TacrineâHydrogen Sulfide Donor Hybrid Ameliorates Cognitive

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Tacrine−Hydrogen Sulfide Donor Hybrid Ameliorates Cognitive Impairment in the Aluminum 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*,† †

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogen Biology and Immunology, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China § Illawarra Health and Medical Research Institute and Faculty of Science, Medicine and Health, University of Wollongong, 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(AChE) inhibitor, was previously a prescribed clinical therapeutic agent for AD, but it was recently withdrawn because it caused widespread hepatotoxicity.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. KEYWORDS: 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 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 post-mortem 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 neuro© XXXX American Chemical Society

inflammation and induce impairments in synaptic plasticity and cognitive function.5,6 Therefore, development of multifunctional compounds with anti-inflammatory as well as antiAChE properties is a promising novel avenue to improve AD treatments. Tacrine is the first potent AChE inhibitor approved by the U.S. Food and Drug Administration for the clinical treatment of AD.7 However, the hepatotoxicity induced by tacrine led to Received: February 25, 2019 Accepted: June 3, 2019 Published: June 3, 2019 A

DOI: 10.1021/acschemneuro.9b00120 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 1. Design strategy and synthetic route of the THS. Reagents and conditions: (a) waterless ZnCl2, reflux, 2 h; (b) NaOH, N2, reflux, room temperature, 24 h; (c) activated iron powder, mechanical stirring, room temperature, 2 h; (d) POCl3, reflux, 3 h; (e) 1-pentanol, propane-1,2diamine, reflux, 18 h; (f) HOBT/EDCI, ACS81, ice-water bath, 1 h.

Figure 2. THS improved cognition and locomotor activity in the AlCl3 mouse model of 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 (D) in the open-field test. Data are reported as mean ± SEM (n = 6). *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.

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, the 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 weight (MW: 198, lower than that of 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 a multifunctional compound that has therapeutic effects without hepatotoxicity.

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, H2SB


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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 AD. THS increased H2S levels in hippocampus of AD mice (C). Results are expressed as mean ± SEM (n = 6). *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.

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 mistakes 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. 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 numbers 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 D). Treatment with THS (15 mmol/kg but not 5 mmol/kg) and tacrine increased locomotor activity in these AD mice (both P < 0.01), back to control levels (Figure 2C and D). 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 D). Overall, THS at a dose of 15 mmol/kg improved memory acquisition and locomotor activity in AlCl3-inducedAD 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 (5 and 15 mmol/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. Hippocampal AChE activity was significantly increased in AD mice compared with controls (P < 0.01) (Figure 3B). AChE activity was decreased in THS (15 mmol/kg) (P < 0.05) and tacrine treated groups (P < 0.01) (Figure 3B), but not in the THS (5 mmol/kg) treated group (P > 0.05) compared to the AD mice. Therefore, THS retains AChE inhibitory effects 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

donating compounds could provide effective AD treatment while 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 aluminum trichloride (AlCl3)-induced mouse of AD model,21 including cognition behavior tests, AChE inhibitory activity, expression of H2S level, proinflammatory cytokines (TNF-α, IL-6, and IL1β) 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 synthesized by anthranilonitrile condensed with cyclohexanone to yield 9amino-1,2,3,4-tetrahydroacridine (1) (THA, Figures S1 and S2). Treatment of diallyl disulfide with trimercaptopropionic acid resulted in ACS81 (2) (Figures S3 and S4). Reduction of the nitro group of o-nitrobenzoic acid resulted in anthranilic acid (3). 3 was condensed with cyclohexanone to yield 9chloro-1,2,3,4-tetrahydro-acridine (4) (THC, Figure S5). Treatment of 4 with propane-1,2-diamine led to 9-propanediamine-1,2,3,4-tetrahydroacridine (5) (THN, Figures S6 and S7), which was acylated by 2 to acquire the target compound THS (6) (Figures S8−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 stepthrough 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 shorter stepthrough latency and a larger number of mistakes in the stepthrough test (both P < 0.001) (Figure 2A and B), suggesting cognitive impairment and shorter memory retention in AlCl3 mice. Both THS (15 mmol/kg, but not 5 mmol/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 C


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Figure 4. THS attenuated inflammation in hippocampus of the AlCl3 mouse model of AD. ACS81 and THS decreased mRNA expressions of TNFα (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 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.

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 (5 mmol/ kg, 15 mmol/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 hippocampus of AD mice. THS Attenuates Inflammation in the Hippocampus of AD Mice. Proinflammatory cytokines (TNF-α, IL-6, and IL1β) 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 IL1β in AD mice was 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−C). THS treatment (15 mmol/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−C). 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−C). Tacrine, however, did not inhibit neuroinflammation in the

hippocampus as compared to AD mice. The results showed that THS (15 mmol/kg) decreased proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in the hippocampus, which may be due to its parental H2S component. 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 postsynaptic 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 mice (both P < 0.05) (Figure 4D and E). THS treatment (15 mmol/kg but not 5 mmol/kg) as well as tacrine increased levels of SYN (P < 0.05) and PSD-95 (P < 0.05) in AD mice (Figure 4D and E). Treatment with the H2S donor, ACS81, increased PSD-95 (P < 0.05) but not SYN (P > 0.05) in the hippocampus (Figure 4D and E). Critically, this has shown that THS (15 mmol/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 proinflammatory 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) D


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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.

study the binding modes of THS to acetylcholinesterase, a Surflex molecular docking study was performed. The binding affinity of THS was 8.87 to human acetylcholinesterase and 8.60 to mouse acetylcholinesterase in the 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.

(Figure 5A−C). Furthermore, there were also significant negative correlations between PSD-95 and the mRNA levels of proinflammatory 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−F). 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 nonH2S parent compound (tacrine) and H2S donor (ACS81). Tacrine caused significant hepatotoxicity and inflammation, evidenced by the increased activity of ALT and AST (both P < 0.001) (Figure 6A and B) 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−E). 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). Binding Modes of THS to Acetylcholinesterase by Molecular Docking. Acetylcholinesterase activity is a marker for degeneration of the central cholinergic system.29,30 To

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DISCUSSION In this study, a H2S-donating tacrine hybrid compound 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 (15 mmol/kg/day) improved cognitive and locomotor activity in AD mice, while also reducing inflammation and increasing synaptic plasticity in the E


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Figure 6. Tacrine, but not THS, shows hepatotoxicity and inflammation in the liver of the AlCl3 mouse model of AD. THS did not increase ALT (A) 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 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.

Figure 7. 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 a 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.

aluminum significantly increased AChE activity in the hippocampus. THS treatment was able to inhibit the AChE levels in the serum and hippocampus 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 stepthrough test and open field test, confirming the suitability of this as an AD model, as widely reported previously.36

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 F


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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.

Importantly, THS treatment at 15 mmol/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 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 antineuroinflammatory property of THS may be involved in its cognitive enhancing effect via inhibition of the NF-κB signaling pathway and proinflammatory cytokines transcription. Pre- and postsynaptic hippocampal proteins, SYN and PSD95, 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 cognition by its depolarizing action (blocking K+ channels) on the postsynaptic neuron40 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. 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 (15 mmol/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 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

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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, cyclohexanone, and propane-1, 2-diamine were purchased from Shandong Xiya Chemical Industry Co., Ltd. (Shandong, China). Aluminum chloride (AlCl3) was purchased from Xuzhou Kete Chemical Glass Instrument Co. Ltd. (Xuzhou, China). Experimental Protocol. 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 (15 mmol/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. First, in groups 2−6, all the mice were given 100 mg/kg AlCl3 (ip, every other day) for 50 days,42−44 while the control group were given saline as vehicle. Groups 3−6 were given ACS81, THS (5 and 15 mmol/kg), or tacrine for 20 days after the 50 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

Figure 8. Schematic figure of the experiment protocol. centrifuging at 3000g for 20 min at 4 °C. Brains and liver were rapidly removed, and the hippocampus was dissected. The samples were stored at −80 °C 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 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 s. In the retention trial, the same test procedure was performed for 5 min. 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. G


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ACS Chemical Neuroscience 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 cm2 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 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 3000g for 20 min at 4 °C. Hippocampal and serum levels of AChE, as well as serum levels 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. Measurement of H2S Levels. The hippocampus of mice was weighed and homogenized with 9 times 100 mM cold PBS buffer solution (pH 7.4) at 4 °C. The homogenate supernatants were used to determine the sulfide concentration as described.23 Briefly, using Na2S as an internal standard (x, x + 5, x + 10, x + 15, x + 20, x + 30 μM), the sulfide concentration in spiked hippocampus homogenates was determined by the addition of 1 μL of 1.0 mM NAP-1 probe (final concentration 10 μM). 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 three times 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 treatment groups was performed by determination of the immunoreactive area of the examined protein/β-actin protein. Quantitative Real-Time PCR (q-PCR). Total RNA was isolated from the hippocampus and liver, using the TRIzol reagent (Invitrogen Co., Carlsbad, CA) 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 10 μL final reaction volume using SYBR green I master mix in a Light Cycle 480 (Roche Applied Science, Germany) instrument. 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 software (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. The 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 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

Table 1. Primer Sequences for q-PCR Analysis target mRNa sequences IL-1β

IL-6

TNF-α

β-actin

primer sequence (5′ to 3′)

annealing Tm (°C)

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

59

59

59

59

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 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).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.9b00120.

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Representative 1H NMR, 13C NMR, and MS spectra for the synthesized compounds; tacrine−hydrogen sulfide donor hybrid (THS) analysis (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yi Liu: 0000-0003-0022-9166 Author Contributions ∥

X.-j.C., J.-x.G., and Y.-p.P. contributed equally to this work.

Funding

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). Notes

The authors declare no competing financial interest.

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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 H


Research Article


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