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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:
wdmzgadyx@hotmail.com *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: sanqiangli2001@163.com
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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
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Lipopolysaccharide (LPS),
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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
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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
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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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a white circular pool (100 cm in diameter and 50 cm high). An escape platform with
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10 cm in diameter was positioned 1 cm below the water and maintained constant
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throughout the training. During 5 days of training phase, each mouse was subjected to
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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
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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
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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.
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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
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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
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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
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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
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■ 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
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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
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produce
25
detrimental
The effects of
Journal of Agricultural and Food Chemistry
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
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Page 19 of 41
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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
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as
AD.
Journal of Agricultural and Food Chemistry
387
Corresponding Author
388
* E-mail: wdmzgadyx@hotmail.com.
389
* E-mail: sanqiangli2001@163.com
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.
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Figure Legends
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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