MOLECULAR MEDICINE REPORTS 17: 7293-7300, 2018 Ameliorative effect of nicergoline on cognitive function through the PI3K/AKT signaling pathway in mouse models of Alzheimer's disease GUOYAO ZANG1, LIZHENG FANG1, LIYING CHEN1 and CHENYAO WANG2 1Department of General Practice, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016; 2Department of Acupuncture and Moxibustion, The Third Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310005, P.R. China Received August 28, 2016; Accepted September 26, 2017 DOI: 10.3892/mmr.2018.8786 Abstract. Alzheimer's disease is one of the most common addition to oxidative stress, were decreased in hippocampal age-associated diseases that frequently leads to memory cells treated with nicergoline. Additionally, amyloid precursor disorders, cognitive decline and dementia. Evidence suggests protein accumulation was cleared in the hippocampal area in that nicergoline serves an important role in the apoptosis of nicergoline-treated mice. Nicergoline inhibited neuronal loss hippocampal cells, memory recovery, cognitive function and and prevented cognitive impairment through the restoration neuronal survival. However, the signaling pathway affected by of learning/memory ability. It was additionally demonstrated nicergoline treatment remains to be elucidated. The purpose in the present study that nicergoline improved motor attention of the present study was to investigate the role of nicergoline impairment and cognitive competence in hippocampal cells in the cognitive competence of a mouse model of Alzheimer's by acting on the PI3K/AKT signaling pathway. Therefore, disease. The apoptosis rates of hippocampal cells were studied memory recovery, cognitive function and neuronal survival in mice with Alzheimer's disease treated with nicergoline were repaired by nicergoline via inhibition of the PI3K/AKT compared with the negative control. Apoptosis-associated signaling pathway, suggesting that nicergoline may be an effi- gene expression levels in hippocampal cells, and hippocampus cient drug for the clinical treatment of patients with Alzheimer's area, were analyzed in the experimental mice. Visual atten- disease. tion and inhibitory control were assessed and neural counting was performed in brain regions of interest. The phosphati- Introduction dylinositol 3-kinase (PI3K)/RAC-α serine/threonine-protein kinase (AKT) signaling pathway was additionally analyzed Senile dementia is a multifactorial syndrome characterized in hippocampal cells following treatment with nicergoline. by cognitive functional disorders and cognitive decline (1). The results of the present study demonstrated that nicergoline Alzheimer's disease is among the most common pathogen- ameliorated apoptosis in hippocampal cells and hippocampus eses leading to senile dementia, with a highest incidence rate tissue in 3xTg-AD mice with Alzheimer's disease. The data among the elderly (2,3). Alzheimer's disease is a progressive indicated that apoptosis-associated genes, including caspase-3, neurodegenerative syndrome characterized by the presence of BCL2 associated X, BH3 interacting domain death agonist neurodegenerative disorders (4). Alzheimer's disease primarily and caspase-9, were downregulated in hippocampal cells impairs hippocampus-associated memory and cognition, and isolated from nicergoline-treated experimental mice. In leads to cognitive functional disorders and impairments in addition, the expression levels of inflammatory factors, in cognition, including memory, language or attention (5,6). Previous studies have suggested that Alzheimer's may directly affect hippocampal cells (7-9). The association between corticocerebral and hippocampal neuronal apoptosis has been demonstrated in a rat model of Alzheimer's disease and in a Correspondence to: Professor Chenyao Wang, Department of clinical study (10,11). Dysfunction and aberrant apoptosis of Acupuncture and Moxibustion, The Third Affiliated Hospital of Zhejiang Chinese Medical University, 219 Moganshan Road, hippocampalcells has been observed in preclinical and clinical Hangzhou, Zhejiang 310005, P.R. China studies (12). Inhibition of neuronal apoptosis may attenuate the E-mail: [email protected] phosphorylation of microtubule-associated protein tau (MAPT) and protect memory, which may be beneficial for the treatment Key words: Alzheimer's disease, nicergoline, hippocampus cells, of Alzheimer's disease (13,14). Absalon et al (15) suggested apoptosis, cognitive competence, phosphatidylinositol 3-kinase/RAC-α that the activation of cell cycle entry, MAPT-phosphorylation serine/threonine-protein kinase and the inhibition of apoptosis in post-mitotic neurons through microRNA-regulated gene expression may contribute to the recovery of mice with Alzheimer's disease. These findings 7294 ZANG et al: NICERGOLINE IMPROVES COGNITIVE FUNCTION VIA THE PI3K/AKT PATHWAY suggested that the apoptosis of hippocampal cells is associated Animal study. A total of twenty 6-week-old 3xTg-AD mice with the progression of cognitive impairment, and thus with (male, 28-35 g) with the Alzheimer's disease were purchased the onset and development of Alzheimer's disease. from Vital River Laboratory Animal Technology Co., Ltd. The apoptosis of hippocampal cells is a diagnostic crite- (Shanghai, China). All animals were housed in a tempera- rion for Alzheimer's disease (16,17). Decreased apoptosis ture‑controlled facility at 23±1˚C and relative humidity of in the hippocampus signifies an improvement in cognitive 50±5% with a 12-h light/dark cycle with free access to food impairment. Previous studies have supported the hypothesis and water. The mice were divided into two group (n=10 in that inflammatory responses are the most important patho- each group) and given intravenous injections of nicergoline genic factors for the initiation and progression of Alzheimer's (10 mg/kg body weight) or PBS (10 mg/kg body weight; both disease (18,19). Although Alzheimer's disease has been from Sigma-Aldrich; Merck KGaA) once a day. The treatment extensively studied, comprehensive assessments have not been lasted 60 days. performed in an animal model. The majority of research in animal models of Alzheimer's disease has focused on the Histological and immunohistochemical analysis. Hippocampal neuroprotective effects of anti‑dementia drugs against inflam- tissues were isolated from the CA1 regions of experimental mation, apoptosis and neuronal excitability (20). Oxidative mice following a 60-day treatment with nicergoline (10 mg/kg stress has been associated with Alzheimer's disease and the body weight) or PBS. The brains were frozen in liquid nitrogen development of neurodegenerative processes (21). Therefore, and following perfusion, fixation and cryoprotection, coronal the development of pharmacological agents targeting apop- sections were cut to 4 µm in a cryostat. After blocking with 5% tosis, inflammation and oxidative stress represents a novel Bull Serum Albumin (Sigma-Aldrich; Merck KGaA) for 1 h at prospect for the treatment of Alzheimer's disease. 37˚C, free‑floating sections were rinsed with PBS and placed Nicergoline is a derivative of ergot produced by semi- in a solution with goat anti-mouse amyloid β (Aβ)-42 (1:1,000; synthesis that exhibits the potential to selectively block cat. no. ab201060; Abcam, Cambridge, UK), Aβ-40 (1:1,000; α-1A adrenergic receptors in the brain and periphery (22). cat. no. ab17295; Abcam) and amyloid precursor protein Nicergoline may improve a number of aging-associated (APP; 1:1,000; cat. no. ab32136; Abcam) for 12 h at 4˚C. diseases, including dysphagia, ischemia and dizziness (23-25). Following incubation the sections were washed and incubated Nicergoline is beneficial for the treatment of cognitive impair- with a secondary rabbit anti-goat antibody conjugated with ment (26-28). At present, nicergolineis a registered drug in horseradish peroxidase (1:500; cat. no. A-11034; Chemicon >50 countries and, for the last 30 years, it has been used for the International; Thermo Fisher Scientific, Inc.) for Aβ-42, treatment of cognitive impairment and behavioral disorders in Aβ‑40 and APP staining for 1 h at 37˚C. The sections were the elderly. Therefore, nicergoline may contribute to the treat- washed and observed using a fluorescent video microscope ment of Alzheimer's disease. (BZ‑9000; Keyence Corporation, Osaka, Japan) at magnifica- In the present study, it was hypothesized that nicergoline tion x40. Immunohistochemical staining was used to examine may be able to protect neurons against apoptosis by control- the abundance of neuroprotection-associated proteins in ling the expression of apoptosis‑associated genes. The efficacy the hippocampus. Immunohistochemical procedures were of nicergoline in the prevention of cognitive impairment was performed as previously described (29). evaluated in a rat model of Alzheimer's disease and determined by the assessment of the learning memory. The molecular Immunofluorescence.Hippocampal CA1 regions were isolated mechanism of nicergoline-mediated improvement in hippo- from experimental mice following treatment with nicergoline. campal cells was studied. Although nicergoline demonstrated Hippocampal neural cells were fixed in situ with bromode- neuroprotective effects via stimulation of the phosphatidylino- oxyuridine and NeuN for 24 h at 4˚C. The impairment of sitol 3-kinase (PI3K)/RAC-α serine/threonine-protein kinase neurogenesis was analyzed under a fluorescence microscope (AKT)