Neurochem Res (2015) 40:767–776 DOI 10.1007/s11064-015-1525-1
Neuroprotective Effect of Hesperidin on Aluminium Chloride Induced Alzheimer’s Disease in Wistar Rats Arokiasamy Justin Thenmozhi • Tharsius Raja William Raja Udaiyappan Janakiraman • Thamilarasan Manivasagam
Received: 27 October 2014 / Revised: 14 January 2015 / Accepted: 20 January 2015 / Published online: 29 January 2015 Ó Springer Science+Business Media New York 2015
Abstract The present study was aimed to evaluate the protective effect of hesperidin (Hes) on aluminium chloride (AlCl3) induced neurobehavioral and pathological changes in Alzheimeric rats. Intraperitonial injection of AlCl3 (100 mg/kg body weight) for 60 days significantly elevated the levels of aluminium (Al), activity of acetylcholinesterase (AChE) and protein expressions of amyloid precursor protein (APP), b amyloid (Ab1–42), b and c secretases as compared to control group in hippocampus and cortex of rat brain. Hes administration orally along with AlCl3 injection for 60 days, significantly revert the Al concentration, AChE activity and Ab synthesis-related molecules in the studied brain regions. Our results showed that aluminum exposure was significantly reduced the spontaneous locomotor and exploratory activities in open field test and enhanced the learning and memory impairments in morris water maze test. The behavioral impairments caused by aluminum were significantly attenuated by Hes. The histopathological studies in the hippocampus and cortex of rat brain also supported that Hes (100 mg/kg) markedly reduced the toxicity of AlCl3 and preserved the normal histoarchitecture pattern of the hippocampus and cortex. From these results, it is concluded that hesperidin can reverse memory loss caused by aluminum intoxication through attenuating AChE activity and amyloidogenic pathway. Keywords Alzheimer’s disease AlCl3 Hesperidin Memory loss Ab synthesis
A. J. Thenmozhi (&) T. R. W. Raja U. Janakiraman T. Manivasagam Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India e-mail: [email protected]
Introduction Alzheimer’s disease (AD) is the most common form of dementia, associated with neuropathological and neurobehavioral changes accompanied by memory and cognitive impairments. It is characterized by excessive production and accumulation of Ab peptide, the pathological product of amyloid precursor protein (APP) by sequential proteolytic action of b- and c-secretases . Aluminum (Al) is the third most abundant metal in the earth’s crust and its role in the etiology and pathogenesis of AD has drawn more perceive, due to the well documented animal experiments  and clinical studies . It is reported that Al is known to accelerate extracellular Ab generation and aggregation [4, 5]. Al acts as a cholinotoxin  and cause alterations on the cholinergic activity, a key event in the neurochemistry of AD . Acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, etc.,), NMDA antagonists (Memantine), antihyperammonemic drugs (Carveditol), anti inflammatory drugs (rofecoxib) and secretase inhibitors (Memoquin) were shown to have neuroprotective effects with considerable side effects and could not be used successfully . Now, the global scenario of therapeutic research is towards herbal or alternate system of medicine. Epidemiological studies showed a link between the consumption of plantderived foods and a range of health benefits, at least partially, to some of the phytochemical constituents including polyphenols . Flavonoids are a broad class of polyphenolic compounds that exhibit neuroprotective properties in experimental models of AD, stroke, PD etc.,  and influence normal cognitive function . Hesperidin (Hes, 3, 5, 7-trihydroxy flavanone-7-rhamnoglucoside) is a biologically and pharmacologically active citrus bioflavonoid, abundantly found in sweet orange and
lemon . Due to its lipophilic nature; Hes can cross the blood brain barrier easily and afford neuroprotection . It is shown to control pathophysiological disturbances of brain excitability in drug abuse, migraine and epilepsy  by the virtue of its antioxidant, anti-inflammatory and antiapoptotic properties. The neuroprotective efficacy of Hes is attributed in neurodegenerative processes including Huntington’s disease, cerebral ischemia/reperfusioninduced memory dysfunction and brain damage induced by stroke [15–17]. Viswanatha et al.  reported the protective effect of Hes against various neurobehavioral alterations induced by immobilization-stress in mice. Based on this background, the present study was carried out to explore the neuroprotective effect of Hes against AlCl3 induced alterations on the level of aluminium, activity of AChE and expressions of APP, Ab1–42, b- and c-secretases in hippocampus and cortex of rats.
Materials and Methods Animals Male Albino Wistar rats (200–225 g) were procured from Central Animal House, Rajah Muthiah Medical College & Hospital, Annamalai University and maintained at 12/12 h light/dark cycle, *22 °C temperature and 60 % humidity with food and water ad libitum. The experimental protocols met with the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Science Academy, New Delhi, 2000) and were approved by the Animal Ethics Committee of the Institute (Reg. No. 160/1999/CPCSEA, Proposal No. 1005). Chemicals Aluminium chloride, hesperidin, rabbit anti- Ab1–42, rabbit anti-c-secretase, rabbit anti-b-secretase, rabbit anti-
Fig. 1 Aluminum concentration increased significantly after AlCl3 exposure. Hes (50, 100 and 200 mg/kg b.w.) treatment was effective in reducing brain aluminum burden. Data are expressed as mean ± SD (oneway ANOVA followed by DMRT). Values not sharing same alphabets differ significantly
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amyloid precursor protein and anti mouse-b-actin, horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG were purchased from Sigma - Aldrich, Bangalore, India and used in this study. All other chemicals used were of analytical grade. Dosage Fixation To determine dose dependent effect of Hes against AlCl3induced experimental model of AD, three different doses of Hes (50, 100, and 200 mg/kg) were used. It was observed that i.p. injection of AlCl3 for 60 days caused a significant and greater aluminium accumulation (Fig. 1) and AChE activity (Fig. 2) in hippocampus and cortex. Treatment with various doses of Hes significantly (P \ 0.05) decreased the Al concentration and AChE activity in AlCl3 intoxicated rats, which was further confirmed by behavioural (Morris water maze test) and histopathological studies. Rats with Al treatment, exhibited longer escape latencies to reach the platform in both acquisition and probe trials (Fig. 3a) as compared with the control group, however, all groups displayed a gradual improvement in performance over the 5 days of testing period. The probe trial studies showed that Al exposed rats spent less time (Fig. 3b) in target (platform) quadrant. But oral administration of Hes significantly reduced the time taken to reach platform in both trials and significantly enhanced time spent in target quadrant. Histopathological staining (Fig. 4) showed that the neurons in the hippocampus and cortex were arranged closely, had a circular shape, nuclear membrane was clear in the control and Hes alone treated group. Neurons had irregular shapes and the nuclear membrane was indistinct in the AlCl3 treated AD model group. Hes dose dependently improved the structural changes induced by AlCl3. It was observed that 100 and 200 mg/kg b.w. of Hes showed similar reduction in Al levels, AChE activity, memory loss and histopathological changes, but more
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Fig. 2 AlCl3 treatment significantly enhanced the activities of AChE as compared to control rats, while Hes (50, 100 and 200 mg/kg b.w.) treatment significantly reduced the activities of AChE as compared to AlCl3 treated group. Data are expressed as mean ± SD (one-way ANOVA followed by DMRT). Values not sharing same alphabets differ significantly
Fig. 3 Rats treated with AlCl3 exhibited a significant increase in the time latency to find the platform and increased time to reach hidden platform area in Moris-water maze test as compared to control group. The probe trial studies showed that Al exposed rats spent less time in target (platform) quadrant. Hes cotreatment attenuated the memory loss induced by AlCl3. Data are expressed as mean ± SD (one-way ANOVA followed by DMRT). Values not sharing same alphabets differ significantly
significant than 50 mg/kg. As a consequence, we have chosen the optimum dose (100 mg/kg) for our further study.
Experimental Design Forty randomly selected rats were divided in four groups containing ten animals in each group. Group I: Rats were treated with saline. Group II: Rats were injected with AlCl3 (100 mg/kg b.w. i.p.) for 60 days .
Group III: Rats were treated with AlCl3 as group II and subsequent oral treatment with hesperidin (100 mg/kg) (one hour prior to AlCl3 injection) for 60 days by oral gavage . Group IV: Rats were treated with hesperidin alone (100 mg/kg). After the end of experimental period, animals were analysed for their movement and activity in the open field test . Rats were sacrificed, brain tissues (cortex and hippocampus) were excised and used for the further studies.
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AlCl3 + Hes (50)
AlCl3 + Hes (100) AlCl3 + Hes (200)
Fig. 4 The neurons were arranged closely, had a circular shape, nuclear membrane was clear in the control and Hes alone treated group. Neurons had irregular shapes and the nuclear membrane was
indistinct in the AlCl3treated AD model group. Hes dose dependently improved the structural changes induced by AlCl3. Same pattern of morphological changes were observed in cortex also
Determination of Aluminium Concentration in Brain Tissues
immersion. The latency from immersion into the pool to escape onto the hidden platform (maximum duration of trial 90 s) was recorded. On day 6, rats were subjected to a 120 s probe trial in which the platform was removed from the pool. The time spent in target quadrant (within 120 s probe test time) was recorded on an electronic time recorder.
Brain tissues (hippocampus and cortex) were weighed and put into polytetrafluoroethene, then added 0.05 ml nitric acid and 0.2 ml H2O2 per 30 mg tissue, and incubated at 120 Æ C for 2 h. The concentration of aluminium was measured by atomic absorption spectrophotometer . Activity of Acetylcholinesterase Brain AChE activity was determined by ELISA method using acetylcholinesterase activity assay kit purchased from BioVision Co, California, USA.
Behavioral Studies Morris Water Maze Morris water maze test was performed as previously described by Morris . The apparatus consists of a large circular pool and a clear Perspex platform (2 cm below the water level). Briefly, the rats were trained twice a day for 5 days. The water was made opaque by addition of 1 l of milk, which prevented visualization of the platform. Four points on the rim of the tank were designated north (N), south (S), east (E) and west (W), thus dividing the pool into four quadrants (NW, NE, SE and SW). On days 1–5, rats were trained for 16 trials (four trials a day) to locate and escape onto the submerged platform. At the start of each trial, the rat was held facing the perimeter of the water tank and dropped into the pool to ensure
Open Field Test The floor of wooden apparatus (W100 cm 9 D100 cm 9 H40 cm) was covered with resin and was divided into 25 (5 9 5) squares. The rats were placed into one corner of open field chamber and its behavior was observed for 5 min. (a) Number of squares explored; only when the animal enters a square with both its forelimb, one count is made. The number of center (central nine squares) and peripheral squares (the 16 squares adjacent to the wall) explored by the animal was noted separately. (b) Numbers of grooming; i.e. consisting of licking the fur, washing face or scratching behavior. (c). Number of rearing; i.e. standing on hind limbs and sometimes leaning on the wall with forelegs, sniffing and looking around . Histological Examination Cortex and hippocampus of control and experimental rats were fixed in 4 % paraformaldehyde and embedded in paraffin. Then it was sliced into 5 lm sections using a section cutter (Leica, Germany). The sections were stained with H&E (hematoxylin and eosin) and examined by a light microscope .
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The brain regions (hippocampus and cortex) were procured and sectioned coronally at 20 lm thickness on a cryostat. Sections were collected in 0.01 M Phosphate buffered saline (PBS)  and incubated with 0.3 % hydrogen peroxide (H2O2) for 10 min at room temperature and then placed in blocking buffer containing 10 % normal goat serum (NGS) with 0.2 % Triton X-100 in 0.01 M PBS (pH 7.2) for 30 min at 37 °C. In each treatment, the slides were washed at least three times with 0.01 M PBS each for 5 min. Sections were incubated for 24 h with primary anti rabbit-APP (1:1,000) in 2 % NGS, 0.2 % Triton X-100 and 0.02 % sodium azide in TBS. After washing with 1 % NGS in TBS, the sections were incubated in anti-mouse IgGHRP conjugated antibody (1:1,000) in 1.5 % NGS for 1 h. APP immunoreactivity was visualized, after incubation in DAB for 2–5 min and analyzed using an Olympus BX51 microscope with a DP71 camera (Olympus, USA) at high magnification (400X). Neuronal numbers were counted by persons who were blind to the treatment and 3 animals were used for cell counts.
Statistical analysis was performed by one-way analysis of variance followed by Duncan’s multiple range test (DMRT) using Statistical Package for the Social Science (SPSS) software package version 12.0. Results were expressed as mean ± SD for ten rats in each group. P \ 0.05 were considered significant.
Results Body Weight Changes Table 1 shows the body weight changes in normal and experimental groups. Rats induced with AlCl3, showed a significant (P \ 0.05) decrease in body weight when compared with control rats. Oral treatment with Hes to AlCl3 induced rats significantly (P \ 0.05) increased the body weight. There are no significant changes in weight gain of Hes alone treated rats when compared with control rats. Open Field Test
Western Blot Analysis Hippocampal and cortical tissues were homogenized in an ice-cold RIPA buffer (1 % Triton, 0.1 % SDS, 0.5 % deoxycholate, 1 mmol/L EDTA, 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 10 mmol/L NaF, and 0.1 mmol/ L phenylmethylsulfonyl fluoride (PMSF)). The homogenate was centrifuged at 12,000 rpm/min for 15 min at 4 °C. Protein concentration was measured by the method of Lowry et al. . Samples containing 50 lg of total cellular protein were loaded and separated on 10 % SDS polyacrylamide gel electrophoresis. The gel was then transferred on to a PVDF membrane (Millipore). The membranes were incubated with the blocking buffer containing 5 % non-fat dry milk powder for 2 h to reduce non-specific binding sites and then incubated in APP, b-amyloid, c- and b-secretases and b-actin (rabbit monoclonal; 1:1,000) 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 (antimouse or anti-rabbit IgG conjugated to horseradish peroxidase) for 2 h at room temperature. The membrane was washed thrice with TBST for 30 min. Immunoreactive protein was visualized by the chemiluminescence protocol (GenScript ECL kit, USA). Densitometric analysis was performed with a computer using a gel image analysis program. The data were then corrected by background subtraction and normalised against b-actin as an internal control.
Locomotion and activities of AlCl3 induced animals were observed by open field test (Fig. 5a, b). The AlCl3 group exhibited a significant decrease (P \ 0.05) in peripheral and central movements and rearing and grooming activities. However the oral administration of Hes (100 mg/kg) to AlCl3 treated rats showed a significant increase in locomotion and activities (P \ 0.05) compared to the AlCl3 group. In addition, no differences in open field test were observed between Hes alone and control group. Immunohistochemical Studies The immunohistochemical analysis (Fig. 6) clearly indicated the distribution of APP in the hippocampus and cortex. Administration of AlCl3 markedly enhanced the Table 1 Gain in body weight were observed among all the groups, whereas aluminium treated groups exhibit a decrease in body weight Groups
Initial body weight (g)
Final body weight (g)
225 ± 16.61
290 ± 25.45a
225 ± 18.80
201 ± 13.28b
AlCl3 ? Hes (100 mg)
235 ± 17.71
258 ± 21.03c
Hes (100 mg)
245 ± 22.13
299 ± 24.36a
Values are represented as mean ± SD a
P-no significant change between control and Hes alone treated rats
P \ 0.05 relative to control P \ 0.05 relative to AlCl3 group
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Fig. 5 Locomotion in terms of number of squares (peripheral and central) crossed and activities (grooming and rearing) were significantly decreased in AlCl3-intoxicated AD rats. Cotreatment with Hes to AD rats significantly increased the locomotion and activity in open field test. Data are expressed as mean ± SD (one-way ANOVA followed by DMRT). Values not sharing same alphabets differ significantly
immunostaining of APP in the rat hippocampus and cortex. Oral treatment of Hes significantly (P \ 0.05) diminished the APP-immunoreactivity in both the tissues. Furthermore, the administration of Hes did not alter the APP-immunoreactivity as compared to control group. In addition, the quantification analysis indicated that the enhanced immunoreactivity of APP in hippocampus and cortex of AD model rats was significantly attenuated by the treatment with Hes. Protein Expression Studies We have performed western blots (Fig. 7a–c) to investigate changes in b- amyloid metabolic markers in our AlCl3 animal models with or without Hes treatment. Chronic administration of AlCl3 showed the enhanced protein expressions of APP, Ab1–42, b and c secretases (Fig. 7a–c) as compared to control group suggesting that Al toxicity would be in favor of Ab formation. Oral administration of Hes to AlCl3 treated rats showed diminished expressions of APP, Ab1–42, b and c secretases.
Discussion Gain in body weight was observed among all the groups except aluminium treated groups, which showed a decrease
in body weight. Abdel-Aal et al.,  reported that the injection of AlCl3 over 60 days botched to gain body weight relative to control rats that may be due to less desire for food intake, which supports our findings. Co-administration of hesperidin to AlCl3 rats showed improvement in the body weight, which is consistent with previous study . Our results showed that i.p. injection of AlCl3 for 60 days caused a significant and greater aluminium accumulation (Fig. 1) in hippocampus and cortex, a finding that is consistent with previous studies [28, 29]. Aluminium enters into the brain via the specific high affinity receptors for transferrin (TfR) expressed in the blood brain barrier  and accumulated in all the regions of rat brain, the maximum being in hippocampus, which is the site of memory and learning . Crapper et al.  also reported that the elevated concentration of Al in brain of AD patients. Flavonoids are effective metal ion chelators and form stable products with beryllium, aluminium, iron and zinc ions . Hydroxyl groups of hesperitin, a metabolite of hesperidin, bind and chelate cadmium (Cd) and enhance the excretion of Cd, which in consequence decrease accumulation of Cd . In vitro studies confirmed the coordination of hesperidin with Al, forms hesperidin-Al complex . In the present study, treatment of hesperidin to AlCl3 rats diminished aluminium levels, which may be due its chelation property.
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AlCl3 + Hes
(P \ 0.05) diminished the AlCl3 induced APP-immunoreactivity in both the tissues. Furthermore, the administration of Hes did not alter the APP-immunoreactivity as compared to control group
Fig. 6 The immunohistochemical analysis clearly indicated the distribution of APP in the hippocampus and cortex. Administration of AlCl3 markedly enhanced the immunostaining of APP in the rat hippocampus and cortex. Co-administration of Hes significantly
Fig. 7 a AlCl3 injections significantly enhanced the expressions of APP, Ab1–42, b and c secretases as compared to control rats, while Hes treatment significantly diminished the expressions of APP, Ab1–42, b and c secretases as compared to AlCl3 treated rats. b, c Immunoblot data are quantified by using b-actin as an internal control and the values are expressed as arbitrary units and given as mean ± SD. b P \ 0.05 compared to the control, cP \ 0.05 compared to the AlCl3 ? Hes-treated rats
C. AlCl3+ Hes
Acetyl choline, a neurotransmitter of cholinergic neurons, is considered to be closely related to short term memory and the degree of Ach reduction was positively correlated with dementia severity . AChE is the key
enzyme involved in the hydrolysis of ACh and forms acetyl coA and choline. Zheng et al.  reported the increased AChE activity in Al-overloaded rats, which corroborate with our results (Fig. 2). The observed elevated activity of
AChE in AlCl3 treated rats might be attributed to the direct effect of Al . It was reported that Al(III) can interact with the peripheral sites of AChE and modify its secondary structure and eventually enhanced its activity . Inhibition of AChE represents an important beneficial strategy in the management of Alzheimer’s disease. AChE inhibitors augment the accessibility of acetylcholine through hang-up of its destruction, thus helps in managing the symptoms of AD, by enhancing cholinergic transmission in the brain [40, 41]. In the present study, treatment with hesperidin offer neuroprotection by diminishing activity of AChE in the aluminium treated rats possibly by reducing aluminum loading in hippocampus and cortex. In this study, we investigated the behavioral changes caused by chronic aluminum exposure and the possible effect of hesperidin treatment using two behavioral tests; open field and Morris water maze tests. In Morris water maze, aluminum exposure was associated with decreased spatial memory and accuracy in both acquisition and probe trials (Fig. 3a, b). In open field test, aluminium treatment significantly decreased movement (peripheral and central crossings) and activities (rearing and grooming) (Fig. 4a, b), which agree with the previous study . Hesperidin treatment ameliorated the AChE activity in the hippocampus and the learning and memory ability of intracerebroventricular-streptozotocin infused mice has been reported earlier . In the present study, improvement of behavioural changes found in AlCl3 exposed rats by hesperidine may be attributed due to its Al reducing and AChE activity lowering ability. APP is a transmembrane glycoprotein with a receptorlike structure that is important in neurite sprouting, branching and elongation . APP can undergo two alternative proteolytic pathways: the nonamyloidosis pathway, which prevents Ab generation and seems to be a protective pathway and the amyloidosis pathway, which leads to Ab generation . Ab is generated from APP via sequential cleavage by b- and c-secretases and the contents of Ab are related to the expression of APP, b- and c-secretases . Chronic exposure to AlCl3 through drinking water or food led to the overexpression of APP in the brain of rats [45, 46]. Results from the present study indicate that the larger amounts of aluminum that accumulate in hippocampal and cortical neurons of AlCl3-deteriorated rat upregulated the expression of the APP and result in greater APP deposition (Fig. 6) than in brain of the controls. The promoter region for the APP gene has multiple binding sites specific for two stress-related transcription factors – NF-jB and hypoxia-inducible factor-1 (HIF-1). Both transcription factors contribute to AD neuropathology and both are inducible by aluminium . Studies by other investigators have shown that APP gene expression is significantly upregulated in human neural cells mediated by nuclear factor-
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kappa B (NF-jB) in response to aluminum exposure [47, 48]. Hes downregulated the expression of NF-jB activity in a PPARc-dependent and independent manner in in vitro studies [49, 50]. Hesperetin suppresses age induced NF-jB expression through the regulation of four signal transduction pathways, NIK/IKK, ERK, p38 and JNK and thereby modulates NF-jB pathway . Therefore we suggested that the beneficial effects of hesperidin in the suppression of Al induced APP expression are probably due to the down regulation of NF-jB expression. We found that injection of aluminium increased the expressions of Ab1–42, b and c- secretases in the hippocampus and cortex of rats, suggesting that Al toxicity would be in favour of Ab formation. Al inhibits the activity of protein kinase C (PKC) , which allows APP cleavage via its non-amyloidogenic a-secretase pathway as opposed to its b-amyloidogenic pathway . Al-inhibition of PKC activity is reported to increase b-amyloidogenesis in cultured neuroblastoma cells . Oral administration of Hes attenuated the Ab burden induced by Al through suppressing the expressions of APP, Ab1–42, b and c- secretases (Fig. 7a–c). Recently, Li et al.  reported that short term hesperidin treatment for 10 days attenuated Ab deposition. However, they did not observe the expression of Ab synthesis related secretases (b and c), which is an interesting outcome, that supports the neuroprotective effect of hesperidin against AD. In vitro studies indicated that various polyphenols such as resveratrol, EGCG, ECG and myricetin prevented the Ab aggregation by increasing the activities of PKC [56, 57]. In the present study, though the effect of hesperidin on the activity of protein kinase C was not analysed, we suggest that the reduced b- secretase expression and Ab deposition might be due to modulation of PKC activity. In conclusion, the present study highlights that the hesperidin treatment attenuated aluminium chloride induced aluminium loading, cholinergic deficit, Ab anabolism and memory loss. However further research is needed to study the precise mechanism of action of hesperidin against Al induced neurotoxicity. Acknowledgments Financial assistance in the form of a major research project from the Department of Science and Technology, New Delhi, is gratefully acknowledged. Conflict of interest Hereby, the authors declare that they have no conflict of interest that might have influenced the views expressed in this manuscript.
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