1]. As the world’s population ages, we face a looming global epidemic of AD; and by 2050 the number of AD patients is estimated to be 106.4 million globally [2]. Although beta-amyloid (Aβ) accumulation and tau protein malformation are generally accepted to be the causal factors of AD, the cellular and molecular pathology and etiology of AD have not been fully understood, largely due to the fact that AD is a multifactorial disease involving genetic, epigenetic, and environmental factors [3]. Furthermore, present treatments available for AD are usually limited to symptomatic management, and no satisfactory therapeutic agents are available for AD patients [4,5,6]. Thus, novel therapies with better efficacy and safety profiles are urgently needed to modify the natural history of AD.
Aluminum, which can cross the blood brain barrier (BBB) [7], is a well-known neurotoxin that has close association with AD pathogenesis [8]. Based on the studies on the hippocampal neurons of AD patients, aluminum was found to be involved in the formation of neurofibrillary tangles in neurons, and therefore considered as a causative factor for AD [9]. Moreover, studies have also shown that exposure to aluminum can cause memory impairment in animal models [10, 11]. The underlying mechanisms for aluminum to induce AD-like behaviors are believed to involve oxidative damage [11], formation of hyperphosphorylated tau [12], aggregation of amyloid beta protein [13], increase in AChE activity [14] and neuronal apoptosis [13]. In addition to neuronal dysfunction, aluminum could also lead to brain dysfunction via glial alterations [15]. Given that aluminum exposure could eventually result in neuropathological and neurobehavioral changes, and impaired learning and memory, AlCl3-induced model is therefore used as an established AD animal model and often used for testing the efficacy of therapeutic agents for AD.
Uncaria rhynchophylla is a component herb of many popular herbal formulae commonly prescribed for the treatment of AD [16, 17]. Recently, many studies have showed that ethanol extract of U. rhynchophylla has neuroprotective effect on different in vivo and in vitro AD models [18, 19]. Isorhynchophylline (IRN, the chemical structure is shown in Fig. 1), a c-22 oxindole alkaloid richly presented in the ethanol extract of U. rhynchophylla, is the main active chemical ingredient for its biological activities [20]. Our previous studies revealed that IRN could ameliorate the neurotoxicity induced by Aβ25–35 on PC12 cells via PI3K/Akt/GSK-3β signaling pathway [21], improve the memory deficits induced by Aβ25–35 in rats via inhibition of neuronal apoptosis and tau protein hyperphosphorylation [22], and reduce the memory deficits induced by d-galactose in mice through enhancing the antioxidant status and anti-inflammatory effect in brain tissues via nuclear factor kappa B (NF-κB) signaling pathway [23]. Moreover, it was found that the LD50 values of IRN in mice are about 510, 217 and 80 mg/kg, respectively, with oral, intraperitoneal and intravenous administration, indicating that IRN is at least 10 times less toxic than other known anti-AD drugs, and is a safer chemical compound for AD treatment [24]. All these studies implied that IRN is a promising neuroprotective agent for AD treatment warranting further investigation. However, whether IRN has therapeutic effect on AlCl3-treated mice and the underlying mechanisms have not been studied yet. The present study aimed to investigate the neuroprotective effects of IRN on cognitive function in the AlCl3-treated mice and to elucidate the underlying mechanisms of action.
Methods
The minimum standards of reporting checklist (Additional file 1) contains details of the experimental design, statistics, and resources used in this study.
Drugs and reagents
Isorhynchophylline (IRN, purity ≥ 98%) was purchased from Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, Sichuan, China). Aluminum chloride hexahydrate (AlCl3) and donepezil hydrochloride (referred to simply as donepezil thereafter) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals used in the study were of analytical grade.
Animals
Fifty male Balb-c mice (4-month-old, weighing 25–30 g) were obtained from the Laboratory Animal Services Center, The Chinese University of Hong Kong. The animals were maintained on a 12 h light/dark cycle under controlled temperature (22 ± 2 °C) and humidity (50 ± 10%), and given standard diet and water ad libitum.
Animal groupings and drug treatment
The mice were randomly assigned into five groups of 10 animals each: (a) control; (b) AlCl3 plus vehicle; (c) and (d) AlCl3 plus IRN (20 and 40 mg/kg); and (e) AlCl3 plus donepezil (5 mg/kg), which was used as the positive control [22]. The dosages of IRN were selected based on our previous studies [22, 23]. AlCl3 was dissolved in sterile physiological saline at the concentration of 5 mg/mL, and subcutaneously injected into the mice at a dosage of 50 mg/kg once a day for 8 consecutive weeks before the behavioral assessments were started. The duration and dosage of aluminum was selected based on the previous published studies with minor modification [25]. In the control group, mice were subjected to the same injection schedule except using physiological saline instead of AlCl3. At the same time, IRN and donepezil were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na), then given to the mice intragastrically daily 30 min before each AlCl3 injection, while the control group and AlCl3 plus vehicle control group were given the same volume of 0.5% CMC-Na. Behavioral assessments were carried out after the AlCl3 injection was finished. During the behavioral assessment, the treatment was ongoing until the working and reference memory tasks test, which was undertaken at day 64. Detailed experimental schedules were depicted in Fig. 2.
Radial arm maze (RAM) test
Spatial learning and memory changes in mice were determined by the RAM test. A modular radial arm maze with SuperMaze V2.0 video tracking software (Shanghai Xinruan Information Technology Co. Ltd, Shanghai, China) was used for this experiment. The maze consisted of eight arms, numbered from 1 to 8 (35 cm long, 5 cm wide and 10 cm high), extending radially from a central platform (22 cm in diameter). The radial arm maze test was conducted as described previously with minor modifications [26, 27]. In the room, several visual extra-maze cues were provided close (10–30 cm) to the maze in a fixed configuration. A circular food well (1 cm in diameter, 0.5 cm deep) was positioned at the end of each arm. During behavioral training and testing, to stimulate hunger, the animals were kept on restricted diet with only water being available ad libitum and the body weight was maintained at about 85–90% of their free-feeding level. Initially, animals received two 10 min habituation trials, 24 h apart, with free access to all arms on the first 2 days. At the beginning of each habituation trial, three or four mice were simultaneously placed in the central platform and all arms were baited with several approximately 10 mg food pellets. Then, mice were trained for 5 consecutive days, one trial per day, to run to the end of the arms and consume the bait. At the beginning of each training trial, only one mouse was placed in the central platform and only four constant arms were randomly selected and baited with one approximately 10 mg food pellet, which was placed in a food cup that prevented visual detection. A trial was ended when all the four baits had been consumed or after a maximum of 10 min had elapsed. An arm entry was counted when all four limbs of the mice were within an arm. At the eighth day, the mice were subjected to working and reference memory tasks, in which the same four arms were baited as each daily training trial, and the following behavioural measurements were recorded: (1) number of reference memory errors (RMEs), i.e. entries into a non-baited arm, (2) number of working memory errors (WMEs), i.e. re-entries into an already visited baited arm, (3) number of total entries to complete the test.
Preparation of brain samples
Twenty-four hours after the RAM test, mice were decapitated under anesthesia and the brain tissues were removed quickly. After washed with normal saline over the ice, brains were immediately stored at − 80 °C until assay.
Measurement of oxidative stress
To evaluate the effect of IRN on oxidative stress status, the levels of malondialdehyde (MDA) and glutathione (GSH) and the activities of superoxide dismutases (SOD) and catalase (CAT) were measured. For these biochemical analyses, SOD assay kit, GSH assay kit, CAT assay kit and MDA assay kit (Cat. A001-3, A006-1, A007-1 and A003-1, respectively, Nanjing Jiancheng Institute, Jiangsu, China) following the manufacturer’s protocols. CAT activity was determined by the rate of decomposition of 1 μM H2O2 and expressed as U/mgprot. SOD activity was determined by the amount of enzyme required to produce 50% inhibition and expressed as U/mgprot. Reduced GSH was determined based on the formation of a yellow colored complex with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and the