1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with cholinergic dysfunction and impaired redox homeostasis in the brain [
1]. The etiology of AD is associated with the deficiency of a neurotransmitter acetylcholine (ACh), which is involved in the communication between neurons in the brain [
2]. Acetylcholinesterase (AChE) is an enzyme in the brain which breaks down ACh into inactive metabolites choline and acetate, and thus the increased activity of this enzyme can result in a deficiency of ACh. Drugs (AChE inhibitors) that were able to restore the appropriate levels of ACh were developed on the rationale of cholinergic hypothesis of AD, wherein these drugs act by inhibiting the action of AChE [
3]. Therefore, AChE inhibitors are still the mainstay of AD treatment though they do not cure the disease, but play a significant role in palliative management of AD [
4]. Another pathological hallmark of AD is the neurofibrillary tangles in the brain which are formed by hyperphosphorylation and abnormal aggregation of tau protein [
5]. It has been hypothesized that the aggregates of tau in the brains of patients with AD disrupts the microtubule formation and maintenance, damaging axonal transport in the neuronal cells, which results in synaptic starvation and neuronal death [
6,
7,
8]. Accumulating evidence from various studies on tau phosphorylation and neurofibrillary tangles have kindled the need to focus attention on tau as a therapeutic target in the treatment of AD and other tauopathies. The current drugs that are based on amyloid beta (Aβ) have not yielded the desired results, prompting clinicians and researchers to look for alternatives drugs, including phosphorylated tau (P-tau) based therapeutics [
5,
9].
D-galactose (D-gal) is present in the human body as a reducing sugar, but when it exceeds its normal level, it is oxidized into hydrogen peroxide (H
2O
2) and aldehydes by galactose oxidase [
10]. Animals, when treated chronically with D-gal, exhibit aging-related changes such as decreased activities of antioxidant enzymes, increased levels of oxidants, and cognitive impairments [
11,
12]. Further, intraperitoneal injection (i.p) of D-gal affects the cholinergic system resulting in increased levels of AChE in the brains of rats [
13]. Aluminum (Al), is a naturally occurring toxic trace element on earth [
12], which has been linked to pathogenesis of AD [
14]. Kumar [
15] reported an upsurge in AChE activities, oxidative damage, and cognitive dysfunction in rats after chronic administration of aluminum chloride (AlCl
3). Accumulating evidence showed that co-administration of D-gal and AlCl
3 to rats impaired their cognitive functions, increased AChE activities, altered oxidative balance, and induced neurodegeneration [
16,
17,
18]. Therefore, rats chronically administered with D-gal and AlCl
3 could be a good model for studying AD-related pathologies and for screening of anti-AD drugs.
Oxidative stress being an essential component in the pathogenesis of AD, designing multifunctional agents including antioxidative properties for targeting AD is crucial. Several researchers have tried to develop and test dual functioning drugs, which comprises of both “oxidative stress suppressing” moiety and an “acetylcholinesterase inhibitory” moiety [
19,
20]. The plant
Centella asiatica (CA) also known as “Icudwane” in South Africa, “Gotu Kola” in India or “Indian pennywort” in USA has several medicinal properties which includes improvement of cognition and wound healing abilities [
21]. The neuroprotective effects of CA which are seen in animal disease models could be attributed to its antioxidant properties [
22]. CA has also shown to have numerous other pharmacological properties like analgesic and anti-inflammatory properties [
23] and anti-hyperglycemic properties on obese diabetic rats [
24]. Kumar and Gupta reported that the aqueous extract of CA has two prominent effect on the brain that is improving learning and memory and antioxidant properties [
25].
Data from previous research indicated that CA attenuated cognitive impairments in D-gal- and AlCl
3-induced rats through the prevention of hippocampal neuronal death and maintenance of its ultrastructure [
26]. Whether CA can also reduce AChE levels and prevent oxidative stress to attenuate cognitive decline in D-gal- and AlCl
3-induced rats remains unknown. Hence, the current work aimed to study the protective properties of CA on cognition by subjecting rats to the Morris water maze (MWM) test. Subsequent to the behavioral tests, the hippocampal and cerebral cortex tissues of the rats were analyzed for AChE, P-tau, and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD) activities, besides the evaluation of the ultrastructure of their prefrontal cortex using transmission electron microscopy (TEM).
4. Discussion
Preceding studies have reported that co-administration of D-gal and AlCl
3 in rats is an easy and inexpensive method of inducing AD-like pathologies and cognitive dysfunction. Some of the observed changes that have been documented include cognitive decline [
16,
37], oxidative stress, cholinergic dysfunction [
12], pathological alteration of astrocytes [
38], accumulation of beta amyloid and P-tau in the brain [
17], and formation of advanced glycation end products (AGEs) [
39], among others. In the current study, D-gal- and AlCl
3-administered rats displayed impaired cognitive abilities, with marked increases of AChE and MDA levels in both hippocampus and cerebral cortex, while the SOD activity was decreased. Further, ultrastructural aberrations were also observed in the neurons of the prefrontal cortex of the rats’ brains. Hence, the current data proposed that D-gal- and AlCl
3-induced rat model could serve as a good alternative for the study of AD-related pathologies and for anti-AD drugs screening.
Behavioural responses in rats depends on external stimuli such as past experience and endogenous factors such as gender, age and physiological state. The cyclical changes of sex hormones like oestrogen, progesterone and prolactin in female rats influence their emotional and cognitive functions [
40,
41,
42,
43]. These hormonal effects might be confounding factor when testing effects of pharmacological substances on behaviour of female rats. Hence, this study used healthy male albino wistar rats in order to test the effects of CA on their cognitive behaviour after being exposed to D-gal and AlCl
3. In the present study, rats chronically co-administered with D-gal and AlCl
3 showed a cognitive decline in MWM test. This is in agreement with preceding works which have reported cognitive impairments in rats administered with D-gal and AlCl
3 [
43,
44]. There was no decrease in latency to locate the submerged escape platform by D-gal- and AlCl
3-induced rats as the training days progressed, whereas co-administration with CA to these rats attenuated the cognitive impairment induced by D-gal and AlCl
3 in rats as demonstrated by decreased latency to locate the submerged escape platform. Additionally, rats co-administered with CA spent more time in the target quadrant searching for the removed escape platform during the probe trial component of the MWM. It is worthy to note that the cognition-enhancing ability of CA is comparable to that of donepezil, as no differences were observed between the CA groups and donepezil-administered group of rats.
The similarities and differences of primary and tertiary structures of AChEs between species is crucial when selecting AChE inhibitory specificity. However, it is only possible to differentiate between mammals and insects, due to the very high structural, functional, and evolutionary similarities of AChEs among mammals [
45]. The tertiary structures of AChEs from
Felis silvestris,
Bos taurus,
Oryctolagus cuniculus, and
Rattus norvegicus have been predicted to be very similar to that of human structure because the identity of the alignment of their primary sequences with that of humans is very high [
45]. Previous studies on anti-AD therapeutics that measured AChE level in the brain used male albino wistar rats [
16,
46,
47]. Therefore, in the present study, male albino wistar rats were preferred to test the effects of CA on AChE levels. ACh is implicated in numerous neuropsychiatric disorders and plays a vital role in cognitive functions [
48,
49]. The cognitive decline observed in AD has been linked to degeneration of cholinergic neurons in the cerebral cortex and hippocampus, which subsequently resulted in a deficit of cholinergic neurotransmission [
12]. In the present study, an impaired cholinergic system was observed in D-gal- and AlCl
3-administered rats as shown by increased AChE levels in their cerebral cortex and hippocampus. Co-administration of CA with D-gal and AlCl
3 significantly reduced AChE levels in the rats’ brains. Inhibitory effects of CA on AChE activity was reported by Arora [
50] in scopolamine-induced amnesic rats. It has also been reported that perindopril lessened the activities of AChE in D-gal- and AlCl
3-administered rats [
12] and streptozotocin-administered rats [
51]. Hence, the present study suggest that the cognition-enhancing ability of CA could be due to its ability to decrease the level of AChE in the rat’s brain. However, some researchers have documented that AChE activities were decreased in AD [
52,
53]. Thus, more studies are needed to know the exact role of AChE in pathogenesis of AD.
The P-tau protein is now receiving much attention in the field of AD research as a potential target for newer therapeutic agents, and this is due to its involvement in synaptic damage and neuronal dysfunction [
5]. The current study showed increased levels of P-tau in the cerebral cortex and hippocampus of D-gal- and AlCl
3-administered rats
. This result is in conformism with previous studies which have reported similar findings [
16,
17]. When the D-gal- and AlCl
3-administered rats were co-administered with donepezil, the P-tau levels in their cerebral cortex and hippocampus were significantly reduced. However, co-administration of CA to D-gal- and AlCl
3-administered rats did not reduce the levels of P-tau, thus giving the impression that CA is exerting its cognition-enhancing effects probably through a different pathway, rather than through P-tau.
Altered levels of oxidant have been found in patients with AD, which is attributed to either overproduction of oxidant or deficits in antioxidant. Indications from preclinical and clinical studies suggested that oxidative stress is linked with etiopathology of AD [
54,
55], resulting in mitochondrial dysfunction [
56], increased Aβ-mediated neurotoxicity [
57], promotion of synaptic dysfunction, and neuron apoptosis [
58]. The common oxidants that participate in redox state includes nitric oxide (NO), hydrogen peroxide (H
2O
2), hydroxyl radical (OH), hydroxyl anion (OH
−), and peroxynitrite (ONOO
−) [
59]. The correlation between the end product of lipid peroxidation, such as thiobarbituric acid-reactive species (TBARS), generally considered as MDA levels, presence of senile plaques, antioxidants enzymes, and accumulation of neurofibrillary tangles in the brains of AD patients, have also been well documented [
60]. Because of this strong correlation between oxidative stress and cognitive dysfunction, the agents that are capable of modulating the reactive oxygen species (ROS) are thought to play a significant role in the attenuation of cognitive deficits in AD. The present study has shown that there is an increased level of MDA and decreased activities of SOD in the cerebral cortex and hippocampus in D-gal- and AlCl
3-administered rats. However, the imbalance of the oxidative stress biomarkers observed were reversed when D-gal- and AlCl
3-administered rats were co-administered with CA. These results are in agreement with preceding studies that have reported on the antioxidative properties of CA [
61,
62,
63].
The prefrontal cortex is the most evolved region of the brain and subserves the highest order for cognitive abilities. However, it is also the most vulnerable region of the brain to the detrimental effects of stress exposure [
64]. In the present study, the ultrastructure of the prefrontal cortex of experimental rats was examined in order to assess the protective effects of CA. A number of ultrastructural morphological abnormalities were observed in rats administered with D-gal and AlCl
3. Prominent abnormalities that were seen include damaged nucleus, disrupted mitochondria, and alterations of synaptic integrity. Similar morphological changes were also observed in the hippocampus of D-gal- and AlCl
3-administered rats [
26]. Nonetheless, co-administration of CA with D-gal and AlCl
3 to the rats attenuated some of the identified morphological aberrations. Hence, results from the present study suggest that CA could be exerting its neuroprotective properties through the maintenance of neuronal morphology for the optimal function of the brain.
Previous studies have reported a good safety margin of CA in rats at 1000 mg/kg [
65,
66] and a lethal dose at 2000 mg/kg [
66]. In our previous study CA extract at highest dose 800 mg/kg/day for 10 weeks does not affect locomotor activities and speed of the rats as observed from the results of open field test [
26]. CA and its phytochemical constituents such as caffeoylquinic acids and tritepenes have been proved to possess a wide range of biological activities beneficial to human health. These activities includes, neuroprotective, neuritogenic, neuroregenerative, synaptogenic and cognitive enhancing abilities [
67,
68,
69]. Using HPLC analysis four major marker compounds have been reported from the CA extract used in this study including, asiatic acid, madecassic acid, asiaticoside and madecassoside [
27]. Therefore, the neuroprotective and the cognitive enhancing effects of CA seen in D-gal and AlCl
3 induced rats could be due to combined or individual effects of these four major compounds quantified from CA.