Geraniin Attenuates Lipopolysaccharide-Induced Cognitive

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Bioactive Constituents, Metabolites, and Functions

Geraniin attenuates lipopolysaccharide-induced cognitive impairment in mice by inhibiting TLR4 activation DONGMEI WANG, XIAOHUI DONG, Bei Wang, Yumei Liu, and Sanqiang Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03977 • Publication Date (Web): 28 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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Journal of Agricultural and Food Chemistry

Geraniin attenuates lipopolysaccharide-induced cognitive impairment in mice by inhibiting TLR4 activation Dongmei Wang †, †

‡

, Xiaohui, Dong †, Bei Wang†, Yumei Liu ‡,Sanqiang Li†

Medical College, Henan University of Science and Technology, Luoyang, China College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, China

*Correspondence: Dongmei Wang, Medical College, Henan University of Science and Technology, No. 263, Kaiyuan Avenue, Luolong District, Luoyang 471023, China.

Fax:

86-0379-64270929;

Tel:

86-0379-64270929;

E-mail:

[email protected] *Correspondence: Sanqiang Li, Medical College, Henan University of Science and Technology, No. 263, Kaiyuan Avenue, Luolong District, Luoyang 471023, China. Fax: 86-0379-64270929; Tel: 86-0379-64270929; E-mail: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT: Geraniin has been reported to possess potent anti-inflammatory

2

properties and to modulate the macrophage polarization. This study sought to evaluate

3

the protective effects and underlying mechanisms of geraniin on lipopolysaccharide

4

(LPS)-induced neuroinflammation and neurobiological alternations as well as

5

cognitive impairment. Daily intragastrical administration with geraniin (20 mg/kg per

6

day) for 14 days significantly prolonged the duration in the target quadrant (26.53 ±

7

2.03 vs 37.09 ± 3.27 %, p < 0.05) and increased crossing-target number (1.93 ±

8

0.22 vs 3.08 ± 0.17, p < 0.01) in the probe test of LPS-treated mice. Geraniin also

9

ameliorated LPS-elicited neural/synaptic impairments and decreased levels of

10

LPS-induced Aβ generation (p < 0.05), amyloid precursor protein (APP) (p < 0.05)

11

and β-site amyloid precursor protein cleavage enzyme 1 (BACE1) (p < 0.05).

12

Furthermore, Geraniin suppressed the production of proinflammatory cytokines

13

including tumor necrosis factor alpha (TNF-α) (9.85 ± 0.58 vs 5.20 ± 0.52 pg/mg

14

protein, p < 0.01), interleukin (IL)-1β (16.31 ± 0.67 vs 8.62 ± 0.46 pg/mg protein,

15

p < 0.01), and IL-6 (12.12 ± 0.45 vs 7.43 ± 0.32 pg/mg protein, p < 0.05), and

16

inhibited glial cell activation. Moreover, geraniin effectively polarized the microglia

17

towards an anti-inflammatory M2 phenotype. Further study revealed that geraniin

18

targeted Toll-like receptor 4 (TLR4)-mediated signaling and decreased the production

19

of proinflammatory cytokines in BV-2 microglial cells. These results indicate that

20

geraniin mitigates LPS-elicited neural/synaptic neurodegeneration, amyloidogenesis,

21

neuroinflammation, as well as cognitive impairment, and suggest geraniin as a

22

therapeutic option for neuroinflammation-associated neurological disorders such as


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Alzheimer's disease.

24

KEY WORDS: geraniin; cognitive; LPS; neuroinflammation; TLR4

25

■ INTRODUCTION

26

Neuroinflammation is well documented to be a part of the neuropathogenesis of

27

cognitive deficit. 1,

28

infections on cognition, and the aging process itself is closely correlated with

29

augmented neuroinflammatory response including the production of proinflammatory

30

cytokines, and the imbalance of microglial M1/M2 polarization. 3 Neuroinflammation

31

also occurs in the brain of patients with Alzheimer's disease (AD). 4, 5 However, the

32

exact mechanism of neuroinflammation on cognition has not yet been thoroughly

33

elucidated.

2

The elderly show vulnerability to the detrimental effects of

34

Recently, the bidirectional networks between the intestinal microbiota and the brain

35

were reported to be maintained through nervous, endocrine, and immune

36

communications.

37

increased gut permeability were observed in AD.

38

which is abundant in the gut, can enter the bloodstream through disrupted tight

39

junctions of intestinal cells and increased gut permeability and induce a systemic

40

inflammatory response. 10, 11 It has been shown that the plasma LPS concentration in

41

AD patients is up to three times higher than that of normal subjects. 12

42

Neuroinflammation induced by systemic injection of LPS persists for about 10

43

months in mice brains and triggers memory impairment. 13 Several studies have

44

confirmed that LPS administration increases Aβ production, induces chronic

6, 7

Altered gut microorganisms, impaired gut barrier function, and 8, 9

1


Lipopolysaccharide (LPS),


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45

neuroinflammation, disrupts synaptic function, and impairs spatial learning

46

performance. 14, 15

47

Anti-inflammatory compounds have been reported to attenuate LPS-induced

48

neuroinflammatory effects and to improve cognitive impairment. 14, 16, 17. Geraniin, the

49

main polyphenolic component of Nephelium lappaceum L, possesses extensive

50

pharmacological effects such as anti-inflammatory, 18 antiviral, 19 antioxidant, 20

51

antihypertensive, 21 and antitumor 22 activities. Previous research demonstrated that

52

geraniin has potential anti-inflammatory effects and protects against LPS-elicited

53

inflammation in animal model 23 and cell lines. 24 Moreover, geraniin modulates the

54

macrophage

55

polarization. 25 Recently, geraniin was reported to exhibit significant β-secretase

56

inhibitory activity, which is implicated in AD. 26 This study was designed to uncover

57

the protective effects of geraniin on an LPS-induced neuroinflammation mouse model

58

by examining the effects of geraniin on neuron damage and cognitive impairment; by

59

investigating the effects of geraniin on LPS-elicited amyloidogenesis and glial

60

overactivation, as well as microglia polarization, and by exploring the mechanisms

61

underlying

immune

response

by

suppressing

its

2


LPS-induced

macrophage

action.

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■ MATERIALS AND METHODS

63

Chemicals. Geraniin (CAS: 60976-49-0) with purity of ≥ 98% was from

64

Bellancom (New Jersey, USA). LPS (Escherichia coli serotype 0111:B4) was

65

obtained from Sigma-Aldrich (St. Louis, USA). Nissl Staining Solution and Cell

66

Counting Kit-8 were offered by Beyotime Institute of Biotechnology (Nanjing, China).

67

Primer sequences for CD16, TNF-α, iNOS, MCP-1, CD206, TGF-β, Arg1 and YM-1

68

were from Sangon Biotech (Shanghai, China). M-MLV Reverse Transcriptase and

69

Opti-MEM medium Lipofectamine 3000 were from Invitrogen (Carlsbad, USA).

70

TLR4 siRNA or control siRNA sequences were from Genepharma (Shanghai, China).

71

NE-PER nuclear extraction kit was obtained from Thermo Fisher Scientific

72

(Pittsburgh, USA). NF-κB activity assay was available from Abcam (Cambridge,

73

USA). Anti-PSD95 antibody (MAB1596) was obtained from Millipore (Temecula,

74

USA).

75

(2859), anti-IκB-α (4814), anti-p-NF-κB (3033), anti-NF-κB (6956), anti-TNF-α

76

(11948) were from Cell Signaling Technology (Danvers, USA). Anti-Iba-1 (ab178846)

77

and anti-APP (ab32136) antibodies were obtained from Abcam (Cambridge, USA).

78

Anti-GFAP (16825-1-AP), anti-TLR4 (66350-1-Ig), anti-Myd88 (23230-1-AP)

79

antibodies, Alexa Fluor 488 conjugated secondary antibody (SA0006-2), and Alexa

80

Fluor 594 conjugated secondary antibody (SA0006-3) were obtained from Proteintech

81

(Chicago, USA).

Anti-Aβ (15126), anti-Synapsin-1 (5297), anti-BACE1 (5606), anti-p-IκB-α

82 83

Animals. Male C57BL/6 mice (18 to 20 g) were maintained in plastic cages on a

3



84

12-hr light/dark cycle at 23 ± 1 ℃ with food and water available ad libitum. All

85

animal experiments were approved by the Institutional Animal Experiment

86

Committee of Henan University of Science and Technology, China.

87 88

Experimental Design. Mice were randomly divided into four groups (n = 12-14):

89

control group, geraniin (20 mg·kg-1·d-1) alone group, LPS group, LPS + geraniin (20

90

mg·kg-1·d-1) group. Mice were administrated intraperitoneal (i.p.) injection of either

91

LPS (250 µg·kg-1·d-1) or its solvent on the 2nd week for 7 days after drug

92

administration. In addition, mice were administered either geraniin (20 mg·kg-1·d-1;

93

i.g.) or its solvent 1 h before LPS treatment for consecutive 14 days. LPS was diluted

94

in PBS and intraperitoneally treated. Geraniin was dissolved in PBS followed by daily

95

intragastrically administration. A preliminary study was carried out with three

96

different dosages of geraniin (10, 20, 40 mg·kg-1·d-1; i.g.). The results showed that

97

either 20 mg·kg-1·d-1 or 40 mg·kg-1·d-1 of geraniin administration remarkably

98

attenuated LPS-induced spatial learning and memory impairment as assessed by

99

Morris water maze (MWM) test. However, no significant difference was found

100

between 20 mg·kg-1·d-1 and 40 mg·kg-1·d-1 of geraniin administration (data not

101

shown). Based on the behavioral results, geraniin (20 mg·kg-1·d-1; i.g.) was selected as

102

the optimal dosage for further experiments.

103 104

Morris Water Maze. The spatial learning and memory of mice was performed as

105

previously reported methods with minor modifications 27. Briefly, mice were tested in

4


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106

a white circular pool (100 cm in diameter and 50 cm high). An escape platform with

107

10 cm in diameter was positioned 1 cm below the water and maintained constant

108

throughout the training. During 5 days of training phase, each mouse was subjected to

109

four trials per day. Each trial was carried out for 60 s until the mouse found and

110

reached the platform and the escape latency was recorded. A probe trial was

111

conducted on the 6th day. The platform was removed, and each mouse swam freely in

112

the water pool for 60 s. The percentage of time in the target quadrant and numbers of

113

crossings through the original platform were recorded. After the probe test, each

114

mouse was trained on a visible platform for 2 days, wherein the platform is clearly

115

visible, to evaluate the visual acuity of the mice. All traces of the mice were recorded

116

using the EthoVisio XT tracking system (Noldus Information Technology,

117

Wageningen, Netherlands).

118 119

Tissue Preparation. Following transcardial perfusion with ice cold normal saline

120

and

4%

paraformaldehyde,

mouse

brains

(n

=

3)

were

postfixed

in

121

4% paraformaldehyde and switched to 30% sucrose phosphate buffer. Serial 20

122

µm-thick coronal sections containing the hippocampus were prepared for

123

immunofluorescence staining. Another mouse brains (n = 3) were postfixed in

124

4% paraformaldehyde and processed to be embedded in paraffin. Serial 5 µm-thick

125

coronal sections were prepared for Nissl staining and immunohistochemistry.

126 127

Nissl Staining. Following deparaffinized and rehydrated , brain sections were

128

stained with Nissl Staining Solution for 15-20 min at room temperature. After rinsed

129

and dehydrated, brain slices were placed in xylene and mounted with Permount. The 5



130

neurons in the hippocampus were observed with a Nikon Eclipse Ti Microscope. At

131

least 4-5 sections per animal were selected and utilized for staining, and representative

132

images captured at 400 × magnification are shown.

133

134

Immunohistochemistry. Following deparaffinized and rehydrated , brain sections

135

were treated with 10 mM citrate buffer for antigen retrieval and blocked using 10%

136

goat serum for 1 h. Brain slices were incubated with GFAP (1:1000) and Iba-1(1:2000)

137

followed by horse radish peroxidase (HRP) conjugated secondary antibodies and

138

DAB staining. The photomicrographs were obtained using a Nikon Eclipse Ti

139

Microscope with a Nikon DS-Fi2 camera and analyzed by the Image-Pro Plus

140

software. At least 8-10 sections per animal were selected and utilized for staining, and

141

representative images captured at 400 × magnification are shown.

142

143

Immunofluorescence Staining. Following blocked with 10% goat serum (0.2%

144

Triton X-100) for 1 h, the brain sections were incubated with the following primary

145

antibodies: Aβ (1:200), p-NF-κB (1:500), Synapsin-1(1:200), and PSD95 (1:300) at

146

4°C overnight. After rinsed, brain slices were incubated with Alexa Fluor 488 or 594

147

conjugated secondary antibodies and nuclear dye. Immunofluorescence images were

148

captured under a ZEISS LSM 800 confocal microscope. The synapses were visualized

149

and determined by reconstructing three-dimensional image of colocalization of

150

Synapsin-1 and PSD95 by using ZEISS-Elements advanced Research software. At

151

least 8-10 sections per animal were selected and utilized for staining, and

152

representative images captured at 400× magnification are shown. 6


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ELISA Measurement. Following homogenizing in RIPA buffer and centrifuging

155

at 4 °C, the supernatants of brain tissue were collected and determined for the

156

concentration of TNF-α, IL-1β, and IL-6 using ELISA kit from R&D Systems

157

(Minneapolis, USA).

158 159

Real-Time RT-PCR. Total RNA was isolated from hippocampus using a Qiagen

160

RNeasy Kit and reverse transcribed into cDNA using M-MLV Reverse Transcriptase

161

following the manufacturer’s instructions. The resultant cDNA was amplified by PCR

162

using the TaqMan Gene Expression Assay kits with an Applied Biosystems

163

StepOnePlus system. Primers for RT-PCR were shown in Table 1. Gene expression

164

was measured using the ddCt method and normalized to GAPDH.

165 166

Cell Culture and Drug Treatment. Immortalized murine microglial (BV-2) cells

167

were grown in DMEM containing 10% FBS and antibiotics (penicillin G and

168

streptomycin). BV-2 cells were cultured to 70–80% confluence and then

169

pre-incubated for 1 h in the absence or presence of geraniin (25, 50, 100 μM) before

170

addition of LPS (1µg/ml) for 24 h. Cell Counting Kit-8 was used to detect the cell

171

viability. After indicated drug treatment, the cells were incubated with CCK-8 for 2 h,

172

and the absorbance at 450 nm was measured using a microplate reader (BioTek

173

Instruments, VT, USA). After indicated drug treatment, the protein was extracted for

174

the desired analyses.

175 176

Small-Interfering RNA Transfection. BV-2 cells were transfected with specific

177

TLR4 siRNA or control siRNA. The siRNA sequences as follows: 5 -CCU CCA

7



178

UAG ACU UCA AUU AT- 3; reverse, 5 -UAA UUG AAG UCU AUG GAG GTT- 3.

179

Both siRNA were transfected into cells using Lipofectamine 3000 in Opti-MEM

180

medium. After transfecting for 48 h, BV-2 cells were treated with geraniin (50 μM)

181

for 1h followed by LPS stimulation for an additional 24 h. Finally, the protein was

182

extracted for the desired analyses.

183 184

NF-κB activity assay. Nuclear and Cytoplasmic proteins were obtained from

185

freshly brain tissue using NE-PER Nuclear and Cytoplasmic Extraction Reagents

186

(Thermo Fisher Scientific Inc.). For the measurement of NF-κB activity, nuclear

187

extracts was applied for NF-κB activity assay following the manufacturer instructions.

188 189

Western Blot Analysis. Equal amounts of protein were separated via SDS-PAGE

190

gel and transferred to a nitrocellulose membrane. Membranes were incubated with

191

primary antibodies at 4 °C overnight. Following incubated with HRP-conjugated

192

secondary antibodies, membranes were visualized using enhanced chemiluminescent

193

detection system. Images were acquired by Bio-Rad Chemidoc Imaging System and

194

the intensity of protein bands was analyzed using Image-Pro Plus software. 28, 29

195 196

Statistical Analysis. All data were expressed as mean ± SEM. One-way analysis of

197

variance (ANOVA) was used for multiple comparisons followed by Bonferroni

198

post-hoc test. Two-way ANOVA with repeated measures was used for the differences

199

in the escape latency among the groups during the training trials. Statistical analyses

200

were conducted using SPSS 13.0, and p < 0.05 was considered significant.

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■ RESULTS

202

Geraniin

Ameliorates

LPS-Induced

Spatial

Learning

and

Memory

203

Impairment in Mice. The effects of geraniin on cognitive deficits induced by LPS

204

were assessed by using the Morris water maze test. The results illustrated that the LPS

205

mice took a longer time to reach the platform from day 4 compared to control mice,

206

whereas administration of geraniin to LPS treated mice significantly decreased the

207

escape latencies, suggesting that the impaired acquisition of the spatial learning ability

208

in LPS mice was rescued by greaniin (Figure 1A).

209

In the probe test, the LPS mice exhibited an obviously reduced swimming time in

210

the target quadrant and a lower number of platform crossings. LPS mice treated with

211

geraniin markedly increased the percentage of time in the target quadrant (Figure 1C)

212

and the crossing-target number (Figure 1D). These results implied that geraniin

213

reversed the spatial memory deficits in LPS mice. To exclude the possibility that

214

different groups of mice may have different swimming abilities, the swimming speed

215

and the path length and were recorded. Our data demonstrated that no significant

216

difference was observed in the velocity (Figure 1E) and the total path length (Figure

217

1F) between the four groups of mice in the probe trial. In the visible platform test,

218

there was no significant difference in the escape latencies between the four groups of

219

mice (Figure 1G). These data further indicated that improvement of learning and

220

memory in geraniin treated LPS mice was not caused by changes in non-cognitive

221

parameters. During the whole test, geraniin alone treated mice showed similar

222

performance to control mice.

9



223 224

Geraniin Attenuates LPS-Elicited Neuron Injury in the Hippocampus of Mice.

225

To investigate the protective effect of geraniin on neurons, Nissl staining was

226

employed to examine the histopathologic changes. We found that Nissl substance in

227

cells from LPS group exhibited significant loss compared to the cells from the

228

controls. Geraniin administration markedly elevated Nissl substance in cells in the

229

hippocampus of adult mice. No significant difference was observed in the Nissl

230

substance between geraniin alone group and control group (Figure 2).

231 232

Geraniin Increases Synaptic Density in the Hippocampus of LPS-treated Mice.

233

Co-localization of synapsin and PSD95 serves as an indicator of a mature excitatory

234

synapse. 30, 31 Therefore, co-staining with these two markers was employed to evaluate

235

the effects of geraniin on the synaptic function. The LPS-treated mice showed an

236

obvious decrease in synaptic density compared to control. However, the synaptic

237

density was remarkably upregulated in the hippocampal CA1 region of geraniin

238

treated LPS mice. There was no significant difference in synaptic density in geraniin

239

alone group as compared to control group (Figure 3).

240 241

Geraniin Inhibits LPS-Induced Amyloidgenesis in LPS-treated Mice. LPS

242

treatment in mice has been reported to promote Aβ generation and accumulation and

243

induce cognitive impairments. To explore the effect of geraniin on LPS-induced

244

amyloidogenesis, Aβ immunofluorescence was carried out for the determination of

10


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245

Aβ accumulation.

Our results observed a higher level of Aβ accumulation in the

246

LPS-injected mice, while a lower level of Aβ accumulation was found in the

247

hippocampus of mice who received geraniin administration (Figure 4A). In addition,

248

geraniin substantially down-regulated the expressions of APP and BACE1 induced

249

by LPS, thereby intervening in the generation of Aβ. No significant difference in Aβ

250

accumulation or the expressions of APP and BACE1 was found in geraniin alone

251

group as compared to control group (Figure 4B).

252 253

Geraniin Suppresses Microglia and Astrocyte Activation in LPS-treated Mice.

254

To

evaluate

the

suppressive

effects

of

geraniin

on

glial

activation,

255

immunohistochemistry staining was performed with Iba-1 and GFAP antibodies. As

256

illustrated in Figure 5A, Iba-1 and GFAP immunoreactivity findings in the

257

hippocampal CA1, CA3, and dentate gyrus regions of LPS-treated mice were

258

obviously increased as compared with in the control. However, geraniin

259

administration dramatically reduced Iba-1 and GFAP immunoreactivity in these

260

regions. Consistently, the western blot results also indicated that geraniin prevented

261

LPS-induced increases in the protein expressions (Figure 5B). In addition, mice

262

administrated with only geraniin exhibited similar neuroinflammatory response as

263

control mice.

264

Geraniin Shifts Microglial Polarization towards M2 Phenotype in LPS-treated

265

Mice. Polarization of microglia from the M1 to the M2 type is linked to

266

proinflammatory and anti-inflammatory activity, respectively. Real-time PCR analysis

11



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267

was performed for M1 and M2 markers to determine microglial polarization in the

268

hippocampus of LPS-treated mice and the effect of geraniin. As shown in Figure 6,

269

The mRNA expression levels of representative M1 markers (CD16, TNF-α, iNOS,

270

MCP-1) increased and the mRNA expression of several M2 markers (CD206, TGF-β,

271

Arg1, YM-1) were decreased dramatically, as compared with normal control,

272

suggesting that the activated microglia were polarized predominantly to an M1

273

phenotype after LPS stimulation. Compared with LPS group, the elevation of M1

274

marker mRNA expression levels and the reduction of M2 marker mRNA expression

275

levels were strongly attenuated after geraniin administration; suggesting geraniin

276

promotes activated microglia from a proinflammatory M1 phenotype to a potentially

277

more beneficial M2 anti-inflammatory phenotype.

278 279

Geraniin

Suppresses

TLR4-Mediated

NF-κB

Signalling

Pathway

in

280

LPS-treated Mice. TLR4 is an important receptor of LPS and the interaction of

281

TLR4 with adaptor MyD88 leads to the activation of downstream NF-κB and

282

subsequent production of proinflammatory cytokines. As illustrated in Figure 7A and

283

B, geraniin administration substantially suppressed the LPS-induced elevation of

284

TLR4 and MyD88 expression, and the phosphorylation of I-κB and NF-κB.

285

Furthermore, geraniin blocked the translocation of NF-κB from the cytosol to the

286

nucleus (Figure 7C) and reduced NF-κB DNA binding activity (Figure 7D) in

287

LPS-treated mice. Consequently, the content of TNF-α, IL-1β, and IL-6 were

288

significantly increased in LPS-treated mice, whereas geraniin remarkably suppressed

12


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289

them (Figure 7E). Geraniin alone treated mice exhibited similar results to control

290

mice.

291 292

Anti-neuroinflammatory Mechanisms of Geraniin against LPS-induced

293

Neuroinflammation in BV-2 Cells. BV-2 cells were used to elucidate the protective

294

mechanism of geraniin against LPS-evoked neuroinflammation. Our results showed

295

that geraniin exerted potent neuroprotective effects following LPS insult by

296

maintaining cell viability (Figure 8A). We also determined that 50 μM of geraniin

297

was the optimum dose for neuroprotective activity. As shown in Figure 8B, geraniin

298

significantly downregulated the expressions of TLR4, p-NF-κB, and TNF-α in

299

LPS-treated BV-2 cells, indicating the anti-inflammatory activity of geraniin. To

300

further acknowledge the role of TLR4 in geraniin against LPS-elicited

301

neuroinflammation, TLR4 expression was knockdowned by using siRNA. The results

302

revealed that pretreatment with geraniin or TLR4 siRNA could suppress the

303

expressions of p-NF-κB and TNF-α in LPS-treated BV-2 cells. Moreover, the

304

combination of geraniin or TLR4 siRNA even lowered their expressions to baseline

305

levels, which suggested that TLR4-mediated signaling might be critical in ensuring

306

the

beneficial

effects

of

geraniin

on

neuroinflammation

13


(Figure

8C).


307

■ DISCUSSION

308

To our knowledge, this is the first work to comprehensively examine the effects of

309

geraniin on neuronal/synaptic function, amyloidogenesis, microgliosis, and cognition.

310

This study shows that geraniin improved systemic LPS-induced learning and memory

311

deficits and attenuated LPS-stimulated neuroinflammation and its associated

312

amyloidogenesis and neuronal/synaptic injury. Furthermore, this study suggested that

313

the inhibition of the TLR4 signalling pathway might mediate the protective effects of

314

geraniin against LPS-evoked neuroinflammation and neurodegeneration.

315

Activation of the TLR4 receptor is the primary event in the induction of

316

inflammation processe. 32 LPS is primarily recognized by TLR4, 33 which is highly

317

expressed on glial cells in the central nervous system. 34 Stimulation of the TLR4

318

extracellular domain by LPS sequentially provokes the intracellular connection of

319

MyD88 with its cytosolic domain. 35 The downstream signal transduction of the

320

interaction of TLR4 with MyD88 activates the NF-κB pathway, resulting in the

321

upregulation of proinflammatory mediators and the release of inflammatory

322

cytokines. 36 LPS has been found to bind TLR4 at the primary level and induce NF-κB

323

signal pathway and subsequent cellular eventss. 37 Consistent with this, our results

324

also demonstrated the occurrence of TLR4 activation and subsequently downstream

325

inflammatory responses in LPS-treated mice, as indicated by the increased interaction

326

of TLR4 with its adaptor molecule MyD88, NF-κB pathway activation, and the higher

327

level of inflammatory cytokines. Previous studies have reported that geraniin

328

suppresses LPS-induced inflammation via inhibiting NF-κB signaling pathways and

14


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329

the production of proinflammatory cytokines in acute lung injury 23 and in RAW

330

264.7 cells. 24 Similarly, our study observed that geraniin markedly inhibited

331

LPS-induced glial activation and the TLR4 expression, as well as the NF-κB

332

transcriptional pathway. Meanwhile, geraniin could suppress the expressions of

333

p-NF-κB and TNF-α in LPS-treated BV-2 cells. Moreover, the combination of

334

geraniin or TLR4 siRNA even lowered their expressions to baseline levels, suggesting

335

that TLR4-mediated signaling is involved in the protective effects of geraniin on

336

neuroinflammation.

337

Interestingly, geraniin was found to promote microglial polarization towards the

338

M2 phenotype in LPS mice in this present study. Activated microglia can be classified

339

into a proinflammatory M1 phenotype and an anti-inflammatory M2 phenotype,

340

respectively. 38

341

pro-inflammatory mediators and aggravate brain injury. On the contrary, activated M2

342

microglia resolve local inflammation, clear cell debris, and improve neuronal

343

injury. Therefore, inhibition the M1 phenotype or/and promotion of the M2 stage

344

constitute a potential strategy for the treatment of neuroinflammatory disorders. 39

345

Geraniin possesses the ability to regulate the macrophage immune response as other

346

polyphenolic compounds 40 and has been shown to inhibit LPS-triggered THP-1

347

macrophages shifting to the M1 phenotype via the NF-κB pathway.

348

geraniin on the modulation of microglia polarization are likely to contribute to

349

lowered inflammatory damage and increased repair of brain cells, which might

350

underlie, in part, the positive effects of geraniin on cognitive impairment.

Generally,

activated

M1

microglia

15


produce

25

detrimental

The effects of


351

LPS-induced glial overactivation and neuronal impairments were associated with

352

amyloidogenesis in rodents. 41,

42

353

activation as a result of LPS administration induce amyloidogenesis via increasing

354

BACE1 activity, 43 which is a critical APP processing enzyme. Furthermore, NF-κB

355

directly binds to the promoter region of BACE1 and upregulates BACE1 transcription,

356

resulting in an increase of Aβ generation. 44 Consistent with this, our results also

357

observed that LPS treatment triggered a TLR4/NF-κB-based neuroinflammation,

358

which unregulated the expression of BACE1 and subsequently Aβ production.

359

Geraniin, however, significantly suppressed the expressions of BACE1 and APP and,

360

thus, reduced Aβ generation, which might be partly explained by the preventive

361

effects of geraniin on TLR4/NF-κB signaling activation.

Proinflammatory cytokines released by microglia

362

Numerous studies have demonstrated the close association between the structure of

363

the pyramidal cells in the hippocampus and cognitive function. 45 Histopathological

364

analysis indicated the existence of injured pyramidal cells in the hippocampus of

365

LPS-treated mice, as evidenced by the extensive loss of Nissl substance. 46 However,

366

geraniin administration could prevent LPS-elicited neural neurodegeneration in the

367

mouse hippocampus. Furthermore, geraniin restored synaptic functionality via an

368

increase in synaptic density.

369

Few limitations of this study along with questions for future research should be

370

noted. First of all, the plasma concentrations and pharmacokinetics profile of geraniin

371

were undetermined in this study. Since sufficient blood samples could not be obtained

372

from mice at different time points, rats instead of mice are selected and

16


Page 18 of 41

Page 19 of 41


373

pharmacokinetic studies is carried out after oral administration of geranine. Moreover,

374

geraniin, a typical ellagitannin, has been shown to be converted to several metabolites

375

by intestinal microflora after oral administration. Theses ellagitannin metabolites

376

possessed several biological activities such as anti-inflammatory, antioxidant, and

377

neuroprotective effects. Therefore, the effects of geraniin and its metabolites on

378

neuron and cognition need further investigations after oral dosing of geraniin.

379

Geraniin ameliorated systemic inflammation-induced neural/synaptic injury and

380

cognitive impairments through preventing amyloidogenesis, suppressing glial

381

overactivation, and promoting microglia polarization from the M1 phenotype to the

382

M2 phenotype, downregulating neuroinflammatory responses in mice. The inhibition

383

of the TLR4-mediated signaling pathway might be the underlying mechanism by

384

which geraniin affects neuroinflammation. Therefore, our research suggests that

385

geraniin is a potential candidate for amyloidogenesis and the management of

386

neuroinflammation

diseases

such

17


as

AD.


387

Corresponding Author

388

* E-mail: [email protected].

389

* E-mail: [email protected]

390

ORCID

391

DONGMEI WANG: 0000-0002-7920-8626

392

SANQIANG LI: 0000-0001-8452-8205

393

Notes

394

The authors declare no competing financial interest

395

■ ACKNOWLEDGMENTS

Page 20 of 41

396

The present work was supported by National Natural Science Foundation of China

397

(81601225 and U1804174), Science and Technology Innovation Talents in the

398

Universities of Henan Province (20HASTIT044), Henan Provincial Key Research and

399

Development and Promotion Project (192102310081), Science &Technology

400

Innovation teams in Universities of Henan Province (18IRTSTHN026), Outstanding

401

Youth of Science and Technology Innovation in Henan Province (184100510006)

402

■ ABBREVIATIONS USED

403

AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; APP, amyloid precursor

404

protein; COX-2, cyclooxygenase-2; CMC, carboxymethylcellulose sodium; iNOS,

405

inducible

406

1-methyl-4-phenyl-1,2,3,6-tetrahydropyride; PD, Parkinson’s disease; TNF-α, Tumor

407

necrosis

nitric

oxide

synthase;

LPS,

lipopolysaccharide;

MPTP,

factor-α.

18


Page 21 of 41


408

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25



551

Figure Legends

552

Figure 1. Effects of geraniin on LPS-induced spatial learning and memory deficits in

553

mice examined by the Morris water maze. Control mice, LPS mice, Geraniin treated

554

LPS mice (LPS + Gera), and Geraniin mice (Gera) were included. Escape latency

555

during the hidden platform tests (A), representative movement tracks during the

556

training phase (5th day) and the probe test (6th day) (B), the percentage of time in the

557

target quadrant in the probe trial (C), the number of platform crossings in the probe

558

trial (D), the velocity in the probe trial (E), the total path length in the probe trial (F),

559

and escape latency during the visible platform test (G) were measured. Data are

560

expressed as mean ± SEM (n = 12-14 mice per group). **p

Geraniin Attenuates Lipopolysaccharide-Induced Cognitive

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