Nutritional Neuroscience An International Journal on Nutrition, Diet and Nervous System
ISSN: 1028-415X (Print) 1476-8305 (Online) Journal homepage: http://www.tandfonline.com/loi/ynns20
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer's disease Arokiasamy Justin Thenmozhi, Tharsius Raja William Raja, Thamilarasan Manivasagam, Udaiyappan Janakiraman & Musthafa Mohamed Essa To cite this article: Arokiasamy Justin Thenmozhi, Tharsius Raja William Raja, Thamilarasan Manivasagam, Udaiyappan Janakiraman & Musthafa Mohamed Essa (2016): Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer's disease, Nutritional Neuroscience To link to this article: http://dx.doi.org/10.1080/1028415X.2016.1144846
Published online: 16 Feb 2016.
Submit your article to this journal
Article views: 12
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ynns20 Download by: [Orta Dogu Teknik Universitesi]
Date: 10 March 2016, At: 06:11
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer’s disease Arokiasamy Justin Thenmozhi 1, Tharsius Raja William Raja 1, Thamilarasan Manivasagam1, Udaiyappan Janakiraman1, Musthafa Mohamed Essa 2,3
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
1
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India, 2Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat, Oman, 3Ageing and Dementia Research Group, Sultan Qaboos University, Muscat, Oman Background/aims: Deregulation of metal ion homeostasis has been assumed as one of the key factors in the progression of neurodegenerative diseases. Aluminium (Al) has been believed as a major risk factor for the cause and progression of Alzheimer’s disease (AD). In our lab, we have previously reported that hesperidin, a citrus bioflavonoid reversed memory loss caused by aluminium intoxication through attenuating acetylcholine esterase activity and the expression of Amyloid β biosynthesis related markers. Al has been reported to cause oxidative stress associated apoptotic neuronal loss in the brain. So in the present study, protective effect of hesperidin against aluminium chloride (AlCl3) induced cognitive impairment, oxidative stress and apoptosis was studied. Methods: Male Wistar rats were divided into control, AlCl3 treated (100 mg/kg., b.w.), AlCl3 and hesperidin (100 mg/kg., b.w.) co-treated and hesperidin alone treated groups. In control and experimental rats, learning and memory impairment were measured by radial arm maze, elevated plus maze and passive avoidance tests. In addition, oxidative stress and expression of pro and anti-apoptotic markers were also evaluated. Results: Intraperitoneal injection of AlCl3 (100 mg/kg., b.w.) for 60 days significantly enhanced the learning and memory deficits, levels of thiobarbituric acid reactive substances and the expression of Bax and diminished the levels of reduced glutathione, activities of enzymatic antioxidants and the expression of Bcell lymphoma-2 (Bcl-2) as compared to control group in the hippocampus, cortex, and cerebellum. Coadministration of hesperidin (100 mg/kg., b.w. oral) for 60 days prevented the cognitive deficits, biochemical anomalies and apoptosis induced by AlCl3 treatment. Conclusion: Results of the present study demonstrated that hesperidin could be a potential therapeutic agent in the treatment of oxidative stress and apoptosis associated neurodegenerative diseases including AD. Keywords: Alzheimer’s disease, Hesperidin, Cognitive dysfunction, Oxidative stress, Apoptosis
Introduction Metal ion homeostasis in the brain is required for normal cognitive performances1 and their deregulation has been obvious as one of the key factors in the progression of neurodegeneration. Nunes et al. 2 reported that various metals such as aluminium, copper, lithium, zinc, lead, silica, fluoride, mercury, and iron might be involved in neurotoxicity. Aluminium (Al) has been reported as a major risk factor for the cause and development of Alzheimer’s disease (AD), amyotrophic lateral sclerosis and Correspondence to: Arokiasamy Justin Thenmozhi, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India. Email: [email protected]
© Taylor & Francis 2016 DOI 10.1080/1028415X.2016.1144846
Parkinson’s disease (PD).3 AD is a progressive neurodegenerative disorder of aged population that is characterized by short-term memory loss in early stage and manifested by confusion, aggression, mood swings, long-term memory loss, and social isolation in advanced stages.4 The hallmarks of AD include the deposition of amyloid beta fibrils in senile plaques and the presence of abnormal tau protein filaments in the form of neurofibrillary tangles.5 Aluminium enters into the brain via diet, antacid, cosmetics, tooth pastes, inhaled fumes/particles, and through drinking water6 and can be deposited in the cortex, hippocampus7 and cerebellum,8 which are responsible for memory and cognition. In these regions, aluminium induces AD associated
Nutritional Neuroscience
2016
1
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
pathologies such as oligomerization and accumulation of amyloid beta (Aβ), aggregation of hyperphosphorylated tau, lipid peroxidation, impaired exchange of calcium ions and apoptosis.9,10 The currently available drugs for AD including galantamine, rivastigmine, donepezil, memantine, carveditol, rofecoxib and memoquin offers only symptomatic relief without preventing the progression of the disease and with considerable side-effects. Phytochemicals may be the good candidates for therapeutic implications of AD due to their ubiquitous nature and less side-effects along with long lasting benefits. Citrus fruits are generally consumed worldwide and are important sources of health-promoting constituents.11 Hesperidin (3,5,7-trihydroxy flavanone-7-rhamnoglucoside), a phytoflavanone that exists abundance in citrus fruits, is reported to cross the blood–brain barrier easily due to its lipophilic nature12 and afford neuroprotection against PD,13 Huntington’s disease,14 immobilization stress,15 cerebral ischemia/reperfusion16 and stroke17 by virtue of its antioxidant, anti-inflammatory, and anti-apoptotic actions. Recently, we reported the neuroprotective effect of hesperidin against aluminium intoxication through attenuating memory impairment, AChE activity and amyloid β biosynthesis related markers.18 Jangra et al. 19 investigated the effect of hesperidin and silibinin against AlCl3-induced AD rats and suggested that the neuroprotection is attributed to the hindrance of oxido-nitrosative stress and inflammation in the hippocampus. However the antiapoptotic role of hesperidin in AlCl3-induced AD rats was not investigated so far. So the present study was designed to assess the protective effect of hesperidin against learning and memory deficits, oxidative stress and apoptosis in the hippocampus, cortex and cerebellum of AlCl3-induced AD like rats.
Materials and methods Animals Male Albino Wistar rats (200–225 g; 10–12 weeks age) were procured from Central Animal House, Rajah Muthiah Medical College & Hospital, Annamalai University and maintained at standard conditions with food and water ad libitum. The experimental protocols were approved by the Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA, Proposal No. 1005) and according to the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Science Academy, New Delhi, 2000).
Chemicals Aluminium chloride, hesperidin, thiobarbituric acid (TBA), reduced glutathione, 5,5-dithiobis-(2-nitrobenzoic acid) and horseradish peroxidase (HRP)
2
Nutritional Neuroscience
2016
conjugated goat anti-rabbit IgG were purchased from Sigma–Aldrich, Bangalore, India. Anti-rabbit Bax, Bcl-2 and β-actin antibodies were obtained from Cell Signaling. All other chemicals used were of analytical grade.
Experimental design The experiment was carried out in two phases: Phase I and II. Injections were given daily at 10:00 AM, while behavioural tests were performed at 01:00 PM. 6/3 animals per cage were maintained.
Phase I Thirty six Rats were randomized and divided into four groups (n = 9) as follows: Group I: Rats were treated with saline. Group II: rats were injected with 0.5 ml of AlCl3 (100 mg/kg., b.w. i.p.) dissolved in saline for 60 days.18 Group III: Rats were treated orally using intragastric tubes with hesperidin (100 mg/kg., b.w.) dissolved in saline (50 mg/ml) (1 hour prior to AlCl3 injection) and subsequently injected with AlCl3 (100 mg/kg., b.w. i.p.) as group II for 60 days.18 Group IV: Rats were orally treated with hesperidin (100 mg/kg) alone for 60 days.
Phase II In phase II, 24 rats were randomized and divided into four groups (n = 6) as Phase I. In Phase I, six rats from each group received 30 trials for radial arm maze test i.e. 2 trials/day, 6 days/week for a total of 2.5 weeks before the end of the experiment. On 61st day, the radial arm maze test was performed. Then six rats were utilized for the estimation of oxidative stress related parameters and three rats for protein expression studies of apoptotic indices. Phase II rats were used for the assessment of elevated plus maze (EPM) test on 61st and 62nd day and then passive avoidance test was performed after 1 day gap.
Behavioural analysis Radial arm maze test The apparatus consists of eight equidistantly spaced arms (15 × 15 × 70 cm), each radiating from an octagonal centre compartment that was 30 cm in diameter and of the same level as the arms. Four fixed arms were baited with sweetened cereals in grooves that were located 2 cm from the ends of the arms. Before the actual training, rats were allowed to explore till the ends of the radiating arms. The baits were gradually limited to grooves. The rat was placed in the central area of the maze during each trial and allowed to explore. Arm choices were recorded only, if the rat travelled half length of the arm. The trial was completed, when the rat chose all baited arms
Justin Thenmozhi et al.
within 10 minutes. The number of entries to unbaited or re-enteries to baited arms were counted as errors. The following parameters were calculated: working memory, measured by counting the number of repeated entries to baited arms, and reference memory, measured by counting the number of entries to unbaited arms. The mean time required to complete the task in all trials was also calculated.20
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Elevated plus maze (EPM) test EPM test is primarily used for the measurement of anxiety in rodents and is modified to evaluate spatial learning and memory.21 EPM consisted of two opposite open arms (50 cm × 10 cm), traversed with two closed walls of the same size with 40 cm high walls. The open arms were connected with a central square (10 cm × 10 cm) and the entire maze was placed 50 cm high above the ground. Animals are tested for acquisition memory at end of the experimental period (61st day). Rats were placed individually at one end of the open arm. The time taken by the animal to move from the open arm to the closed arm in the maze was recorded as the initial transfer latency (ITL). After recording the ITL animals were allowed to explore the maze for 20 seconds and were then returned to the home cages. By placing the rat in an open arm, retention of memory was assessed and the retention latency was noted on 62nd day.
Passive avoidance task The apparatus consisted of two chambers (an illuminated and a dark) with a metal grid floor. These chambers were divided by a wall and contains execute door. The test was performed on two consecutive days. In the acquisition trial, each and every rat was independently placed in the illuminated chamber. Soon after entering into the dark chamber, an electric shock (40 V, 0.5 mA for 1 second) was passed to the feet of the rat through the floor grid. The rat was immediately taken out from the apparatus and returned to the cage. Rat was placed again in the illuminated chamber after 24 hours and the time taken to the access into the dark chamber was recorded as step-through latency. If the animal did not enter the dark chamber within a 5-minute test period, the test was terminated and the step-through latency was recorded as 300 seconds.20
Tissue preparation for biochemical estimation (TBA reactive substances (TBARS) and antioxidants) After completing the behavioural experiments, the rats were sacrificed to procure brain tissues such as hippocampus, cortex and cerebellum for the biochemical analysis and protein expression studies.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Dissected hippocampus, cortex and cerebellum were homogenized in 10 mM Tris–HCl buffer ( pH 7.0) containing 10 μl/ml protease inhibitor to get 5% (w/v) homogenate and centrifuged (800 × g for 5 minutes at 4°C) to separate the nuclear fragments. The supernatant 1 (S1) was used for estimation of TBARS content. The remaining pellet was further centrifuged (10 500 × g for 30 minutes at 4°C) and the post-mitochondrial supernatant (PMS) was obtained for the estimation of antioxidants.
Biochemical estimation Estimation of TBARS Briefly, the brain tissue extracts were added to 0.2 ml phenyl methosulphate and kept in water bath shaker (37°C). 0.4 ml of 5% trichloroacetic acid and 0.4 ml of 0.67% TBA were added to the reaction mixture after 1 hour. Then it was centrifuged at 3000 × g for 15 minutes and the supernatant was heated for 10 minutes. After cooling, changes in absorbance were observed spectrophotometrically at 532 nm and the rate of lipid peroxidation was expressed as nmol of TBARS formed/g tissue.22
Assay of SOD SOD activity was assayed using an indirect inhibition assay, in which xanthine and xanthine oxidase serve as a superoxide generator, and nitro blue tetrazolium (NBT) is used as a superoxide indicator. To 20 μl of the brain supernatant, 960 μl of sodium carbonate buffer ( pH 10.2) having xanthine, NBT and ethylene-diamine-tetraacetic acid and 20 μl of xanthine oxidase were added. Changes in optical density were read at 560 nm and its activity was expressed as units/min/mg protein.23
Determination of the catalase activity Catalase activity was assayed by measuring the rate of decomposition of hydrogen peroxide at 240 nm. The assay mixture consisted of 50 μl of 1 M Tris–HCl buffer ( pH 8.0) containing 5 mM EDTA, 900 μl of 10 mM H2O2, 30 μl of MQ water and 20 μl of the brain tissue supernatant. The rate of the decomposition of hydrogen peroxide was observed at 240 nm. The enzyme activity was measured as nmol of hydrogen peroxide decomposed/min/mg protein.24
Assay of GPx
To 20 μl of the brain extract, 100 μl of 1 M Tris–HCl ( pH 8.0) having EDTA, 20 μl of 0.1 M GSH, 100 μl of glutathione reductase (10 U/ml), 100 μl of 2 mM NADPH, 10 μl of 7 mM hydroperoxide and 650 μl of distilled water were added. Oxidation of NADPH was determined at 340 nm and the amount of GPx required to oxidize l mol of NADPH per min was termed as one unit of activity.25
Nutritional Neuroscience
2016
3
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Estimation of GSH The level of GSH in the hippocampus, cortex and cerebellular homogenate was measured by the method described by Jollow et al. 26 The homogenate was centrifuged (16 000 × g for 15 minutes at 40°C) and the supernatant (0.5 ml) was added to 4 ml of 0.1 mM solution of 5,5-dithiobis [2-nitrobenzoic acid] in 1 M phosphate buffer ( pH 8). The changes in absorbance were measured at 412 nm.
Western blot analysis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Brain tissues were homogenized with a teflon homogenizer in cold suspension buffer (20 mM Hepes–KOH ( pH 7.5) containing 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 2 mg/ml aprotinin, 10 mg/ml leupeptin, 5 mg/ml pepstatin and 12.5 mg/ml of N-acetyl-Leu-Leu-Norleu-Al. Then it was centrifuged (750 × g at 4°C for 10 minutes) to separate the nuclear debris and then at 10 000 × g for 20 minutes at 4°C to isolate the mitochondrial fraction. Then the pellets were resuspended in cold buffer without sucrose and used as the mitochondrial fraction. Protein concentration was measured by the method of Lowry et al. 27 Samples containing 50 μg of total cellular protein were loaded on 10% sodium docecyl sulphate polyacrylamide gel electrophoresis and separated. Then the gel was transferred on to a polyvinylidene difluoride membrane (Millipore). The membranes were processed28 and then incubated in Bax, Bcl-2 and β-actin (rabbit monoclonal; 1:100) in 5% BSA in Tris-buffered saline and 0.05% Tween-20 (TBST) with gentle shaking overnight at 4°C. After this, membranes were incubated with their corresponding secondary antibodies conjugated to HRP for 2 hours at room temperature and washed. According to the chemiluminescence protocol (GenScript ECL kit, USA), immunoreactive protein was visualized and the densitometric analysis was performed using a gel image analysis program with a computer. The data were then corrected by background subtraction and normalized against β-actin as an internal control.
Results Hesperidin ameliorates AlCl3-induced contextual and exteroceptive memory impairments In radial arm maze test, the animals injected with AlCl3 exhibited more errors both in the reference and working memory task and required more time to end the maze as compared to control group. Cotreatment of hesperidin to AlCl3-treated rats significantly (P < 0.05) reversed the learning and memory deficits as compared to AlCl3 alone treated animals. Hesperidin alone treated rat showed no significant
4
Nutritional Neuroscience
2016
differences in above said indices as compared to control animals (Fig. 1). In elevated plus maze test, the animals treated with AlCl3 exhibited significant (P < 0.05) increase in retention transfer latency as compared to control group. Treatment with hesperidin significantly (P < 0.05) reduced retention transfer latency as compared to aluminium alone treated group. Hesperidin alone treated rat showed no significant differences in retention transfer latency in comparison to control animals (Fig. 2). Performance in passive avoidance task (Fig. 3) showed a decrease in step-through latency in aluminium exposure group as compared to control group, whereas co-administration of hesperidin significantly reversed the AlCl3-induced memory and learning deficits as compared to AlCl3-treated rats. Hesperidin alone treated rat showed no significant differences in mean step-through latency in comparison to control animals.
Figure 1 Effect of hesperidin on radial arm maze performance of AlCl3-induced neurotoxicity in rats. A significant increase in the mean number of errors in measuring (A) reference and (B) working memory and (C) the time required to end the task were observed in AlCl3-treated animals, whereas coadministration of hesperidin to AlCl3treated animals attenuated those performance deficits. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Figure 2 Effect of hesperidin in elevated plus maze performance of AlCl3-induced neurotoxicity in rats. A significant increase in RTL was observed in AlCl3-treated animals, whereas coadministration of hesperidin to AlCl3-treated animals diminished the RTL as compared to AlCl3-treated animals. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
Hesperidin attenuates AlCl3-induced oxidative stress Fig. 4A–E shows the levels of TBARS and GSH and the activities of SOD, catalase, and GPx in the hippocampus, cortex, and cerebellum of control and experimental rats. The levels of TBARS were significantly elevated and the levels of GSH and the activities of SOD, catalase, and GPx were reduced significantly in the hippocampus, cortex, and cerebellum of AlCl3-treated animals (group II) as compared to control. Co-treatment of hesperidin significantly decreased the levels of TBARS and enhanced the levels of GSH and the activities of SOD, catalase, and GPx in the above said brain regions as compared to AlCl3-treated animals.
Hesperidin prevents AlCl3-induced apoptosis We have performed western blots to investigate changes in pro- and anti-apoptotic markers in control and
Figure 3 Effect of hesperidin in passive avoidance test of AlCl3-induced neurotoxicity in rats. A significant increase in the step-through latency was observed in AlCl3-treated animals, whereas coadministration of hesperidin to AlCl3treated animals improved performance in the passive avoidance test. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
experimental groups (Fig. 5). Animals treated with a chronic AlCl3 regimen manifested significant inductions in the expressions of Bax and depletion in Bcl-2 in the hippocampus, cortex and cerebellum. Meanwhile, co-treatment with hesperidin significantly attenuated the expression of pro- and anti-apoptotic markers. Hesperidin alone treated rats did not exhibited significant alterations as compared to control rats.
Discussion We found that hesperidin co-treatment attenuated learning and memory impairments, oxidative stress, and apoptosis caused by aluminium administration in the hippocampus, cortex and cerebellum of the rat. Plenty of learning and memory assessment tests have been used to study the pathogenesis of AD in animal models. In radial arm maze, AlCl3 injection exhibited a significant increase in the time required to end the task, which indicates the decline in reference and working memory.29 Elevated plus maze test is served to evaluate learning and memory in rodents. The present results indicated that rats treated with AlCl3 showed impaired performance in passive avoidance task as evidenced by decreased step-through latencies and enhanced retention transfer latency in elevated plus maze test. The passive avoidance learning is believed to be based on contextual memory in which an adaptive response to a stressful experience, serves as a measure of learning.30 Acetylcholine is very well related with learning and memory processes. Al causes disturbances in cholinergic neurotransmission, which may be associated with altered memory and learning processes. Previous study from our lab indicated that the co-administration of hesperidin to AlCl3 injected animals showed the reverse in memory loss caused by aluminium intoxication through attenuating AChE activity and amyloidogenic pathway.18
Nutritional Neuroscience
2016
5
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Justin Thenmozhi et al.
Figure 4 Effect of hesperidin administration on the levels of (A) lipid peroxidation (TBARS) and (B) reduced glutathione (GSH), activities of (C) SOD, (D) catalase, and (E) GPx of AlCl3induced rat hippocampus, cortex and cerebellum. AlCl3 treatment increased the levels of TBARS and decreased the levels of GSH and activities of SOD, catalase and GPx, whereas cotreatment of hesperidin attenuated the AlCl3 mediated oxidative stress. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
6
Nutritional Neuroscience
2016
The brain is more susceptible to free radical damages due to its low content of glutathione, the high proportion of polyunsaturated fatty acids in its membranes, high iron content and its ability to consume about 20% of total body oxygen.31 Excessive formation of reactive oxygen species subsequently attacks almost all cell components including membrane lipids and causes lipid peroxidation.32 Although aluminium is relatively a low redox mineral, it can induce iron and non iron-mediated lipid peroxidation processes.33 Lipid peroxidation is a consequence of ROS production and is an indirect measure of a metal induced oxidation. AlCl3 treatment significantly increased brain TBARS levels, a sensitive marker of lipid peroxidation process. Previous experiments reported that the aluminium exposure leads to an increased lipid peroxidation and oxidative damage in specific areas of brain including cerebral hippocampus,34,36 cerebellum,35,37 cortex,34,35 35 medulla oblongata, hypothalamus,35 and brain 37 stem, which support our results. SOD is considered as the first line of defence against oxidative stress, as it converts toxic superoxide anion to less toxic H2O2 and O2. H2O2 is then converted to H2O by catalase and GPx at the expenditure of GSH. Therefore, the increased TBARS levels in AlCl3-treated rats may be due to the inhibition of SOD, catalase, and GPx activities and other antioxidants levels. SOD and catalase are protective enzymes and both function in very close association for the detoxification of highly reactive free radicals. Glutathione in its reduced form is the most abundant intracellular antioxidant, which directly scavenges free radicals or serving as a substrate for the glutathione peroxidase enzyme. In our study, treatment of hesperidin to AlCl3 rats decreased the levels of TBARS and increased the levels of GSH and activities of enzymatic antioxidants are in corroborate with earlier studies, where flavonoids with antioxidant properties had been used for the treatment of different type of neurodegenerative diseases including AD.32,38,39 Since the B ring of hesperidin is not conjugated with carbonyl group on the C ring, it enhanced pharmacological functions as compared to other types of flavonoids.40 Hesperidin can accesses easily into the cell, because of its lipophilic nature.41 All these attributed properties, strengthens the antioxidant potential of hesperidin. Recently, Wang et al. 42 demonstrated the protective effects of hesperidin against Aβinduced cognitive dysfunction, oxidative damage and mitochondrial dysfunction in APPswe/PS1dE9 transgenic mice. Al causes neuronal degeneration in the hippocampus and loss of cholinergic terminals in the cortical regions by down-regulating the anti-apoptotic mediators and up-regulating the pro-apoptotic
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Figure 5 Effect of hesperidin administration on the expressions of (A) Bax and Bcl-2 of AlCl3-induced rat hippocampus, cortex and cerebellum. AlCl3 treatment increased the expression of Bax and decreased the expression of Bcl-2, whereas cotreatment of hesperidin attenuated the AlCl3 mediated apoptosis. Immunoblot data of (B) Bax and (C) Bcl-2 were quantified by using β-actin as an internal control and the values are expressed as arbitrary units and given as mean ± SEM for three rats in the hippocampus, cortex, and cerebellum. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
factors.43 Chaudhary et al. 44 revealed that the purkinje cells in the cerebellum was the most affected cell population and their number was decreased, after Al treatment. B-cell lymphoma 2 family of proteins (Bcl-2) play a key role in the regulation of mitochondrial mediated apoptosis. At least, 20 Bcl-2-related proteins are present in mammalian cells, which are classified into two subgroups: the anti-apoptotic (Bcl-2 and Bcl-XL) and the pro-apoptotic (Bax and Bak) proteins. Thus, the balance between antagonistic family members performs a key role in determining cell survival or death. In our study, chronic AlCl3 treatment significantly increased Bax and reduced Bcl-2 expressions, suggesting that Al toxicity would be in favour of apoptosis. Bcl-2 may function as a counter acting force to reduce damage by reducing lipid peroxidation triggered by cytotoxic stimuli such as ROS.45 Bcl-2 was also found to prevent the release of cytochrome c. In contrast, Bax regulates apoptosis, not only by dimerizing with anti-apoptotic Bcl-2 proteins, but also by regulating cytochrome c release and subsequent caspase-3 activation, that finally leads to
cell death.46 However, treatment with hesperidin prevents Al induced apoptosis by reducing the expression of Bax and increasing the expression of Bcl-2. Hesperidin attenuated the apoptosis against experimental autoimmune encephalomyelitis,47 AD,48 global cerebral ischemia/reperfusion49 and H2O2induced cytotoxicity.50 Interestingly, neohesperidin, another flavanone glycoside of citrus fruits, inhibited the middle cerebral artery occlusion induced upregulation of Bax, cytochrome c and cleaved caspase-9 and -3, as well as the downregulation of Bcl-2 by its antioxidant properties.51 To conclude, as the progression of AD is multi-factorial involving various deleterious processes including mitochondrial dysfunction, inflammation and signal transduction pathways and further extensive research is needed to demonstrate the neuroprotective effect of hesperidin.
Acknowledgments Financial assistance in the form of a major research project from the Department of Science and Technology, New Delhi, is gratefully acknowledged.
Nutritional Neuroscience
2016
7
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Disclaimer statements Contributors All authors contributed equally. Funding This work was supported by Department of Science and Technology, Newdelhi, India [grant number SB/FT/LS-348/2012]. Conflict of interest The authors declare that they have no conflict of interest that might have influenced the views expressed in this manuscript. Ethics approval The experimental protocols were approved by the Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA, Proposal No. 1005).
References Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
1 Nelson N. Metal ion transporters and homeostasis. EMBO J 1998;18:4361–71. 2 Nunes PV, Forlenza OV, Gattaz WF. Lithium and risk for Alzheimer’s disease in elderly patients with bipolar disorder. Br J Psychiatry 2007;190:359–60. 3 Singla N, Dhawan DK. Zinc modulates aluminium-induced oxidative stress and cellular injury in rat brain. Metallomics 2014;6: 1941–50. 4 Waldemar G, Dubois B, Emre M, Georges J, McKeith IG, Rossor M, et al. Recommendations for the diagnosis and management of Alzheimer’s disease and other disorders associated with dementia: EFNS guideline. Eur J Neurol 2007;14:1–26. 5 Monczor M. Diagnosis and treatment of Alzheimer’s disease. Curr Med Chem Cent Nerv Syst Agents 2005;5:1–9. 6 Enas AK. Study of possible protective and therapeutic influence of coriander (Coriandrum sativum L.) against neurodegenerative disorders and Alzheimer’s disease induced by aluminum chloride in cerebral cortex of male albino rats. Nat Sci 2010;8: 202–13. 7 Miu AC, Benga O. Aluminum and Alzheimer’s disease: a new look. J Alzheimers Dis 2006;10:179–201. 8 Linardaki ZI, Orkoula MG, Kokkosis AG, Lamari FN, Margarity M. Investigation of the neuroprotective action of saffron (Crocus sativus L.) in aluminum-exposed adult mice through behavioral and neurobiochemical assessment. Food Chem Toxicol 2013;52:163–70. 9 Savory J, Herman MM, Ghribi O. Intracellular mechanisms underlying aluminum-induced apoptosis in rabbit brain. J Inorg Biochem 2003;97:151–4. 10 Goma AA, Mahrous UE. Ethological problems and learning disability due to aluminum toxicity in rats. Anim Vet Sci 2013;1:12–7. 11 Benavente-Garcisa O, Castillo J, Marin FR, Ortuno A, Del Rio JA. Uses and properties of citrus flavonoids. J Agric Food Chem 1997;45:4505–15. 12 Salem HRA, El-Raouf AA, Saleh EM, Shalaby KAF. Influence of hesperidin combined with Sinemet on genetical and biochemical abnormalities in rats suffering from Parkinson’s disease. Life Sci J 2012;9:930–45. 13 Tamilselvam K, Braidy N, Manivasagam T, Essa MM, Prasad NR, Karthikeyan S, et al. Neuroprotective effects of hesperidin, a plant flavanone, on rotenone-induced oxidative stress and apoptosis in a cellular model for Parkinson’s disease. Oxid Med Cell Longev 2013;2013:11. 14 Kumar P, Kumar A. Protective effect of hesperidin and naringin against 3-nitropropionic acid induced Huntington’s like symptoms in rats: possible role of nitric oxide. Behav Brain Res 2010;206:38–46. 15 Viswanatha GL, Shylaja H, Sandeep Rao KS, Santhosh Kumar VR, Jagadeesh M. Hesperidin ameliorates immobilization stressinduced behavioral and biochemical alterations and mitochondrial dysfunction in mice by modulating nitrergic pathway. ISRN Pharmacol 2012;479570. idoi: 10.5402/2012/479570. 16 Ikemura M, Sasaki Y, Giddings JC, Yamamoto J. Preventive effects of hesperidin, glucosyl hesperidin and naringin on hypertension and cerebral thrombosis in stroke-prone spontaneously hypertensive rats. Phytother Res 2012;26:1272–7.
8
Nutritional Neuroscience
2016
17 Raza SS, Khan MM, Ahmad A, Ashafaq M, Khuwaja G, Tabassum R, et al. Hesperidin ameliorates functional and histological outcome and reduces neuroinflammation in experimental stroke. Brain Res 2011;1420:93–105. 18 Justin Thenmozhi A, Raja TR, Janakiraman U, Manivasagam T. Neuroprotective effect of hesperidin on aluminium chloride induced Alzheimer’s disease in Wistar rats. Neurochem Res 2015;40:767–76. 19 Jangra A, Kasbe P, Pandey SN, Dwivedi S, Gurjar SS, Kwatra M, et al. Hesperidin and silibinin ameliorate aluminuminduced neurotoxicity: modulation of antioxidants and inflammatory cytokines level in mice hippocampus. Biol Trace Elem Res 2015;168:462–71. 20 Abdel-Aal RA, Assi AA, Kostandy BB. Rivastigmine reverses aluminum-induced behavioral changes in rats. Eur J Pharmacol 2011;659:169–76. 21 Itoh J, Nabeshima T, Kameyama T. Utility of an elevated plusmaze for the evaluation of memory in mice: effects of nootropics scopolamine and electroconvulsive shock. Psychopharmacology 1990;101:27–33. 22 Bhattacharya A, Ghosal S, Bhattacharya SK. Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. J Ethnopharmacol 2001;74:1–6. 23 Oberley LW. Inhibition of tumor cell growth by overexpression of manganese-containing superoxide dismutase. Age 1998;21: 95–7. 24 Aebi H. Catalase invitro. Methods Enzymol 1984;105:121–6. 25 Yamamoto Y, Takahashi K. Glutathione peroxidase isolated from plasma reduces phospholipid hydroperoxide. Arch Biochem Biophys 1993;305:541–5. 26 Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. Bromobenzene induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 1974;11:151–69. 27 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. 28 Mathiyazahan DB, Justin Thenmozhi A, Manivasagam T. Protective effect of black tea extract against aluminium chloride-induced Alzheimer’s disease in rats: a behavioural, biochemical and molecular approach. J Funct Foods 2015;16:423–35. 29 Ichitani Y, Okaichi H, Yoshikawa T, Ibata Y. Learning behaviour in chronic vitamin E deficient and supplemented rats: radial arm maze learning and passive avoidance response. Behav Brain Res 1992;51:157–64. 30 Tsuji M, Takeda H, Matsumiya T. Modulation of passive avoidance in mice by the 5-HT1A receptor agonist flesinoxan: comparison with the benzodiazepine receptor agonist diazepam. Neuropsychopharmacology 2003;28:664–74. 31 Gupta VB, Anitha S, Hegdea ML, Zecca L, Garruto RM, Ravid R, et al. Aluminium in Alzheimer’s disease: are we still at a crossroad?. Cell Mol Life Sci 2005;62:143–58. 32 El-Sayed el SM, Abo-Salem OM, Abd-Ellah MF, Abd-Alla GM. Hesperidin, an antioxidant flavonoid, prevents acrylonitrile-induced oxidative stress in rat brain. J Biochem Mol Toxicol 2008;22:268–73. 33 Exley C. The pro-oxidant activity of aluminum. Free Radic Biol Med 2004;36:380–7. 34 Abd-Elghaffar SKh, El-Sokkary GH, Sharkawy AA. Aluminum-induced neurotoxicity and oxidative damage in rabbits: protective effect of melatonin. Neuro Endocrinol Lett 2005;26:609–16. 35 Nehru B, Bhalla P, Garg A. Further evidence of centrophenoxine mediated protection in aluminium exposed rats by biochemical and light microscopy analysis. Food Chem Toxicol 2007;45: 2499–505. 36 Nedzvetsky VS, Tuzcu M, Yasar A, Tikhomirov AA, Baydas G. Effects of vitamin E against aluminium neurotoxicity in rats. Biochemistry (Mosc) 2006;71:239–44. 37 Dua R, Gill KD. Effect of aluminium phosphide exposure on kinetic properties of cytochrome oxidase and mitochondrial energy metabolism in rat brain. Biochim Biophys Acta 2004;1674:4–11. 38 Cho J. Antioxidant and neuroprotective effects of hesperidin and its aglycone hesperetin. Arch Pharm Res 2006;29:699–706. 39 Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastawa P, Khan MB, et al. Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009;1292:123–35.
Justin Thenmozhi et al.
46 Cai YL, Cui S, Li ZQ, Wang HX, Ji LH, Chai KX. Studies on apoptosis and caspase-8 and caspase-9 expressions of bone marrow cells in chronic mountain sickness. Zhonghua Xue Ye Xue Za Zhi 2011;32:762–5. 47 Ciftci O, Ozcan C, Kamisli O, Cetin A, Basak N, Aytac B. Hesperidin, a citrus flavonoid, has the ameliorative effects against experimental autoimmune encephalomyelitis (EAE) in a C57BL/J6 mouse model. Neurochem Res 2015;40:1111–20. 48 Banji OJ, Banji D, Ch K. Curcumin and hesperidin improve cognition by suppressing mitochondrial dysfunction and apoptosis induced by D-galactose in rat brain. Food Chem Toxicol 2014;74:51–9. 49 Oztanir MN, Ciftci O, Cetin A, Aladag MA. Hesperidin attenuates oxidative and neuronal damage caused by global cerebral ischemia/reperfusion in a C57BL/J6 mouse model. Neurol Sci 2014;35:1393–9. 50 Hwang SL, Yen GC. Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells. J Agric Food Chem 2008;56:859–64. 51 Wang JJ, Cui P. Neohesperidin attenuates cerebral ischemiareperfusion injury via inhibiting the apoptotic pathway and activating the Akt/Nrf2/HO-1 pathway. J Asian Nat Prod Res 2013;15:1023–37.
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
40 Heim KE, Tagliafero AR, Bobilya DJ. Flavonoid antioxidants: chemistry, metabolism and structure activity relationship. J Nutr Biochem 2002;13:572–84. 41 Spencer JP, Abd-el-Mohsen MM, Rice-Evans C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch Biochem Biophys 2004;423:148–61. 42 Wang D, Liu L, Zhu X, Wu W, Wang Y. Hesperidin alleviates cognitive impairment, mitochondrial dysfunction and oxidative stress in a mouse model of Alzheimer’s disease. Cell Mol Neurobiol 2014;34:1209–21. 43 Niu Q, Wang LP, Chen YL, Zhang HM. Relationship between apoptosis of rat hippocampus cells induced by aluminum and the copy of the bcl-2 as well as bax mRNA. Wei Sheng Yan Jiu 2005;34:671–3. 44 Chaudhary M, Joshi DK, Tripathi S, Kulshrestha S, Mahdi AA. Docosahexaenoic acid ameliorates aluminum induced biochemical and morphological alteration in rat cerebellum. Ann Neurosci 2014;21:5–9. 45 Akifusa S, Kamio N, Shimazaki Y, Yamaguchi N, Nishihara T, Yamashita Y. Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species dependent pathway involving Bcl-2. Free Radic Biol Med 2009;46:1308–16.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Nutritional Neuroscience
2016
9
ISSN: 1028-415X (Print) 1476-8305 (Online) Journal homepage: http://www.tandfonline.com/loi/ynns20
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer's disease Arokiasamy Justin Thenmozhi, Tharsius Raja William Raja, Thamilarasan Manivasagam, Udaiyappan Janakiraman & Musthafa Mohamed Essa To cite this article: Arokiasamy Justin Thenmozhi, Tharsius Raja William Raja, Thamilarasan Manivasagam, Udaiyappan Janakiraman & Musthafa Mohamed Essa (2016): Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer's disease, Nutritional Neuroscience To link to this article: http://dx.doi.org/10.1080/1028415X.2016.1144846
Published online: 16 Feb 2016.
Submit your article to this journal
Article views: 12
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ynns20 Download by: [Orta Dogu Teknik Universitesi]
Date: 10 March 2016, At: 06:11
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer’s disease Arokiasamy Justin Thenmozhi 1, Tharsius Raja William Raja 1, Thamilarasan Manivasagam1, Udaiyappan Janakiraman1, Musthafa Mohamed Essa 2,3
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
1
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India, 2Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat, Oman, 3Ageing and Dementia Research Group, Sultan Qaboos University, Muscat, Oman Background/aims: Deregulation of metal ion homeostasis has been assumed as one of the key factors in the progression of neurodegenerative diseases. Aluminium (Al) has been believed as a major risk factor for the cause and progression of Alzheimer’s disease (AD). In our lab, we have previously reported that hesperidin, a citrus bioflavonoid reversed memory loss caused by aluminium intoxication through attenuating acetylcholine esterase activity and the expression of Amyloid β biosynthesis related markers. Al has been reported to cause oxidative stress associated apoptotic neuronal loss in the brain. So in the present study, protective effect of hesperidin against aluminium chloride (AlCl3) induced cognitive impairment, oxidative stress and apoptosis was studied. Methods: Male Wistar rats were divided into control, AlCl3 treated (100 mg/kg., b.w.), AlCl3 and hesperidin (100 mg/kg., b.w.) co-treated and hesperidin alone treated groups. In control and experimental rats, learning and memory impairment were measured by radial arm maze, elevated plus maze and passive avoidance tests. In addition, oxidative stress and expression of pro and anti-apoptotic markers were also evaluated. Results: Intraperitoneal injection of AlCl3 (100 mg/kg., b.w.) for 60 days significantly enhanced the learning and memory deficits, levels of thiobarbituric acid reactive substances and the expression of Bax and diminished the levels of reduced glutathione, activities of enzymatic antioxidants and the expression of Bcell lymphoma-2 (Bcl-2) as compared to control group in the hippocampus, cortex, and cerebellum. Coadministration of hesperidin (100 mg/kg., b.w. oral) for 60 days prevented the cognitive deficits, biochemical anomalies and apoptosis induced by AlCl3 treatment. Conclusion: Results of the present study demonstrated that hesperidin could be a potential therapeutic agent in the treatment of oxidative stress and apoptosis associated neurodegenerative diseases including AD. Keywords: Alzheimer’s disease, Hesperidin, Cognitive dysfunction, Oxidative stress, Apoptosis
Introduction Metal ion homeostasis in the brain is required for normal cognitive performances1 and their deregulation has been obvious as one of the key factors in the progression of neurodegeneration. Nunes et al. 2 reported that various metals such as aluminium, copper, lithium, zinc, lead, silica, fluoride, mercury, and iron might be involved in neurotoxicity. Aluminium (Al) has been reported as a major risk factor for the cause and development of Alzheimer’s disease (AD), amyotrophic lateral sclerosis and Correspondence to: Arokiasamy Justin Thenmozhi, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamilnadu 608 002, India. Email: [email protected]
© Taylor & Francis 2016 DOI 10.1080/1028415X.2016.1144846
Parkinson’s disease (PD).3 AD is a progressive neurodegenerative disorder of aged population that is characterized by short-term memory loss in early stage and manifested by confusion, aggression, mood swings, long-term memory loss, and social isolation in advanced stages.4 The hallmarks of AD include the deposition of amyloid beta fibrils in senile plaques and the presence of abnormal tau protein filaments in the form of neurofibrillary tangles.5 Aluminium enters into the brain via diet, antacid, cosmetics, tooth pastes, inhaled fumes/particles, and through drinking water6 and can be deposited in the cortex, hippocampus7 and cerebellum,8 which are responsible for memory and cognition. In these regions, aluminium induces AD associated
Nutritional Neuroscience
2016
1
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
pathologies such as oligomerization and accumulation of amyloid beta (Aβ), aggregation of hyperphosphorylated tau, lipid peroxidation, impaired exchange of calcium ions and apoptosis.9,10 The currently available drugs for AD including galantamine, rivastigmine, donepezil, memantine, carveditol, rofecoxib and memoquin offers only symptomatic relief without preventing the progression of the disease and with considerable side-effects. Phytochemicals may be the good candidates for therapeutic implications of AD due to their ubiquitous nature and less side-effects along with long lasting benefits. Citrus fruits are generally consumed worldwide and are important sources of health-promoting constituents.11 Hesperidin (3,5,7-trihydroxy flavanone-7-rhamnoglucoside), a phytoflavanone that exists abundance in citrus fruits, is reported to cross the blood–brain barrier easily due to its lipophilic nature12 and afford neuroprotection against PD,13 Huntington’s disease,14 immobilization stress,15 cerebral ischemia/reperfusion16 and stroke17 by virtue of its antioxidant, anti-inflammatory, and anti-apoptotic actions. Recently, we reported the neuroprotective effect of hesperidin against aluminium intoxication through attenuating memory impairment, AChE activity and amyloid β biosynthesis related markers.18 Jangra et al. 19 investigated the effect of hesperidin and silibinin against AlCl3-induced AD rats and suggested that the neuroprotection is attributed to the hindrance of oxido-nitrosative stress and inflammation in the hippocampus. However the antiapoptotic role of hesperidin in AlCl3-induced AD rats was not investigated so far. So the present study was designed to assess the protective effect of hesperidin against learning and memory deficits, oxidative stress and apoptosis in the hippocampus, cortex and cerebellum of AlCl3-induced AD like rats.
Materials and methods Animals Male Albino Wistar rats (200–225 g; 10–12 weeks age) were procured from Central Animal House, Rajah Muthiah Medical College & Hospital, Annamalai University and maintained at standard conditions with food and water ad libitum. The experimental protocols were approved by the Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA, Proposal No. 1005) and according to the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Science Academy, New Delhi, 2000).
Chemicals Aluminium chloride, hesperidin, thiobarbituric acid (TBA), reduced glutathione, 5,5-dithiobis-(2-nitrobenzoic acid) and horseradish peroxidase (HRP)
2
Nutritional Neuroscience
2016
conjugated goat anti-rabbit IgG were purchased from Sigma–Aldrich, Bangalore, India. Anti-rabbit Bax, Bcl-2 and β-actin antibodies were obtained from Cell Signaling. All other chemicals used were of analytical grade.
Experimental design The experiment was carried out in two phases: Phase I and II. Injections were given daily at 10:00 AM, while behavioural tests were performed at 01:00 PM. 6/3 animals per cage were maintained.
Phase I Thirty six Rats were randomized and divided into four groups (n = 9) as follows: Group I: Rats were treated with saline. Group II: rats were injected with 0.5 ml of AlCl3 (100 mg/kg., b.w. i.p.) dissolved in saline for 60 days.18 Group III: Rats were treated orally using intragastric tubes with hesperidin (100 mg/kg., b.w.) dissolved in saline (50 mg/ml) (1 hour prior to AlCl3 injection) and subsequently injected with AlCl3 (100 mg/kg., b.w. i.p.) as group II for 60 days.18 Group IV: Rats were orally treated with hesperidin (100 mg/kg) alone for 60 days.
Phase II In phase II, 24 rats were randomized and divided into four groups (n = 6) as Phase I. In Phase I, six rats from each group received 30 trials for radial arm maze test i.e. 2 trials/day, 6 days/week for a total of 2.5 weeks before the end of the experiment. On 61st day, the radial arm maze test was performed. Then six rats were utilized for the estimation of oxidative stress related parameters and three rats for protein expression studies of apoptotic indices. Phase II rats were used for the assessment of elevated plus maze (EPM) test on 61st and 62nd day and then passive avoidance test was performed after 1 day gap.
Behavioural analysis Radial arm maze test The apparatus consists of eight equidistantly spaced arms (15 × 15 × 70 cm), each radiating from an octagonal centre compartment that was 30 cm in diameter and of the same level as the arms. Four fixed arms were baited with sweetened cereals in grooves that were located 2 cm from the ends of the arms. Before the actual training, rats were allowed to explore till the ends of the radiating arms. The baits were gradually limited to grooves. The rat was placed in the central area of the maze during each trial and allowed to explore. Arm choices were recorded only, if the rat travelled half length of the arm. The trial was completed, when the rat chose all baited arms
Justin Thenmozhi et al.
within 10 minutes. The number of entries to unbaited or re-enteries to baited arms were counted as errors. The following parameters were calculated: working memory, measured by counting the number of repeated entries to baited arms, and reference memory, measured by counting the number of entries to unbaited arms. The mean time required to complete the task in all trials was also calculated.20
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Elevated plus maze (EPM) test EPM test is primarily used for the measurement of anxiety in rodents and is modified to evaluate spatial learning and memory.21 EPM consisted of two opposite open arms (50 cm × 10 cm), traversed with two closed walls of the same size with 40 cm high walls. The open arms were connected with a central square (10 cm × 10 cm) and the entire maze was placed 50 cm high above the ground. Animals are tested for acquisition memory at end of the experimental period (61st day). Rats were placed individually at one end of the open arm. The time taken by the animal to move from the open arm to the closed arm in the maze was recorded as the initial transfer latency (ITL). After recording the ITL animals were allowed to explore the maze for 20 seconds and were then returned to the home cages. By placing the rat in an open arm, retention of memory was assessed and the retention latency was noted on 62nd day.
Passive avoidance task The apparatus consisted of two chambers (an illuminated and a dark) with a metal grid floor. These chambers were divided by a wall and contains execute door. The test was performed on two consecutive days. In the acquisition trial, each and every rat was independently placed in the illuminated chamber. Soon after entering into the dark chamber, an electric shock (40 V, 0.5 mA for 1 second) was passed to the feet of the rat through the floor grid. The rat was immediately taken out from the apparatus and returned to the cage. Rat was placed again in the illuminated chamber after 24 hours and the time taken to the access into the dark chamber was recorded as step-through latency. If the animal did not enter the dark chamber within a 5-minute test period, the test was terminated and the step-through latency was recorded as 300 seconds.20
Tissue preparation for biochemical estimation (TBA reactive substances (TBARS) and antioxidants) After completing the behavioural experiments, the rats were sacrificed to procure brain tissues such as hippocampus, cortex and cerebellum for the biochemical analysis and protein expression studies.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Dissected hippocampus, cortex and cerebellum were homogenized in 10 mM Tris–HCl buffer ( pH 7.0) containing 10 μl/ml protease inhibitor to get 5% (w/v) homogenate and centrifuged (800 × g for 5 minutes at 4°C) to separate the nuclear fragments. The supernatant 1 (S1) was used for estimation of TBARS content. The remaining pellet was further centrifuged (10 500 × g for 30 minutes at 4°C) and the post-mitochondrial supernatant (PMS) was obtained for the estimation of antioxidants.
Biochemical estimation Estimation of TBARS Briefly, the brain tissue extracts were added to 0.2 ml phenyl methosulphate and kept in water bath shaker (37°C). 0.4 ml of 5% trichloroacetic acid and 0.4 ml of 0.67% TBA were added to the reaction mixture after 1 hour. Then it was centrifuged at 3000 × g for 15 minutes and the supernatant was heated for 10 minutes. After cooling, changes in absorbance were observed spectrophotometrically at 532 nm and the rate of lipid peroxidation was expressed as nmol of TBARS formed/g tissue.22
Assay of SOD SOD activity was assayed using an indirect inhibition assay, in which xanthine and xanthine oxidase serve as a superoxide generator, and nitro blue tetrazolium (NBT) is used as a superoxide indicator. To 20 μl of the brain supernatant, 960 μl of sodium carbonate buffer ( pH 10.2) having xanthine, NBT and ethylene-diamine-tetraacetic acid and 20 μl of xanthine oxidase were added. Changes in optical density were read at 560 nm and its activity was expressed as units/min/mg protein.23
Determination of the catalase activity Catalase activity was assayed by measuring the rate of decomposition of hydrogen peroxide at 240 nm. The assay mixture consisted of 50 μl of 1 M Tris–HCl buffer ( pH 8.0) containing 5 mM EDTA, 900 μl of 10 mM H2O2, 30 μl of MQ water and 20 μl of the brain tissue supernatant. The rate of the decomposition of hydrogen peroxide was observed at 240 nm. The enzyme activity was measured as nmol of hydrogen peroxide decomposed/min/mg protein.24
Assay of GPx
To 20 μl of the brain extract, 100 μl of 1 M Tris–HCl ( pH 8.0) having EDTA, 20 μl of 0.1 M GSH, 100 μl of glutathione reductase (10 U/ml), 100 μl of 2 mM NADPH, 10 μl of 7 mM hydroperoxide and 650 μl of distilled water were added. Oxidation of NADPH was determined at 340 nm and the amount of GPx required to oxidize l mol of NADPH per min was termed as one unit of activity.25
Nutritional Neuroscience
2016
3
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Estimation of GSH The level of GSH in the hippocampus, cortex and cerebellular homogenate was measured by the method described by Jollow et al. 26 The homogenate was centrifuged (16 000 × g for 15 minutes at 40°C) and the supernatant (0.5 ml) was added to 4 ml of 0.1 mM solution of 5,5-dithiobis [2-nitrobenzoic acid] in 1 M phosphate buffer ( pH 8). The changes in absorbance were measured at 412 nm.
Western blot analysis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Brain tissues were homogenized with a teflon homogenizer in cold suspension buffer (20 mM Hepes–KOH ( pH 7.5) containing 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 2 mg/ml aprotinin, 10 mg/ml leupeptin, 5 mg/ml pepstatin and 12.5 mg/ml of N-acetyl-Leu-Leu-Norleu-Al. Then it was centrifuged (750 × g at 4°C for 10 minutes) to separate the nuclear debris and then at 10 000 × g for 20 minutes at 4°C to isolate the mitochondrial fraction. Then the pellets were resuspended in cold buffer without sucrose and used as the mitochondrial fraction. Protein concentration was measured by the method of Lowry et al. 27 Samples containing 50 μg of total cellular protein were loaded on 10% sodium docecyl sulphate polyacrylamide gel electrophoresis and separated. Then the gel was transferred on to a polyvinylidene difluoride membrane (Millipore). The membranes were processed28 and then incubated in Bax, Bcl-2 and β-actin (rabbit monoclonal; 1:100) in 5% BSA in Tris-buffered saline and 0.05% Tween-20 (TBST) with gentle shaking overnight at 4°C. After this, membranes were incubated with their corresponding secondary antibodies conjugated to HRP for 2 hours at room temperature and washed. According to the chemiluminescence protocol (GenScript ECL kit, USA), immunoreactive protein was visualized and the densitometric analysis was performed using a gel image analysis program with a computer. The data were then corrected by background subtraction and normalized against β-actin as an internal control.
Results Hesperidin ameliorates AlCl3-induced contextual and exteroceptive memory impairments In radial arm maze test, the animals injected with AlCl3 exhibited more errors both in the reference and working memory task and required more time to end the maze as compared to control group. Cotreatment of hesperidin to AlCl3-treated rats significantly (P < 0.05) reversed the learning and memory deficits as compared to AlCl3 alone treated animals. Hesperidin alone treated rat showed no significant
4
Nutritional Neuroscience
2016
differences in above said indices as compared to control animals (Fig. 1). In elevated plus maze test, the animals treated with AlCl3 exhibited significant (P < 0.05) increase in retention transfer latency as compared to control group. Treatment with hesperidin significantly (P < 0.05) reduced retention transfer latency as compared to aluminium alone treated group. Hesperidin alone treated rat showed no significant differences in retention transfer latency in comparison to control animals (Fig. 2). Performance in passive avoidance task (Fig. 3) showed a decrease in step-through latency in aluminium exposure group as compared to control group, whereas co-administration of hesperidin significantly reversed the AlCl3-induced memory and learning deficits as compared to AlCl3-treated rats. Hesperidin alone treated rat showed no significant differences in mean step-through latency in comparison to control animals.
Figure 1 Effect of hesperidin on radial arm maze performance of AlCl3-induced neurotoxicity in rats. A significant increase in the mean number of errors in measuring (A) reference and (B) working memory and (C) the time required to end the task were observed in AlCl3-treated animals, whereas coadministration of hesperidin to AlCl3treated animals attenuated those performance deficits. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Figure 2 Effect of hesperidin in elevated plus maze performance of AlCl3-induced neurotoxicity in rats. A significant increase in RTL was observed in AlCl3-treated animals, whereas coadministration of hesperidin to AlCl3-treated animals diminished the RTL as compared to AlCl3-treated animals. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
Hesperidin attenuates AlCl3-induced oxidative stress Fig. 4A–E shows the levels of TBARS and GSH and the activities of SOD, catalase, and GPx in the hippocampus, cortex, and cerebellum of control and experimental rats. The levels of TBARS were significantly elevated and the levels of GSH and the activities of SOD, catalase, and GPx were reduced significantly in the hippocampus, cortex, and cerebellum of AlCl3-treated animals (group II) as compared to control. Co-treatment of hesperidin significantly decreased the levels of TBARS and enhanced the levels of GSH and the activities of SOD, catalase, and GPx in the above said brain regions as compared to AlCl3-treated animals.
Hesperidin prevents AlCl3-induced apoptosis We have performed western blots to investigate changes in pro- and anti-apoptotic markers in control and
Figure 3 Effect of hesperidin in passive avoidance test of AlCl3-induced neurotoxicity in rats. A significant increase in the step-through latency was observed in AlCl3-treated animals, whereas coadministration of hesperidin to AlCl3treated animals improved performance in the passive avoidance test. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
experimental groups (Fig. 5). Animals treated with a chronic AlCl3 regimen manifested significant inductions in the expressions of Bax and depletion in Bcl-2 in the hippocampus, cortex and cerebellum. Meanwhile, co-treatment with hesperidin significantly attenuated the expression of pro- and anti-apoptotic markers. Hesperidin alone treated rats did not exhibited significant alterations as compared to control rats.
Discussion We found that hesperidin co-treatment attenuated learning and memory impairments, oxidative stress, and apoptosis caused by aluminium administration in the hippocampus, cortex and cerebellum of the rat. Plenty of learning and memory assessment tests have been used to study the pathogenesis of AD in animal models. In radial arm maze, AlCl3 injection exhibited a significant increase in the time required to end the task, which indicates the decline in reference and working memory.29 Elevated plus maze test is served to evaluate learning and memory in rodents. The present results indicated that rats treated with AlCl3 showed impaired performance in passive avoidance task as evidenced by decreased step-through latencies and enhanced retention transfer latency in elevated plus maze test. The passive avoidance learning is believed to be based on contextual memory in which an adaptive response to a stressful experience, serves as a measure of learning.30 Acetylcholine is very well related with learning and memory processes. Al causes disturbances in cholinergic neurotransmission, which may be associated with altered memory and learning processes. Previous study from our lab indicated that the co-administration of hesperidin to AlCl3 injected animals showed the reverse in memory loss caused by aluminium intoxication through attenuating AChE activity and amyloidogenic pathway.18
Nutritional Neuroscience
2016
5
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Justin Thenmozhi et al.
Figure 4 Effect of hesperidin administration on the levels of (A) lipid peroxidation (TBARS) and (B) reduced glutathione (GSH), activities of (C) SOD, (D) catalase, and (E) GPx of AlCl3induced rat hippocampus, cortex and cerebellum. AlCl3 treatment increased the levels of TBARS and decreased the levels of GSH and activities of SOD, catalase and GPx, whereas cotreatment of hesperidin attenuated the AlCl3 mediated oxidative stress. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
6
Nutritional Neuroscience
2016
The brain is more susceptible to free radical damages due to its low content of glutathione, the high proportion of polyunsaturated fatty acids in its membranes, high iron content and its ability to consume about 20% of total body oxygen.31 Excessive formation of reactive oxygen species subsequently attacks almost all cell components including membrane lipids and causes lipid peroxidation.32 Although aluminium is relatively a low redox mineral, it can induce iron and non iron-mediated lipid peroxidation processes.33 Lipid peroxidation is a consequence of ROS production and is an indirect measure of a metal induced oxidation. AlCl3 treatment significantly increased brain TBARS levels, a sensitive marker of lipid peroxidation process. Previous experiments reported that the aluminium exposure leads to an increased lipid peroxidation and oxidative damage in specific areas of brain including cerebral hippocampus,34,36 cerebellum,35,37 cortex,34,35 35 medulla oblongata, hypothalamus,35 and brain 37 stem, which support our results. SOD is considered as the first line of defence against oxidative stress, as it converts toxic superoxide anion to less toxic H2O2 and O2. H2O2 is then converted to H2O by catalase and GPx at the expenditure of GSH. Therefore, the increased TBARS levels in AlCl3-treated rats may be due to the inhibition of SOD, catalase, and GPx activities and other antioxidants levels. SOD and catalase are protective enzymes and both function in very close association for the detoxification of highly reactive free radicals. Glutathione in its reduced form is the most abundant intracellular antioxidant, which directly scavenges free radicals or serving as a substrate for the glutathione peroxidase enzyme. In our study, treatment of hesperidin to AlCl3 rats decreased the levels of TBARS and increased the levels of GSH and activities of enzymatic antioxidants are in corroborate with earlier studies, where flavonoids with antioxidant properties had been used for the treatment of different type of neurodegenerative diseases including AD.32,38,39 Since the B ring of hesperidin is not conjugated with carbonyl group on the C ring, it enhanced pharmacological functions as compared to other types of flavonoids.40 Hesperidin can accesses easily into the cell, because of its lipophilic nature.41 All these attributed properties, strengthens the antioxidant potential of hesperidin. Recently, Wang et al. 42 demonstrated the protective effects of hesperidin against Aβinduced cognitive dysfunction, oxidative damage and mitochondrial dysfunction in APPswe/PS1dE9 transgenic mice. Al causes neuronal degeneration in the hippocampus and loss of cholinergic terminals in the cortical regions by down-regulating the anti-apoptotic mediators and up-regulating the pro-apoptotic
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Figure 5 Effect of hesperidin administration on the expressions of (A) Bax and Bcl-2 of AlCl3-induced rat hippocampus, cortex and cerebellum. AlCl3 treatment increased the expression of Bax and decreased the expression of Bcl-2, whereas cotreatment of hesperidin attenuated the AlCl3 mediated apoptosis. Immunoblot data of (B) Bax and (C) Bcl-2 were quantified by using β-actin as an internal control and the values are expressed as arbitrary units and given as mean ± SEM for three rats in the hippocampus, cortex, and cerebellum. Values not sharing the same letters differ significantly – bP < 0.05 compared to all other groups, cP < 0.05 compared to all other groups.
factors.43 Chaudhary et al. 44 revealed that the purkinje cells in the cerebellum was the most affected cell population and their number was decreased, after Al treatment. B-cell lymphoma 2 family of proteins (Bcl-2) play a key role in the regulation of mitochondrial mediated apoptosis. At least, 20 Bcl-2-related proteins are present in mammalian cells, which are classified into two subgroups: the anti-apoptotic (Bcl-2 and Bcl-XL) and the pro-apoptotic (Bax and Bak) proteins. Thus, the balance between antagonistic family members performs a key role in determining cell survival or death. In our study, chronic AlCl3 treatment significantly increased Bax and reduced Bcl-2 expressions, suggesting that Al toxicity would be in favour of apoptosis. Bcl-2 may function as a counter acting force to reduce damage by reducing lipid peroxidation triggered by cytotoxic stimuli such as ROS.45 Bcl-2 was also found to prevent the release of cytochrome c. In contrast, Bax regulates apoptosis, not only by dimerizing with anti-apoptotic Bcl-2 proteins, but also by regulating cytochrome c release and subsequent caspase-3 activation, that finally leads to
cell death.46 However, treatment with hesperidin prevents Al induced apoptosis by reducing the expression of Bax and increasing the expression of Bcl-2. Hesperidin attenuated the apoptosis against experimental autoimmune encephalomyelitis,47 AD,48 global cerebral ischemia/reperfusion49 and H2O2induced cytotoxicity.50 Interestingly, neohesperidin, another flavanone glycoside of citrus fruits, inhibited the middle cerebral artery occlusion induced upregulation of Bax, cytochrome c and cleaved caspase-9 and -3, as well as the downregulation of Bcl-2 by its antioxidant properties.51 To conclude, as the progression of AD is multi-factorial involving various deleterious processes including mitochondrial dysfunction, inflammation and signal transduction pathways and further extensive research is needed to demonstrate the neuroprotective effect of hesperidin.
Acknowledgments Financial assistance in the form of a major research project from the Department of Science and Technology, New Delhi, is gratefully acknowledged.
Nutritional Neuroscience
2016
7
Justin Thenmozhi et al.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Disclaimer statements Contributors All authors contributed equally. Funding This work was supported by Department of Science and Technology, Newdelhi, India [grant number SB/FT/LS-348/2012]. Conflict of interest The authors declare that they have no conflict of interest that might have influenced the views expressed in this manuscript. Ethics approval The experimental protocols were approved by the Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA, Proposal No. 1005).
References Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
1 Nelson N. Metal ion transporters and homeostasis. EMBO J 1998;18:4361–71. 2 Nunes PV, Forlenza OV, Gattaz WF. Lithium and risk for Alzheimer’s disease in elderly patients with bipolar disorder. Br J Psychiatry 2007;190:359–60. 3 Singla N, Dhawan DK. Zinc modulates aluminium-induced oxidative stress and cellular injury in rat brain. Metallomics 2014;6: 1941–50. 4 Waldemar G, Dubois B, Emre M, Georges J, McKeith IG, Rossor M, et al. Recommendations for the diagnosis and management of Alzheimer’s disease and other disorders associated with dementia: EFNS guideline. Eur J Neurol 2007;14:1–26. 5 Monczor M. Diagnosis and treatment of Alzheimer’s disease. Curr Med Chem Cent Nerv Syst Agents 2005;5:1–9. 6 Enas AK. Study of possible protective and therapeutic influence of coriander (Coriandrum sativum L.) against neurodegenerative disorders and Alzheimer’s disease induced by aluminum chloride in cerebral cortex of male albino rats. Nat Sci 2010;8: 202–13. 7 Miu AC, Benga O. Aluminum and Alzheimer’s disease: a new look. J Alzheimers Dis 2006;10:179–201. 8 Linardaki ZI, Orkoula MG, Kokkosis AG, Lamari FN, Margarity M. Investigation of the neuroprotective action of saffron (Crocus sativus L.) in aluminum-exposed adult mice through behavioral and neurobiochemical assessment. Food Chem Toxicol 2013;52:163–70. 9 Savory J, Herman MM, Ghribi O. Intracellular mechanisms underlying aluminum-induced apoptosis in rabbit brain. J Inorg Biochem 2003;97:151–4. 10 Goma AA, Mahrous UE. Ethological problems and learning disability due to aluminum toxicity in rats. Anim Vet Sci 2013;1:12–7. 11 Benavente-Garcisa O, Castillo J, Marin FR, Ortuno A, Del Rio JA. Uses and properties of citrus flavonoids. J Agric Food Chem 1997;45:4505–15. 12 Salem HRA, El-Raouf AA, Saleh EM, Shalaby KAF. Influence of hesperidin combined with Sinemet on genetical and biochemical abnormalities in rats suffering from Parkinson’s disease. Life Sci J 2012;9:930–45. 13 Tamilselvam K, Braidy N, Manivasagam T, Essa MM, Prasad NR, Karthikeyan S, et al. Neuroprotective effects of hesperidin, a plant flavanone, on rotenone-induced oxidative stress and apoptosis in a cellular model for Parkinson’s disease. Oxid Med Cell Longev 2013;2013:11. 14 Kumar P, Kumar A. Protective effect of hesperidin and naringin against 3-nitropropionic acid induced Huntington’s like symptoms in rats: possible role of nitric oxide. Behav Brain Res 2010;206:38–46. 15 Viswanatha GL, Shylaja H, Sandeep Rao KS, Santhosh Kumar VR, Jagadeesh M. Hesperidin ameliorates immobilization stressinduced behavioral and biochemical alterations and mitochondrial dysfunction in mice by modulating nitrergic pathway. ISRN Pharmacol 2012;479570. idoi: 10.5402/2012/479570. 16 Ikemura M, Sasaki Y, Giddings JC, Yamamoto J. Preventive effects of hesperidin, glucosyl hesperidin and naringin on hypertension and cerebral thrombosis in stroke-prone spontaneously hypertensive rats. Phytother Res 2012;26:1272–7.
8
Nutritional Neuroscience
2016
17 Raza SS, Khan MM, Ahmad A, Ashafaq M, Khuwaja G, Tabassum R, et al. Hesperidin ameliorates functional and histological outcome and reduces neuroinflammation in experimental stroke. Brain Res 2011;1420:93–105. 18 Justin Thenmozhi A, Raja TR, Janakiraman U, Manivasagam T. Neuroprotective effect of hesperidin on aluminium chloride induced Alzheimer’s disease in Wistar rats. Neurochem Res 2015;40:767–76. 19 Jangra A, Kasbe P, Pandey SN, Dwivedi S, Gurjar SS, Kwatra M, et al. Hesperidin and silibinin ameliorate aluminuminduced neurotoxicity: modulation of antioxidants and inflammatory cytokines level in mice hippocampus. Biol Trace Elem Res 2015;168:462–71. 20 Abdel-Aal RA, Assi AA, Kostandy BB. Rivastigmine reverses aluminum-induced behavioral changes in rats. Eur J Pharmacol 2011;659:169–76. 21 Itoh J, Nabeshima T, Kameyama T. Utility of an elevated plusmaze for the evaluation of memory in mice: effects of nootropics scopolamine and electroconvulsive shock. Psychopharmacology 1990;101:27–33. 22 Bhattacharya A, Ghosal S, Bhattacharya SK. Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. J Ethnopharmacol 2001;74:1–6. 23 Oberley LW. Inhibition of tumor cell growth by overexpression of manganese-containing superoxide dismutase. Age 1998;21: 95–7. 24 Aebi H. Catalase invitro. Methods Enzymol 1984;105:121–6. 25 Yamamoto Y, Takahashi K. Glutathione peroxidase isolated from plasma reduces phospholipid hydroperoxide. Arch Biochem Biophys 1993;305:541–5. 26 Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. Bromobenzene induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 1974;11:151–69. 27 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. 28 Mathiyazahan DB, Justin Thenmozhi A, Manivasagam T. Protective effect of black tea extract against aluminium chloride-induced Alzheimer’s disease in rats: a behavioural, biochemical and molecular approach. J Funct Foods 2015;16:423–35. 29 Ichitani Y, Okaichi H, Yoshikawa T, Ibata Y. Learning behaviour in chronic vitamin E deficient and supplemented rats: radial arm maze learning and passive avoidance response. Behav Brain Res 1992;51:157–64. 30 Tsuji M, Takeda H, Matsumiya T. Modulation of passive avoidance in mice by the 5-HT1A receptor agonist flesinoxan: comparison with the benzodiazepine receptor agonist diazepam. Neuropsychopharmacology 2003;28:664–74. 31 Gupta VB, Anitha S, Hegdea ML, Zecca L, Garruto RM, Ravid R, et al. Aluminium in Alzheimer’s disease: are we still at a crossroad?. Cell Mol Life Sci 2005;62:143–58. 32 El-Sayed el SM, Abo-Salem OM, Abd-Ellah MF, Abd-Alla GM. Hesperidin, an antioxidant flavonoid, prevents acrylonitrile-induced oxidative stress in rat brain. J Biochem Mol Toxicol 2008;22:268–73. 33 Exley C. The pro-oxidant activity of aluminum. Free Radic Biol Med 2004;36:380–7. 34 Abd-Elghaffar SKh, El-Sokkary GH, Sharkawy AA. Aluminum-induced neurotoxicity and oxidative damage in rabbits: protective effect of melatonin. Neuro Endocrinol Lett 2005;26:609–16. 35 Nehru B, Bhalla P, Garg A. Further evidence of centrophenoxine mediated protection in aluminium exposed rats by biochemical and light microscopy analysis. Food Chem Toxicol 2007;45: 2499–505. 36 Nedzvetsky VS, Tuzcu M, Yasar A, Tikhomirov AA, Baydas G. Effects of vitamin E against aluminium neurotoxicity in rats. Biochemistry (Mosc) 2006;71:239–44. 37 Dua R, Gill KD. Effect of aluminium phosphide exposure on kinetic properties of cytochrome oxidase and mitochondrial energy metabolism in rat brain. Biochim Biophys Acta 2004;1674:4–11. 38 Cho J. Antioxidant and neuroprotective effects of hesperidin and its aglycone hesperetin. Arch Pharm Res 2006;29:699–706. 39 Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastawa P, Khan MB, et al. Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009;1292:123–35.
Justin Thenmozhi et al.
46 Cai YL, Cui S, Li ZQ, Wang HX, Ji LH, Chai KX. Studies on apoptosis and caspase-8 and caspase-9 expressions of bone marrow cells in chronic mountain sickness. Zhonghua Xue Ye Xue Za Zhi 2011;32:762–5. 47 Ciftci O, Ozcan C, Kamisli O, Cetin A, Basak N, Aytac B. Hesperidin, a citrus flavonoid, has the ameliorative effects against experimental autoimmune encephalomyelitis (EAE) in a C57BL/J6 mouse model. Neurochem Res 2015;40:1111–20. 48 Banji OJ, Banji D, Ch K. Curcumin and hesperidin improve cognition by suppressing mitochondrial dysfunction and apoptosis induced by D-galactose in rat brain. Food Chem Toxicol 2014;74:51–9. 49 Oztanir MN, Ciftci O, Cetin A, Aladag MA. Hesperidin attenuates oxidative and neuronal damage caused by global cerebral ischemia/reperfusion in a C57BL/J6 mouse model. Neurol Sci 2014;35:1393–9. 50 Hwang SL, Yen GC. Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells. J Agric Food Chem 2008;56:859–64. 51 Wang JJ, Cui P. Neohesperidin attenuates cerebral ischemiareperfusion injury via inhibiting the apoptotic pathway and activating the Akt/Nrf2/HO-1 pathway. J Asian Nat Prod Res 2013;15:1023–37.
Downloaded by [Orta Dogu Teknik Universitesi] at 06:11 10 March 2016
40 Heim KE, Tagliafero AR, Bobilya DJ. Flavonoid antioxidants: chemistry, metabolism and structure activity relationship. J Nutr Biochem 2002;13:572–84. 41 Spencer JP, Abd-el-Mohsen MM, Rice-Evans C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch Biochem Biophys 2004;423:148–61. 42 Wang D, Liu L, Zhu X, Wu W, Wang Y. Hesperidin alleviates cognitive impairment, mitochondrial dysfunction and oxidative stress in a mouse model of Alzheimer’s disease. Cell Mol Neurobiol 2014;34:1209–21. 43 Niu Q, Wang LP, Chen YL, Zhang HM. Relationship between apoptosis of rat hippocampus cells induced by aluminum and the copy of the bcl-2 as well as bax mRNA. Wei Sheng Yan Jiu 2005;34:671–3. 44 Chaudhary M, Joshi DK, Tripathi S, Kulshrestha S, Mahdi AA. Docosahexaenoic acid ameliorates aluminum induced biochemical and morphological alteration in rat cerebellum. Ann Neurosci 2014;21:5–9. 45 Akifusa S, Kamio N, Shimazaki Y, Yamaguchi N, Nishihara T, Yamashita Y. Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species dependent pathway involving Bcl-2. Free Radic Biol Med 2009;46:1308–16.
Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis
Nutritional Neuroscience
2016
9
