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ORIGINAL ARTICLE
Year : 2018  |  Volume : 8  |  Issue : 2  |  Page : 35-40

Attenuation of Aluminum-Induced Neurotoxicity by Tannoid Principles of Emblica officinalis in Wistar Rats


Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India

Date of Web Publication26-Apr-2018

Correspondence Address:
Arokiasamy Justin Thenmozhi
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamilnadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijnpnd.ijnpnd_23_18

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   Abstract 

Introduction: In our earlier studies, we reported the neuroprotective effect of tannoid principles of Emblica officinalis (EoT) against aluminum chloride (AlCl3)-induced neurotoxicity in the hippocampus and cortex of rats. The present study is focused on the efficacy of EoT in the mitigation of AlCl3-induced toxicity in the cerebellum. Materials and Methods: Rats were randomly divided into four groups and treated with AlCl3 (100 mg/kg, b.w. i.p.), EoT (100 mg/kg, b.w. oral), and simultaneously with AlCl3 and EoT daily for 60 days. Results: AlCl3 treatment altered the body weight, brain and cerebellum weight, Al levels, acetylcholinesterase activity, and the expression of apoptotic markers. Treatment with EoT attenuated these physiological, biochemical, and molecular indices. Conclusion: The results of the present study strengthen the fact that the nutritional supplement of EoT in AlCl3-treated rats might improve the adverse effects by its pharmacological properties.

Keywords: Aluminum, apoptosis, cerebellum, tannoid principles of Emblica officinalis


How to cite this article:
Dhivya Bharathi M, Justin Thenmozhi A. Attenuation of Aluminum-Induced Neurotoxicity by Tannoid Principles of Emblica officinalis in Wistar Rats. Int J Nutr Pharmacol Neurol Dis 2018;8:35-40

How to cite this URL:
Dhivya Bharathi M, Justin Thenmozhi A. Attenuation of Aluminum-Induced Neurotoxicity by Tannoid Principles of Emblica officinalis in Wistar Rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2018 [cited 2018 Aug 21];8:35-40. Available from: http://www.ijnpnd.com/text.asp?2018/8/2/35/231269


   Introduction Top


Alzheimer’s disease (AD) is an age-related and irreversible brain disease characterized by short term memory loss and in advanced stages; it is manifested by confusion, aggression, mood changes, long-term memory loss and social withdrawal.[1] Cholinergic neurons are project mainly to the hippocampal and cortical regions that are concerned with cognitive functions including memory and learning processes.[2] The cerebellum plays a role in the learning of procedural memory, and motor learning, such as skills requiring co-ordination and fine motor control.[3] Aluminum (Al) produced the pathological state clinically resembled like AD.[4] Al administration impairs long term potential (LTP), which strengthens synaptic information storage and also damages various enzymes involved in the synthesis and degradation of neurotransmitters thereby affecting the neurotransmitter levels.[5] Cholinergic neurons and acetylcholine (ACh) are linked mainly to learning and memory processes in the brain.[6] Al decreases the functions of cholinergic system which is reflected by the measurement of acetylcholinesterase (AChE)[7],[8],[9],[10] and choline acetyltransferase activities, the enzymes implicated in the ACh metabolism.[11] An imbalanced expression of proapoptotic (Bax, Bak, and Bad), antiapoptotic (Bcl-xL and Bcl-2) proteins, initiator caspases-8, 9, and effector caspases-3, 6 were reported in the brain of rats induced with AlCl3.[12],[13],[14],[15]

Numerous synthetic and natural compounds have been shown to offer neuroprotection against AD, for example, by preventing the accumulation of Aβ and hyperphosphorylation of tau protein, inhibiting the activity of AChE, reducing the damage caused by reactive oxygen, suppressing the inflammatory reaction and neuronal apoptosis, and so on. Unfortunately, to date no pharmacological interventions that effectively prevent or stall AD have been developed. While a single-target therapeutic strategy seems to produce only suboptimal results, a broader neuroprotective approach, at least theoretically, appears more appealing. Recently, many experiments and clinical trials have shown that traditional herbal medicine, which has multiple targets, can provide effective treatment of neurological diseases including AD.

Emblica officinalis (EoT) Gaertn or Indian Amla (Family: Euphorbiaceae) is considered as a best rejuvenating herbs in various medicinal systems including Ayurveda and Unani. The fruits are considered as the best among the “rasayana herbs” and “sour fruits” in Ayurveda and used as a tonic for heart and brain in Unani medicine. It has been used to promote intelligence, memory, freedom from disease, longevity and strength of the senses.[16] Recent studies from our lab indicated the neuroprotective effect of tannoids principles of EoT against aluminum chloride (AlCl3) intoxication in hippocampus and cortex through its pharmacological properties.[8],[12] However, till date, no report available in demonstrating the neuroprotective role of EoT on cerebellar neurodegeneration following AlCl3 exposure. Therefore, the present study was aimed to investigate the protective role of EoT on cerebellar Al, AChE levels, and the expressions of apoptotic markers against AlCl3-induced rat model of AD.


   Materials and Methods Top


Animals

Male Albino Wistar rats (200–225 g) of age 10–12 weeks were acquired from Central Animal House, Rajah Muthiah Medical College & Hospital, Annamalai Nagar and maintained under standard conditions.[7] The experimental protocols were approved by the Animal Ethics Committee of the Institute (Reg. No. 160/1999/CPCSEA, Proposal No. 1016).

Chemicals

AlCl3, horseradish peroxidase (HRP) conjugated goat antirabbit antibodies were procured from Sigma–Aldrich, Bangalore, India. Antirabbit Bcl-2, Bax, cytochrome c, Apaf-1, β-actin primary IgG were obtained from Cell Signaling, Danvers, Massachusetts, USA. All other chemicals used were of analytical grade.

Tannoid principles of Emblica officinalis

Tannoid principles of EoT were obtained from Indian Herbs Research & Supply Company (Saharanpur, India), which was synthesized according to Ghosal et al.[17]

Experimental design

Forty eight rats were divided into four groups (n = 12) as follows:
  1. Group I: Rats were treated with saline.
  2. Group II: Rats were injected with AlCl3 (100 mg/kg, b.w. i.p.) for 60 days.[7]
  3. Group III: Rats were orally treated with EoT (100 mg/kg) (1 h prior to AlCl3 injection) and subsequently injected with AlCl3 (100 mg/kg, b.w. i.p.) as group II for 60 days.[8],[12]
  4. Group IV: Rats were treated orally with EoT alone (100 mg/kg, b.w.).


At the end of the experimental period, novel object recognition (NOR) test was conducted in control and experimental animals to study the memory and cognitive functions. After the behavioral assessment, body weight was measured and animals were sacrificed. Then the brain weight was measured, and cerebellum was procured and utilized for the biochemical analysis and protein expression studies.

Novel object recognition test

The apparatus consists of a square box (100 cm × 100 cm × 100 cm) made of grey painted wood. Two familiar objects to be distinguished (A1 and A2) were placed inside the box and fixed with white cement, so that rats unable to move them. Another object, which is generally consistent in height and volume, but are different in shape and appearance, is used as a novel object, B. The test was performed in three phases: habituation, training, and test session. On day one to habituate the rats were pre-exposed to the testing chamber for 10 min. Next day, rat was positioned inside the box in between two similar objects (A1 and A2) and was allowed to explore the objects for 5 min. After 5 min, rat was removed from the object recognition box and returned to its home cage. Objects A1 and A2 were also removed from the box. After 20 min (test phase), rat was exposed to one new object (B) and one of the old object for 3 min. The time spent exploring each object and the discrimination index percentage was recorded. The discrimination index is an index of the measure of discrimination between the familiar and the novel objects corrected for exploratory activity. It is calculated as: (time spent on novel object−time spent on familiar object)/(time spent on novel object + time spent on familiar object). The discrimination index can range from −1 to 1, with −1 indicating complete preference for the familiar object, 0 indicating no preference for either object, and 1 indicating complete preference for the novel object.[18]

Western blot analysis

Proteins from the mitochondrial and cytosolic fractions were extracted as described previously.[19] Tissues from the entire cerebellum were gently homogenized, using a teflon homogenizer in 7 volume of cold suspension buffer [20 mM Hepes–potassium hydroxide (KOH) (pH 7.5), 250 mM sucrose, 10 mM potassium chloride (KCl), 1.5 mM magnesium chloride (MgCl), 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethylene glycol-bis (2-aminoethyl ether) tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 0.1 mM phenylmethane sulfonyl fluoride (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]. The homogenates were centrifuged at 750×g at 4°C for 10 min to first isolate the nuclear fraction, and then at 10,000×g for 20 min at 4°C to separate the mitochondria from the soluble fraction. The supernatant was further centrifuged at 100,000×g for 60 min at 4°C and was then used as cytosolic fraction. Protein concentration was measured by the method of Lowry et al.[20]

About 50 μg of total cellular protein were loaded on 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis BBB - Blood brain barrier (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (Millipore) after separated. Membranes were incubated with the blocking buffer (with 5% nonfat dry milk powder) for 2 h to reduce nonspecific binding sites and then incubated with cyto-c, Bax, Bcl-2, Apaf-1 and β-actin antibodies in 5% bovine serum albumin in tris-buffered saline and 0.05% Tween-20 (TBST) and placed in a shaker at 4°C for overnight. Then membranes were hatched with secondary antibodies (IgG conjugated to HRP) at room temperature for 2 h. For 30 min, membranes were washed thrice with TBST. Final results were visualized by the chemiluminescence protocol (GenScript ECL Kit, Piscataway, NJ, USA). Gel image analysis program was used for the densitometry analysis. The data were normalized using β-actin as a loading control.[14],[21]

Data analysis

All data were expressed as mean ± standard error (SEM) of number of experiments. The statistical significance was calculated by using one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences version 15.0 software (SPSS Inc., Chicago, IL, USA), and the individual comparisons were made by using Duncan’s multiple range test (DMRT). A value of P < 0.05 was considered to indicate a significant difference between groups, and the values having symbols differ significantly with each other.


   Results Top


[Figure 1] shows the changes in the body weight and weights of whole brain and cerebellum 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 EoT to AlCl3-induced rats significantly (P < 0.05) increased the body weight and there are no significant changes in weight gain of EoT alone treated rats when compared with control rats. The relative whole brain [Figure 2] and cerebellum [Figure 3] weights were also decreased significantly in AlCl3 than in the control group, and administration of EoT in parallel with AlCl3 produced a recovery in relative whole brain and cerebellum as compared to AlCl3-treated animals.
Figure 1: Effect of EoT on the changes in body weight of control and AlCl3-treated rats. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing same symbols differ significantly as compared to the initial weight

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Figure 2: Effect of EoT on the changes in weight of whole brain in control and AlCl3-treated rats. Data are expressed as mean ± SEM (one-way ANOVA followed by DMRT) for six rats in each group. Values not sharing same symbols differ significantly (P < 0.05); (*) compared to control group, (#) compared to AlCl3-treated group

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Figure 3: Impact of EoT on the changes in weight of cerebellum in control and AlCl3-treated rats. Results given are mean ± SD (n = 6); values not sharing common symbols differ significantly − *P < 0.05 compared to the control, #P < 0.05 compared to the AlCl3-treated rats, one-way ANOVA followed by DMRT

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EoT administration attenuates aluminum chloride-induced memory deficits

[Figure 4]A and [Figure 4]B revealed the memory and movement co-ordination functions of control and experimental rats. Chronic AlCl3 induction significantly reduced the sniffing time and discrimination index for new object in NOR test as compared to control rats. Coadministration of EoT significantly attenuated these memory and behavioral deficits as compared to AlCl3 alone treated rats. Moreover, no significant changes were observed between control and EoT alone treated rats.
Figure 4: Role of EoT on the sniffing time (A) and discrimination index (B) of novel object test in control and AlCl3 injected animals. Results given are mean ± SD (n = 6); values not sharing common symbols differ significantly – *P < 0.05 compared to the control, #P < 0.05 compared to the AlCl3-treated rats, one-way ANOVA followed by DMRT

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EoT treatment ameliorates aluminum chloride-induced aluminum overloading

Administration of AlCl3 significantly increased the Al concentration in cerebellum [Figure 5] than the control rats. However, EoT cotreatment significantly diminished the Al overloading in cerebellum of brain than control animals. No significant differences were found in the levels of Al in control and EoT alone treated rats.
Figure 5: Impact of EoT on the levels of Al in control and AlCl3-treated rats. Results given are mean ± SD (n = 6); values not sharing common symbols differ significantly − *P < 0.05 compared to the control, #P < 0.05 compared to the AlCl3-treated rats, one-way ANOVA followed by DMRT

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EoT administration nullifies the aluminum chloride-mediated acetylcholinesterase hyperactivity

AlCl3 administration significantly enhanced the AChE activity [Figure 6] in cerebellum as compared to untreated rats. EoT cotreatment significantly attenuated the AlCl3-induced AChE hyperactivity in cerebellum of brain as compared to control rats. No significant differences were found in the activity of AChE in control and EoT alone treated rats.
Figure 6: Impact of EoT on the activities of AChE in control and AlCl3-treated rats. Results given are mean ± SD (n = 6); values not sharing common symbols differ significantly − *P < 0.05 compared to the control, #P < 0.05 compared to the AlCl3-treated rats, one-way ANOVA followed by DMRT

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EoT treatment attenuates aluminum chloride-induced apoptosis

Elevated protein expressions of cytosolic cyto c, Bax, Apaf-1, and diminished expression of Bcl-2 were found in cerebellum of AlCl3-treated rats as compared to control rats, but in contrast, EoT cotreatment attenuated their expressions significantly as compared to AlCl3-treated rats. EoT alone treatment did not cause significant changes in the control animals [Figure 7].
Figure 7: Effect of EoT on the expressions of Bax, Bcl-2, cytochrome c and Apaf-1 in control and AlCl3-treated rats. Results given are mean ± SD (n = 3); values not sharing common symbols differ significantly − *P < 0.05 compared to the control, #P < 0.05 compared to the AlCl3-treated rats, one-way ANOVA followed by DMRT

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   Discussion Top


Results of the present study showed that the AlCl3 administration significantly diminished the body weight and weights of whole brain and cerebellum as compared to control rats. Decreased water and food intake, transient diarrhea, and reduced efficacy in converting feed-to-body weight gain leads to reduction in body mass of Al treated animals as compared to controls.[22],[23] EoT cotreatment significantly enhanced the body weight and weights of whole brain and cerebellum as compared to AlCl3 alone treated group.

In our study, Al loading in cerebellum of AlCl3-treated rats was attenuated by EoT administration. Once absorbed into the body, Al is carried in the blood bound to transferin (∼80%) and citrate (∼20%) and transported to tissues. It has been estimated that bones, lungs and brain contains ∼50, ∼25 and ∼1%, respectively, of the total Al burden.[24] This low level of Al distribution in the brain indicates that the nervous tissue is a susceptible target tissue and even at low levels, it could induce aberrant pathophysiological changes. Mobilization of Al in brain can be performed by iron chelator through enhancing carrier mediated mechanism and BBB efflux.[25] Experimental studies have shown that the oral administration of EoT fruit juice enriched with emblicanin A and emblicanin B was effective in reducing the iron,[26] cadmium,[27] and arsenic[28] induced toxicities through its chelation properties, which also supports our present findings.

AlCl3 administration showed a significant enhancement in the brain AChE activity, which is supported by our previous and other studies.[7],[8],[29] AChE is an important biological component of the membrane and contributes to its integrity. Al induces lipid peroxidation and plasma membrane damage by interacting with plasma membrane lipids, results in the release of membrane associated enzymes, including AChE; thereby, it enhances AChE levels and activity.[30] Enhanced brain AChE activity following Al exposure of rats was attributed to an allosteric interaction between Al with the peripheral anionic site of the enzyme molecule.[31] Coadministration of EoT to AlCl3 intoxicated rats showed its neuroprotective effect by reducing AChE activity. The ability of EoT to directly suppress the enhanced AChE activity following Al intoxication in the current study could be explained with the finding of an in vitro study that demonstrated a potential anticholinesterase property of Emblica.[32] Inhibition of AChE activities increased the ACh levels thereby enhancing cognitive events.[33] Our results also indicated that a reduction in cognition of NOR test during AlCl3 injection reflected the response of an animal to an unfamiliar environment. EoT treatment improved the capacity to recognize new objects in NOR test, which might be due its AChE inhibiting activity.[7]The Bcl-2 family regulates the mitochondria-dependent intrinsic apoptotic pathways, which are grouped into two boarder classes: the antiapoptotic proteins (Bcl-xL and Bcl-2) and the proapoptotic proteins (Bak, Bax, and Bad). The expression of pro- and antiapoptotic proteins establishes the balance between neuronal survival or death. Bcl-2 is shown to inhibit apoptosis, whereas the Bax promotes it by suppressing or triggering cyto c release, respectively.[34] AlCl3 administration induces mitochondrial cyto c discharge, caspase-3, 8 and 9 activation, and enhanced Bax expression with Bcl-2 downregulation, which is clearly indicating the apoptotic progression. Administration of Al maltolate downregulated the Bcl-2 and upregulated the Bax, cytosolic cyto c and Apaf-1 expressions strengthening our results.[15] Coadministration of EoT in Al induced rats, slow down cyto c discharge and the expressions of Bax and Apaf-1 augment the Bcl-2 expressions are in consistence with previous reports.[12],[35]


   Conclusion Top


The results of the previous[8],[12] and present studies indicated that Al administration adversely affected the cerebellum by altering biochemical and molecular indices and concomitant administration of EoT could protect against such hazardous effects through its pharmacological activities.

Acknowledgements

We gratefully acknowledge the Indian Herbs Research & Supply Company, Saharanpur, India for the generous supply of standardized extract of EoT tannoids.

Financial support and sponsorship

Financial assistance in the form of a major research project from the University Grants Commission, India (42–664/2013(SR)/22.03.2013) is gratefully acknowledged.

Conflicts of interest

There are no conflicts of interest.

 
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