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Reversal of diabetes-induced behavioral and neurochemical deficits by cinnamaldehyde.

ABSTRACT

Background: Chronic hyperglycemia during diabetes is associated with altered cognitive function. Cinnamaldehyde showed to have many pharmacological activities indicating anti-diabetic, cognitive enhancer, antiinflammatory etc. In the present study, we have investigated the effects of cinnamaldehyde (CA) on diabetes-induced cognitive deficits.

Methods: Diabetes was induced in Sprague Dawley rats using high fat diet followed by streptozotocin (35 mg/kg, i.p.). High fat diet feeding was continued for 18 week after STZ administration. CA was administered daily during the last 3 weeks (week 16-18) at a doses of 10, 20 and 40 mg/kg (p.o.). Animals were subjected to behavioral tests during 18th week. Neurotransmitter levels (glutamate and GABA), acetylcholine esterase (AChE) activity and inflammatory markers (TNF-[alpha] and IL-6) were assessed in the hippocampus and cortex.

Results: Vehicle-treated diabetic rats showed impaired behavior in open field, elevated plus maze and water maze test compared to age-matched control rats. Cinnamaldehyde showed significant reduction in blood glucose levels at dose of 20 and 40 mg/kg. Three weeks treatments of cinnamaldehyde showed significant amelioration of behavioral deficits in diabetic rats. Chronic treatment with cinnamaldehyde showed improvement in brain ChE activity, neurotransmitter levels and reduction in IL-6 and TNF-[alpha] levels.

Conclusion: The present study demonstrates that treatment with cinnamaldehyde reverse neuroinflammation and changes in neurotransmitter levels, and consequently improves behavioral deficits in diabetic rats.

Keywords:

Diabetes

Cognititon

Cinnamaldehyde

GABA

Glutamate

IL-6

TNF-[alpha]

Introduction

Diabetes is one of the most common metabolic disorder in human. 382 million people are presently suffering from diabetes which is expected to rise to 592 million by 2035 (Guariguata et al., 2014). Type-2 diabetes (T2D) adversely affects major regulating systems of body to which central nervous system is not an exception. Studies have demonstrated an association between diabetes and a variety of brain alterations including cognitive decline, anxiety, depression, Alzheimer's disease, and Parkinson's disease. 17.5% of total T2D population face moderate to severe behavioral deficits moreover elderly people of age above 65 are more prone to develop diabetes-induced cognitive deficits (DACD) and dementia. (Strachan et al., 2011). In an earlier study, we demonstrated that 15 weeks duration of diabetes significantly impaired the cognitive function in high fat diet/streptozotocin induced rat model of diabetes (Datusalia and Sharma, 2014). Diabetic rats showed significant impairment in spatial memory and avoidance response. Synaptic plasticity in hippocampus has been studied in rat model of diabetes to elucidate the pathology behind cognitive decline. Recent studies on various animal models have given new hope for the treatment of DACD with enalapril, insulin, tocotrienol, C-peptide replacement, vitamine E, sesamol, lycopene, resveratrol, curcumin, PARP inhibitors but none is nearby to definitive treatment (Kuhad et al., 2009; Sima et al., 2004; Tiwari et al., 2009). Moreover, most studies focus vascular theory of cognitive decline, taupathy and oxidative stress dependent CNS damage. However, 90% population among the diabetics are suffering from T2DM but still there is a significant gap to address the cognitive deficits in T2DM (American Diabetes Association, 2014).

Cinnamomum verum J.Presl, Cinnamomum zeylanicum Blume (Lauraceae) and other species of the genus Cinnamomum are used worldwide as household spices and food and condiment additives. In addition to its culinary uses, cinnamon bark has been used in medieval times to treat various conditions such as coughing, arthritis, sore throats and gynecological aliments (Ranasinghe et al., 2013). In current clinical and predinical research, this spice has been shown to have potential as cognitive enhancer, anti-diabetic, anti-hypertensive, immunomodulatory, anti-arthritic, anti-inflammatory, anti-microbial and anti-parasitic activities and in reducing risk of colonic cancer (Frydman-Marom et al., 2011; Leach and Kumar, 2012; Ranasinghe et al., 2013). Cinnamon bark contained essential oil with trons-cinnamaldehyde (Figure 1) as the major component 60-90% of the total essential oil (Wong et al., 2014). Cinnamaldehyde has gained recognition for the management of diabetes, lipid disorders and diabetes-induced hypertension (Li et al., 2012). The treatment of diabetic subjects with cinnamon was investigated in several clinical trials and its insulin-like effects were present in type-2 diabetic patients. In addition, cinnamaldehyde showed improvement of brain insulin sensitivity in mouse models of obesity (Sartorius et al., 2014). Interestingly, it was also described as beneficial in Alzheimer's disease by reducing /1-amyloid oligomerization and cognitive decline, and further prevented glutamate-induced neuronal death in cultured cerebellar granule cells (Frydman-Marom et al., 2011; Shimada et al., 2000). These reports provide evidences that, cinnamaldehyde (a principal component of cinnamon oil) is of high ethnopharmacological importance and exerts a variety of biological-pharmacological effects including diabetes and brain disorders. However, the effect of cinnamaldehyde on diabetes-induced change in neurobehavior and neurochemistry has not yet explored.

In the present study, we have explored the effect of cinnamaldehyde in diabetes-induced behavioral deficits using open field, elevated plus maze, passive avoidance paradigms and morris water maze. We have also investigated the effects of cinnamaldehydye against the diabetes-induced alteration in the inflammatory cytokine (IL-6), acetylcholinesterase activity and neurotransmitter levels (glutamate and GABA) in the hippocampus and cerebral cortex of diabetic rats.

Materials and methods

Animals

Studies were carried out in male Sprague-Dawley rats (weighing around 130-160 g; 5-6 weeks of age). Animals were procured from central animal facility, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar and provided with standard diet and water ad libitum. The experimental study protocol was approved by the Institutional Animals Ethics Committee (1AEC), NIPER, and all the guidelines of Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), Govt, of India were followed. Animals were housed in room maintained at approximately 24[+ or -]1[degrees]C temperature and humidity of 55 [+ or -] 5% with 12 h light/dark cycle. Animals were acclimatized for at least 7 days before the initiation of the experiment and were observed for any sign of disease. Animal's body weight was determined on a weekly basis.

Drugs and chemicals

Cinnamaldehyde (C80687; [greater than or equal to] 99%; Lot no. STBC3013), streptozotocin, acetylthiocholine, phenylmethanesulfonyl chloride, L-tyrosine, o-phthalaldehyde were precured from Sigma Aldrich, USA. All other chemicals used in the study were of analytical grade. The composition of high fat diet is given in Supplementary Table 1.

Induction of diabetes and experimental design

Type-2 diabetes was induced by combination of high fat diet (HFD) feeding and low dose of streptozotocin (STZ) treatment as described elsewhere (Srinivasan et al., 2005). Rats were fed with HFD for two weeks and then injected with single low dose of STZ (35 mg/kg, i.p.) to induce type-2 diabetes. Plasma glucose level was analysed at the end of 2 weeks of STZ administered and only those rats with plasma glucose level of >250mg/dl were considered diabetic and selected for the study.

At the end of 15 weeks of STZ administration animals were randomized and divided into following groups; (1) Control (NPD) (n = 8), (2) diabetic Control (HFD + STZ + Veh) (n = 8), (3) Normal + Cinnamaldehyde (40 mg/kg) (n = 6), (4) Diabetic + Cinnamaldehyde (10 mg/kg) (n = 7), (5) Diabetic + Cinnamaldehyde (20 mg/kg) (n = 7), (6) Diabetic + Cinnamaldehyde (40 mg/kg) (n = 7). Cinnamaldehyde treatment was given for three weeks as daily single oral dose, from 16th week to 18th week. Olive oil was used as a vehicle. Animals were assessed for behavioural parameters during 18th week of STZ administration.

Behavioral studies

Open field test

Open field test was used for evaluating animal's spontaneous behaviour in response to a novel environment. An open field arena was constructed of plywood. Dimensions of arena was 70 x 70 x 37 cm and divided into two zone as peripheral and central zone. Experiments were carried out in diffused light at day time. All the experiments were recorded and analysed using ANYMAZE software. Movement of rat in peripheral and central zone was recorded (Eilam, 2003).

Elevated plus maze

The elevated plus maze (EPM) apparatus consisted of a fourarm cross-formed wooden maze with two opposite open arms (15 x 46 cm) and two opposite closed arms (15 x 46 x 23 cm), which all extended at an angle of 90[degrees] from a central area (15 x 16 cm). The EPM was placed on a support frame located 70 cm above the floor and was placed in the center of an experimental room with a distance of at least 1 m to the adjacent walls. Experiments were carried out in diffused light and analysed using ANYMAZE software. At the time of testing, the rat was placed in the centre of the plus maze facing a closed arm and was allowed to explore the maze for 5 min, after that it was removed from the maze. Time spent in open vs. closed arms, time spent in central area and the number of entries to open vs. closed arms was monitored (Eilam, 2003).

Morris water maze test

Water maze is abundantly used to assess cognitive function in rodents. It consist of, a pool divided into four equal quadrants, partially filled with water and in one of the quadrants hidden platform was placed in south-west quadrant. In acquisition session, animals were kept in one quadrant facing to wall and given 120 s trial to escape and find the platform. Similar, four trials were given each day to each animal (intra-trial intervals is 10 min) for five consecutive days. All experiments were recorded and analyzed using ANYMAZE software. Latency to reach platform and swimming speed were calculated for each trial and each animal. Probe trial was also performed on 6th day for spatial memory assessment and time spent in respective quadrants was recorded (Datusalia and Sharma, 2014).

Biochemical estimations

Plasma glucose levels

Blood was collected from tail vein in micro centrifuge tubes containing heparin. Glucose levels were measured using Glucose oxidase-peroxidase kit manufactured by Accurex, India. HbAlc levels were measured using ion exchange resin kit (Accurex, India) as per manufacturer instructions.

Acetylcholinesterase activity

Brain regions (hippocampus and cortex) were homogenized in 0.1 M phosphate buffer (pH 8). To a cuvette containing 100 [micro]l of DTNB and 2.6 ml of phosphate buffer (0.1 M, pH 8), 0.4 ml aliquot of the homogenate was added. Thorough mixing of the contents was done by bubbling. Substrate (20 [micro]l of 0.075 M acetylthiocholine) was added. Change in absorbance for a period of 10 min was measured at 412 nm. Using the formula, R = 5.74 x [10.sup.-4] x A/CO; where, R = Rate in moles of substrate hydrolysed/min/gm tissue; A = Change in absorbance/min; CO = Original concentartion of the tissue (mg/ml), enzyme activity was calculated (Lakshmana et al., 1998).

Brain TNF-[alpha] and IL-6 levels

Brains tissues (hippocampus and cortex) were homogenized in PBS buffer containing PMSF (phenylmethanesulfonyl chloride) and protease inhibitor cocktail. Homogenate was sonicated and centrifuged at 10.000 rpm for 10 min at 4[degrees]C and supernatant fraction was collected for estimations. Proinflammatory cytokines IL-6 and TNF-[alpha] levels were measured using sandwiched ELISA method according to manufacturing instructions (eBioscience, USA). Protein levels were measured by Bradford assay. IL-6 and TNF-[alpha] levels were calculated as pg/mg of protein.

Neurotransmitters estimation

HPLC-EC method was used to measure GABA and glutamate levels. An ice-cold petri dish was used to keep isolated brains. Hippocampus were removed and homogenization was carried out using ice cold perchloric acid (0.05 M) containing L-tyrosine (0.1 mg/ml). Samples were centrifuged and the collected supernatant were derivatized using o-phthalaldehyde (OPA). Samples were derivatized with 30:50 ratio of sample and derivatizing agent. Contents of the mobile phase were; 10.92 g of [Na.sub.2]HP[O.sub.4] and 148.8 mg of EDTA in 500 ml of ultrapure millipore water. After that 1.5 ml of tetrahydrofuran and 450 ml of HPLC grade methanol were added. pH was adjusted to 5.25 with o-phosphoric acid, and volume was made to 1000 ml. Auto sampler was used to inject 20 [micro]l of derivatized samples into column (Neucleosil[R]: RP18, 5 [micro]m, 4.6 x 250, 30[degrees]C). Run time of samples was 35 min and flow was maintained as 1 ml/min. Corresponding standards were used to identify glutamate and GABA and quantification was done using internal standard method (Datusalia and Sharma, 2014).

Statistical analysis

Results are expressed as mean [+ or -] S.E.M. Significance of difference between the two groups were evaluated using Student's t-test. For the multiple comparisons analysis of variance (ANOVA) was used. Morris water maze learning performance data were analysed using two way repetitive measure ANOVA followed by post hoc Bonferroni correction to examine the differences between individual treatments (GraphPad Prism 5). p < 0.05 was considered statistically significant.

Results

Effect of cinnamaldehyde on body weight, plasma glucose and HbAlc levels

After fifteen weeks of diabetes induction, diabetic animals exhibited significantly (p<0.001) high plasma glucose (357.2 [+ or -] 31.4mg/dl) and HbAlc (14.7 [+ or -] 1.6% of Hb) levels as compared with normal control animals (104.4 [+ or -] 1.4 mg/dl and 6.3 [+ or -] 1.2% respectively). Three doses of cinnamaldehyde 10, 20 and 40 mg/kg were selected and treatment was given for three weeks. Out of three doses, 20 and 40 mg/kg significantly lowered plasma glucose levels (from 329.1 [+ or -] 15.6 to 163.2 [+ or -] 18.3 and 302.2 [+ or -] 12.1 to 155.5 [+ or -] 16.3 mg/dl respectively) in diabetic rats. Three weeks cinnamaldehyde treatment also produced a significant reduction in the HbAlc levels at a dose of 20 (from 14.2 [+ or -] 1.6 to 8.6 [+ or -] 0.9% of Hb) and 40mg/kg (from 13.7 [+ or -] 1.6 to 8.2 [+ or -] 1.1% Hb) while the effect of treatment on body weight of animals was not significant (Table 1).

Behavioral tests

Open field test

To investigate the effects of cinnamaldehyde on anxiety-like behavior in diabetic rats, the open-field test was carried out. Time spent in central and peripheral arena during 5 min were calculated. Diabetic animals spent significantly less time (p < 0.001) in central zone compared to control animals (Fig. 2A). Cinnamaldehyde induced improvement in altered open field behaviour in diabetic animals. Diabetic animals treated with 40 mg/kg dose significantly spent higher time (119.7 [+ or -] 17.2 s; p < 0.001) in central zone compared to non-treated diabetic animals (39.26 [+ or -] 12.14 s). Cinnamaldehyde treatment at 10 and 20 mg/kg failed to produce significant effect in open field behaviour in diabetic rats. Cinnamaldehyde treatment per se at 40 mg/kg did not affect animal open field behaviour as compared to control animals and spent 115.6 [+ or -] 15.4 s in central zone.

Elevated plus maze test

Time spent in open and closed arms during a 5 min trial were calculated. Diabetic animals spent significantly less time (p< 0.001) in open arms compared to control animals (Fig. 2B). Diabetic animals treated with cinnamaldehyde at 40 mg/kg spent significantly higher time (19.4 [+ or -] 3.0 s p < 0.01 respectively) in open arm as compared to non-treated diabetic animals (6.1 [+ or -] 1.3 s). Treatment with 10 and 20 mg/kg failed to show any significant effect. Cinnamaldehyde (40 mg/kg) did not alter the time spent in open arm in control animals.

Morris water maze test

In spatial aquisition trial a significant difference was observed between normal and diabetic animals for the latency to find the hidden platform (Fig. 2C). Repeated measure ANOVA determined that there was significant learning between days ([F.sub.(4700)] = 56.86; p< 0.001). Further diabetic group showed significant impaired ([F.sub.(1,4)] = 9.57; p < 0.01) learning compared to control animals. Post-hoc tests using Bonferroni correction revealed that treatment with cinnamaldehyde have significant ([F.sub.(4700)] = 2.93; p < 0.001) effect in learning compared to diabetic group. Mean swim speed was also measured throughout the test to exclude differences in navigation speed, which might have led to differences in the previous parameters. We did not observe significant difference in swimming speed among the groups ([F.sub.(4700)] = 1.68, p = 0.44). During the probe trial, control animals showed tendencies to swim in platform quadrant (SW) as compared to other quadrants. Diabetic animals did not exhibit any significant difference in time spent in platform quadrant and in other three quadrants. Cinnamaldehyde treatment to non diabetic animals did not significantly alter the performance in probe trial. However, diabetic animals treated with 20 and 40 mg/kg dose showed significant difference (p < 0.01 and p< 0.001 respectively) between time spent in platform quadrant and in other quadrants as shown in Fig. 2D.

Cinnamaldehyde treatment reversed diabetes-induced AChE activity and neurotransmitter level changes

Effect of cinnamaldehyde on AChE activity in diabetic rat brain

AChE activity was assessed in brain areas such as, hippocampus and cortex as measure of cholinergic dysfunction. After 18 weeks of diabetes induction variable effect was observed on brain AChE activity (Fig. 3A). Hippocampus of diabetic rats (9.8 [+ or -] 0.56 activity levels as per methods) showed significantly (p < 0.05) increased AChE activity as compared to control rats (6.5 [+ or -] 0.57 activity levels). However, AChE activity in cortex of diabetic rats was not significantly altered as compared to control group (6.3 [+ or -] 0.4 and 6.5 [+ or -]0.13 activity levels respectively). Cinnamaldehyde (20 mg/kg and 40 mg/kg) treatment showed significant (p < 0.05 and p < 0.01 respectively) reduction in AChE activity in hippocampus. In cortex, cinnamaldehyde at 20 and 40 mg/kg showed significant decrease (p < 0.05) in AChE activity.

Effect of cinnamaldehyde on brain glutamate and GABA levels in diabetic rats

Glutamate levels were found to be significantly (p<0.05) reduced in hippocampus of diabetic rats compared to normal animals (Fig. 3B). Treatment of cinnamaldehyde at a dose of 20 and 40 mg/kg showed significant (p < 0.05 and p < 0.01 respectively) improvement in glutamate levels in hippocampus of diabetic rats. There was no significant alteration in glutamate levels in cortex of diabetic rats (0.013 [+ or -] 0.0004 [micro]M/g of tissue) compared to normal (0.015 [+ or -] 0.0016 [micro]M/g of tissue). In cortex, cinnamaldehyde did not alter glutamate levels significantly at a dose of 10 and 20 mg/kg. Increased glutamate levels (0.016 [+ or -] 0.0007 [micro]M/g of tissue) in cortex was observed at a dose of 40mg/kg cinnamaldehyde compared to diabetic animals (0.013 [+ or -] 0.0004 [micro]M/g of tissue). Further, diabetic rats brain showed significantly increased GABA levels in hippocampus (0.0057 [+ or -] 0.0005 [micro]M/g of tissue; p < 0.01) and cortex (0.0051 [+ or -] 0.0004 [micro]M/g of tissue; p < 0.05) compared to age matched normal control animals (0.003 [+ or -] 0.0007 and 0.004 [+ or -] 0.0002 [micro]M/g of tissue respectively). Treatment with cinnamaldehyde induced significant decrease in GABA levels in both hippocampus and cortex of diabetic rats. Higher dose of cinnamaldehyde (40mg/kg) decreased GABA levels significantly in hippocampus (0.0013 [+ or -] 0.0001 [micro]M/g of tissue) and cortex (0.0009 [+ or -] 0.0001 [micro]M/g of tissue) of diabetic animals as compared to control rats (Fig. 3C).

Cinnamaldehyde treatment decreased diabetes-induced inflammation in brain of diabetic rats

As an impact of diabetes, increased IL-6 and TNF-[alpha] levels were observed in hippocampus and cortex of diabetic rats. Compared to normal animals hippocampus and cortex of diabetic animals showed two and three times of IL-6 levels, respectively (Fig. 4A). Cinnamaldehyde at doses of 20 and 40 mg/kg significantly (p<0.01) reduced IL-6 levels in cortex (37.7 [+ or -] 4.2 and 37.5 [+ or -] 4.8 pg/mg of protein as compared to 77.5 [+ or -] 9.1 pg/mg of protein in diabetic animals) in cortex. In hippocampus, only 40 mg/kg dose of cinnamaldehyde reduced IL-6 to a significance level (28.6 [+ or -] 3.6 pg/mg of protein whereas 50.3 [+ or -] 0.5 pg/mg of protein) in diabetic animals. Further, treatment of diabetic rats with cinnamaldehyde also reduced the TNF-[alpha] levels in both hippocampus and cortex. Higher dose (40 mg/kg) showed marked reduction in TNF-[alpha] levels as compared to diabetic animals (Fig. 4B).

Discussion

Diabetes showed negative impact on brain in both type-1 and type-2 and patients with diabetes are susceptible to complications, such as deficits of cognition, psychomotor function, learning and memory. Although several factors such as vascular complications, metabolic disturbances, inflammation and the release of free radicals are implicated, the mechanisms underlying these complications are not fully understood. Diabetes-induced cognitive deficits are the consequences of prolonged uncontrolled hyper glycemia and impaired insulin function . As per earlier published report by our group, cognitive deficits develop after 15 weeks of T2D rats (Datusalia and Sharma, 2014). In the present study, cognitive and behavioral deficits were evident by altered performance in various behavioral test such as MWM test, open field and elevated plus maze. Significant impairment of learning and retrieval of platform location was observed in the MWM test in diabetic animals. Diabetic animals showed a significant higher levels of anxiety-like behavior compared to control animals in elevated pluz maze and open field test. Three weeks cinnamaldehyde (10, 20 and 40 mg/kg) treatment significantly lowered blood glucose in T2D rats. This could be due to pharmacological actions of cinnamaldehyde action such as insulin-like action, increase in insulin sensitivity and extra-pancreatic action that reduces carbohydrate absorption from intestine. (Li et al., 2012) also reported antidiabetic effect of cinnamaldehyde in line to present study. Moreover, cinnamaldehyde was also reported to increase the expression and translocation of GLUT4 and modulate of peroxisome proliferator-activated receptor gamma (PPAR-[gamma]) and 5' adenosine monophosphate-activated protein kinase (AMPK) signaling pathways (Anand et al., 2010; Khare et al., 2016). Even, crude cinnamon extract and traditional medicine containing cinnamon is used for its beneficial property in Chinese and Indian system of medicine from ancient time and proved to effective in management of diabetes and hypertension in animal models and clinical condition (Li et al., 2012).

Insulin resistance and T2D are closely linked with advance aging and Alzheimer's disease. Reducing blood levels, oxidative stress and inflammation by cinnamaldehyde has been proven for its potential as anti-diabetic (Kumar et al., 2012). It has been reported earlier that cholinergic dysfunction is observed in dementia and neurological disorders. Membrane bound AChE is an enzyme which degrades acetylcholine, which is associated with learning and memory (Datusalia and Sharma, 2014). The present study demonstrates that hippocampus of diabetic rats showed significant increase in AChE activity, which was reversed by cinnamaldehyde at a dose of 20 and 40 mg/kg. Further, glutamate, a major excitatory neurotransmitter in CNS was found to decrease in the hippocampus and cortex of diabetic rats. Glutamate synthesis and metabolism in CNS are maintained by glutamate-glutamine cycle and the decrease in glutamate is linked with altered cognition (Sickmann et al., 2012). Impaired glutamate-glutamine cycle may be responsible for reduction in glutamate levels. Cinnamaldehyde treatment restored the glutamate pool in hippocampus and cortex. Moreover, GABA, an inhibitory neurotransmitter is also associated with GABA-glutamate-glutamine cycle which has negative correlation with cognition and learning. Studies have been carried out to reduce GABAergic inhibition which led to restored cognitive functions in mouse model of Down syndrome (Potier et al., 2014). Thus, drugs which are linked with modulation of GABAergic system at various stages like chloride ion channels, GABA synthesis and degradation have positive impact on cognition and memory (Brioni, 1993). Increased GABA levels were observed in hippocampus of diabetic rats and this may be responsible for impaired cognition of diabetic animals. Increased GABA levels in T2D model can be taken as indicative of maintenance of ratio of excitatory and inhibitory neurotransmitters by brain glycogen (Datusalia and Sharma, 2014). These findings agree with a previous study by (Banuelos et al., 2014) in which age-related increase in the inhibitory tone in prefrontal cortex was found to be associated with impairment in working memory. Higher level anxiety phenotype has also been reported to be positively associated with increased GAD mRNA expression in HAB-mice (Tasan et al., 2011). Moreover, increase in extracellular GABA levels was found to be associated with compensatory decrease in the GABAb receptor expression and leads to nervousness behavior in the rats (Chiu et al., 2005). Consistent with the relationship between the perturbed inhibitory and excitatory tone and behavioral deficits, cinnamaldehyde restore the GABA-glutamate homeostasis with improved performance in behavioral tasks. Neuroinflammation was also reported to be linked with GABA-glutamate-glutamine cycle (Datusalia and Sharma, 2014). Increased inflammation in hippocampus and cortex of diabetic rats may also have contributory effect in alteration of neurotransmitter homeostasis and cognitive deficits. Three week cinnamaldehyde treatment showed significant decrease in IL-6 levels in brain of diabetic animals which may have contributory role in maintainance of GABA-glutamate-glutamine cycle. Anti-inflammatory effect of cinnamaldehyde might be due to inhibition of NF-[kappa]B mediated transcription of pro-inflammatory cytokine release (Ho et al., 2013). Cinnamaldehyde also induces BDNF expression and suppression of AGE-induced biological responses mediated by inactivating the JAK2-STAT1/STAT3 cascade or activating the NO pathway (Huang et al., 2015). Moreover, a link between the peripheral nerve injury and cognitive deficits in animals models was reported (Kodama et al., 2011). Chronic pain has a crucial influence on hippocampal plasticity related to cognitive function with dysfunction in glycine transporters (Kodama et al., 2011). Moreover, astrocyte TRPA1 channels contribute to astrocyte-neuron interactions by mainitaning basal [Ca.sup.2+] levels and NMDA receptor-dependent LTP and increase the miniature excitatory postsynaptic currents through glutamate release (Shigetomi et al., 2013). Genetic deletion or pharmacological blockade of the TRPA1 produce inhibitory activity in mouse models of anxiety and depression and suggests targeting TRPA1 as an innovative strategy for the treatment of neurological disorders (de Moura et al., 2014). Cinnamaldehyde also acts as a dual modulator of TRPAl(Alpizar et al., 2013). There is now overwhelming evidence that TRP channels might play a significant role in the regulation of inflammation and peripheral pain perception (Nilius et al., 2007). Previously it has been reported that TRPA1 are involved in nerve damage and neuropathic complications (Caspani et al., 2009), however the role of TRPA1 channels in diabetes related CNS complications is yet to be explored. These findings have broad relevance for the study by demonstrating that how cinnamaldehyde, a dual modulator of TRPA1 channel, can improve diabetes-induced memory deficits.

In conclusion, results of our study demonstrate the protective effect of cinnamaldehyde in diabetes-induced cognitive deficits, which may be attributed through improved glycemic control, reduction in neuroinflammation and AChE activity and maintenance of GABA-glutamine-glutamate cycle.

http://dx.doi.org/10.1016/j.phymed.2016.04.008

ARTICLE INFO

Article history:

Received 8 December 2015

Revised 25 April 2016

Accepted 26 April 2016

Conflict of interest

There are no financial or other relationships that might lead to a conflict of interest.

Acknowledgments

This study was supported by the Department of Pharmaceuticals, Govt of India by the funding to Prof. Shyam S. Sharma, National Institute of Pharmaceutical Education and Research (NIPER). Ashok Kumar Datusalia was the recipient of research fellowship from the University Grant Commission (UGC), New Delhi.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.04.008.

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Abbreviations: AChE, Acetylcholine esterase; CA, cinnamaldehyde; DACD, Diabetes-induced cognitive deficits; DTNB, 5,5-dithiobis-2-nitrobenzoic acid; EPM, Elevated plus maze; GABA, [gamma]-amino butyric add; GLUT4, Glucose transporter 4; HAB, high anxiety-related behaviour; HbAlc, Glycated haemoglobin; HFD, high fat diet; IL-6, Interleukin-6; LTP, long-term potentiation; MWM, Morris water maze; NPD, Normal palate diet; STZ, streptozotocin; T2D, Type-2 diabetes; TNF-[alpha], tumour necrosis factor-[alpha]; TRPA1, transient receptor potential cation channel A1.

Akshay Jawale (a,1), Ashok Kumar Datusalia (a,1), Mahendra Bishnoi (b,1), Shyam S. Sharma (a,1), *

(a) Molecular Neuropharmacology Lab, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SA.S. Nagar -160062, Punjab, India

(b) National Agri-Food Biotechnology Institute (NABI), SA.S. Nagar (Mohali)- 160071, Punjab, India

* Corresponding author.

E-mail addresses: jawalead9970@gmail.com (A. Jawale), ashokdatusalia@gmail.com (A.K. Datusalia), mbishnoi@nabi.res.in (M. Bishnoi), sssharma@niper.ac.in (S.S. Sharma).

(1) Author contributions: AJ performed research; AJ and AKD analyzed data; SSS, AKD and MB designed hypothesis and wrote the paper.

Table 1
Effect of cinnamaldehyde on body weight and biochemical parameters.

                      Control              Diabetes

Body Weight (g)
15th week             337.4 [+ or -] 6.0      311 [+ or -] 5.8
18th week             379.5 [+ or -] 4.8    327.8 [+ or -] 6.0

Plasma Glucose
  (mg/dl)
15th week             104.4 [+ or -] 1.4    357.2 [+ or -] 31.4 ***
18th week              96.1 [+ or -] 6.7   391.45 [+ or -] 33.3 ***

Glycated
  Haemoglobin (%Hb)
15th weeks              6.3 [+ or -] 1.2     14.7 [+ or -] 1.6 ***
18th weeks              6.7 [+ or -] 0.9     15.9 [+ or -] 1.9 ***

                      CIN40                  D+CIN10

Body Weight (g)
15th week             335.44 [+ or -] 7.9    320.21 [+ or -] 13.1
18th week              369.4 [+ or -] 9.9    343.25 [+ or -] 16.1

Plasma Glucose
  (mg/dl)
15th week              102.5 [+ or -] 11.4    344.7 [+ or -] 18.4 ***
18th week               94.6 [+ or -] 3.12   317.52 [+ or -] 24.67 ***

Glycated
  Haemoglobin (%Hb)
15th weeks            --                       14.8 [+ or -] 2.5 ***
18th weeks            --                       13.2 [+ or -] 1.7 **

                      D+CIN20

Body Weight (g)
15th week             332.71 [+ or -] 9.0
18th week              349.4 [+ or -] 9.0

Plasma Glucose
  (mg/dl)
15th week              329.1 [+ or -] 15.6 ***
18th week              163.2 [+ or -] 18.3 **, ###

Glycated
  Haemoglobin (%Hb)
15th weeks              14.2 [+ or -] 1.6 ***
18th weeks               8.6 [+ or -] 0.9 ##

                      D [+ or -] CIN40

Body Weight (g)
15th week             325.4 [+ or -] 11.1
18th week               341 [+ or -] 8.4

Plasma Glucose
  (mg/dl)
15th week             302.2 [+ or -] 12.1 ***
18th week             155.5 [+ or -] 16.3 **, ###

Glycated
  Haemoglobin (%Hb)
15th weeks             13.7 [+ or -] 1.6 ***
18th weeks              8.2 [+ or -] 1.1 ##

Data are expressed as mean [+ or -] SEM. There were 6-8 animals in
experimental groups at each time point.

** p < 0.01 and *** p < 0.001 vs respective control value (t-test).

## p < 0.01 and

### p < 0.001 vs 15th weeks (paired t-test).


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Author:Jawale, Akshay; Datusalia, Ashok Kumar; Bishnoi, Mahendra; Sharma, Shyam S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Aug 15, 2016
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