Cognitive effects of vanillic acid against streptozotocin-induced neurodegeneration in mice

Abstract

Context: Vanillic acid (VA), a flavoring agent used in food and drug products, obtained naturally from the plant Angelica sinensis (Oliv.) Diels (Apiaceae), used in the traditional Chinese medicine. It is reported to possess strong antioxidant, anti-inflammatory, and neuroprotective effects. However, the pharmacological effects on oxidative stress-induced neurodegeneration are not well investigated.

Objective: This study investigates the neuroprotective effect of VA on streptozotocin (STZ)-induced neurodegeneration in mice through behavioral and biochemical parameters.

Materials and methods: The behavioral effects were determined using the Y-maze and open-field habituation memory. In biochemical parameters, acetylcholinesterase (AChE), corticosterone, tumor necrosis factor (TNF)-α, and antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase) were measured. Five groups of animals used were of control, negative control, and three separate groups treated with 25, 50, and 100 mg/kg of VA, respectively, for 28 d. Intracerebroventricular (ICV) injections of STZ were performed for all groups except control on 14th and 16th of 28 d of VA treatment.

Results: VA improved spatial learning and memory retention by preventing oxidative stress compared with control animals. VA at 50 and 100 mg/kg dose significantly (p < 0.001) improved the habituation memory, decreased the AChE, corticosterone, TNF-α, and increased the antioxidants (p < 0.001). VA (100 mg/kg) exhibited dose-dependent effect in all parameters with p < 0.001 except antioxidants in which VA showed the significance of p < 0.01.

Discussion and conclusion: VA exhibited reduction in AChE, TNF-α, and corticosterone with improved antioxidants to contribute neuroprotection and could be an effective therapeutic agent for treating neurodegenerative disorders.

• Acetylcholinesterase
• Alzheimer’s disease
• corticosterone
• oxidative stress
• tumor necrosis factor-alpha

Introduction

Oxidative stress is caused by imbalance between the production of reactive oxygen species and biological system's ability to detoxify the reactive intermediates or its incapability to restore the resulting damage (Sies, 1997; Storz & Imlay, 1999). Oxidative stress was concerned in the pathophysiology of several neurodegenerative disorders characterized by progressive cognitive deficits (Coyle & Puttfarcken, 1993; Olanow, 1993). Moreover, exposure to high levels of glucocorticoids or chronic stress may lead to oxidative injury of various parts of the brain including the hippocampus, which may impair learning and memory functions (Behl et al., 1997; McIntosh et al., 1998a,b; Sato et al., 2010; You et al., 2009).

In Alzheimer’s type of dementia (AD), one of the perceptible transforms was elevated level of acetylcholinesterase (AChE). The AChE is an enzyme which hydrolyzes the acetylcholine in cholinergic neurons and non-cholinergic neurons in brain. In AD brain, AChE enhances the conversion of Aβ peptide into the form of fibrils, which are highly neurotoxic than that of Aβ peptides (Alvarez et al., 1998). Evidence implicates that the over expression of AChE was initiated during the generation of free radicals in neurodegeneration elicited by the Aβ peptide. The free radicals generated in neuronal membrane initiates the peroxidation of unsaturated fatty acids to release malondialdehyde (MDA) and 4-hydroxy-2,3-neonal compound (4-HNE). Additionally Aβ has been accounted to provoke the production of H₂O₂ and LPO in rat brain hippocampal neuronal cells (Yatin et al., 2000). Intracerebroventricular (ICV) injection of streptozotocin (STZ) in mice impairs brain biochemistry, cerebral glucose and energy metabolism, cholinergic transmission, and increases generation of free radicals, ultimately leading to cognitive deficits (Hoyer et al., 1999; Hoyer & Lannert, 2008; Ishrat et al., 2009). Collectively, these effects are similar to sporadic dementia of Alzheimer's type in humans (Hoyer, 1991). Since oxidative damage is implicated in the etiology of neurological complications, treatment with antioxidants is used as a therapeutic approach in various types of neurodegenerative disease (Javed et al., 2011). Polyphenols, a subclass of phytochemicals known as flavanoids, mainly derived from diets and Chinese herbs, have received much attention due to their biological properties including anti-oxidative, anti-inflammatory, anti-apoptotic, and neuroprotective effects (Dragsted, 2003; Scalbert & Williamson, 2000; Schroeter et al., 2001).

Vanillic acid (VA) is a benzoic acid derivative that is used as a flavoring agent. It is an oxidized form of vanillin. At present, the mechanisms by which VA exerts its anti-oxidant and neuroprotective effects are incompletely understood. In this study, we attempted to determine the neuroprotective effects of VA on STZ-induced dementia in mouse.

Materials and methods

Reagents and chemicals

VA, acetylthiocholine iodide, eserine, dexamethasone, glutathione oxidized (GSSG), reduced (GSH), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), thiobarbituric acid (TBA), 2,4-dinitrophenyhydrazine (DNPH), STZ, adenosine 5-triphosphate (ATP), and pyrogallol were purchased from Sigma-Aldrich, St. Louis, MO. All other chemicals were of analytical grade from S. D Fine Chemicals LTD, Mumbai, India. Mouse TNF-α kit was purchased from Ray Biotech, Norcross, GA.

Animals

Swiss albino male mice (20–22 g) of age 4–5 weeks were used in the study. They were kept in the central animal house of School of Pharmacy, Anurag Group of Institutions (JNTU University, Hyderabad, Telangana) in colony cages at an ambient temperature of 25 ± 2 °C and relative humidity 45–55% with a 12 h light/dark cycle. They had free access to standard rodent pellet diet and provided water ad libitum. The animals were used in accordance with the CPCSEA guidelines for laboratory animal facility. The experimental protocol was duly approved by institutional animal ethics committee (IAEC) (Protocol no. I/IAEC/LCP/0035/2012/SM/30). Animals were divided into five groups of each six separately for the pharmacological study. Group I: vehicle treated (PBS); group II: treated with only two doses of STZ (3 mg/kg) ICV; groups III, IV, and V: treated with two doses of STZ (3 mg/kg) and VA, 25, 50, and 100 mg/kg p.o., respectively, for 28 d. Doses of vanillic acid were selected according to the previous studies (Leal et al., 2011). Prior to a week of ICV injection, the animals were trained for the behavioral parameters. The neurotoxic STZ ICV administrations were performed on 14th and 16th days of VA treatment.

ICV administration of STZ

Mice were anesthetized with anesthetic ether for ICV administrations. ICV injections were made by identifying the bregma point with a hypodermic needle of 0.4 mm external diameter attached to a 50 μl Hamilton microlitre syringe (Top Syringe, Mumbai, India). In brief, bregma was identified approximately 1–3 mm rostral to the line drawn through anterior base of ears. Then at 45° angle, the needle was inserted 2 mm lateral to midline and STZ (3 mg/kg) was injected in a volume of 10 μl. STZ was dissolved in phosphate-buffered saline solution, made freshly. Two doses of STZ (3 mg/kg) were administered by ICV injection bilaterally. The second dose was administered after 48 h of first dose (Haley & McCormick 1957; Laursen & Belknap, 1986).

Behavioral testing

Behavioral tests were carried out at the end of second week after the induction of neurotoxicity (27th day). The experiment was performed between 9.00 and 16.00 h in the laboratory at established optimal conditions.

Open-field habituation

The exploratory behavior of the mice was evaluated by the open-field habituation task method. Mice were placed in a 40 cm × 50 cm × 60 cm open field whose brown linoleum floor was divided into 12 equal squares by white lines and left to explore it freely for 5 min. The number of line crossings and head dipping were counted (Ramirez et al., 2005).

Video tracking in Y maze

The Y-maze task was used to measure the spatial working memory in mice. The maze is made of gray plastic. Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converged at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze for 8 min. Mice tend to explore the maze systematically, entering each arm in turn. The ability to alternate requires that the mice know which arm they have already visited. The series of arm entries, including possible returns into the same arm, is recorded by a video-tracking system (VJ Instruments, Washim, Maharastra, India). Alteration is defined as the successive entries into the three arms, on overlapping triplet sets. The percentage of alteration is calculated as the ratio of actual alterations to possible alterations, defined as the total number of arm entries minus two, and multiplied by hundred. Typically, mice exhibit an alteration percentage of 60–70%, and perform 25–35 arm entries within the 8 min session (Reddy, 1997).

Biochemical analysis

After the completion of behavioral studies on 27th day, the animals were treated further on 28th day with VA and sacrificed after ½ h of the drug administration for the determinations of biochemical parameters. All the biochemical parameters were determined on whole brain tissue homogenate except corticosterone, which was carried out in plasma.

Estimation of acetylcholinesterase

The brain acetylcholinesterase activity was measured by the formation of thiocholine with slight modifications (Ellman et al., 1961; Koladiya et al., 2008). This was measured on the basis of yellow color formation due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylthiocholine iodide in the presence of brain cholinesterase was measured using a spectrophotometer at 420 nm (UV-1800, Shimadzu, Kyoto, Japan).

Estimation of corticosterone

Blood collection was performed during morning hours (9.00–11.00 h) by carotid bleeding in tubes containing heparin while sacrificing the animals and centrifuged at 1000 × g for 20 min at 4 °C. HPLC/UV system (SPD-20 A, Shimadzu, Kyoto, Japan) was used for quantification of plasma corticosterone according to Woodward and Emery (1987) with minor modifications using dexamethasone as an internal standard. Plasma was separated; 50 µl of plasma containing known quantity of dexamethasone (1 µg) was extracted with 5 ml of dichloromethane (DCM). The DCM extract was evaporated to dryness and dissolved in 100 µl of mobile phase. The extract (20 µl) was injected into the HPLC system for quantification. The mobile phase consisted of methanol:water (70:30); at a flow rate of 1.2 ml/min, corticosterone was detected at 250 nm using a UV detector (Shimadzu, Kyoto, Japan) (Sheikh et al., 2007; Woodward & Emery, 1987).

Estimation of TNF-α

Brain tissue was homogenized in 1 ml ice cold buffer (pH 7.2, 4 °C) having 50 mM Tris, 1 mM EDTA, 6 mM MgCl₂, 5% (w/v) protease inhibitor cocktail (Sigma P8340, Sigma-Aldrich, St. Louis, MO). Then the samples were sonicated and centrifuged at 20 800 × g for 20 min in cooling centrifuge. The supernatants were used for the determination of TNF-α using commercially available ELISA kit obtained from Ray Biotech, Norcross, GA (Boring et al., 1996). The procedures were followed as per the manufacturer’s instructions. All data are expressed as pg/mg total protein.

Estimation of antioxidant parameters

Superoxide dismutase (SOD) activity was determined by the pyrogallol oxidation method (Marklund & Marklund, 1974). This is an indirect method that is based on the ability of the enzyme to inhibit the auto-oxidation of pyrogallol. SOD activity was determined by monitoring the rate of oxidation of pyrogallol by superoxide radicals. The reaction is initiated by adding pyrogallol and the change in optical density at 420 nm is recorded for 3 min. One unit SOD activity is defined as the amount of enzyme that inhibits the rate of auto-oxidation of pyrogallol by 50%. Glutathione peroxidase (GPx) activity was assayed based on the oxidation of glutathione (GSH) to GSSG catalyzed by GPx, which is then coupled to the recycling of GSSG back to GSH utilizing GR and NADPH (Lawrence & Burk, 1976). The decrease in NADPH absorbance measured at 340 nm during the oxidation of NADPH to NADP⁺ is indicative of GPx activity, since GPx is the rate limiting factor of the coupled reactions. CAT activity was determined in 50 µl of sample mixed with 50 µl of substrate for 60 s, then 100 µl of 32.4 mM ammonium molybdate solution was added, and the absorbance change was measured at 405 nm (Goth, 1991). One unit of the enzyme was defined as mill moles of H₂O₂ degraded/min/mg of protein.

Statistical analysis

Results are expressed as mean ± SEM. Statistical analysis was done by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. The p-value < 0.05 was considered as statistically significant.

Results

Behavioral estimations

Open-field explorations

In the open-field behavioral test, injection of STZ in group II significantly (p < 0.001) reduced the habituation memory depicted by line crossings and head dips. Treatment of VA at the dose of 50 and 100 mg/kg (groups IV and V), respectively, improved the exploratory behavior with significance of p < 0.001 when compared with the negative control group, whereas the 25 mg/kg treated animals (group III) exhibited significant effect only in line crossings but not in head dips. The high dose of VA (100 mg/kg) treatment exhibited dose-dependent effect significantly with p < 0.001 and p < 0.01 on comparison with the low dose (25 mg/kg) and intermediate dose (50 mg/kg) treated groups, respectively (Table 1).

Table 1. Effect of vanillic acid on behavioral activity.

	Open field exploration
Groups	Line crossings	Head dips	Y-maze (% alterations)
Group I (PBS)	142.13 ± 3.44	23.56 ± 0.83	48.72 ± 1.73
Group II (STZ)	85.51 ± 3.19^a	5.82 ± 1.02^a	26.48 ± 1.70^a
Group III (STZ + VA 25 mg/kg)	105.62 ± 3.43^b	9.64 ± 0.83	32.19 ± 2.18
Group IV (STZ + VA 50 mg/kg)	118.18 ± 3.32^c	14.17 ± 1.53^c	37.13 ± 1.42^b
Group V (STZ + VA 100 mg/kg)	135.65 ± 2.83^c,d,e	19.69 ± 1.49^c,d,e	44.42 ± 1.71^c,d

Values are expressed as mean ± SEM (n = 6). Symbol represents the statistical significance done by ANOVA, followed by Tukey’s multiple comparison tests. ^ap < 0.001 indicates comparison of the negative control with group I, ^bp < 0.01 and ^cp < 0.001 indicate the significant difference of vanillic acid-treated group with group II, ^dp < 0.001 and ^ep < 0.05 indicate the dose-dependent activity on comparison of the high dose (100 mg/kg) with low dose and intermediate (25 and 50 mg/kg) dose of the VA, respectively.

Y-maze

The percentage alteration in the neurotoxicity-induced group (II) was significantly (p < 0.001) reduced when compared with the control group. In the treatment groups, the low-dose (25 mg/kg) treated animals did not shown any significant improvement in percentage alteration, whereas the intermediate (50 mg/kg) and high doses (100 mg/kg) exhibited significant improvement with p < 0.01 and p < 0.001, respectively. There is also a significant dose-dependent effect exhibited by 100 mg/kg on comparing with 25 mg/kg treated animals (Table 1).

Biochemical studies

Acetylcholinesterase

The negative control animals (group II) exhibited a significant higher level of AChE when compared with the control animals (p < 0.001) which indicates the intensity of neurotoxicity. In the treatment groups, the AChE levels in brain homogenate were decreased significantly (p < 0.001) in groups III, IV, and V when compared with group II. On comparison, 25 and 50 mg/kg with 100 mg/kg dose, the 100 mg/kg treated animals exhibited a significant (p < 0.01 and p < 0.001) dose-dependent effect, respectively (Table 2).

Table 2. Effect of vanillic acid on AChE, TNF-α, and corticosterone.

Groups	Ach E (µ mol/min/mg)	Corticosterone (ng/ml plasma)	TNF-α (pg/ml)
Group I (PBS)	14.62 ± 0.20	111.92 ± 3.63	271.64 ± 30.78
Group II (STZ)	22.34 ± 0.41^a	255.85 ± 6.25^a	802.83 ± 49.22^a
Group III (STZ + VA 25 mg/kg)	18.87 ± 0.39^c	225.66 ± 6.28^b	603.58 ± 22.36^c
Group IV (STZ + VA 50 mg/kg)	16.92 ± 0.29^c,d	207.51 ± 4.60^c	485.16 ± 23.14^c
Group V (STZ + VA 100 mg/kg)	15.52 ± 0.23^c,e	187.87 ± 6.28^c,e	370.27 ± 14.21^c,e

Values are expressed as mean ± SEM of six animals. Symbol represents the statistical significance done by ANOVA, followed by Tukey’s multiple comparison tests. ^ap < 0.001 indicates comparison of the negative control with group I, ^bp < 0.01 and ^cp < 0.001 indicate the significant difference on comparison with group II, ^dp < 0.01 indicates the dose-dependent activity on comparison of the intermediate dose with low dose (25 mg/kg) of the vanillic acid, ^ep < 0.001 indicates the dose-dependent activity on comparison of the high dose with the respective low dose (25 mg/kg) of the vanillic acid.

Corticosterone level

Table 2 shows the serum corticosterone level in all experimental groups and indicates the significant differences among the groups. Comparison of group ІІ as against group І indicated that the chronic stress significantly (p < 0.001) increased plasma corticosterone. In contrast, the corticosterone level in groups IV and V were significantly (p < 0.001) reduced after the treatment of VA. Of the treatment groups, significant (p < 0.001) dose-dependent reduction in corticosterone was found only in high dose (100 mg/kg) treated animals on comparing with low dose (25 mg/kg) treated group. These findings indicate that VA did significantly prevent stress-induced elevation of corticosterone level.

TNF-α

The induction of neurotoxicity in group II significantly (p < 0.001) increased the TNF-α level which was compared with the control group. After the treatment with VA, groups III, IV, and V at the doses 25, 50, and 100 mg/kg attenuated the elevated levels of TNF-α. The reduction in TNF-α exhibited by the three doses was compared with negative control and found to be effective with significance of p < 0.001. The high-dose-treated group with 100 mg/kg exhibited dose-dependent activity on comparing with low-dose-treated group with 25 mg/kg (Table 2).

Antioxidants

The antioxidant parameters (SOD, catalase, and GPx) were significantly (p < 0.001) lowered in STZ-induced animals compared with the control group. The treatment of VA at the dose of 25 mg/kg (group III) did not show any significant improvement in antioxidant parameters. In contrast, there was a significant (p < 0.001) improvement in all antioxidants at 50 and 100 mg/kg except catalase at the dose of 50 mg/kg with p < 0.01. It was noted that there was a significant dose-dependent effect in high dose with p < 0.01 for SOD and catalase and p < 0.001 for GPx, respectively (Table 3).

Table 3. Effect of vanillic acid on antioxidants enzymes.

Groups	SOD (U/min/mg protein)	Catalase (U/mg protein)	GPx (U/min/mg protein)
Group I (PBS)	16.87 ± 0.83	1.82 ± 0.10	33.86 ± 1.38
Group II (STZ)	7.51 ± 1.03^a	0.76 ± 0.12^a	20.84 ± 0.47^a
Group III (STZ + VA 25 mg/kg)	10.82 ± 0.68	1.05 ± 0.06	23.84 ± 0.62
Group IV (STZ + VA 50 mg/kg)	13.20 ± 0.73^c	1.23 ± 0.08^b	27.93 ± 1.11^c,d
Group V (STZ + VA 100 mg/kg)	15.88 ± 0.68^c,e	1.54 ± 0.04^c,e	31.06 ± 0.71^c,f

Values are expressed as mean ± SEM of six animals. Symbol represents the statistical significance done by ANOVA, followed by Tukey’s multiple comparison tests. ^ap < 0.001 indicates comparison of the negative control with group I, ^bp < 0.05 and ^cp < 0.001 indicate the significant difference of vanillic acid-treated group with group II, ^dp < 0.05 indicates the dose-dependent activity on comparison of the intermediate dose (50 mg/kg) with the respective low dose (25 mg/kg) of the vanillic acid, ^ep < 0.01 and ^fp < 0.001 indicate the dose-dependent activity on comparison of the high dose with the respective low dose (25 mg/kg) of the vanillic acid.

Discussion

The main findings of the present study are that STZ impairs spatial learning and memory and induces oxidative stress. These harmful effects of chronic stress can be prevented by VA pretreatment, suggesting that VA have potential therapeutic application protecting against the detrimental effect of chronic stress on cognitive function. The impairing effects of chronic oxidative stress on learning and memory are mainly mediated via activation of the HPA axis, which culminates in the production of glucocorticoids in rodents causing dendritic atrophy in hippocampal CA3 pyramidal neurons (Conrad et al., 2007; Magarinos & McEwen, 1995), inhibit neurogenesis (Gould et al., 1997), and cause hippocampal volume loss (McEwen, 2000). These changes in the hippocampus after chronic stress or elevation of glucocorticoids have been related to changes in spatial learning and memory (McEwen, 2001). Oxidative stress could be one of the mechanisms by which chronic stress or glucocorticoids negatively affect learning and memory (Abidin et al., 2004) and induce neuronal damage (Abraham et al., 2001; McIntosh & Sapolsky, 1996a,b; Patel et al., 2002). Oxidative stress can be increased by an increased production of reactive oxygen species or by a decrease in antioxidant enzymes (Storz & Imlay, 1999). Brain cells are at particular risk of being damaged by free radicals because the brain has a high oxygen turnover, and central nervous system neuronal membranes are rich in polyunsaturated fatty acids that are potential targets for lipid peroxidation (Anderson et al., 1985; Metodiewa & Koska, 2000). Previous studies suggest that the intracerebral injection of STZ induces chronic decline (10–30%) in glucose and glycogen metabolism in different parts of brain such as cerebral cortex and hippocampus, which impairs the learning and memory. The involvement of reduced brain oxidative metabolism is the prime factor for degenerative changes in the STZ model (Duelli et al., 1994). Moreover STZ (intracerebral) injected twice 48 h apart in mice resulted in a persistent significant deficit loss of memory evidently found in the performance of passive avoidance and Morris water maze tests (Saxena et al., 2008).

Our findings revealed that the ICV injection of STZ increased the plasma glucocorticoids, TNF-α and AChE in brain tissue as well as induced the oxidative stress in hippocampus and impaired spatial learning and memory. From this investigation, it was revealed that pretreatment with VA abolished the deleterious effect of oxidative stress induced by STZ on learning and memory. These finding are well co-relatable with previous studies of VA (Mattila & Kumpulainen, 2002). The present data allow two interpretations: (a) VA may prevent cognitive deficits by interacting with the mechanism that causes spatial learning and memory impairment in STZ-injected animals. This hypothesis predicts that oxidative stress and VA may act on similar neurological substrates. (b) Alternatively VA-treated mice may have enhanced spatial learning and memory compared with control animals treated with only STZ. In fact, previous studies have observed different effects on antioxidant enzymes following glucocorticoid administration. Rats treated with glucocorticoids showed decreased activities of SOD and GPx in the brain, and glucocorticoids prevented induction of antioxidant enzymes after kainic acid administration in the hippocampus (McIntosh et al., 1998a). Corticosterone has been shown to decrease GPx activity and reduce GSH levels in hippocampus cell cultures (McIntosh & Sapolsky, 1996a). In this study design, it was found that there was a reduction in antioxidants after the induction of neurotoxicity. Thereafter it was observed that, with the treatment of VA, the GPx, SOD, and catalase were increased, which implicates the possible adaptations to chronic stress.

Beside oxidative stress, there is decreased activity of glycolytic enzymes in the STZ model of memory deficit resulting in the reduction of acetylcholine level (Blokland & Jolles., 1994; Plaschke & Hoyer, 1993; Weinstock et al., 2001) which is intricately associated with cognition. Acetylcholine is degraded by the enzyme acetylcholinesterase and AChE inhibitors are the most effective pharmacological approach for the symptomatic treatment of AD (Racchi et al., 2004). In our study, we found an increased AChE activity in the brain of STZ-treated mice. This finding is in conformity with the previous studies showing an increase in AChE expression (Lester-Coll et al., 2006) and activity following central STZ injection (Agrawal et al., 2009; Saxena et al., 2007; Tota et al., 2009). Anti-AChE activity in the present study is dose dependent; this may be due to amelioration of disturbed glucose metabolism and insulin signaling induced by STZ. Laboratory evidence implicate TNF-α in inflammatory molecular mechanisms producing neurotoxicity, neuronal death, or neuronal dysfunction involving both TNF-glutamate (De et al., 2005; Stellwagen et al., 2005; Taylor et al., 2005) or TNF-amyloid interactions (Craft, 2006; Floden et al., 2005; Koenigsknecht & Landreth, 2005; Mbebi et al., 2005; Pickering et al., 2005; Ralay Ranaivo et al., 2006). In the brain slice culture model, TNF-α was found to potentiate glutamate neurotoxicity, with TNF-α and glutamate acting synergistically to induce neuronal cell death (Zou & Crews, 2005). In this investigation, the induction of neurotoxicity with STZ increased the TNF-α activity and indicating potential influence of pro-inflammatory cytokines in oxidative stress-induced neurodegeneration. The treatment of VA in different doses attenuated the aggravation of pro-inflammatory cytokine.

Conclusion

In our study, we found that the reduction in pro-inflammatory cytokine (TNF-α) and AChE with increased levels of antioxidants remarkably played a central role in cognitive improvement on neurotoxicity and memory deficit induced by STZ. This hypothesis revealed that VA exerts specific anti-inflammatory and antioxidant effects that down-regulate the neuroinflammatory process and could be used as a potential drug for therapeutic benefit in AD. Further studies are required to validate the influence of VA on interleukins, and other cytokines with neuroimmune modulation, brain oxidative stress and inflammation in amyloidogenic phenotype of a transgenic mouse model (Tg2576).

Acknowledgements

The authors would like to thank Dr. P. Rajeshwar Reddy, Chairman, and the management of Anurag Group of Intuitions for their support in providing research facilities.

Declaration of interest

The authors report no declarations of interest.