Tacrine-Hydrogen Sulfide Donor Hybrid Ameliorates Cognitive Impairment in the Aluminum Chloride Mouse Model of Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.9b00120

Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by progressive loss of memory and cognitive function, and is associated with the deficiency of synaptic acetylcholine, as well as chronic neuroinflmmation. Tacrine, a potent acetylcholinesterase (AChE) inhibitor, was previously a prescribed clinical therapeutic agent for AD, but it was recently withdrawn because it caused widespread hepatotoxicity. Hydrogen sulfide (H₂S) has neuroprotective, hepatoprotective, and anti-inflammatory effects. In this study, we synthesized a new compound, a tacrine-H₂S donor hybrid (THS) by introducing H₂S-releasing moieties (ACS81) to tacrine. Subsequently, pharmacological and biological evaluations of THS were conducted in the aluminum trichloride (AlCl₃)-induced AD mice model. We found that THS (15 mmol/kg) improved cognitive and locomotor activity in AD mice in the step-through test and open field test, respectively. THS showed strong AChE inhibitory activity in the serum and hippocampus of AD mice and induced increased hippocampal H₂S levels. Furthermore, THS reduced mRNA expression of the proinflammatory cytokines, TNF-α, IL-6, and IL-1β and increased synapse-associated proteins (synaptophysin and postsynaptic density protein 95) in the hippocampus of AD mice. Importantly, THS, unlike tacrine, did not increase liver transaminases (alanine transaminase and aspartate transaminase) or proinflammatory cytokines, indicating THS is much safer than tacrine. Therefore, the multifunctional effects of this new hybrid compound of tacrine and H₂S indicate it is a promising compound for further research into the treatment of AD.

Full text of DOI:10.1021/acschemneuro.9b00120

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Article
A Tacrine-Hydrogen Sulfide Donor Hybrid Ameliorates Cognitive
Impairment in the Aluminium Chloride Mouse Model of Alzheimer’s Disease
Xiao-jing Cheng, Jing-xue Gu, Yi-peng Pang, Jiao Liu, Ting Xu, Xin-rui Li,
Yu-zhou Hua, Kelly A. Newell, Xu-Feng Huang, Yinghua Yu, and Yi Liu
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00120 • Publication Date (Web): 03 Jun 2019
Downloaded from http://pubs.acs.org on June 10, 2019
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A Tacrine-Hydrogen Sulfide Donor Hybrid Ameliorates Cognitive Impairment
in the Aluminium Chloride Mouse Model of Alzheimer’s Disease
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Xiao-jing Cheng1, #, Jing-xue Gu1, #, Yi-peng Pang1, #, Jiao Liu1, Ting Xu1, Xin-rui Li1,
Yu-zhou Hua1，Kelly A. Newell3, Xu-Feng Huang2, 3, Yinghua Yu2, 3* and Yi Liu1,*
1Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou
Medical University, Xuzhou, Jiangsu 221004, China
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2Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen
Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu 221004,
China
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3 Illawarra Health and Medical Research Institute and Faculty of Science, Medicine
and Health, University of Wollongong, NSW 2522, Australia
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Abstract
Alzheimer’s disease (AD) is a neurodegenerative disorder, characterized by
progressive loss of memory and cognitive function, and is associated with the
deficiency of synaptic acetylcholine, as well as chronic neuroinflmmation. Tacrine, a
potent acetylcholinesterase (AChE) inhibitor, was previously a prescribed clinical
therapeutic agent for AD, but was recently withdrawn because it caused widespread
hepatotoxicity. Hydrogen sulfide (H2S) has neuroprotective, hepatoprotective and
anti-inflammatory effects. In this study, we synthesized a new compound, a tacrine-
H2S donor hybrid (THS) by introducing H2S-releasing moieties (ACS81) to tacrine.
Subsequently, pharmacological and biological evaluations of THS were conducted in
the aluminium trichloride (AlCl3)-induced AD mice model. We found that THS
(15mmol/kg) improved cognitive and locomotor activity in AD mice in the
step-through test and open field test respectively. THS showed strong AChE
inhibitory activity in the serum and hippocampus of AD mice and induced increased
hippocampal H2S levels. Furthermore, THS reduced mRNA expression of the
proinflammatory cytokines, TNF-α, IL-6, and IL-1β and increased synapse-associated
proteins (synaptophysin and postsynaptic density protein 95) in the hippocampus of
AD mice. Importantly, THS, unlike tacrine, did not increase liver transaminases
(alanine transaminase and aspartate transaminase) or proinflammatory cytokines,
indicating THS is much safer than tacrine. Therefore, the multifunctional effects of
this new hybrid compound of tacrine and H2S indicate it is a promising compound for
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further research into the treatment of AD.
Key words tacrine; H2S donor; AChE; hepatotoxicity; neuroinflammation; synaptic
plasticity
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Introduction
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory
loss and cognitive decline. The deficiency of synaptic acetylcholine (ACh), chronic
neuroinflammation and the emergence of the toxic amyloid plaques are considered the
main pathogenesis of AD [1, 2]. ACh is degraded by acetylcholinesterases (AChEs),
while AChE inhibitors have been used as mainstream drugs for clinical treatment of
AD [3]. Growing evidence from postmortem and in vivo studies support that the
pathogenesis of AD is also closely related to neuroinflammation [4]. The
proinflammatory cytokines, TNF-α, IL-6 and IL-1β secreted from glia cells
exacerbate neuroinflammation and induce impairments in synaptic plasticity and
cognitive function [5, 6]. Therefore, development of multifunctional compounds with
anti-inflammatory as well as anti-AChE properties, is a promising novel avenue to
improve AD treatments.
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Tacrine is the first potent AChE inhibitor approved by the FDA for the clinical
treatment of AD [7]. However, the hepatotoxicity induced by tacrine led to its
removal from the market [8]. The primary amino group in the tacrine molecule has
been considered to contribute to the hepatotoxicity, with modification of this amino
group decreasing hepatotoxicity [9, 10]. For example, NO donor at the 9-NH2 of
tacrine hybrid compounds have shown to reduce hepatotoxicity and improve
cognition in a rat study of scopolamine-induced cognitive impairment [11]. The
structure of tacrine is simple with high AChE inhibition activity, low molecular
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weight (MW: 198, lower than other approved AChE inhibitors) and good endurance
against structural modification [10, 12]. Therefore, it is practical to use tacrine as a
scaffold for the development of multifunctional compound that has therapeutic effects
without hepatotoxicity.
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Hydrogen sulfide (H2S) is an endogenous signaling gasotransmitter molecule
produced in various parts of the body such as the brain and liver [13]. Levels of H2S
and activity of its synthesized, enzyme cystathione β–synthase (CBS), are severely
reduced in the brain of AD patients [14] and plasma H2S levels are negatively
correlated with the severity of AD [15]. Treatment with H2S or a H2S donor inhibits
proinflammatory cytokines (TNF-α, IL-6, and IL-1β) [16] and improves cognitive
function in AD patients and rat models [17, 18]. Furthermore, H2S has shown
hepatoprotective effects in multiple models of hepatic diseases [19]. For example,
intraperitoneal injection of NaHS (14 µmol/kg) significantly attenuated the severity of
liver injury induced by ischemia-reperfusion [20]. Therefore, H2S-donating
compounds could provide effective AD treatment whilst also attenuating side effects
of tacrine on the liver.
In the present study, we described the synthesis of a new compound,
tacrine-hydrogen sulfide donor hybrid (THS) by introducing H2S-releasing moieties
(ACS81) to the primary amino group in tacrine. Furthermore, the pharmacological
and biological effects of THS were examined in the aluminium trichloride
(AlCl3)-induced mouse of AD model [21], including cognition behavior tests, AChE
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inhibitory activity, expression of H2S level, pro-inflammatory cytokines (TNF-α, IL-6,
and IL-1β) and synaptic proteins in the hippocampus as well as hepatotoxicity studies.
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Results
Compound design and chemistry
The synthesis of THS is described in Figure 1. Tacrine was is synthesized by
anthranilonitrile condensed with cyclohexanone to yield 9-Amino-1, 2, 3,
4-tetrahydroacridine (number 1) (THA, Fig. S1 and S2). Treatment of diallyl disulfide
with trimercaptopropionic acid resulted in ACS81 (number 2) (Fig. S3 and S4).
Reduction of the nitro group of o-nitrobenzoic acid resulted in anthranilic acid
(number 3). 3 was condensed with cyclohexanone to yield 9-Chloro-1, 2, 3,
4-tetrahydro-acridine (number 4) (THC, Fig. S5). Treatment of 4 with propane-1,
2-diamine led to 9- propanediamine -1, 2, 3, 4- tetrahydroacridine (number 5) (THN,
Fig. S6 and S7), which was acylated by 2 to acquire the target compound THS
(number 6) (Fig. S8, S9 and S10).
THS improves cognition and locomotor activity in AD mice
To gain more insight into the therapeutic potential of the hybrid compound,
behavior was assessed using the step-through and open field tests.
The step-through test was performed to evaluate memory acquisition of the passive
avoidance response in AD mice. The AlCl3-induced AD mice showed significantly
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shorter step-through latency and a larger number of mistakes in the step-through test
(both P < 0.001) (Figure 2A and 2B), suggesting cognitive impairment and shorter
memory retention in AlCl3 mice. Both THS (15mmol/kg, but not 5mmol/kg) and
tacrine treated groups showed memory improvement compared with the untreated
AD mice, with increased step-through latencies (both P < 0.01) (Figure 2A) and
decreased number of mistakes (both P < 0.001) (Figure 2B); importantly, these two
groups were not significantly different compared to controls. This indicates that THS
has similar effects as tacrine in overcoming the effects of AlCl3·on cognitive
impairment. The number of mistake was decreased in ACS81 treated mice (P < 0.01)
(Figure 2B), but the step-through latency was not increased (P > 0.05) (Figure 2A),
suggesting the H2S donor improved cognition to some degree.
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Locomotor and exploratory activity were evaluated by measurements of horizontal
activity (number of crossing squares) and vertical activity (number of rearing up onto
the hind legs) in the open-field test [22]. The number of crossings and rearings in AD
mice were significantly decreased compared with the control group (P < 0.001 and P
< 0.01 respectively) (Figure 2C and 2D). Treatment with THS (15mmol/kg but not
5mmol/kg) and tacrine increased locomotor activity in these AD mice (both P < 0.01),
back to control levels (Figure 2C and 2D). Furthermore, the number of crossings and
rearings in the AD mice significantly increased with ACS81 (P < 0.01 and P < 0.05
respectively) (Figure 2C and 2D).
Overall, THS at a dose of 15mmol/kg improved memory acquisition and locomotor
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activity in AlCl3-induced AD mice, showing similar effects as tacrine.
THS retains AChE inhibitory activity in the serum and hippocampus of AD mice
Serum AChE activity was significantly increased in AD mice compared to control
(P < 0.01) (Figure 3A). AChE activity was significantly decreased in THS (5mmol/kg
and 15mmol/kg; both P < 0.05) and tacrine (P < 0.01) treated AD mice compared to
AD mice (Figure 3A). No significant difference was observed between AD mice with
ACS81 treated AD mice.
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Hippocampal AChE activity was significantly increased in AD mice compared with
controls (P < 0.01) (Figure 3B). AChE activity was decreased in THS (15mmol/kg) (P
< 0.05) and tacrine treated groups (P < 0.01) (Figure 3B), but not in the THS
(5mmol/kg) treated group (P > 0.05) compared to the AD mice.
Therefore, THS retains AChE inhibitory effects in in the serum and hippocampus
of AD mice similar to its parent compound, tacrine.
THS increases H2S levels in the hippocampus of AD mice
H2S levels in the hippocampus were examined by using the hydrogen sulfide
fluorescence probe NAP-1[23]. H2S levels were significantly deceased in the
hippocampus of AD mice compared with the control group (P < 0.001) (Figure 3C),
Compared with the AD mice, H2S levels in the hippocampus were increased in
ACS81 (P < 0.01) and the THS (5mmol/kg, 15mmol/kg;both P < 0.05) treated group
(Figure 3C), but not in the tacrine treated group (P > 0.05). These results show that
ACS81 as a H2S donor, is responsible for THS-induced increase in H2S levels in the
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hippocampus of AD mice.
THS attenuates inflammation in the hippocampus of AD mice
Pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) are important factors
contributing to the pathogenesis and development of cognitive impairment. mRNA
expression of TNF-α, IL-6, and IL-1β in the hippocampus were investigated using
Q-PCR. The mRNA expression of TNF-α, IL-6, and IL-1β in AD mice were
significantly increased in the hippocampus of AD mice compared to the control group
(TNF-α, P < 0.001; IL-6, P < 0.05; IL-1β, P < 0.001) (Figure 4A, 4B and 4C). THS
treatment (15mmol/kg) significantly inhibited the expression of IL-1β, IL-6, and
TNF-α in AD mice (TNF-α, P < 0.05; IL-6, P < 0.05; IL-1β, P < 0.01) (Figure 4A, 4B
and 4C). ACS81 treatment had similar inhibitory effects on these cytokines (TNF-α, P
< 0.01; IL-6, P < 0.05; IL-1β, P < 0.001) (Figure 4A, 4B and 4C). Tacrine, however,
did not inhibit neuroinflammation in the hippocampus as compared to AD mice. The
results showed that THS (15mmol/kg) decreased pro-inflammatory cytokines (TNF-α,
IL-6, and IL-1β) in the hippocampus, which may be due to its parental H2S
component.
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THS improves synaptic plasticity in the hippocampus of AD mice
Synaptic proteins are essential for synaptic plasticity and cognitive function [24].
Therefore, we examined pre and post synaptic proteins, Synaptophysin (SYN) and
PSD-95, in the hippocampus of AD mice and the various treatment groups. SYN and
PSD-95 levels were decreased in the hippocampus of AD mice compared with control
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mice (both P < 0.05) (Figure 4D and 4E). THS treatment (15mmol/kg but not
5mmol/kg) as well as tacrine, increased levels of SYN (P < 0.05) and PSD-95 (P <
0.05) in AD mice (Figure 4D and 4E). Treatment with the H2S donor, ACS81,
increased PSD-95 (P<0.05), but not SYN (P > 0.05) in the hippocampus (Figure 4D
and 4E). Critically, this has shown that THS (15mmol/kg) and tacrine both improved
the deficit in synaptic proteins that occurs in in the hippocampus of AD mice.
Pearson’s correlations revealed significant negative correlations between SYN and
the mRNA levels of pro-inflammatory cytokines in the hippocampus (TNF-α:
r=-0.5902, P < 0.01; IL-1β: r=-0.4800, P < 0.05; IL-6: r=-0.5499, P < 0.05) (Figure
5A, 5B and 5C). Furthermore, there were also significant negative correlations
between PSD-95 and the mRNA levels of pro-inflammatory cytokines in the
hippocampus (TNF-α: r=-0.6188, P < 0.01; IL-1β: r=-0.5086, P < 0.05; IL-6:
r=-0.6025, P < 0.01) (Figure 5D, 5E and 5F).
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Tacrine, but not THS, shows hepatotoxicity and inflammation in the liver
Hepatotoxicity is the main limitation of using tacrine in the clinic [25, 26]. It has
been considered that drug-induced liver injury occurs through innate or adaptive
immune responses inducing hepatic inflammation [27]. Moreover, H2S was recently
found to exhibit anti-inflammatory effects [28]. To determine whether the
introduction of a H2S donor could improve the safety of the parent compound, the
effects of THS on hepatotoxicity and liver inflammation were examined and
compared with the non-H2S parent compound (tacrine) and H2S donor (ACS81).
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Tacrine caused significant hepatotoxicity and inflammation, evidenced by the
increased activity of ALT and AST (both P<0.001) (Figure 6A and 6B) as well as
increased mRNA expression of pro-inflammatory cytokines in the liver of AD mice
(TNF-α, P<0.05; IL-6, P<0.01; IL-1β, P < 0.05) (Figure 6C, 6D and 6E). In contrast,
THS did not show hepatotoxicity or liver inflammation as there was no increase in
ALT, AST or and proinflammatory cytokines (TNF-α, IL-6 and IL-1β).
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Binding modes of THS to acetylcholinesterase by molecular docking
Acetylcholinesterase activity is a marker for degeneration of the central cholinergic
system [29, 30]. To study the binding modes of THS to acetylcholinesterase, Surflex
molecular docking study was performed. The binding affinity of THS was 8.87 to
human acetylcholinesterase and 8.60 to mouse acetylcholinesterase in docking score.
Furthermore, THS, was well accommodated into the binding site of human
acetylcholinesterase and formed two hydrogen bonds with the side chains of Tyr124
and Tyr 337 (Figure 7A). The “Tacrine” part of compound THS had strong
hydrophobic interactions with the residues Trp86, Asn87, Pro88, Trp117, Tyr119,
Leu130, Tyr133, His447 and Ile451. It is likely that compound THS has distinct
binding position in the mouse acetylcholinesterase, in which THS formed two
hydrogen bonds with the oxygen atom of backbone of Trp86 and the side chain of
Ser125 (Figure 7B). In addition, the “Tacrine” part of THS had π-π interactions with
the residues Tyr337 and Tyr341.
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Discussion
In this study, a H2S-donating tacrine hybrid compounds was designed and
synthesized as a novel multifunctional AChE inhibitor. The Tacrine-Hydrogen sulfide
donor hybrid (THS) retained strong AChE inhibitory activity in the serum and
hippocampus of AD mice and induced increased hippocampal H2S levels. Importantly,
THS (15mmol/kg/day) improved cognitive and locomotor activity in AD mice, whilst
also reducing inflammation and increasing synaptic plasticity in the hippocampus.
Furthermore, hepatotoxicity studies confirmed that THS was much safer than tacrine.
ACh plays a critical role in memory and cognition, especially in
hippocampus-dependent cognition processes. The cholinergic system is severely
impaired in AD [31]. Aluminum, a potent cholinotoxin, alters the blood brain
barrier,elevates AChE and decreases ACh transmission [32-34]. Consistent with
previous studies [32, 35], we found that the neurotoxic effect of aluminum
significantly increased AChE activity in the hippocampus. THS treatment was able to
inhibit the AChE levels in the serum and hippocamps of these AD mice with
comparable effects to tacrine. Furthermore, we showed that chronic administration of
AlCl3 led to the deterioration of memory and locomotor activity, as evidenced by the
step-through test and open field test, confirming the suitability of this as an AD model,
as widely reported previously [36]. Importantly, THS treatment at 15mmol/kg was
able to reverse the memory deficit and correct locomotion and exploratory behavior
impairments, similar to the effects seen with its parent compound, tacrine. In addition
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to its AChE properties, a novel aspect of THS is that, unlike its parent compound
tacrine, THS is a H2S donor hybrid.
H2S has shown to have anti-neuroinflammatory and neuroprotective properties
when tested in various neurodegenerative disorders such as stroke, AD, and vascular
dementia [6, 15, 37]. In the present study, the H2S donor, ACS81, and the H2S donor
hybrid, THS inhibited hippocampal inflammation as evidenced by decreased the
mRNA expression of proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in AlCl3
induced AD mice. Tacrine, however, did not show these anti-neuroinflammatory
properties, suggesting it is the novel H2S donor addition to the THS compound that is
responsible for its anti-neuroinflammation effects. It is reported that H2S inhibits the
NF-κB signaling pathway, which activates the transcription of TNF-α, IL-6 and IL-1β
[28, 38]. Neuroinflammation is a critical driver of cognitive deficits associated with
neurodegenerative diseases, such as AD [4, 5]. Therefore, the anti-neuroinflammatory
property of THS may be involved in its cognitive enhancing effect via inhibition of
the NF-κB signaling pathway and proinflammatory cytokines transcription.
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Pre-and post-synaptic hippocampal proteins, SYN and PSD-95 are important for
synaptic plasticity and cognition. Deficits in SYN and PSD-95 are correlated with
cognitive decline in AD [39]. In the present study, presynaptic SYN and postsynaptic
PSD-95 were decreased in the hippocampus of AlCl3-induced AD mice. The
AlCl3-induced deficits in synaptic proteins may contribute to the cognitive decline
observed in these mice in the present study. ACh facilitates synaptic plasticity and
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cognition by its depolarizing action (blocking K+ channels) on the postsynaptic neuron
[40] and increasing NMDA mediated postsynaptic plasticity [41]. Importantly, in our
study, THS and tacrine, which have similar AChE inhibitory effects, comparably
prevented the reduction of SYN and PSD-95 in the hippocampus of mice induced by
AlCl3, while the preventive effects of the H2S donor, ACS81, were restricted to
PSD-95. Therefore, the AChE inhibitory property of THS is the main component
required for its improving cognition.
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Tacrine is ability to elevate ACh levels provides an effective strategy for treating
the cholinergic deficiency, which is associated with the brain lesions in patients with
AD. The lack of ability to use tacrine in the clinic is mainly related to its serious
hepatotoxicity side effects, in which elevated liver transaminase levels are induced by
the primary amino group of tacrine [10]. In this study, we confirmed that tacrine
induced hepatotoxicity and liver inflammation in AD mice. However, THS, the new
compound with introduction of ACS81 to block the primary amino group in tacrine,
did not show evidence of hepatotoxicity or liver inflammation as measured by hepatic
transaminases and proinflammatory cytokines. Therefore, the introduction of H2S
donor at the 9-position, ensured THS (15mmol/kg/day) treated animals were free of
hepatotoxicity.
In summary, a H2S-donating tacrine hybrid compound has been designed and
synthesized as a novel multifunctional AChE inhibitor. Using the AlCl3-induced
cognitive impairment animal model, THS induced significant improvements in
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cognition and locomotion. In further examination of the underlying mechanisms, THS
showed strong AChE inhibitory activity in the serum and hippocampus of AD mice.
THS also increased H2S levels, decreased inflammation and improved synaptic
plasticity in the hippocampus. Furthermore, in hepatotoxicity studies, THS did not
increase liver transaminase ALT, AST or proinflammatory cytokines (TNF-α, IL-6
and IL-1β) indicating it is much safer than tacrine. Altogether, the multifunctional
effects of this new hybrid compound qualify it as potential candidate anti-AD drug
and a promising compound for further research.
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Materials and Methods
Animals
Experiments were performed in adult male Kunming mice of similar size， which
were obtained from the Experimental Animal Center of Xuzhou Medical University
(Xuzhou, China, SYXK (Su) 2010-0011). The mice were housed with ad libitum
access to food and water under temperature- and humidity-controlled conditions with
a 12 h light/dark cycle. All procedures were approved by the Animal Ethics
Committee, Xuzhou Medical University, China, and complied with the National
Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Drugs
Anthranilonitrile, cyclohexanone, diallyl disulfide, trimercaptopropionic acid ，
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cyclohexanone and propane-1, 2-diamine were purchased from Shandong Xiya
Chemical Industry Co., Ltd. (Shandong, China). Aluminium chloride (AlCl3) was
purchased from Xuzhou Kete Chemical Glass Instrument Co. Ltd. (Xuzhou, China).
Experimental protocol
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The mice were randomly assigned into six groups (n =15 per group): 1) control
group; 2) AlCl3 induced AD group; 3) H2S donor ACS81 treatment group
(15mmol/kg/day, i.p.); 4) THS (5 mmol/kg/day, i.p.) treatment group; 5) THS (15
mmol/kg/day, i.p.) treatment group; 6) Tacrine (15 mmol/kg/day, i.p.) treatment group.
Firstly, in Groups 2-6, all the mice were given 100 mg/kg AlCl3 (ip, every other day)
for fifty days [42-44], while the control group were given saline as vehicle. Groups
3-6 were given ACS81, THS (5mmol/kg), THS (15mmol/kg) or tacrine for twenty
days after the fifty days, while the AlCl3 induced AD group was given 0.5% (w/v)
sodium carboxymethylcellulose (CMC-Na) solution as vehicle treatment. After the
treatment period, animals were tested for cognition and locomotor activity in
step-through and open-field test (Figure 8). Blood was collected and serum was
separated by centrifuging at 3000 ×g for 20 mins at 4oC. Brains and liver were rapidly
removed, and the hippocampus was dissected. The samples were stored at -80oC for
the pharmacological and biological evaluations.
Behavioral tests
The step-through test was operated to examine memory acquisition as anteriorly
described [45]. Briefly, the test included a training trial on the first day and a retention
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trial on the second day. The apparatus consisted of a compartment with two chambers
(a light chamber stocked with a dark and an illuminator chamber) and an
interconnecting semicircular door. In the training trial, the mice were placed in the
light chamber for 3 min. After the door opened, the mice moved to the dark chamber
and got electric shocks for 1 sec. In the retention trial, the same test procedure was
performed for 5 minutes. The latency between the placement in the light chamber and
the entry into the dark chamber was recorded as step-through latency reflecting
memory retention. The entering events during the testing trial were recorded as the
number of mistakes.
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The open field test was performed as previously described to assess general
movement (locomotor and behavioral activity) [22]. Observations were recorded in a
white open-field square box (50 cm× 50 cm × 50 cm) that was divided into 10 × 10
cm equal squares. Each mouse was allowed to freely explore for 5 min. All behaviors
were recorded using an infrared camera and video system, followed by analysis of the
number of square crossings and rearings by a mouse behavior analysis system
(SLY-ETS, Beijing Shuo Lin Yuan Technology, Beijing, China).
Enzyme linked immunosorbent assays (ELISAs)
The refrigerant hippocampal tissues were homogenized in ice-cold normal saline
o
and centrifuged at 12,000 rpm at 4 C for 20 min. The supernatants were then
collected. Blood was collected and serum was separated by centrifuging at 3000 ×g
for 20 mins at 4oC. Hippocampal and serum levels of AChE, as well as serum levels
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of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were
evaluated by the use of enzymatic kits (Shun ran Biotechnology, Shanghai, China),
following the instructions of the manufacturer. The concentration of AChE, AST and
ALT in the samples was determined by comparison to a standard curve.
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Measurement of H2S Levels
The hippocampus of mice was weighed and homogenized with 9 times 100mM
cold PBS buffer solution (pH 7.4) at 4 oC. The homogenate supernatants were used to
determine the sulphide concentration as described [23]. Briefly, using Na2S as an
internal standard (X, X + 5, X + 10, X + 15, X + 20, X + 30μM), the sulphide
concentration in spiked hippocampus homogenates was determined by the addition of
1μL 1.0mM NAP-1 probe (final concentration 10μM). All fluorescence measurements
were made on a Hitachi F4600 fluorescence spectrophotometer (Tokyo, Japan).
Western blot
As we reported earlier [46], membranes containing the separated proteins, were
finally incubated with the following primary antibodies overnight: SYN (1:1000)
(RRID:AB_1904154), PSD-95 (1:1000) (RRID:SCR_013516) and β-actin (1:1000)
(RRID:AB_2560935). Membranes were washed thrice with TBST over 15 min and
incubated with secondary antibodies (ZSGB-BIO, Beijing, China) in TBST. The
membranes were exposed to BCIP/NBT alkaline phosphatase color developing
reagent (Beyotime Institute of Biotechnology, Shanghai, China) for 15 min. All
quantitative analyses were normalized to β-actin. Comparison between different
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treatment groups was performed by determination of the immunoreactive area of the
examined protein/β-actin protein.
Quantitative Real-time PCR (q-PCR)
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Total RNA was isolated from the hippocampus and liver, using the TRIzol® reagent
(Invitrogen Co., Carlsbad, CA, USA), and reverse transcribed into cDNA using a
High Capacity RNA-to-cDNA kit (TOYOBO, Japan), according to the
manufacturer’s instructions. PCRs were carried out in a 10μL final reaction volume
using SYBR green I master mix in a Light Cycle 480 (Roche Applied Science,
Germany). Amplification was performed with a thermal profile of 10 min at 95 °C
followed by 45 cycles of 15 s at 95 °C, 30 s at 60 °C, a melting curve of 15 s at 95 °C,
60 s at 1 min, heating to 95 °C, and cooling for 30 s at 4 °C. The results were
analyzed using Roche LightCycler 480 soft-ware (version 1.5, Roche Applied Science,
Germany). Relative levels of mRNA were analyzed using the ΔΔCt method.[47]. The
primers used are detailed in Table 1.
Molecular docking studies
Surflex molecular docking module in Sybyl X-2.1 (SYBYL_X2.1 is available from
Tripos Associates Inc., S Hanley Rd., St. Louis, MO 631444, USA) was used for
conducting molecular docking to investigate the binding modes of THS to
acetylcholinesterase. The 3D structure of THS was built in Sybyl X2.1 and charged
by the Gasteiger-Huckel method. Energy minimization was performed on THS using
the Tripos force field with energy optimization gradient convergence criterion of
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0.001 kcal/mol•Å and a maximum optimal step size of 1000. The crystal structure of
human acetylcholinesterase was obtained from the RCSB Protein Data Bank, with
PDB ID of 4M0E. The crystal structure of mouse acetylcholinesterase was 4B82. The
Protomol module was used to define the binding site of THS. During the preparation
of receptor protein, all water molecules were removed and the missing hydrogen
atoms were added by biopolymer module. The docking parameters were kept as
default values and the binding affinities were evaluated by docking score as described
in previous studies [48, 49].
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Statistical analysis
The data were analyzed using One-way ANOVA followed by LSD post hoc
comparisons for data with equal variances, or by Dunnett’s T3 for data with unequal
variances. All values are showed as mean ± SEM. All analyses were performed using
SPSS16.0. P values of less than 0.05 were regarded as statistically significant. Graphs
were produced using Prism5 (RRID:SCR_002798).
Abbreviations H2S: Hydrogen sulfide; THS: Tacrine-Hydrogen sulfide Donor Hybrid;
AChE: Acetyl-cholinesterase; AD: Alzheimer’s disease; ip: Intraperitoneal; IL-1β:
Interleukin-1β; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; SYN:
synaptophysin; PSD-95: postsynaptic density protein 95.
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ASSOCIATED CONTENT
Supporting Information
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Figure S1-11 Representative H NMR, C NMR and MS spectra for the synthesized
compounds. Figure S9-11 for the structure of Tacrine-Hydrogen sulfide Donor Hybrid
(THS) analysis.
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AUTHOR INFORMATION
*Corresponding Author.
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Professor Yi Liu PhD, Jiangsu Key Laboratory of New Drug Research and Clinical
Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China. Email:
cbpeliuyinew@163.com
Dr. Yinghua Yu, PhD. Jiangsu Key Laboratory of Immunity and Metabolism,
Department of Pathogen Biology and Immunology, Xuzhou Medical University,
Xuzhou, Jiangsu 221004, China. Email: yinghua@uow.edu.au
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Author Contributions
#These authors contributed equally to this work.
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Funding and Disclosure
The authors declare that they have no competing interests. This work was supported
by Chinese National Natural Science Foundation （ 81671069 ） and the Open
Research Project of Jiangsu Key Laboratory of Immunity and Metabolism
(JSKIM201802).
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Figure legends
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Table.1 Primer sequences for q-PCR analysis
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Figure 1 The design strategy and synthetic route of the THS. Reagents and
conditions: (a) waterless ZnCl2, reflux, 2h; (b) NaOH, N2, reflux, room temperature,
24 h; (c) activated iron powder, mechanical stirring, room temperature, 2 h; (d)
POCl3, reflux, 3h; (e) 1-pentanol, propane-1, 2-diamine, reflux, 18 h; (f)
HOBT/EDCI, ACS81, ice-water bath, 1 h.
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Figure 2 THS improved cognition and locomotor activity in the AlCl3 mouse model
of Alzheimer’s disease (AD) as measured via step-through latency (A) and number of
mistakes (B) in the step-through test. Number of crossings (C) and number of rearings
*
**
558
(D) in the open-field test. Data are reported as mean ± SEM (n= 6). P < 0.05, P <
#
##
559
560
0.01, ***P < 0.001, as compared with the control group; P < 0.05, P < 0.01, ###P <
0.001, as compared with the model group.
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Figure 3 THS retained the acetylcholinesterase (AChE) inhibitory activity in the
serum (A) and hippocampus (B) of the AlCl3 mouse model of Alzheimer’s disease
(AD). THS increased H2S levels in hippocampus of AD mice (C). Results are
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*
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expressed as mean ± SEM (n=6). P < 0.05, P < 0.01, ***P < 0.001, as compared
#
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with the control group; P < 0.05, ##P < 0.01, ###P < 0.001, as compared with the
model group.
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Figure 4 THS attenuated inflammation in hippocampus of the AlCl3 mouse model of
Alzheimer’s disease (AD). ACS81 and THS decreased mRNA expressions of TNF-α
(A), IL-6 (B), and IL-1β (C) in the hippocampus. THS and tacrine restored synaptic
proteins, SYN (D) and PSD-95 (E) in the hippocampus of AD mice. Results are
*
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expressed as mean ± SEM (n=3). P < 0.05, P < 0.01, ***P < 0.001, as compared
#
574
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with the control group; P < 0.05, ##P < 0.01, ###P < 0.001, as compared with the
model group.
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Figure 5 Correlations between synapse-associated proteins and mRNA expressions of
TNF-α, IL-1β and IL-6. There were negative correlations between synapse-associated
proteins (SYN and PSD-95) and TNF-α, IL-1β and IL-6 (A–F).
Figure 6 Tacrine, but not THS, shows hepatotoxicity and inflammation in the liver of
the AlCl3 mouse model of Alzheimer’s disease (AD). THS did not increase ALT (A)
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or AST (B) levels in the serum. Results are expressed as mean ± SEM (n=6). THS did
not increase mRNA levels of TNF-α (C), IL-6 (D) or IL-1β (E) in the liver of the
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**
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AlCl3-induced AD mice. Results are expressed as mean ± SEM (n=3). P < 0.05, P
< 0.01, ***P < 0.001, as compared with the control group; #P < 0.05, ##P < 0.01, ###P <
0.001, as compared with the model group.
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Figure 7 The simulated binding modes of four representative compounds. THS was
formed two hydrogen bonds with the side chains of Tyr124 and Tyr 337 in human
acetylcholinesterase (A). The “Tacrine” part of compound THS had strong
hydrophobic interactions with the residues Trp86, Asn87, Pro88, Trp117, Tyr119,
Leu130, Tyr133, His447 and Ile451. THS had distinct binding pose in the mouse
acetylcholinesterase (B). THS formed two hydrogen bonds with the oxygen atom of
backbone of Trp86 and the side chain of Ser125. The “Tacrine” part of THS had π-π
interactions with the residues Tyr337 and Tyr341.
Figure
8
Schematic
figure
of
the
experiment
protocol
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Tabel 1
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Target mRNA
Primer sequence
Annealing
Tm (°C)
59
sequences
（5′ to 3′）
IL-1β
R-CTT CTC CAC AGC CAC ATT GA
F-AGT TGA CGG ACC CCA AAA G
R-ATT TCC ACG ATT TCC CAG AG
F-CGG AGA GGA GAC TTC ACA GAG
R-CAG TAG ACA GAA GAG CGT GGT G
F-AGG CAC TCC CCC AAA AGA T
R- TTT AAT GTC ACG CAC GAT TTC
F- CCC ATC TAT GAG GGT TAC GC
IL-6
59
59
59
TNF-α
β-actin
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