INTRODUCTION:

Alzheimer’s disease (AD) is a neurodegenerative disorder seen in elderly individuals. The pathological feature of AD is mainly due to the accumulation of neurofibrillary tangles (NFTs), amyloid plaques and the selective loss of neurons and synapses in the cerebral cortex and hippocampus in the brain^[1-5].60 to 70% cases of dementia are possibly due to Alzheimer disease^[6,7]..AD affects more often the geriatric population, which leads to social and economic burden on the society^[8,9].

Development of AD may be due to various risk factors such as ageing, oxidative stress, environmental and socioeconomic conditions, genetics, and neuroinflammation^[10].Neuroinflammation plays a vital role in developing AD^[11].Females are more prone to develop AD than males^[12,13].

Two types of AD can be found–familial (Late-onset AD) and sporadic (early-onset)^[^8]. Familial AD or FAD is mainly due to the mutation in three genes - presenilin-1, presenilin-2 and Aβ protein precursor genes^[14]. The main gene involved in late-onset AD is Apolipoprotein E (APOE)^[15,16].People in the age group between 30 to 60y accounts for less than 5% of all cases of early-onset AD. When there are multiple factors like–advancing age, female-sex, APOE allele, oxidative stress and a family history of dementia^[17].It is classified as sporadic AD, and it accounts for 95% of all the cases of AD and is expected to rise to 131.5 million by 2050^[18].

Aluminium is the common neurotoxic, non-essential metal present in the environment which can easily get access via water, food, air, utensils, food additives, deodorants and drugs to the human body^[19,20].Aluminium is considered to be one of the most important environmental risk factor causing AD^[21,22,23]. Because of the higher affinity of aluminiumto transferrin receptors in the blood-brain barrier, it crosses the blood-brain barrier and makes the brain vulnerable to aluminium exposure^[^24]. Some of the epidemiological studies have proven the relative relationship between AD and aluminium content in the drinking water and people who are working in aluminium industries are more prone to have impaired cognitive functions^[2].

Aluminium(Al) inside the brain impairs glycol-metabolism and generation of ATP^[2,25].It induces the generation of ROS and also causes mitochondrial dysfunction, which in turn leads to the death of neurons. Al also impairs cholinergic function by enhancing the activity of acetylcholinesterase enzyme, which in turns leads to cognitive impairment and memory loss^[26].Aluminium causes the overexpression of harmful proteins like ß-secretase, γ-secretase and amyloid precursor protein (APP) and amyloid-beta (Aß). Aluminium poisoning leads to misfolding of cytoskeleton proteins which results in the formation of amyloid-beta plaques and tau neurofibrillary tangles in the brain. Al impairs slow and fast axonal transport and inhibits long-term potentiation and hence can cause structural and synaptic changes and induction of inflammatory response which may result in neurodegeneration^[2,27].Various animal models have proved that prolonged exposure of aluminium increases the aluminium content in the brain and also lead to various neurobehavioral, neurochemical and neuropathological changes which in turn causes cognitive impairment and memory loss^[28,29].

Artemisinin is the active principle of the Chinese herb Artemisia annua. Artemether, artesunate and dihydroartemisinin etc. are its derivatives. Artesunate and its active congener dihydroartemisinin are used for the treatment and management of drug-resistant Plasmodium falciparum malaria^[30,31,32].

Neuroinflammation plays a major key role in the pathogenesis of AD, whereas the inflammatory response is mainly a secondary response which in turn is mediated by primary response mediators like trauma or injury, infection etc.Hence, inflammation is an important mechanism inneurodegenerative disorderslike AD ^[33,34]^.Neuroinflammation and behavioural deficits are two important neuropathophysiological features of Alzheimer disease; theyare prevented by inhibiting or blocking the inflammatory pathway. In detail, the mainkey domain is a beta-secretase -1 (BACE 1) expression which is expressed after activation of nuclear factor Kappa B (NF-kB) signaling path way.^[35,36,37].NF-kB signalling pathway factor plays an important role in the activation of the NALP3inflammasome complex which plays an important role in the pathogenesis of AD. Moreover, in another study, it has been proved that by inhibiting inflammatory pathway, we can protect the behavioural and neuropathology in mice model of AD^[36,38,39].

Recent study showed that single dose of artesunate administered in cerebral malaria not only restricted the systemic parasitesbut also regulated inflammatory mediated responses in the brain by reducing the production of local proinflammatory cytokines and other mediators which are involved in cognitive impairment. It also caused different pattern of cytokine expression in a key area related to learning and memory such as frontal cortex and hippocampus^(31).Another study has proved the antiepileptic activity of artesunate in Wistar rats by using MES and PTZ model, where it has been shown to have activity against MES- induced seizures. The anti-epileptic activity of artesunate, can be explained by inhibiting Na+ channel or by inhibiting or blocking glutamatergic excitation neurotransmitter through N-methyl-D-aspartate (NMDA) receptors. NMDA receptor’s antagonists are highly effective in reducing MES- Induced seizures, as compared to PTZ/ Picrotoxin –induced seizures. Hence the role of artesunate on NMDA receptors, the mechanism involved in this regard should be explored in detail, which is being widely used in hospitals for chloroquine-resistant falciparum malaria^[40].

In our previous study, the authors have studied the effect of the combination of artesunate with standard drugs used in AD-like rivastigmine and memantine^[41]. But there were no studies to show the effect of artesunate alone in AD. Therefore, artesunate being water-soluble and more potent with less adverse effects profile compared to artemisinin, the present study has been planned to evaluate the role of artesunatealone on cognition in an Aluminium chloride induced animal model of AD in Wistar rats.

MATERIALS AND METHODS:

Animals:

Thirty adult male albino Wistar rats weighing (150-250g) were collected after obtaining approval from Institutional Animal Ethics Committee (IAEC/KMC/56/2016). Standard laboratory conditions were maintained for keeping the animals throughout the experiment. All the animals were fed with the standard diet and water ad libitumin sterile cages with paddy husk. Standard guidelines were followed throughout the experiment.

Chemicals and Reagents:

All the chemicals required were purchased of standard analytical grade from the Durga laboratory suppliers, Mangalore. Artesunate was purchased from Shaanxi top pharm chemical co., Ltd, AlCl₃was purchased from Merck life sciences private limited, and other drugs were obtained from Sun pharma laboratories limited.

Study Design:

Thirty adult albino Wistar rats were divided into five groups with 6 rats in each group. AlCl₃and drugs were administered orally in their respective groups as shown in (Table 1). AlCl₃ was administered at a dose of 100mg/kg bwin distilled water. Artesunate (28mg/kgbw), Rivastigmine (1mg/kgbw) and Memantine (20mg/kgbw). All the drugs were dissolved in 0.5 % sodium carboxymethylcellulose (CMC). Doses of Aluminium chloride, Artesunate, Rivastigmine, Memantine were selected from the previous studies ^{[8,42,43,44].}

After the drug treatment, all the rats were observed for behavioural changes by passive avoidance test and then sacrificed for observing histological changes in the hippocampus by Hand E staining.

Table 1. Study design

Groups	Treatment
Group 1-Control	Distilled water (8 weeks)
Group 2-Lesion (AlCl₃₎	Aluminium chloride (AlCl₃₎100mg/kg (8 weeks)
Group 3-Artesunate	Artesunate (28mg/kg) (4-8^th week)+AlCl₃ (100mg/kg (8 weeks)
Group 4-Rivastigmine	Rivastigmine (1mg/kg (4-8^th week)+AlCl₃(100mg/kg) (8 weeks)
Group 5-Memantine	Memantine (20mg/kg) (4-8^th week)+AlCl₃(100mg/kg) (8 weeks)

Behavioural studies:

Passive avoidance test:

Passive avoidance testing is a behavioural model used in laboratory rats to assess fear-induced avoidance learning and memory. Passive avoidance testing was conducted according to Rai et al. 2001^[45]. It was conducted in three phases- exploration, learning and retention. The experiment was conducted by following the protocol from the author’s previous study ^[41] and other studies^[46,47,48].

Histopathological analysis:

After the behavioural experiments, rats were sacrificed by a high dose of ether and then the brain was removed after perfusion with ice-cold phosphate-buffered saline and was stored in 10% formalin. The standard protocol was followed for Haematoxylin and Eosin staining to observe the histological changes microscopically in the light microscope (10x/40x)^[49].

Statistical Analysis:

The values were expressed as Mean ± SEM, and all the groups were analysed by one-way analysis of variance (ANOVA) and followed by post hoc Tukey's test using graph pad prism trial version 5.0. The values P<0.05 was considered as statistically significant.

RESULTS:

Artesunate attenuates AlCl₃inducedcognitive dysfunction in Wistarrats.

The effect of Artesunateon cognitive impairment was assessed by Passive avoidance test after 30days of treatment (31^st to 60^th day) with Artesunate, Rivastigmine and Memantine along with aluminium chloride (60daystreatment). The data was compared with normal control (Saline) and aluminium chloride (AlCl₃) (1^st to 60^th day) and with standard drugs Rivastigmine and Memantine. In the exploration phase, there was no significant difference observed between the groups in all the parameters as is shown in figure (1,2,3). In retention phase,24hrs after an electric shock, our first parameter was latency to enter dark compartment where animals in the lesion (AlCl₃) group took very less time (14.50±11.64s) in seconds and showed a significant decrease (p<0.001) compared to control group(126.3±20.76s) where as drug treatments Artesunate (89.83±34.79s), Rivastigmine (81.50±49.95s) and Memantine (133±52.80s) took significantly more time and showed more latency time compared to lesion (AlCl₃) group.(p<0.05,Artesunate, Rivastigmine vs. AlCl₃), (p< 0.001 Memantine vs. AlCl₃)as shown in (Fig.1)

Fig. 1. Time taken to entry into the dark compartment (Latency) in passive avoidance test. A represents Exploration, B represents retention. Values are expressed as mean ± SEM (n=6). Data was analysed by using one-way analysis of variance (ANOVA) followed by tukey’s multiple comparison test. *P<0.05, **P<0.01, and ***P<0.001 vs control and @P<0.02, @@ P<0.01, and @@@P<0.001 vs Lesion Group.

Next parameter in the passive avoidance test was time spent in the dark compartment. During the retention phase, time spent in the dark compartment was more in lesion group (AlCl₃) (120.7±47.16s) compared to the control group (5.50±2.07s)(p<0.001, lesion vs control group). Where as the groups with drug treatments Artesunate (6.5s±4.7s), Rivastigmine (5±3.4s) and Memantine (5.66±4.6s) has spent lesser time in the dark compartment in comparison to Lesion (AlCl₃) group. Since they could remember the electric shock during the learning phase compared to lesion (AlCl₃) group (p<0.001 vs AlCl₃₎ as shown in (Fig. 2.)

Fig.2. Time spent in the dark compartment in passive avoideance test. A represents Exploration, B represents retenion. Values are expressed as mean ± SEM(n=6). Data was analysed by using one-way analysis of variance (ANOVA) followed by tukey’s multiple comparison test. *P<0.05, **P<0.01, and ***P<0.001 vs Control group and @ P<0.05, @@P<0.01, and @@@P< 0.001 vs Lesion Group.

Next parameter wasthe number of crossingsbetween two compartments. During the retention phase, in the lesion(AlCl₃) (2.83±1.16) group number of crossings were more compared tothe control(1.16±0.75) group. Whereas in drug treatments, Artesunate (1.16±0.98), Rivastigmine (1.83±0.98) and Memantine (1.16±1.13)groups, number of crossings were less compared to the lesion (AlCl₃).Though there was no statistically significant difference among the groups.(Fig.3.)

Fig.3. Number of Crossings in passive avoidance test.

A represents Exploration, B represents retention. Values are expressed as mean ± SEM (n=6). Data was analysed by using one-way analysis of variance (ANOVA) followed by tukey’s multiple comparison test. *P<0.05, **P<0.01, and P<0.001 vs Control group and @P<0.05, @@P<0.01, and @@@P<0.001 vs Lession Group.

Artesunate attenuated the cognitive impairment caused by aluminium chloride in the fear-related memory by passive avoidance test and the effect of artesunate on cognition was comparable to the standard drugs Rivastigmine and Memantine in aluminium chloride induced AD.

Effect of artesunate on histopathological changes in AlCl₃ induced AD.

After the behavioural study, animals were sacrificed by decapitation and intracardiac perfusion was done, the brain was removed and processed for histopathological observation by H and E staining, and hippocampal CA1 and CA3 region was observedunder light microscopy. Theneurons of the control group were normal with clear cytoplasm and round cell body with a prominent nucleus in the centre of the cell body both in 10x and 40x and in lesion(AlCl₃)group neurons there was no clear cytoplasm and pyknoticnucleus and cell body was also irregular shaped andappearedlike degenerated neurons(Fig. 4.)

Artesunate, rivastigmine and memantine neurons appear to be normal with some of the degenerated neurons and appeared to look like normal neurons in CA1 region of the hippocampus and by this observation, we can conclude that artesunate and other drugs attenuated the effect of AlCl₃ in the region of the hippocampus. In the CA3 region of the hippocampus (Fig.5.)

The effect of treatment groups appears same as in the CA1 region and the treatment groups attenuated the effect of AlCl₃in both the regions of the hippocampus.

DISCUSSION:

The results of the present study evaluated the effect of Artesunate at a dose of 28mg/kgbw in AlCl₃induced behavioural and histopathological changes in Wistar rats. Significant results were shown in behavioural observations (passive avoidance test) and histopathological changes when compared tothe control, and AlCl₃ groups and also results were comparable with standard drugs. Drug treatment with Artesunate, Rivastigmine and Memantine group of rats was able to increase the latency and decrease the time spent in the dark compartment by remembering the electric shock after 24 hrs compared to the lesion (AlCl₃) group and the results were also comparable to the control. Because of cognitive dysfunction, rats of the lesion (AlCl₃) group did not remember the electric shock and showed less latency and spent more time in the dark compartment when compared to the control group.

The histopathological changes observed were comparable to behavioural observations where the neurons lost their round cell body, and also clear cytoplasm and nucleus were not seen due to the effect of AlCl_3.

Artesunate has improved learning and memory by its anti-inflammatory activity by inhibiting pro-inflammatory cytokines and reactive oxygen species and also by inhibiting other inflammatory mediators. Artesunate showed anti-inflammatory action by inhibiting Nf-kb and NLRP3 inflamma some complex, which in turn decreased the production of cytokines like TNF- α, IL-6 and IL-1β. Therefore, from this previous study it has been proved that NF-kB and NALP3 inflamma some play an important role in the pathogenesis of AD. Moreover, reports from other studies also have shown that the inhibition of inflammatory path way can protect the behavioural and neuropathology in mice model of AD^[36,38,39]. Artemisinin compounds by inhibiting NF-kB signalling pathway prevents the formation of amyloid plaques in AD^[50,51].Artesunate also showed to have antiepileptic activity by acting on NMDA receptors^[40]^. AlCl₃also induces the generation of ROS and proinflammatory cytokines and also causes mitochondrial dysfunction which in turn leads to the death of neurons^[52].Artesunate also shown to modulateneurotrophic factors like BDNF which plays a role in neuron survival and neuronal regeneration. In the present study neurons appear to be normal with round cell body, clear cytoplasm and nucleus in the Centre and were almost comparable to control and standard drugs. Therefore, Artesunate has shown to have attenuated the effect of AlCl₃induced cognitive impairment and neuronal degeneration. Further studies are required to prove the mechanism of neuroprotective activity of artesunate.

CONCLUSION:

Artesunate has shown to have a neuroprotective effect against cognitive impairment caused by AlCl₃at a dose of 28mg/kg. Further study has been carried out to explore the activity of artesunate in different models of cognitive impairment with the possible underlying molecular mechanism responsible for the attenuating effect of artesunate in the neurodegenerative disorder.

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Received on 16.01.2020 Modified on 11.03.2020

Research J. Pharm. and Tech. 2021; 14(2):633-639.

DOI: 10.5958/0974-360X.2021.00113.X