The effects of astaxanthin and docosahexaenoic acid-acylated

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Functional Structure/Activity Relationships

The effects of astaxanthin and docosahexaenoic acid-acylated astaxanthin on Alzheimer's disease in APP/PS1 double transgenic mice Hongxia Che, Qian Li, Tiantian Zhang, Dandan Wang, Lu Yang, Jie Xu, Teruyoshi Yanagita, Changhu Xue, Yaoguang Chang, and YuMing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00988 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

The effects of astaxanthin and docosahexaenoic acid-acylated astaxanthin on Alzheimer's disease

2

in APP/PS1 double transgenic mice

3

Hongxia Che, † Qian Li,† Tiantian Zhang,† Dandan Wang,† Lu Yang,† Jie Xu,† Teruyoshi Yanagita,‡ Changhu Xue,†,

4

⊥

⊥,*

Yaoguang Chang,†, * Yuming Wang†,

5

6

†

7

Shandong, China

8

‡

9

Saga University, Saga, 840-8502, Japan

10 11

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003,

Laboratory of Nutrition Biochemistry, Department of Applied Biochemistry and Food Science,

⊥

Qingdao National Laboratory for Marine Science and Technology, Laboratory of Marine Drugs

& Biological products, Qingdao 266237, Shandong, China

ACS Paragon Plus Environment


12

ABSTRACT

13

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with the characteristics of

14

senile plaques, neuro-inflammation, neurofibrillary tangles and destruction of synapse structure

15

stability. Previous studies have verified the protective effects of astaxanthin (AST). However,

16

whether synthesized docosahexaenoic acid-acylated AST diesters (AST-DHA) could delay AD

17

pathogenesis remains unclear. In the present study, APP/PSEN1 (APP/PS1) double transgenic

18

mice were administrated with AST and AST-DHA for 2 months. The results of radial 8-arm maze

19

and Morris water maze tests showed that AST-DHA exerted more significant effects than AST in

20

enhancing learning and memory levels of APP/PS1 mice. Further mechanical studies suggested

21

that AST-DHA was superior to AST in regulating the parameters of oxidative stress, reducing Tau

22

hyper-phosphorylation, suppressing neuro-inflammation and regulating inflammasome expression

23

and activation in APP/PS1 mice. The findings suggested AST-DHA attenuated cognitive disorders

24

by reducing pathological features in APP/PS1 mice, suggesting AST-DHA might be a potential

25

therapeutic agent for Alzheimer's disease.

26

KEYWORDS: Alzheimer's disease, astaxanthin, cognitive disorder, DHA-acylated AST esters,

27

neuro-inflammation

28


Page 2 of 30

Page 3 of 30


29

INTRODUCTION

30

The main pathological features of Alzheimer's disease (AD) are senile plaques,

31

neuro-inflammation, and destruction of synapse structure stability 1. There are very few AD drugs

32

on the present market, and these drugs only provide minimal symptomatic relief rather than

33

changes in disorder progression. Therefore, there is a great need for the therapeutic agents to

34

modify AD.

35

Astaxanthin (AST, Fig.1A), one of natural carotenoids, is widely present in marine organisms

36

such as shrimp, crab, krill, salmon, and microalgae 2. Importantly, the hydroxyl and keto moieties

37

on each ionone ring in AST imply its unique properties, especially, the ability to be esterified with

38

fatty acids to increase stability. Usually, AST occurs in esterified forms in nature, which is

39

chemically bound to various types of fatty acids such as oleic, eicosanoic, palmitic and stearic acid.

40

The AST in red crab langostilla (Pleuroncodes planipes) is comprised of about 70% diesters, 12%

41

monoesterified and 10% unesterified AST 3. Interestingly, it has been reported that the main

42

existing form of AST in Atlantic salmon is unesterified AST 4.

43

The bioactivity of AST is usually related to the reduced markers of oxidative damage.

44

Notably, recent evidence has emerged to indicate AST has a broad range of bioactivities including

45

anti-inflammatory, anti-apoptotic properties5. Moreover, AST plays a vital role in reducing

46

neurotoxicity in cell models of AD. In addition, AST could protect PC12 cells from Aβ-induced

47

cytotoxicity by up-regulating heme oxygenase-1 expression via ERK1/2 pathway5-6. Lobos et al.

48

provided further evidence to indicate that AST protected neurons from the noxious effects of Aβ

49

on mitochondrial ROS production and calcium dysregulation 7. Moreover, Cheong et al. have

50

verified that krill oil could enhance cognitive capability and modulate proteomic alterations in

51

brain of D-galactose induced aging mice 8.

52

Docosahexaenoic acid (DHA) is well known for various bioactivities 9. However, few study

53

focused on the protective effects of DHA-acylated AST ester. The APP/PSEN1 (APP/PS1)

54

transgenic mice, co-expressing the mutated Swedish APP gene and the exon-9-deleted variant of

55

the presenilin-1 (PS1) gene, are a successfully established transgenic animal models for AD


10

.


56

This model displays age-related plaque pathology, inflammatory response, oxidative damage,

57

age-related memory deficits11-12. In the present study, DHA-acylated AST diesters (AST-DHA)

58

was prepared, and APP/PS1 mice were used to compare the neuroprotective actions of AST and

59

AST-DHA and illustrate the possible underlying mechanisms.

60

MATERIALS AND METHODS

61

Chemicals and Reagents. Astaxanthin was obtained from Xinxiang Kesen Food Additives

62

Corporation (Tongxiang, Zhejiang, China). Aβ42 and Aβ40 ELISA kits were purchased from

63

Wuhan Uscn Life Science, Inc. (Wuhan, Hubei, China). The superoxide dismutase (SOD) assay

64

kit and nitric oxide (NO) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute

65

(Nanjing, Jiangsu, China). The Nitric Oxide Synthase ELISA kit was from R&D System

66

(Minneapolis, MN, USA). p-Tau (Ser396) antibody, Tau antibody, p- GSK-3β (Y216+Y279)

67

antibody, GSK-3β antibody, CD11b antibody, GFAP antibody, IL-1β antibody, TNF-α antibody,

68

NLRP3 antibody, Caspase-1 antibody, Bax antibody, Bcl-2 antibody, caspase-9 antibody and

69

β-actin antibody were from Abcam (Cambridge, UK). Caspase-3 antibody and cleaved caspase-3

70

antibody were purchased from Cell Signaling Technology (Boston, USA).

71

Preparation of AST-DHA. AST-DHA (Fig.1A) was synthesized by 4-dimethylaminopyridine

72

catalyzed reaction of AST and DHA in the presence of 1-(3-Dimethylaminopropyl)-

73

3-ethylcarbodiimide hydrochloride system with nitrogen protection and light isolation according

74

to our previous study

75

25 ℃. The organic phase is recovered following the subsequent cleaning by 1 M hydrochloric acid,

76

saturated sodium bicarbonate and saturated sodium chloride. The organic mixture was evaporated

77

to dryness to obtain the AST-DHA (the purity > 90%), which was confirmed by HPLC-DAD

78

according to our previously published study.

79

Animals and Treatments. The animal study protocol was proved by the Animal Ethics

80

Committee of College of Food Science and Engineering of Ocean University of China. All the

81

animals were housed at the Laboratory Animal Facility at the Ocean University of China. The

82

research was conducted in accordance with the Guide for the Care and Use of Laboratory Animals

13

. Dichloromethane was added to the system after the reaction for 3 h at


Page 4 of 30

Page 5 of 30


83

(8 th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research

84

Council, National Academy of Sciences, Washington DC). APP/PS1 transgenic mice (half male

85

and half female, weight of 20-25 g, aged 3 months) and eight of their wild-type littermates as

86

normal control group (Control) were obtained from Vital River Laboratories (Beijing, China). All

87

the mice were acclimatized for 1 week under a 12 h/12 h light/dark cycle at 23 °C with 60 ± 10%

88

humidity and provided with food and water ad libitum. The APP/PS1 transgenic mice were

89

randomly divided into Model group, AST group and AST-DHA group. All the mice were

90

supplemented with AIN-93G diet. The mice in AST and AST-DHA group were supplemented

91

with 0.2% AST and AST-DHA for 60 days, respectively. Then the water maze and radial 8-arm

92

maze tests were used to determine learning and memory levels. After that, the mice were

93

sacrificed by rapid decapitation. The cerebral cortex and hippocampus were separated from the

94

whole brain and weighed, then frozen with liquid nitrogen and stored at -80 °C until use.

95

Radial 8-Arm Maze Test. Spatial learning ability was determined by the radial 8-arm maze test

96

according to a previous method

97

radiating arms was placed at 1 m above the floor. A food cup was located at the end of each

98

radiating arm. Before training, the mice were deprived of half of the diet during the test. The baits

99

were restricted to the food cups. On the first two training days, the food pellet (45 mg) were

100

located at the food cups (eight trails) and central octagonal plate, the mice of the same group were

101

allowed to explore for food for 10 minutes together. On the third and fourth days, every mouse

102

was allowed to explore for food for 10 minutes. On the last day, four identical arms were baited

103

with a single 45 mg food pellet. Each trial continued until all four baits had been consumed or

104

until 10 minutes had elapsed. The numbers of reference memory errors and working memory

105

errors were conducted.

106

Water Maze Test. A circular stainless-steel pool with water (21-23 °C) was divided into four

107

quadrants. A circular black escape platform was located in the center of one quadrant. The mice

108

were trained to find the platform for five consecutive days. The time of finding the platform was

109

recorded as latency. The swim paths, distances, and latencies taken to the platform were

110

monitored with a video camera. Probe test without platform on the sixth day was used to

14

. The apparatus composed of a central octagonal plate and 8



111

determine spatial memory retention. The mice were placed in a position opposite the platform

112

location and allowed to swim for 60 sec. The number crossing over the previous position of the

113

platform and the time spent in the target quadrant were recorded as measures for spatial memory.

114

Immunohistochemistry. For paraffin sections, mice were killed and systemically perfused with

115

phosphate buffered saline (PBS) and 4% buffered paraformaldehyde through the left ventricle to

116

wash out blood cells. Then brain samples were collected, ehydrated and embedded in paraffin

117

using standard techniques. Sections (5 µm) were cut and deparaffinized. After incubation in

118

methanol containing 3% H2O2 for 15 minutes to block endogenous peroxidase activity,

119

nonspecific binding was done with universal blocking reagent for 30 minutes at room temperature.

120

The sections were incubated with the primary antibody against anti-Aβ (1:100; Servicebio),

121

anti-Iba1 (1:500; Servicebio) and anti-GFAP (1:500; Servicebio) at 4 °C for overnight followed by

122

several washing steps in PBS. Following incubation with biotinylated goat anti-rabbit IgG (1/200)

123

for 50 minutes, staining was done through incubation with peroxidase streptoavidin and

124

diaminobenzidine (DAB) at room temperature. Specific primary antibody was omitted in negative

125

control of the reactions. After counter staining nuclei with Mayer’s haematoxylin, the

126

immunopositive amyloid plaques in cortex and hippocampus were observed and counted. The

127

images were taken using a 5 MP Canon A95 camera integrated to the microscope and were

128

evaluated using image-J analysis.

129

Determination of Aβ Concentration by ELISA. Soluble and insoluble Aβ were extracted by the

130

previous method 15. Briefly, the right hippocampus was homogenized in TBS (pH8.0) containing

131

protease inhibitors. Samples were sonicated and centrifuged. The supernatant was used to

132

determine soluble Aβ40 and Aβ42 concentrations, whereas the TBS-insoluble pellet was firstly

133

sonicated in 2% SDS. To analyze the insoluble Aβ content, the SDS-insoluble pellet was

134

dissolved and sonicated in 70% formic acid. The extract was neutralized with 0.5 M Tris before

135

loading on the ELISA plate. The soluble/insoluble Aβ40 and Aβ42 levels were determined by

136

ELISA kits according to the manufacture’s instruction.

137

The Measurement of Oxidative Stress Parameters. The brain was prepared as a tissue

138

homogenate in 0.9% saline solution for the determination of protein concentration using a BCA


Page 6 of 30

Page 7 of 30


139

protein assay kit. The concentrations of nitric oxide (NO), inducible nitric oxide synthase (NOS)

140

and the activity of superoxide dismutase (SOD) were measured by the manufacture’s instruction.

141

Western Blot Analysis. The total protein and RNA of hippocampus were extracted by the

142

total-DNA-RNA-Protein kit. Equal amounts of protein were separated on 5-12% SDS-PAGE gels

143

and transferred to poly membranes. The membranes were incubated with antibodies against p-Tau

144

antibody (1:5000), Tau antibody (1:2000), p-GSK3β antibody (1:1000), GSK-3β antibody (1:500),

145

CD11b antibody (1:500), GFAP antibody (1:5000), IL-1β antibody (1:5000), TNF-α antibody

146

(1:200), NLRP3 (1:2000), Caspase 1 (1:1000) at 4 °C for overnight. After this, membranes were

147

incubated with specific horse radish peroxidase (HRP)-conjugated secondary antibodies (1:3000)

148

at room temperature 2 h and the blots were evaluated with chemiluminescent horseradish

149

peroxidase substrate. Then the blots were visualized by enhanced chemiluminescence (ECL)

150

substrate with UVP Auto Chemi Image system. Protein loading was evaluated by anti-β-actin

151

antibody (1:2000)..

152

Statistical Analysis. Data were expressed as mean ± standard deviation (SD). Statistical analyses

153

were evaluated by Student’s t test and Tukey’s test using SPSS 18.0. P < 0.05 was considered

154

statistically significant. Different letters indicated significant differences between each group.

155

RESULTS

156

AST and AST-DHA Improved Cognitive Disorder in APP/PS1 Mice. The spatial learning was

157

measures by radial 8-arm maze and Morris water maze tests. The radial 8-arm maze results

158

showed that APP/PS1 transgenic mice remarkably enhanced the number of reference memory

159

errors and working memory errors in comparison with non-APP/PS1 transgenic mice (Fig 1B and

160

C). Interestingly, AST and AST-DHA obviously reduced the number of reference memory errors

161

and working memory errors in APP/PS1 transgenic mice (Fig 1B and C), and no remarkable

162

difference was observed between these two groups.

163

The results of escape latency showed that the mice of control group had better performance

164

than APP/PS1 transgenic mice from day 1 to day 5, suggesting APP/PS1 transgenic mice

165

exhibited significant deficiency in spatial learning ability (Fig. 1D). The treatment with AST and



166

AST-DHA significantly improved the behaviors of APP/PS1 transgenic mice. Interestingly,

167

AST-DHA had a more significant effect than AST in alleviating spatial deficits of APP/PS1

168

transgenic mice.

169

The spatial memory results showed that APP/PS1 transgenic mice in model group spent less

170

time in the target quadrant and less crossing number than the control group (Fig. 1E and F). The

171

mice treated with AST and AST-DHA exerted similar effects with no statistical difference in

172

improving the time spent in the target quadrant. Interestingly, AST-DHA was superior to AST in

173

improving the number of crossing platform.

174

AST and AST-DHA Regulated Aβ Levels in APP/PS1 Mice. Brain sections were

175

immunostained with anti-Aβ antibody to show the Aβ deposition in the cortex and hippocampus

176

(Fig.2A), which was analyzed the number and area of plaques (Fig.2B and C). Compared with

177

control group, the amyloid plaques number in the cortex and hippocampus of APP/PS1 transgenic

178

mice were remarkably increased. Interestingly, AST and AST-DHA treatment could reduce

179

amyloid plaques number in cortex and hippocampus of APP/PS1 transgenic mice. Importantly,

180

AST-DHA was superior to AST in suppressing the number of amyloid plaques in cortex. The

181

results of quantitative analysis of Aβ showed that AST and AST-DHA suppressed the Aβ load.

182

AST-DHA exhibited more statistical effects than AST in reducing the Aβ load in hippocampus.

183

The soluble/insoluble Aβ40 and Aβ42 in the hippocampus were determined by ELISA kits,

184

and the results were shown in Fig. 3 A-D. The APP/PS1 transgenic mice exhibited higher soluble

185

and insoluble Aβ40 and Aβ42 levels than control group. Supplementation of AST and AST-DHA

186

exhibited a remarkable decrease of soluble and insoluble Aβ40 and Aβ42 levels in a certain degree.

187

Notably, AST-DHA exhibited more statistical effects than AST in reducing soluble Aβ40/Aβ42

188

levels. Unexpectedly, AST showed a better effect in reducing insoluble Aβ40 level than

189

AST-DHA group. No significant difference between AST and AST-DHA was observed in

190

inhibiting insoluble Aβ42 generation.

191

Following the soluble/insoluble Aβ40 and Aβ42 measurement, ADAM10, BACE1 and

192

Nicastrin were detected by western blotting. Compared with control group, the ADAM10 level of


Page 8 of 30

Page 9 of 30


193

model group was significantly decreased, and BACE1 and Nicastrin expression were obviously

194

increased. Notably, AST and AST-DHA treatment exerted similar effects in decreasing Nicastrin

195

level. However, there were no significant improvement for AST and AST-DHA in protein

196

expressions of ADAM10 and BACE1.

197

Effects of AST and AST-DHA on Oxidative Stress. Oxidative stress plays a central role in the

198

physiopathology of AD. Thus, we further investigated the effects of AST and AST-DHA on

199

oxidative stress to confirm the neuroprotective effects of AST and AST-DHA against AD. The

200

indexes of oxidative stress including SOD, NO, NOS were detected (Fig.4). Compared with

201

control group, the SOD activity in model group was significantly decreased, meanwhile, NOS

202

activity and NO level were obviously increased. Importantly, AST and AST-DHA treatment

203

significantly recovered the activity of SOD as well as declining NO and NOS levels, in which

204

AST-DHA was superior to AST in up-regulating SOD activity and down-regulating NO and NOS

205

levels.

206

The Effects of AST and AST-DHA on the Expression of p-GSK-3β and p-Tau. The effects of

207

AST and AST-DHA treatment on GSK-3β activity and p-Tau protein expression were evaluated

208

by Western blotting (Fig. 5A). Compared with the control group, the expression of p-GSK-3β

209

level in model group was obviously increased (p < 0.05, Fig. 5B). AST-DHA significantly

210

suppressed the expression of GSK-3β phosphorylation. Notably, AST treatment had no effects in

211

regulating the p-GSK-3β expression.

212

Following GSK3β activity, we also investigated the effects of dietary supplements of AST

213

and AST-DHA on p-Tau protein expression. The expression of p-Tau in model group was

214

remarkably increased compared with control group (Fig. 5C). Notably, dietary supplementation of

215

AST and AST-DHA could obviously reverse these changes, and AST-DHA exerted more

216

significant effects than AST in reducing the expression of p-Tau.

217

Inhibitory Effects of AST and AST-DHA on Neuro-inflammation in APP/PS1 Mice. The

218

neuro-inflammation induced by activated microglia and astrocytes is related to AD development.

219

To test the effects of AST and AST-DHA on neuro-inflammatory processes in APP/PS1



220

transgenic mice, immunohistochemical analysis and western blotting assay of astrogliosis and

221

microgliosis were performed using the astroglial marker (GFAP) and the microglial marker (Iba1

222

and CD11b, Fig. 6 A-D). The results showed that GFAP, Iba1 and CD11b were markedly

223

increased in the APP/PS1 transgenic mice but were significantly reduced in the AST and

224

AST-DHA treated mice both in immunohistochemical analysis and western blotting assay.

225

Importantly, AST-DHA was superior to AST in regulating the activation of microglia and

226

astrocytes.

227

Cytokines secreted by activated microglia and astrocytes are crucial in the inflammatory

228

processes of AD. The levels of IL-1β and TNF-α in AD mice were determined to investigate the

229

effects of AST and AST-DHA on cytokine production (Fig. 6 B, E and F). Compared with

230

no-transgenic mice, the expression of TNF-α in the APP/PS1 transgenic mice was markedly

231

increased. Notably, AST and AST-DHA supplementation could significantly reduce the

232

expression of TNF-α, and AST-DHA was superior to AST. Only AST-DHA significantly

233

suppressed the expression of IL-1β, and no statistical difference was found between AST group

234

and model group.

235

Inhibitory Effects of AST and AST-DHA on Inflammasome Activation in APP/PS1 Mice.

236

The NLRP3 inflammasome activation initiates an inflammatory response through caspase-1

237

activation, resulting in inflammatory cytokine IL-1β℃maturation and secretion. Therefore,

238

western blotting assay was performed to detect the NLRP3 inflammasome activation-related

239

proteins (Fig. 7). Unexpectedly, no statistical difference was observed in the expression of NLRP3

240

in these four groups. Compared with the control group, the expression of ASC in APP/PS1

241

transgenic mice was significantly reduced. However, AST and AST-DHA supplementation further

242

decreased the expression of ASC protein. Compared with the non-transgenic mice, the expression

243

of Caspase 1 and Pro-IL-1β in APP/PS1 transgenic mice was remarkably increased. Interestingly,

244

AST and AST-DHA supplementation obviously reversed the increase of Caspase 1 and Pro-IL-1β,

245

and AST-DHA exerted more significant effects than AST.

246

AST and AST-DHA Suppressed Apoptosis in APP/PS1 Mice. Western blotting assay was

247

performed to detect the apoptosis-related proteins in APP/PS1 mice (Fig. 8). Unexpectedly, AST


Page 10 of 30

Page 11 of 30


248

and AST-DHA supplementation had no corresponding improvement in regulating Bcl-2 and Bax

249

protein expression. The relative densities of Caspase-9 and Caspase-3 in APP/PS1 transgenic mice

250

were remarkably increased compared with the mice in the control group. Following AST and

251

AST-DHA administration, the protein levels of Caspase-9 and Caspase-3 were obviously reduced.

252

Notably, AST-DHA was superior to AST in reducing the protein expressions of Caspase-9/-3. Both

253

AST and AST-DHA could obviously suppress cleaved Caspase-3 expression with a similar degree.

254

DISCUSSION

255

The pathological characteristics of AD brains mainly include extracellular Aβ plaques,

256

intracellular

257

neuro-inflammation 1. APP/PS1 transgenic mouse is one of well-established AD animal model16-17.

258

To illustrate the possibility of false positivity, we determined the efficacy of AST and AST-DHA in

259

8-Arm maze and Morris water maze test in the present study. Both AST and AST-DHA treatment

260

could improve spatial learning ability in APP/PS1 mice by 8-arm maze apparatus, which were

261

further corroborated by the Morris water maze test. The results showed that both AST and

262

AST-DHA could improve the learning and memory skills, in which the AST-DHA was superior to

263

AST in Morris water maze test. The different result of these two behavior tests may be attributed

264

to that 8-Arm maze test is appetitive reinforcement, and Morris water maze test is aversive

265

reinforcement. Furthermore, the learned behavior in these two tests is also different among

266

learning tasks. The Morris water maze test usually learns a spatial task, in which rats have to learn

267

complex behavioral strategies to recognize the platform to escape from water.

268

neurofibrillary

tangles,

massive

neuronal

cell

and

synapse

loss,

and

The accumulation and deposition of Aβ are considered to play an important role in the 18

269

pathogenesis of AD, which is the dominant theory of AD in the past few decades

270

produced by the proteolytic processing of amyloid precursor protein (APP). An important way to

271

process APP is the nonamyloidogenic pathway, in which α-secretase cleaves the Aβ domain in

272

APP, thereby precluding the formation of intact Aβ. However, under normal circumstances, a

273

small amount of APP is processed via the amyloidogenic pathway, in which Aβ is released from

274

APP by β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretases. ADAM10

275

and Nicastrin is one of the components of α-secretase and γ-secretases, respectively. Aβ as a


. Aβ is


276

monomer readily aggregates to form multimeric complexes 19. These complexes are composed of

277

soluble Aβ ranging from oligomers to protofibrils and insoluble Aβ such as amyloid plaques. The

278

immunohistochemistry results showed that AST-DHA treatment showed better effects than AST in

279

decreasing the number of senile plaques and Aβ plaques load in the cortex and hippocampus of

280

APP/PS1 mice, which was consistent with the results of ELISA kits. Interestingly, AST and

281

AST-DHA only showed notable effects in regulating the expression of Nicastrin instead of

282

BACE1 and ADAM10. The Aβ decrease might partly depend on the clearance mechanism, as it

283

has been reported that n-3 LCPUFAs significantly promoted interstitial Aβ clearance from the

284

brain to resist Aβ injury 20.

285

AST treatment is usually related to the reduced markers of oxidative damage. AST may

286

increase the levels of endogenous antioxidant enzymes including superoxide dismutase and

287

catalase

288

indispensable role of DHA in AST-DHA.

21

. AST-DHA exhibited more effective antioxidant capacity than AST, indicating the

289

Increasing evidence has indicated Aβ induced hyper-phosphorylation of Tau protein by

290

GSK3β activation 22-23. The results showed that AST and AST-DHA significantly decreased p-Tau

291

and p-GSK3β, and AST-DHA was superior to AST. The cognitive improvement of AST-DHA on

292

APP/PS1 transgenic mice are mainly attributed to the neuroprotective effects on GSK3β activation

293

and tau hyper-phosphorylation.

294

Uncontrolled microglia and astrocytes activation, and sustained inflammatory responses may

295

contribute independently to neurodegeneration 24-25. It can not only be a consequence but also be a

296

trigger of pathology

297

APP/PS1 transgenic mice, which was accompanied by a strong increase of TNF-α rather than

298

IL-1β compared with the non-transgenic mice. Following AST and AST-DHA treatment,

299

pro-inflammatory cytokine levels were greatly reduced, especially in the AST-DHA group,

300

indicating that the anti-inflammatory effects of AST and AST-DHA may account for the reduced

301

Aβ level and cognitive improvement in APP/PS1 transgenic mice.

26

. The activation of microglia and astrocytes were found in the brain of


Page 12 of 30

Page 13 of 30


302

Neuro-inflammatory cascades depend on the activation of NLRP3 inflammasome, which was 27

303

crucial in neurodegenerative diseases

304

NLRP3 inflammasome, process IL-1β and IL-18, and finally induce AD pathology and tissue

305

damage 28. Moreover, in AD transgenic mouse model, the inhibition of NLRP3 can largely protect

306

memory loss and decrease Aβ deposition. A chronic administration of AST and AST-DHA in

307

APP/PS1 transgenic mice led to a remarkable alteration in NLRP3 inflammasome, in which

308

AST-DHA was superior to AST. The effects of AST and AST-DHA on glial activation and NLRP3

309

inflammasome confirmed the importance of AST-DHA in regulating inflammatory responses.

. It has been proved that the toxicity of Aβ can activate

310

Numerous studies on mitochondria dysfunction revealed that the mitochondria were the

311

central of oxidative stress induced apoptosis29-30. Bcl-2 is an anti-apoptotic protein, while Bax has

312

the opposite function. Caspase-9/-3 are the initiator and executioner, respectively, which typically

313

predominate in neurodegenerative diseases

314

decrease the expression Caspase-9/-3 and cleaved Caspase-3, and the effect of AST-DHA was

315

superior to AST. However, no expected effects of AST and AST-DHA on reducing Bax and

316

improving Bcl-2 were observed in APP/PS1 transgenic mice. Apoptosis is a complex and precisely

317

controlled process, which is regulated by multiple pathways. We speculated that the decreased

318

Caspase-9 and Caspase-3 were not only regulated by the Bcl-2/Bax, other pathways might be

319

related to regulating Caspase family.

31

. Both AST and AST-DHA could significantly

320

In summary, both AST and AST-DHA could improve AD in different degrees. AST-DHA

321

exhibited better actions than AST in improving learning and memory abilities by the behavior

322

experiments. The further mechanical research indicated AST-DHA exerted more remarkable

323

functions than AST on inhibiting Aβ generation, regulating oxidative stress, suppressing the

324

hyperphosphorylation of Tau and GSK-3β, and reducing neuroglial activation and

325

neuro-inflammation (Fig. 9). Therefore, AST and AST-DHA may be applied as food supplements

326

and/or functional ingredients to relieve neurodegenerative disease.

327

Author Information

328

* E-mail: [email protected]; phone: +86 0532 82032597; fax: +86 0532 82032468



329

*E-mail: [email protected]; phone: +86 0532 82032597; fax: +86 0532 82032468

330

Author contributions

331

Hongxia Che and Qian Li designed and conducted the research. Qian Li, Dan-dan Wang and Lu

332

Yang analyzed the data; Hongxia Che wrote the manuscript; Tian-tian Zhang, Jie Xu and

333

Teruyoshi Yanagita revised the manuscript. Yuming Wang, Changhu Xue and Yaoguang Chang

334

had primary responsibility for the final content. All authors read and approved the final

335

manuscript.

336

Funding

337

This work was supported by grants from State Key Program of National Natural Science of China

338

(No. 31330060) and National Natural Science Foundation of China-Shandong Joint Fund for

339

Marine Science Research Centers (U1606403), and the Fundamental Research Funds for the

340

Central Universities (No. 201762028).

341

Conflicts of interest

342

All the authors declare that there is no conflict of interest for any of them.

343

List of Abbreviations

344

AD, Alzheimer’s disease; AST, Astaxanthin; AST-DHA, Docosahexaenoic acid-acylated AST

345

diesters; APP, amyloid precursor protein; PS1, presenilin; Aβ, β-amyloid; SOD, superoxide

346

dismutase; NOS, inducible nitric oxide synthase; NO, nitric oxide; GFAP, glial fibrillary acidic

347

protein; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1beta; Bcl-2, B-cell lymphoma 2; Bax,

348

Bcl-2-associated X Protein; NLRP3, nucleotide-binding oligomerization domain (NOD)-like

349

receptor containing pyrin domain 3; ASC, apoptosis-associated speck-like protein containing a

350

CARD


Page 14 of 30

Page 15 of 30


351

REFERENCES

352

1.

353

Neuroprotective effects of bajijiasu against cognitive impairment induced by amyloid-β in APP/PS1

354

mice. Oncotarget. 2017, 54, 92621.

355

2.

356

and its commercial applications--a review. Mar. Drugs. 2014, 1, 128-52.

357

3.

358

planipes): optical R/S isomers and fatty acid moieties of astaxanthin esters. Comp. Biochem. Phys. B.

359

2002, 3, 437.

360

4.

361

canthaxanthin in raw and smoked Atlantic salmon (Salmo salar) during frozen storage. Food Chem.

362

1998, 3, 313-317.

363

5.

364

with potential in human health and nutrition. J. Nat. Prod. 2006, 3, 443-449.

365

6.

366

heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against

367

beta-amyloid-induced cytotoxicity in SH-SY5Y cells. Brain Res. 2010, 1, 159-167.

368

7.

369

Paula-Lima, A. Astaxanthin Protects Primary Hippocampal Neurons against Noxious Effects of

370

Aβ-Oligomers. Neural. Plast.. 2016, 1, 3456783.

371

8.

372

neurocognitive functions and modulates proteomic changes in brain tissues of d-galactose induced

373

aging mice. Food Funct. 2017, 5, 2038-2045.

374

9.

375

Annu. Rev. Food Sci. T. 2018, 9, 345-381.

Cai, H.; Wang, Y.; He, J.; Cai, T.; Wu, J.; Fang, J.; Zhang, R.; Guo, Z.; Guan, L.; Zhan, Q.

Ambati, R. R.; Moi, P. S.; Ravi, S. Astaxanthin: sources, extraction, stability, biological activities

Coral-Hinostroza, G. N.; Bjerkeng, B. Astaxanthin from the red crab langostilla (Pleuroncodes

Sheehan, E. M.; O'Connor, T. P.; Pja, S.; Buckley, D. J.; Fitzgerald, R. Stability of astaxanthin and

Hussein, G., Sankawa, U., Goto, H., Matsumoto, K., Watanabe, H. Astaxanthin, a carotenoid

Wang, H. Q.; Sun, X. B.; Xu, Y. X.; Zhao, H.; Zhu, Q. Y.; Zhu, C. Q. Astaxanthin upregulates

Lobos, P.; Bruna, B.; Cordova, A.; Barattini, P.; Galaz, J. L.; Adasme, T.; Hidalgo, C.; Munoz, P.;

Cheong, L. Z.; Sun, T.; Li, Y.; Zhou, J.; Lu, C.; Li, Y.; Huang, Z.; Su, X. Dietary krill oil enhances

Shahidi, F.; Ambigaipalan, P. Omega-3 Polyunsaturated Fatty Acids and Their Health Benefits.



376

10. Reiserer, R. S.; Harrison, F. E.; Syverud, D. C.; Mcdonald, M. P. Impaired spatial learning in the

377

APPSwe + PSEN1DeltaE9 bigenic mouse model of Alzheimer's disease. Genes Brain Behav. 2007, 1,

378

54-65.

379

11. Xu, Z.; Na, X.; Chen, Y.; Huang, H.; Marshall, C.; Gao, J.; Cai, Z.; Wu, T.; Gang, H.; Ming, X.

380

Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits.

381

Mol. Neurodegener. 2015, 1, 58.

382

12. Huang, H.; Nie, S.; Cao, M.; Marshall, C.; Gao, J.; Xiao, N.; Hu, G.; Xiao, M. Characterization of

383

AD-like phenotype in aged APPSwe/PS1dE9 mice. Age. 2016, 4, 1-20.

384

13. Yang, L.; Xuemin, L. I.; Zhou, Q.; Zhaojie, L. I.; Jie, X. U.; Xue, C. Synthesis and Mass

385

Spectrometry Analysis of Docosahexaenoic Acid Astaxanthin Ester. Food Sci. 2017, 2, 220-226.

386

14. Enomoto, T.; Ishibashi, T.; Tokuda, K.; Ishiyama, T.; Toma, S.; Ito, A. Lurasidone reverses

387

MK-801-induced impairment of learning and memory in the Morris water maze and radial-arm maze

388

tests in rats. Behav. Brain res. 2008, 2, 197.

389

15. Che, H.; Zhou, M.; Zhang, T.; Zhang, L.; Ding, L.; Yanagita, T.; Xu, J.; Xue, C.; Wang, Y.

390

Comparative study of Phosphatidylcholine rich in DHA or EPA on Alzheimer's Disease and the

391

possible involved mechanisms in CHO-APP/PS1 cell and SAMP8 mice. Food Funct. 2017, 2, 197-207.

392

16. Deng, M.; Huang, L.; Ning, B.; Wang, N.; Zhang, Q.; Zhu, C.; Fang, Y. β-asarone improves

393

learning and memory and reduces Acetyl Cholinesterase and Beta-amyloid 42 levels in APP/PS1

394

transgenic mice by regulating Beclin-1-dependent autophagy. Brain Res. 2016, 1652, 188-194.

395

17. Jiang, T.; Yu, J. T.; Zhu, X. C.; Tan, M. S.; Wang, H. F.; Cao, L.; Zhang, Q. Q.; Shi, J. Q.; Gao, L.;

396

Qin, H. Temsirolimus promotes autophagic clearance of amyloid-β and provides protective effects in

397

cellular and animal models of Alzheimer's disease. Pharmacol. Res. 2014, 81, 54-63.

398

18. Takahashi, R. H.; Nagao, T.; Gouras, G. K. Plaque formation and the intraneuronal accumulation

399

of β-amyloid in Alzheimer's disease. Pathology. International. 2017, 4, 185.

400

19. Riqiang Yan, R. V. Targeting the β secretase BACE1 for Alzheimer's disease therapy. Lancet

401

Neurology. 2014, 3, 319-29.


Page 16 of 30

Page 17 of 30


402

20. Ren, H.; Luo, C.; Feng, Y.; Yao, X.; Shi, Z.; Liang, F.; Kang, J. X.; Wan, J. B.; Pei, Z.; Su, H.

403

Omega-3 polyunsaturated fatty acids promote amyloid-β clearance from the brain through mediating

404

the function of the glymphatic system. Faseb J. 2017, 1, 282.

405

21. Grimmig, B.; Kim, S. H.; Nash, K.; Bickford, P. C.; Shytle, R. D. Neuroprotective mechanisms of

406

astaxanthin: a potential therapeutic role in preserving cognitive function in age and neurodegeneration.

407

Geroscience. 2017, 1, 1-14.

408

22. Vossel, K. A.; Xu, J. C.; Fomenko, V.; Miyamoto, T.; Suberbielle, E.; Knox, J. A.; Ho, K.; Kim, D.

409

H.; Yu, G. Q.; Mucke, L. Tau reduction prevents Aβ-induced axonal transport deficits by blocking

410

activation of GSK3β. J. Cell. Biol. 2015, 3, 419-433.

411

23. Simic, G.; Leko, M. B.; Wray, S.; Harrington, C.; Delalle, I.; Natasa, J. M.; Bazadona, D.; Buee,

412

L.; Silva, R. D.; Giovanni, G. D.; Wischik, C. Tau Protein Hyperphosphorylation and Aggregation in

413

Alzheimer's Disease and Other Tauopathies, and Possible Neuroprotective Strategies. Biomol. 2016, 1,

414

6.

415

24. Heppner, F. L.; Ransohoff, R. M.; Becher, B. Immune attack: the role of inflammation in

416

Alzheimer disease. Nat. Rev. Neurosci. 2015, 6, 358-72.

417

25. McGeer, E. G.; McGeer, P. L. Inflammatory processes in Alzheimer's disease. Prog.

418

Neuro-Psychoph. 2003, 5, 741-749.

419

26. Liao, W.; Liu, Z.; Zhang, T.; Sun, S.; Ye, J.; Li, Z.; Mao, L.; Ren, J. Enhancement of

420

Anti-Inflammatory Properties of Nobiletin in Macrophages by a Nano-Emulsion Preparation. J. Agr.

421

Food Chem. 2017, 1, 91-98.

422

27. Zhou, K.; Shi, L.; Wang, Y.; Chen, S.; Zhang, J. Recent Advances of the NLRP3 Inflammasome in

423

Central Nervous System Disorders. J. Immunol. Res. 2016, 2, 1-9.

424

28. Tan, M. S.; Yu, J. T.; Jiang, T.; Zhu, X. C.; Tan, L. The NLRP3 inflammasome in Alzheimer's

425

disease. Mol. neurobio. 2013, 3, 875-82.



426

29. Liao, W.; Lai, T.; Chen, L.; Fu, J.; Sreenivasan, S. T.; Yu, Z.; Ren, J. Synthesis and

427

Characterization of a Walnut Peptides Zinc Complex and Its Antiproliferative Activity against Human

428

Breast Carcinoma Cells through the Induction of Apoptosis. J. Agr. Food Chem. 2016, 7, 849-859.

429

30. Liao, W.; Zhang, R.; Dong, C.; Yu, Z.; Ren, J. Novel walnut peptide-selenium hybrids with

430

enhanced anticancer synergism: facile synthesis and mechanistic investigation of anticancer activity. Int.

431

J. Nanomed. 2016, 11, 1305-1321.

432

31. Che, H.; Fu, X.; Zhang, L.; Xiang, G.; Min, W.; Lei, D.; Xue, C.; Jie, X.; Wang, Y.

433

Neuroprotective Effects of n-3 Polyunsaturated Fatty Acid-Enriched Phosphatidylserine Against

434

Oxidative Damage in PC12 Cells. Cell. Mol. Neurobio. 2017, 1-12.


Page 18 of 30

Page 19 of 30


435

Figure Captions

436

Fig.1 Structure and effects of AST and AST-DHA on spatial learning and memory deficiency.

437

Structure of AST and AST-DHA (A). The number of reference memory errors in radial 8-Arm

438

maze (B), number of working memory errors in radial 8-Arm maze (C). Time needed to reach the

439

hidden platform in the Morris maze (D). The time spent in the target quadrant. (E) and the number

440

of crossing platform (F) were measured for analysis of spatial memory function. Data were

441

presented as mean ± SD (n = 8), *P< 0.05 was considered statistically significant. Different letters

442

indicated significant difference between each group.

443

Fig.2 Represent photo and quantitative analysis of amyloid plaques in cortex and hippocampus of

444

APP/PS1 transgenic mice (A). Plaques number of Aβ in cortex and hippocampus (B). Relative of

445

quantitative analysis of Aβ load in cortex and hippocampus (C). Scale bar = 100 µm.

446

Fig.3 Effects of AST and AST-DHA on the regulation of Aβ concentration in APP/PS1 transgenic

447

mice. Levels of insoluble Aβ40 (A), soluble Aβ40 (B), insoluble Aβ42 (C) and soluble Aβ42 (D)

448

were measured by ELISA. Representative western blots (E) and densitometry of ADAM10 (F),

449

BACE1 (G) and Nicastrin (H). Data were presented as mean ± SD (n=8); *P< 0.05 was

450

considered statistically significant. Different letters indicated significant difference between each

451

group.

452

Fig.4 Effects of AST and AST-DHA on the SOD activity (A), NO concentration (B) and NOS

453

activity (C) in brain. Data were expressed as mean± SD (n = 8), *P< 0.05 was considered

454

statistically significant. Different letter indicated significant difference between each group.

455

Fig.5. Effects of AST and AST-DHA on tau and GSK-3β hyper-phosphorylation. (A) Western

456

blot analysis of p-GSK3β and p-Tau. Densitometry analysis of p-GSK3β (B) and p-Tau (C).

457

Values were indicated as the mean ± SD (n = 8), *P< 0.05 was considered statistically significant.

458

Different letter indicated significant difference between each group APP/PS1 transgenic mice.

459

Fig.6 Effects of AST and AST-DHA on neuroglial activation and neuro-inflammation. (A) Effects

460

of AST and AST-DHA on glial markers were analyzed by immunohistochemistry mmunostaining



461

of the microglial marker Iba1astroglial marker GFAP and the Scale bar = 10 µm. (B) Western blot

462

analysis of CD11b, GFAP, IL-1β and TNF-α. Densitometry analysis of CD11b (C), GFAP (D),

463

IL-1β (E) and TNF-α (F). Values were indicated as the mean ± SD (n = 8), *p< 0.05 was

464

considered statistically significant. Different letter indicated significant difference between each

465

group.

466

Fig.7 Effects of AST and AST-DHA on inflammasome expression and activation. (A) Western

467

blot analysis of NLPR3, ASC, Caspase 1 and Pro-IL-1β. Densitometry analysis of NLRP3 (B),

468

ASC (C), Caspase 1 (D), and Pro-IL-1β (D). Values were indicated as the mean ± SD (n = 8), *p

The effects of astaxanthin and docosahexaenoic acid-acylated

Recommend Documents