Cognition enhancing abilities of vitamin D, epalrestat and their combination in diabetic rats with and without scopolamine induced amnesia

Diabetes Mellitus (DM) is a dynamic metabolic disease that can have debilitating effects on numerous organ systems in the body, including the nervous system, through a variety of mechanisms (Kodl and Seaquist 2008). One of the well-recognized but less addressed complications of diabetes is cognitive dysfunction leading to impairment of learning and memory. Period of cognitive dysfunction in DM is thought to be linked to increased apoptosis and reduced neuronal density in hippocampus diabetic rats (Li et al. 2002). Factors which play possible important role in pathogenesis of diabetes induced learning and memory deficit are (I) oxidative stress, (II) metabolic impairment in the form of diabetic hyperglycaemia due to insulin resistance and (III) vascular complications (Hasanein and Shahidi 2010). Oxidative stress is generally recognized as the primary mediator for the production and progression of diabetic complications. Oxidative stress is characterized as a chain of events caused by an imbalance between oxidative substances and antioxidant ability, followed by an exacerbated development of reactive oxygen species and weakened antioxidant defences (Li et al. 2016). The hippocampus and the amygdala are important in the consolidation of learning and memory. Hippocampus is mainly involved in consolidation of declarative memory (Halbach 2007). Short-term memory conversion into long-term memory is severely affected when hippocampus is damaged. Spatial memory is also affected in case of hippocampal damage (Burgess et al. 2001).

Diabetic hyperglycaemia can cause a polyol pathway that converts glucose to sorbitol by a rate-limiting enzyme called aldose reductase. Sorbitol can be converted to fructose using fructose reductase, but the absence of fructose kinase in peripheral nervous tissue causes sorbitol to build up intracellular hypertonia and inhibits inositol uptake (Kodl and Seaquist 2008). Intracellular inositol is depleted, resulting in oedema, demyelination and necrosis of nerve cells. These mechanisms may be operational in the brain and may contribute to alterations in cognitive function that have been identified in patients with diabetes (Kodl and Seaquist 2008). Epalrestat (5-[(1Z, 2E)-2-methyl-3-phenyl propenylidene]-4-oxo-2-thioxo-3-thiazolidine acetic acid) is an aldose reductase inhibitor (ARI) authorized in Japan for the treatment of diabetic neuropathy and considered to have limited adverse effects with the well-proven antioxidant and anti-inflammatory properties (Hotta et al. 2006). Anti-inflammatory and antioxidant effects are recorded with rat Schwann cell and human neuroblastoma cell line (Yama et al. 2016; Sato et al. 2013; Ohmura et al. 2009). 1, alpha-25 dihydroxyvitamin D3 (1, 25-(OH)₂ D₃) is associated with the development and functioning of the brain (Mathieu and Badenhoop 2005; Garcion et al. 2002). Some vitamin D target gene products and their regulated processes are known to be involved in the critical functions necessary for cognition and behaviour. Vitamin D can also be involved in the planning, processing and formation of learning and memory (Garcion et al. 2002; McCann and Ames 2008; Moghadamnia et al. 2015; Buell et al. 2009). Thus both vitamin D and epalrestat have found to have the positive effect on learning and memory, therefore this study aims to combine vitamin D and epalrestat to assess the effect of this combination and compare it with donepezil which is a cerebro-selective inhibitor of acetyl-cholinesterase (AChE).

The present study was conducted to screen and determine the neuroprotective, neuro modulatory and anti-oxidant activities of Vitamin D, epalrestat and its combination against persistent hyperglycaemia; with and without scopolamine induced amnesia in rodents. In humans, it is a known fact that diabetes status, oxidative stress, ageing and cholinergic nerve degeneration; triggering unique pathogenic pathways and mechanisms which are interlaced and proven to be deterrent in maintaining sound cognitive functions. There is no reason why a prolific researcher should not be focused in exploring any active pharmacological moieties or repositioning the drug in preventing cognition ailments. We found that amnesia produced by (STZ + HFD) induced diabetic rats treated with and without scopolamine was reversed by donepezil, Vitamin D, epalrestat and (Vitamin D + epalrestat) combination. Streptozotocin induced destruction of pancreatic beta cells and HFD induced obesity synergistically produce persistent hyperglycaemia. This uncontrolled hyperglycaemia has been related to lowered verbal memory, processing speed, episodic memory, instant recall, delayed recall, executive function, visual perception, and responsiveness (Kodl and Seaquist 2008). Bruce et al. (2003) found that 17.5% of elderly patients with type 2 diabetes had moderate to severe deficits in activities of daily living, 11.3% had cognitive impairment, and 14.2% had depression. Multiple reports have shown an inverse association between blood sugar levels and working memory, learning, and complex neurocognitive output in type 2 diabetic patients, encouraging the hypothesis that deteriorating glucose regulation contributes to declining cognitive performance (Kodl and Seaquist 2008). Diabetic rats were also treated separately with scopolamine, which is one of the gold standard models for inducing cognitive decline and memory difficulties. The antiamnesic activity of test drugs was compared to donepezil.

In the current study, type-2 diabetic rats exhibited marked impairment in learning and memory that was revealed with various behavioural parameters in MWM tasks and PA test. MWM is a hippocampus-dependent memory task that is commonly used in rodents to show cognitive deficiencies in permanent spatial learning ability and reference memory (Vittalrao et al. 2019). The PA model is the most frequently used fear-aggravated test for screening potential cognition enhancers in central nervous system disorders, with reproducible and dependable results (Vittalrao et al. 2019). In the probe test session of MWM, percentage of time spent and percentage of distance travelled in target quadrant by groups treated with donepezil, vitamin D, epalrestat and (vitamin D + epalrestat) was markedly higher in comparison with diabetic and (diabetic + scopolamine) groups of rats. However, it was maximum in donepezil followed by (vitamin D + epalrestat) treated rats. This justifies that the rats treated with donepezil and (vitamin D + epalrestat) displayed an improvement in the intensity and precision of recalling the previous navigation memory. Common mechanisms used for "visuospatial navigation" in rats also relate significantly to day-to-day human cognitive functions (Terry 2009). The PA model, rats showed deleterious behavioural effects of diabetic and (diabetic + scopolamine) treatment by a decrease in the avoidance latency compared to control rats. However, rats given donepezil, vitamin D, epalrestat, and a combination of the two (vitamin D + epalrestat) were able to successfully learn that the dark compartment was electrified, resulting in a foot shock, as evidenced by a significant increase in the time it took to enter the dark compartment or step through latency (STL). Hence the latency time to enter the dark compartment was notably increased and percentage of time spent was decreased. This suggests that rats treated with (vitamin D + epalrestat) and standard drug donepezil facilitated PA responding by increasing the latency to enter the dark compartment and also prevented cognitive impairment caused by chronic hyperglycaemia with and without scopolamine. The present results support the notion that chronic diabetic state and scopolamine impaired the learning of the inhibitory response primarily by acting on the consolidation and retention phases of memory formation. Our test drug (vitamin D + epalrestat) and standard drug donepezil prevented memory impairment in both training and retention phases, most probably by alleviating oxidative stress and facilitating cholinergic transmission respectively thus improving the learning and memory processes in rats. These results concord that cognitive impairment in STZ-induced diabetic rats, is associated with hippocampal dysfunction.

Another well-known causative element in the pathophysiology of dementia and age-related neurodegenerative illnesses, such as Alzheimer's disease, is oxidative stress. Because of its high oxygen consumption (20%), metal concentration, polyunsaturated fatty acids, and limited anti-oxidant capacity, brain tissue is particularly sensitive to oxidative stress. The brain contains only low to moderate levels of antioxidant enzymes, such as catalase, SOD (superoxide dismutase), and GPx (glutathione peroxidase), compared with other organs, and GSH is the most prevalent anti-oxidant in most brain cells (Hafez et al. 2017). Hyperglycaemia increases flux through the polyol pathway in nervous tissue. In the study conducted by Kodl CT. STZ-treated hyperglycaemic rats, showed increased sorbitol in cranial nerves, sciatic nerve, cerebral cortex, and retina (Kodl and Seaquist 2008). Increased intracellular sorbitol in neurons causes tremendous increase in oxidative stress and release of reactive oxygen species (ROS). Greater oxidative injury was seen in the brains of experimentally induced hyperglycemic rats. Oxidative stress induces inflammation and, consequently, raises inflammatory mediators. Inflammation is regarded as an effective pathophysiological phenomenon in type 2 diabetes which could possess a part to perform in the vulnerability of type 2 diabetes patients to neurodegenerative diseases (Jaiswal et al. 2018b). Glutathione (GSH), the most important antioxidant, has a vital activity in maintaining the cellular redox state. Thalamus and hypothalamus are more susceptible to free radical damage as evidenced by the decrease in GSH levels in these regions. Hippocampus is mainly responsible for the collection, consolidation and retrieval of various aspects of learning, memory and spatial navigation (Jaiswal et al. 2018b). Elevated hippocampal oxidative stress corresponding to reduced content of glutathione and raised levels of MDA are considered as reliable indices of oxidative stress and our study showed deranged cognition most probably due to the oxidative damages observed in hippocampus.

In the current study, rats given donepezil, vitamin D, epalrestat, or the combination (vitamin D + epalrestat) significantly improved memory deficits caused by diabetic rats with and without scopolamine, as well as significantly alleviating oxidative stress by lowering MDA and increasing GSH-level activities; which corresponds to the results of previous studies conducted by Jaiswal S, and Kodl CT (Kodl and Seaquist 2008; Jaiswal et al. 2018a). Other, neurochemical changes, including decreased acetylcholine have been observed (Kodl and Seaquist 2008). The modulating AChE activity could affect the underlying neuronal processes which encouraged us to measure AChE levels. The pretreatment with donepezil, vitamin D, epalrestat, or the combination (vitamin D + epalrestat) significantly reduced the AChE activity and the loss of cholinergic activity in the hippocampus as compared with the diabetic rats with and without scopolamine. The inhibitory activity of (vitamin D + epalrestat) comparable to standard drug donepezil. The diabetic rats with and without scopolamine possibly inflicted severe deficits in cholinergic neuron functionality, augmented AChE activity and expression in the hippocampus enhancing the neurodegeneration in the brain. The possible neuro-modulatory mechanism by test donepezil and (vitamin D + epalrestat) drugs could be due to inhibition of AChE activity, increasing the release of ACh and prolonging the duration of acetylcholine in the brain. These finding could be attributed to central cholinergic neuro-modulatory properties of vitamin D similar to donepezil (cerebro-selective anti-cholinesterase) (Alrefaie and Moustafa 2020).

These findings corroborate to the fact that the test drugs (vitamin D + epalrestat) and donepezil treated group of rats showed significant neuro-protective activities against diabetic rats treated with and without scopolamine; probably by modulating neuro inflammatory processes, learning memory processes, anti-oxidant, and maintaining neuronal structural integrity.

Vitamin D is a steroid hormone that plays an important role in the central nervous system and has neuroprotective properties. There is enough cumulative evidence suggesting vitamin D receptors and important enzymes involved in its metabolism are scattered throughout the brain of rats, particularly in areas afflicted by neurodegenerative illnesses. Vitamin D has been proposed to cross through the BBB and have a direct effect on the central nervous system. Animal studies have shown that the function of the hippocampus, a pivotal region of the brain involved in cognition, is adversely affected by obesity. Neuronal hippocampal inflammation, neurotrophin decrease, and alteration of cerebral vascular function, such as BBB integrity in HFD fed mice, are some of the postulated underlying processes. Our study results were partly in agreement with G Hajiluian et al. who reported that Vitamin D significantly increased time spent in MWM probe test, oxidative stress and reversed HFD-induced cognitive impairments via reduction of NF-κB concentrations, BBB permeability and elevation of BDNF (Brain-derived neurotrophic factor) in the hippocampus (Hajiluian et al. 2017).

Diabetic neuropathy is caused by an increase in the activity of aldose reductase, a major enzyme in the polyol pathway. Epalrestat (EPS) is a reversible aldose reductase inhibitor that lowers intracellular sorbitol, which is thought to be the aetiology of diabetic neuropathy. However, it has no effect on glycaemic control. The improvement in sorbitol levels and Na⁺/K⁺ ATPase activity that leads to better nerve conduction velocity has been demonstrated in animal tests (Ramirez and Borja 2008; Steele et al. 1993).

The anti-oxidant, neuro modulatory and nerve cell protective role of epalrestat underscored by the recent studies; heme oxygenase (HO)-1 possesses significant antioxidant and anti-inflammatory activities, according to earlier results; nevertheless, epalrestat activates Nrf2 (Nuclear factor erythroid 2-related factor 2) and upregulates HO-1, dismutase, and catalase, implying that it has a favourable influence on the improvement of numerous neurological illnesses (Yama et al. 2016). Epalrestat lowers intracellular sorbitol accumulation in hyperglycemic situations, which has been linked to the pathophysiology of diabetic complications (Steele et al. 1993). Epalrestat is quickly absorbed by brain tissue and effectively inhibits aldose reductase with few side effects. A recent study found that treating diabetic neuropathy with EPS at an early stage avoided the onset/progression of retinopathy and nephropathy (Hotta et al. 2012). Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH, the most abundant non-protein thiol antioxidant in cells, is important for protection against oxidative stress (Yama et al. 2015).

On histopathological analysis, it was perceived that in STZ and (STZ + scopolamine) group, neuronal cells suffered from inflammatory damage due to increased oxidative stress caused by diabetes, leaving behind ruptured flame shaped cells with no nuclei and disintegrated cell membrane. Due to scopolamine injection, the damage was strikingly more with maximum number of apoptotic dead cells. In groups treated with vitamin D and epalrestat, reasonable number of neurons were preserved despite the damage due to inflammation. The (vitamin D + epalrestat) offered better protection against inflammatory damage and managed to preserve significantly more number of neurons. Donepezil being a standard drug managed to keep the oxidative stress in check. These findings support the fact that the test drugs (vitamin D + epalrestat) and donepezil-treated rats showed significant neuro-protective activity against diabetic rats treated with and without scopolamine, possibly by modulating neuro-inflammatory processes, anti-oxidant activity, and maintaining neuronal structural integrity.

The present study demonstrated that vitamin D and epalrestat, individually as well as in combination possess cognition enhancing ability against scopolamine induced amnesia in diabetic rat model by inhibiting lipid peroxidation and augmenting endangered antioxidants. In addition to this, vitamin D also exhibited reduction in acetylcholinesterase activity in brain which was also displayed by the combination of vitamin D and epalrestat. Ultimately, our study indicated that cognition enhancing ability of combination of vitamin D and epalrestat is comparable with that of the standard drug donepezil. However, further studies are warranted to detect the potential use of the combination of vitamin D and epalrestat for cognitive enhancement in humans.