Research Article Cite This: ACS Chem. Neurosci. 2019, 10, 2903−2914
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Selenepezil, a Selenium-Containing Compound, Exerts Neuroprotective Effect via Modulation of the Keap1−Nrf2−ARE Pathway and Attenuates Aβ-Induced Cognitive Impairment in Vivo Jun Yan,† Yanqing Pang,‡ Jialing Zhuang,† Haibiao Lin,† Qiaoxuan Zhang,† Liqiao Han,† Peifeng Ke,† Junhua Zhuang,*,† and Xianzhang Huang*,†
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†
Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China ‡ Department of Integrated Chinese medicine immunization, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510120, China S Supporting Information *
ABSTRACT: Oxidative stress is a major risk factor for neurodegenerative disease. The Kelch-like ECH-associated protein 1 (Keap1)−nuclear factor erythroid 2 related factor 2 (Nrf2)−antioxidant response element (ARE) pathway is one of the most potent defensive systems against oxidative stress. Selenepezil, a selenium-based compound, was previously found to exhibit excellent acetylcholinesterase (AChE) inhibition, to mimic endogenous glutathione peroxidase (GPx) activity, and to exhibit scavenging activity for hydrogen peroxide in vitro. However, none of these activities have been evaluated in a cellular model, and detailed molecular mechanisms are not elucidated. Moreover, whether selenepezil ameliorates memory deficits in vivo remains unknown. This study validated the cytoprotective effect of selenepezil against 6-hydroxydopamine (6-OHDA)- or H2O2-induced SH-SY5Y cell damage via alleviation or neutralization of intracellular microtubule disorder, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and cell apoptosis. Our study clearly demonstrated that selenepezil pretreatment exhibited remarkable cytoprotective effect in a Nrf2-dependent manner via activating the Keap1−Nrf2−ARE pathway and stimulating the transcription of Nrf2−ARE-regulated cytoprotective genes. Moreover, selenepezil·HCl exerts neuroprotective effect via attenuating Aβ-induced cognitive impairment in Alzheimer’s disease (AD) rat and was more active than the reference drug donepezil. In summary, selenepezil deserves further consideration for AD therapy. KEYWORDS: selenium-based compound, Alzheimer’s disease, oxidative stress, neuroprotective effect, Keap1−Nrf2−ARE pathway aggravate the oxidative stress response.5 Therefore, oxidative stress induced by reactive oxygen species (ROS) and the subsequent cell damage are pathogenic causes of neurodegenerative disorders.6,7 Cellular endogenous antioxidant defense systems are actively mobilized under pathological conditions to counter various oxidative stresses. Nuclear factor erythroid 2 related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and antioxidant response elements (AREs) are three primary components of the inducible cell defense system, which mediates the expression of over 100 oxidative stress-related genes.8,9 Nrf2 is anchored by Keap1 to form a complex in the
1. INTRODUCTION Alzheimer’s disease (AD), one of the most horrible and extensive central nervous system (CNS) diseases, is characterized by progressive memory loss and cognitive impairments.1 The pathogenesis of AD is related to a complex and correlative network of multiple factors and etiological hallmarks such as abnormal deposits of β-amyloid (Aβ) peptides, hyperphosphorylation of tau protein, low levels of acetylcholine, neuro-inflammation, metal homeostasis imbalance, and oxidative stress.2 Among these etiologies, oxidative stress seems to play a central role in the network of the disease course and prior to the formation of the pathology.3 Oxidative stress promotes Aβ and tau-mediated neurotoxicity and aggravates the deposits of Aβ and phosphorylation of tau protein, which further leads to the presence of senile plaques and neurofibrillary tangles (NFTs).4 This, in turn, will further © 2019 American Chemical Society
Received: February 18, 2019 Accepted: April 29, 2019 Published: April 29, 2019 2903
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 1. Cytoprotective effect of selenepezil against 6-OHDA- or H2O2-induced cell death.14 (A) Chemical structure of compound selenepezil. (B) Cytotoxicity of selenepezil or donepezil at 10, 15, 25, or 50 μM for 24 h toward SH-SY5Y cells using a CCK-8 assay. (C) Cell damage caused by 6-OHDA or H2O2 at 100, 200, 400, and 800 μM for 12 h. (D) Cell damage caused by 200 μM 6-OHDA or 400 μM H2O2 for 6, 12, and 24 h. (E, F) Dose-dependent protective effects of selenepezil or donepezil. Cells were pretreated with selenepezil or donepezil at 2.5, 5, or 10 μM for 24 h and then exposed to 200 μM 6-OHDA or 400 μM H2O2 for another 12 h. Cell damage was evaluated by CCK-8 assay. All values are shown as mean ± SD (standard error) of at least three independent experiments. **P < 0.01 and ***P < 0.001 vs the vehicle group; #P < 0.05 and ###P < 0.005 vs 6-OHDA- or H2O2-treated group.
(taken off the market because of hepatotoxicity), rivastigmine, donepezil, memantine, and galantamine.23 Among them, donepezil is considered as the most effective drug for AD treatment.24 Selenepezil (Figure 1A), derived from donepezil, designed and first reported by our group, is a typical seleniumcontaining compound that exhibited excellent potency for inhibition of human acetylcholinesterase (hAChE), mimicking the activity of GPx, and exhibiting scavenging activity for hydrogen peroxide in vitro.25 However, these activities were only tested in buffer solution; none of them were evaluated in a cellular model, and detailed molecular mechanisms were also not elucidated. Moreover, whether selenepezil ameliorates memory deficits in vivo remains unknown. Herein, to continue our previous research, the neuroprotective effect of selenepezil against oxidative damage to SH-SY5Y cells was investigated and its potent effect as Nrf2 activator to modulate the Keap1− Nrf2−ARE signaling pathway was also first demonstrated in this study. Furthermore, notable improvements of cognitive and spatial memory in Aβ-induced AD rat model implied its potential therapeutic value for Alzheimer’s disease.
cytoplasm and degraded via ubiquitin-26S proteolysis under normal conditions.10 Sustained ROS generation promotes Nrf2 to escape from degradation by Keap1, translocate into the nucleus, and activate the expression of ARE-dependent genes and proteins, such as NAD(P)H, quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and thioredoxin reductase 1 (TrxR1).11 Therefore, the Keap1−Nrf2−ARE pathway is the primary cytoprotective system to defend against neurodegenerative disorders.12,13 Various small molecule modulators of the Keap1−Nrf2− ARE pathway were discovered from natural sources or designed using synthetic methods during the past decades. Based on the chemical structure or interaction mode with sulfhydryl groups of Keap1, these small molecular modulators are classified as Michael acceptors,14,15 polyenes,16 isothiocyanates (ITCs),17 organosulfur compounds,18 oxidizable diphenols,19 vicinal dimercaptans,20 trivalent arsenicals,21 heavy metal species, and selenium-containing compounds.8,22 So far, only five drugs have been approved by Food and Drug Administration (FDA) for AD therapy, namely, tacrine 2904
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 2. Protective effect of selenepezil against 6-OHDA- or H2O2-induced morphological alterations (A) and intracellular microtubule disorder (B) in SH-SY5Y cells.26 SH-SY5Y cells were pretreated with 10 μM selenepezil for 24 h and exposed to 200 μM 6-OHDA or 400 μM H2O2 for another 6 h. The cell morphology was observed using a phase-contrast microscope (Olympus, Tokyo, Japan). The image obtaining of intracellular microtubules was performed using an LSM 570 laser confocal microscope (Carl Zeiss, Germany). The experiments were performed at least three times, and the representative results are shown.
2. RESULTS AND DISCUSSION Increasing evidence implicates the substantial detriment of ROS-induced oxidative stress in the development and progression of neurodegenerative disease. Our previous report25 demonstrated that selenepezil possessed a rapid scavenging activity for H2O2 and peroxynitrite, mimicked the activity of GPx, and served as a substrate of mammalian TrxR. These activities may be attributed to its antioxidant capacity and are considered as selenium-related pharmacological effects. Has selenepezil maintained its antioxidant activity in cellular models and in AD rat models? If it indeed has, what is the detailed molecular mechanism? Keeping these two questions in mind, we established two kinds of cellular oxidative stress models and the Aβ-induced cognitive impairment animal model to evaluate the neuroprotective effect of selenepezil in vitro and in vivo. Selenepezil Inhibits 6-OHDA- and H2O2-Induced SHSY5Y Cell Death. At first, the cytotoxicity of selenepezil was initially determined using CCK-8 assay (Figure 1A). Donepezil was used as reference compound. Selenepezil exhibited obvious toxicity toward SH-SY5Y cells at a relatively high concentration (50 μM), but no apparent toxicity up to 25 μM (Figure 1B). Two cellular models of neurodegenerative disorders,14 6-OHDA- and H2O2-induced SH-SY5Y cell damage models, were established to evaluate the protective effect of selenepezil. Obvious cell death was observed after exposure to 200 μM 6-OHDA or 400 μM H2O2 for 12 h (Figure 1C,D). Pretreatment with selenepezil for 24 h considerably increased the population of viable cells in a dose-dependent manner. Particularly, 10 μM selenepezil almost completely reversed the cytotoxic effects of 6-OHDA and H2O2. The reference compound donepezil displayed negligible activity against 6-OHDA- or H2O2-induced cell damage, which might be due to its minimal effect on the intracellular oxidation−reduction system. Even though selenepezil was derived from donepezil, besides the target of AChE,
selenepezil, the multitarget-directed ligand, also displayed antioxidative capacity, which further confirmed the positive anti-AD effect of introduction of antioxidative activity. These results demonstrated that selenepezil exerted a significant neuroprotective effect and neutralized the cell damage induced by 6-OHDA and H2O2. Selenepezil Protected SH-SY5Y Cells from 6-OHDAor H2O2-Induced Cell Morphology Alteration and Intracellular Microtubule Disorder. To observe the protective effect of selenepezil more intuitive, phase-contrast microscopy was used to observe the morphological alterations in SH-SY5Y cells. 6-OHDA- or H2O2-treated cells displayed significant morphological changes, including cell rounding and shrinkage and loss of adherence leading to detachment from the plates and floating in the medium (Figure 2A). Treatment with selenepezil alleviated 6-OHDA- or H2O2-induced cell injuries in a dose-dependent manner. Microtubules are critical cytoskeleton components throughout the cytoplasm that are involved in various cellular processes, such as maintenance of cell shape, secretory vesicle transportation, and cell movement and division.26 Stimulated with 6-OHDA or H2O2 for 6 h, the intracellular tubulin− microtubule system was heavily disturbed; the originally slim and fibrous microtubules significantly shrank around the center of the cell nucleus and displayed distinct abnormalities, as evidenced by some dotted disorder formations. These abnormal properties may further induce cell cycle arrest and eventually lead to programmed cell death. Cells pretreated with selenepezil at 10 μM for 24 h exhibited microtubule networks that were regularly assembled and exhibited normal filiform structure wrapped around uncondensed cell nuclei. Selenepezil Alleviated 6-OHDA- or H2O2-Induced Intracellular ROS Accumulation and Mitochondrial Dysfunction. The neurotoxic effects of 6-OHDA and H2O2 are likely relevant to the generation of oxidative stress or interfere with mitochondrial function. Considering the cytoprotective effect of selenepezil was confirmed above, and 2905
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 3. Selenepezil alleviated 6-OHDA- or H2O2-induced intracellular ROS accumulation (A, B) and mitochondrial dysfunction (C, D). SHSY5Y cells were pretreated with 2.5, 5, 10 μM selenepezil for 24 h and then exposed to 200 μM 6-OHDA or 400 μM H2O2 for another 12 h. (A, B) Cell samples were incubated with 10 μM DCFH-DA at 37 °C for 30 min. ROS quantification was detected by flow cytometry using EXPO32 ADC Analysis software, and cell images for intracellular ROS were acquired using fluorescence microscope (Life Technologies, EVOS FL Auto, USA). (C, D) Cell samples were incubated with JC-1 solution for 30 min, and then the intracellular MMP was monitored by fluorescence microscopy and flow cytometry.27 Representative images from three independent experiments are shown.
81.5%, 53.6%, and 7.91%, respectively, in the 6-OHDA-treated group. Similarly, the percentages of ROS decreased to 92.2%, 61.5%, and 26.4% in the H2O2-treated group. Mitochondria are the major generators of ROS, while sustained ROS accumulation leads to mitochondrial dysfunction characterized by decreased mitochondrial membrane potential (MMP, ΔΨm), which can be monitored by JC-1.27 Under normal conditions, living cells exhibit bright red fluorescence, which represents JC-1 aggregates, and relatively weak green fluorescence, which represents JC-1 monomers (Figure 3C). Stimulated with 6-OHDA or H2O2 for 6 h, the fluorescence changes to bright green and weak red fluorescence, which
we have some reasons to hypothesized that it may also alleviate oxidative stress induced by 6-OHDA or H2O2. Stimulation with 6-OHDA or H2O2 for 6 h led to the burst of ROS generation, as evidenced by the appearance of a bright green fluorescence (Figure 3A). Pretreatment with selenepezil at 2.5, 5, and 10 μM for 24 h remarkably and dose-dependently reduced ROS accumulation. ROS accumulation in the 10 μMtreated group was negligible. This effect of selenepezil was also quantified by flow cytometry analysis (Figure 3B). Stimulated with 6-OHDA or H2O2 for 12 h, ROS production was increased to 84.3% and 94.8%, respectively. Pretreatment with selenepezil at 2.5, 5, and 10 μM decreased ROS levels to 2906
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 4. Selenepezil alleviated 6-OHDA- and H2O2-induced cell apoptosis. SH-SY5Y cells were pretreated with 10 μM selenepezil for 24 h and exposed to 200 μM 6-OHDA or 400 μM H2O2 for an additional 12 h. (A) The flow cytometry analysis of apoptosis detected by Annexin V−FITC/ PI staining: (upper left quadrant) necrotic cells; (upper right quadrant) late apoptotic cells; (bottom left quadrant) live cells; (bottom right quadrant) early apoptotic cells. Quantitative analyses were performed using EXPO32 ADC Analysis software. (B) Western blot analysis of Bax, Bad, Bcl-2, and Bcl-xl protein expression. Representative images from three independent experiments are shown. Densitometric quantification was analyzed using Quantity One (Biorad Laboratories) software. Data are presented as the mean ± SEM.
Selenepezil Activated the Keap1−Nrf2−ARE Pathway and Induced Antioxidant Gene Expression. Concluded from the results obtained above, pretreatment of SH-SY5Y cells with selenepezil exerted a neuroprotective effect against 6OHDA- and H2O2-induced cell damage, alleviated ROS accumulation and mitochondrial dysfunction, and thus relieved cell apoptosis. However, which intracellular signaling pathways were involved? This is the main problem to be solved in the following study. The Keap1−Nrf2−ARE pathway is the defense system against oxidative stress triggered by internal or external stimuli.29 Selenium-based compounds are a major category of Nrf2 activators. Therefore, we hypothesized that selenepezil would activate the cellular ARE response. The nuclear and cytoplasmic Nrf2 expression and the subcellular localization of Nrf2 were first examined. Nuclear Nrf2 accumulation was initiated within 3 h, increased maximally at 6 h, and declined after 12 h (Figure 5A). A corresponding decline was observed in cytoplasmic Nrf2 expression. The expression of Keap1, a negative repressor of Nrf2, decreased gradually after selenepezil (10 μM) pretreatment. Immunofluorescence analyses also demonstrated that selenepezil strikingly induced the translocation of Nrf2 from cell cytosol to nucleus (Figure 5B). qRT-PCR analysis demonstrated that exposure of SH-SY5Y cells to 2.5−10 μM selenepezil for 6 h resulted in a sharp increase of the Nrf2-regulated genes (HO-1, NQO1, GCLM, TrxR1, and GCLC) in a dose-dependent manner (Figure 5C). The mRNA levels for HO-1, NQO1, GCLM, and GCLC were maximal after a 3-h treatment with the highest concentration of selenepezil (10 μM), and the mRNA level for TrxR1 reached a maximum at 6 h. The corresponding proteins levels examined by Western blot analysis were also consistent with these results. Treatment
indicates a decrease in MMP. The addition of selenepezil for 24 h nearly reversed the MMP collapse. The quantitative analysis of percentages of JC-1 aggregates or monomers was evaluated using flow cytometry (Figure 3D), and these results were consistent with the results of Figure 3C. Oxidative stress, arising from excessive production and accumulation of ROS, is the primary and most common cause of neurodegenerative diseases. Preventing ROS accumulation and maintaining normal mitochondrial function in neuronal cells may underlie the cytoprotection effect of selenepezil. Selenepezil Relieved the 6-OHDA- and H2O2-Induced Cell Apoptosis. The ROS overproduction and accompanied MMP collapse is of crucial importance in cell apoptosis. Incubated with 200 μM 6-OHDA or 400 μM H2O2 for 12 h, SH-SY5Y cells underwent significant apoptosis (88.2% and 93.6% apoptotic cells, respectively). Pretreatment with selenepezil at indicated concentrations for 24 h remarkably reduced the cell apoptosis (Figure 4A). The B-cell lymphoma 2 (Bcl-2) family, composed of pro-apoptotic proteins (Bax, Bad, Bak, Bid, and Bim) and antiapoptotic proteins (Bcl-2, BclW, and Bcl-xL), are major regulators of mitochondrial stressinduced cellular apoptosis.28 As illustrated in Figure 4B, 6OHDA and H2O2 induced the remarkable downregulation of expression of antiapoptotic proteins (Bcl-2, Bcl-xL) and the corresponding upregulation of expression of the pro-apoptotic proteins (Bax, Bad) (Figure 4B). The expression alterations of these apoptosis-related proteins were offset by pretreatment with selenepezil in a dose-dependent manner. Altogether, selenepezil relieved 6-OHDA- or H2O2-induced cell apoptosis via regulation of the balance of antiapoptotic and proapoptotic members of the Bcl-2 family. 2907
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 5. Selenepezil activated the Keap1−Nrf2−ARE pathway and induced antioxidant gene expression. (A) Effect of selenepezil (10 μM) on the nuclear translocation of the Nrf2 protein and expression of the Keap1 protein. (B) Immunofluorescence staining of Nrf2 at the indicated times in the SH-SY5Y cells treated with selenepezil. Image detection was performed using an LSM 570 laser confocal microscope (Carl Zeiss, Germany). (C) mRNA expression of antioxidant genes HO-1, NQO-1, GCLM, TrxR1, and GCLC induced by selenepezil at 2.5, 5, 10 μM for 3, 6, 12 h in each group detected by quantitative real-time PCR. (D) Western blot analysis of antioxidant proteins HO-1, NQO-1, GCLM, TrxR1, and GCLC after cells were treated with selenepezil at 2.5, 5, 10 μM for 6, 12, and 24 h. Densitometric quantification was analyzed using Quantity One (Biorad Laboratories) software. Data are presented as the mean ± SEM. The experiments were performed at least three times, and the representative results are shown.
with 2.5, 5, or 10 μM selenepezil for 12 h up-regulated HO-1, NQO1, GCLM, TrxR1, and GCLC protein expression in a
dose-dependent manner (Figure 5D) Altogether, these results demonstrated that treatment of SH-SY5Y cells with selenepezil 2908
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 6. Selenepezil exerted cytoprotective effects in a Nrf2-dependent manner. (A) SH-SY5Y cells were first treated with 200 nM brusatol for 1 h, followed by treatment with selenepezil (2.5, 5, or 10 μM) for 24 h and exposure to 200 μM 6-OHDA or 400 μM H2O2 for another 12 h. Cell viability was evaluated by CCK-8 assay. (B) Western blotting of Nrf2 protein expression in SH-SY5Y-shNT and SH-SY5Y-shNrf2 cells. (C) Cytoprotective effect of selenepezil (10 μM) in SH-SY5Y-shNT and SH-SY5Y-shNrf2 cells exposed to 200 μM 6-OHDA or 400 μM H2O2 for 12 h. All values are shown as the mean ± SD (standard error) of at least three independent experiments. ***P < 0.001 vs the vehicle group; ∧∧P < 0.01 and ∧∧∧P < 0.001 vs the 6-OHDA- or H2O2-treated group.
effect in SH-SY5Y-shNT cells as in wild-type SH-SY5Y cells. However, knockdown of Nrf2 completely suppressed the cytoprotective effect of selenepezil against 6-OHDA- or H2O2induced cell damage. These results validated the definite role of Nrf2 in the cytoprotective effects of selenepezil in SH-SY5Y cells. Selenepezil·HCl Ameliorates Aβ25−35-Induced Cognitive Impairment. Aβ peptide deposition is reported to be the main pathogenesis associated with senile plaques (SPs) and neurofibrillary tangles of patients’ brain. Meanwhile, numerous research works have verified that ROS promote the assembly of Aβ into fibrils and accelerate deposition of Aβ peptide, which, in turn, will also further aggravate ROS accumulation. Moreover, AChE also induces Aβ peptide aggregation through directly binding to its peripheral binding site. In the previous study, it was confirmed that selenepezil exhibited excellent effect on AChE-induced Aβ aggregation.25 Hence, in the present study, the Aβ-induced cognitive impairment animal model was established to investigate whether selenepezil has ability to attenuate Aβ-induced cognitive impairments in vivo. The cognitive ability evaluation was conducted using Morris water maze test in 5 consecutive training days. Long-term oral administration with selenepezil·HCl for 30 days did not cause significant weight changes (Figure 7A), which indicated its low toxicity. During the training period, the mean escape latency of rats in each group declined gradually, while the rats in the Aβ25−35-injected model group displayed relatively longer
effectively suppressed Keap1 protein expression and promoted Nrf2 nuclear translocation, thus activating the transcription of numerous cytoprotective genes and ultimately leading to the activation of the Keap1−Nrf2−ARE signaling pathway to maintain cellular homeostasis. Selenepezil Exerted Cytoprotective Effects in a Nrf2Dependent Manner. It has been verified that selenepezil exerted neuroprotective effects via activating the Keap1− Nrf2−ARE pathway. The well-known Nrf2 inhibitor, brusatol, was used to further define the role of Nrf2 in the cytoprotective effects of selenepezil in response to oxidative stress. Cells transfected with shNrf2 were applied. Brusatol enhances the degradation of Nrf2 at a very low concentration.30 SH-SY5Y cells were pretreated with 200 nM brusatol for 1 h, followed by pretreatment with selenepezil (2.5, 5, or 10 μM) for 24 h and exposure to 200 μM 6-OHDA or 400 μM H2O2 for another 12 h. Pretreatment with selenepezil protected SH-SY5Y cells from 6-OHDA- or H2O2-induced cell death obviously (Figure 6A). However, the addition of brusatol (200 nM) prior to selenepezil treatment neutralized the cytoprotective effect of selenepezil. SH-SY5Y cells transfected with an shRNA plasmid specifically targeted Nrf2 in order to obtain stable transformants of SH-SY5Y cells (SHSY5Y-shNrf2 cells). The protein levels of Nrf2 in SH-SY5YshNrf2 cells were dramatically down-regulated (P < 0.001) (Figure 6B). The results in Figure 6C exhibited that selenepezil treatment produced nearly the same cytoprotective 2909
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 7. Treatment with selenepezil·HCl attenuated Aβ25−35 induced cognitive deficits in rats. (A) Body weight record of rats during drug administration period. (B) Escape latency time of each group during the training trial period. (C−H) Effect of memory retention on the spatial probe trial in the Morris water maze test. Swimming speed (C), number of platform crossings (D), path in virtual platform (E) or effective region (F), and time in virtual platform (G) and effective region (H) were record to evaluate the memory retention using a spatial probe trial, with the platform removed and 2-times the diameter of the platform set as the effective region. The results were expressed as the mean ± SD (n = 8). Statistical significance was analyzed by two-way ANOVA: ns P > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 vs the model group.
escape latency, which indirectly indicated the success of model establishment. Treatment with selenepezil·HCl (10 (mg/kg)/ day) or donepezil (10 (mg/kg)/day) led to a decline in escape latency (Figure 7B). Twenty-four hours after the last training trial, the average swimming speed of rats nearly reached the same level as that in controls, which further proved almost the same motility and exploratory activities of rats in each group (Figure 7C). Besides for the swimming speed, compared to the sham group, other indicators including the number of platform crossings and path and time in virtual platform or effective region in the model group were significantly decreased, which strongly demonstrated intrahippocampal injection of Aβ25−35 induced cognitive deficits in rats. The administration of selenepezil·HCl significantly improved the cognitive and spatial memory of rats, which implied its potential therapeutic value for AD (Figure 7D−H). Selenepezil·HCl Strengthens Antioxidative Capacity in Vivo. In order to verify the antioxidative capacity of selenepezil·HCl in vivo, some oxidative stress indicators such as TEAC, SOD activity, GPx activity, and MDA content were detected in brain tissue after behavior testing. As illustrated in Figure 8A, the total antioxidant capacity was tested using Trolox as a standard and expressed as Trolox-Equivalent Antioxidant Capacity (TEAC). In the model group, the TEAC
value was significantly compromised after Aβ treatment. After the administration of donepezil or selenepezil·HCl, the total antioxidant capacity improved obviously (Figure 8A). Additionally, significant increase in SOD and GPx activity in brain tissue were observed following donepezil or selenepezil·HCl treatment after Aβ exposure, while the MDA contents were correspondingly decreased (Figure 8B−D). These results collectively indicated that the neuroprotective effect of selenepezil·HCl against Aβ-induced damage in rats was mediated by its antioxidant capacity.
3. METHODS Chemistry. Compound selenepezil·HCl, the hydrochloride form of selenepezil (chemical code was 7d in our published literature),25 was prepared as follows: compound selenepezil (4.61 g, 10 mmol) was dissolved in 100 mL of CH2Cl2, and HCl gas was bubbled into the solution. After being stirred at room temperature for 2−4 h, the mixture was filtered to obtain a yellow precipitate and then washed with CH2Cl2 and dried under reduced pressure as yellow solid. Yield, 85%. Mp 225.4−226.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 7.75 (s, 1H), 7.63−7.55 (m, 2H), 7.45 (t, J = 3.2 Hz, 3H), 7.22 (s, 1H), 4.22 (d, J = 5.3 Hz, 2H), 3.82 (d, J = 4.6 Hz, 6H), 3.71 (t, J = 6.7 Hz, 2H), 3.28 (d, J = 12.1 Hz, 2H), 2.83 (q, J = 10.5 Hz, 2H), 1.91 (d, J = 11.4 Hz, 2H), 1.83−1.64 (m, 1H), 1.56−1.43 (m, 4H). 13C NMR (126 MHz, DMSO) δ 166.72, 152.52, 148.58, 132.38, 131.90, 130.28, 129.83, 129.15, 120.94, 108.84, 108.74, 59.40, 56.19, 2910
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience
Figure 8. Antioxidative capacity of selenepezil·HCl in vivo. The TEAC content (A), SOD activity (B), GPx acitivity (C), and MDA content (D) in rat brain tissues were determined by the colorimetry method using a microplate reader (Molecular Devices, Flex Station 3). The results were expressed as the mean ± SD (n = 8). Statistical significance was analyzed by two-way ANOVA: ***p < 0.001 vs the sham group, *p < 0.05, **p < 0.01 vs the model group. Cell Viability Evaluation. SH-SY5Y cells grown to the logarithmic phase were seeded in 96-well plates for 24 h and incubated in the presence or absence of different concentrations of selenepezil or brusatol for indicated times. After treatment, 10 μL of CCK-8 solution (Keygen Biotech, Nanjing, China) was added and cells were incubated for another 2 h. The absorbance at 450 nm was recorded by a multifunction microplate reader (Molecular Devices, Flex Station 3).14 Cells for the 6-OHDA and H2O2 injury models were plated for 24 h and pretreated with selenepezil for the indicated times. After replacing with the fresh medium containing indicated concentrations of 6-OHDA or H2O2 for indicated times, the cell viability was determined by the CCK-8 assay as described above. Each experiment was repeated at least in triplicate. Morphological Observations. SH-SY5Ycells for live cell microscopy were seeded into 6-well plates to adhere for 1 day and pretreated with selenepezil for different times. Cell morphology was obtained using a phase-contrast microscope (Olympus, Tokyo, Japan) after replacement with fresh medium containing the indicated concentrations of 6-OHDA or H2O2 for 12 h. Immunofluorescence of Intracellular Microtubules. Immunofluorescence detection was conducted under a previously published procedure.31,32 Briefly, SH-SY5Y cells were plated in confocal culture dishes (NEST Biotechnology, China) for 24 h, pretreated with 10 μM selenepezil for 24 h, and then exposed to 200 μM 6-OHDA or 400 μM H2O2 for another 6 h. The cells were fixed, incubated with the tubulin antibody at room temperature for 2 h, and subsequently incubated with the IgG/Alexa-Fluor 488 antibody (Invitrogen, USA) for 1 h. Image detection was conducted with an LSM 570 laser confocal microscope (Carl Zeiss, Germany). Apoptosis Assay with FITC-Labeled Annexin V and PI Staining. SH-SY5Y cell apoptosis was detected by the FITC-labeled Annexin V/PI staining Apoptosis Detection Kit (Keygen Biotech, Nanjing, China) according to our published procedure.31,32 The prepared SH-SY5Y cell samples were incubated with FITC and PI staining solution according to the manufacturer’s instruction. Almost
56.05, 51.76, 36.62, 30.98, 28.92. The full NMR spectra are provided in the Supporting Information. Materials. The biological activities in cellular experiments were evaluated using selenepezil, while the in vivo animal experiment was performed using selenepezil·HCl due to the increased solubility. Cell Counting Kit-8 (CCK-8) an d Annexin-V/FITC apoptosis kit were purchased from Keygen Biotechnology Company (Nanjing, Jiangsu, China). Mouse anti-α-tubulin antibody, rabbit anti-Bcl-2, Bcl-xl, Bax, Bad, β-actin, GCLC, and TrxR1 monoclonal antibodies, and HRPconjugated goat anti-mouse IgG secondary antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies to Nrf2, Keap1, HO-1, NQO1, and GCLM were acquired from Proteintech (Wuhan, Hubei, China). The GoScript Reverse Transcriptase system and Go Taq qPCR Master mix were obtained from Promega (Madison, Wisconsin, USA). PCR primers specific to HO-1, NQO1, GCLM, TrxR1, GCLC, and GADPH genes were synthesized by Invitrogen (Camarillo, California, USA). Total RNA was extracted by RNAiso plus (Takara, Shiga, Japan). Total protein was quantified by BCA protein assay kit (Thermo Fisher Scientific, Rockford, Illinois, USA). Predesigned shRNA against human Nrf2 and control scrambled sequence (sh-NT) were obtained from Biomics (Biomics, China). Aβ25−35 was purchased from Millipore (Massachusetts, USA). Cell Culture. SH-SY5Y cells, obtained from the laboratory animal center of Sun Yat-sen University (Guangzhou, China) were cultured in a mixture of 1:1 of Ham’s F12 and Dulbecco’s modified Eagle medium (DMEM). The medium was supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 2 mM glutamine, and cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cell Treatment. SH-SY5Y cells in indicated density grown to the logarithmic phase were seeded in plates for 24 h and divided into five groups: control, 6-OHDA, H2O2, 6-OHDA + selenepezil, and H2O2 + selenepezil treatment groups.14 The cells in the control group were cultured normally. Cells were pretreated with indicated concentrations of selenepezil for 12 h and then exposed to 200 μM 6-OHDA or 400 μM H2O2 for indicated times. 2911
DOI: 10.1021/acschemneuro.9b00106 ACS Chem. Neurosci. 2019, 10, 2903−2914
Research Article
ACS Chemical Neuroscience 10 000 events were collected, detected by flow cytometry (Beckman Coulter, Epics XL), and the percentage of apoptotic cells was analyzed by the EXPO32 ADC Analysis software. Determination of Intracellular ROS. The prepared cell samples were incubated with DCFH-DA (10 μM) in blank medium at 37 °C for 0.5 h. Cells for intracellular ROS quantification were washed with PBS three times and immediately detected by flow cytometry.27 Almost 10 000 events were analyzed by flow cytometry (Beckman Coulter, Epics XL). Cell images for intracellular ROS detection were acquired using a fluorescence microscope (Life Technologies, EVOS FL Auto, USA). MMP Measurement. A lipophilic cationic dye named JC-1 (Beyotime, China) was adopted to detect MMP levels according to a published procedure.31,32 The prepared cell samples were incubated with JC-1 work solution for 30 min. The JC-1-loaded samples were detected by flow cytometry (Beckman Coulter, Epics XL) with the excitation wavelength at 488 nm, the emission wavelength at 590 nm (JC-1 aggregates) and 525 nm (JC-1 monomers). Image acquisition was performed using a Zeiss LSM 570 laser scanning confocal microscope. Western Blot Analysis. SH-SY5Y cells were pretreated with or without different concentrations of selenepezil for 12 h. Total proteins were extracted after further treatment with 200 μM 6-OHDA or 400 μM H2O2 for 12 h and quantified by a BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, Illinois, USA). Cytosolic and nuclear proteins were extracted from cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, Illinois, USA), respectively, according to manufacturer’s instructions with moderate modifications. Western blotting analysis was performed according to the published procedure.31−33 Blot images were detected using a LAS4000 imager (GE Healthcare, Waukesha, Wisconsin, USA). The quantification of blot intensities was performed using Quantity One (Biorad Laboratories) software. Quantitative Real-Time PCR. SH-SY5Y cells were treated with selenepezil for 0, 3, 6, and 12 h. Total RNA was isolated using RNAiso plus (Takara, Shiga, Japan) under the manufacturer’s instruction and quantified using 260/280 absorbance on a Trace quantitative instrument (Nanodrop2000, Thermo Fisher, USA). First strand cDNA was synthesized using the GoScript Reverse Transcriptase system (Promega, Wisconsin, USA). RT-PCR was conducted in a CFX96-PCR system (Biorad, USA) using the Go Taq qPCR Master mix (Promega, Wisconsin, USA). The mRNA levels were normalized to GAPDH expression level, which was used as control. The following specific PCR primers were used: HO-1, 5′gccctggaagaggagatagag-3′ and 5′-tagtgctgtgtggctggtgt-3′; NQO1, 5′tcaccactctactttgctccaa-3′ and 5′-ttttctgctcctcttgaacctc-3′; GCLM, 5′ggcacaggtaaaacccaatagt-3′ and 5′-ttcaatgtcagggatgctttct-3′; GCLC, 5′caaggacaagaacacaccatct-3′ and 5′-cagcactcaaagccataacaat-3′; TrxR1, 5′-actgctcaatccacaaacagc-3′ and 5′-ccacggtctctaagccaatagt-3′; GAPDH, 5′-cagtgccagcctcgtctcat-3′ and 5′-aggggccatccacagtcttc-3′. Knockdown of Nrf2 Expression. shNrf2-842, a short hairpin RNA specifically targeting the rat Nrf2 gene, and scrambled sequence (shNT) were used for knockdown of Nrf2 expression. When grown to 60−80% confluence state in culture medium, the SH-SY5Y cells were transfected with shNrf2 or shNT using a transfection reagent under the manufacturer’s instructions. After incubation for 24 h, the transfection solution was discarded and subsequently replaced with fresh DMEM medium containing 10% FBS and 2 mM glutamine. G418 (0.5 mg/mL) was used to select stable transformants of cells with silent Nrf2 expression (SH-SY5Y-shNrf2 cells). The knockdown efficiency was validated using Western blotting. Establishment of AD Models. Chemicals and Reagents. Due to the poor solubility (
