H) and then

496

recovered with the complete growth medium for 24 h. The cleavage of caspase-3 protein was 20


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497

assessed by Western blotting (A). Apoptotic cells were analyzed by flow cytometry (B). Data

498

are presented as mean ± SEM (n=4). *:P < 0.05 vs control. #P < 0.05 vs hypoxia/reperfusion

499

group.

500 501

Fig. 5. Effects of hypoxia/reperfusion on phosphorylation of Akt and mTOR and

502

autophagy in microglia. BV2 cells were incubated in hypoxia chamber (O2 < 1%) for 6

503

hours (hypoxia, H) and then recovered with the complete growth medium for 0.5-24 h (A) or

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3 and 6 h (B) in the presence or absence of PI3K/Akt inhibitor LY294002 (LY, 10 µM; B-a)

505

or mTOR inhibitor rapamycin (Rapa, 0.5 µM; B-b). The protein expressions of LC3-II/LC3-I,

506

phospho-mTOR, and phospho-AKT were determined by Western blotting. Data are presented

507

as mean ± SEM (n=4). #: P < 0.05 vs hypoxia/reperfusion group.

508 509

Fig. 6. Effects of catechin on phosphorylation of Akt and mTOR, autophagy, apoptosis,

510

and cell viability in microglia during hypoxia/reperfusion. BV2 cells were treated with

511

hypoxia/reperfusion procedure for 3, 6, and 24 h in the presence or absence of catechin (100

512

µM; A and B) or LY294002 (LY, 10 µM; B) or rapamycin (Rapa, 0.5 µM; B). The protein

513

expressions of phospho-Akt, phospho-mTOR, and LC3-II/LC3-I were determined by Western

514

blotting (A). Cell apoptosis was determined by flow cytometry (B-a). Cell viability was

515

determined by MTT assay (B-b). Data are presented as mean ± SEM (n=4). *P < 0.05 vs

516

control. #P < 0.05 vs hypoxia/reperfusion group.

517 518

Fig. 7. Scheme representing the proposed molecular mechanism underlying the action of

519

catechin in inhibiting the oxidative stress and autophagy-activated apoptosis in microglial

520

cells during hypoxia/reperfusion through the upregulation of the PI3K/Akt/mTOR axis.

521 21



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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Reperfusion-Evoked Oxidative

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