The Ayurvedic drug Ksheerabala (101) ameliorates alcohol-induced neurotoxicity by down-regulating the expression of transcription factor (NFkB) in rat brain.

INTRODUCTION: Most of the pharmaceutical effects of alcohol are due to its accumulation in the brain. Ksheerabala (101) an Ayurvedic formulation mainly used against central nervous system disorders. AIM: To determine the antioxidant and neuroprotective effect of Ksheerabala (101) on alcohol-induced oxidative stress in rats.
MATERIALS AND METHODS: Male Albino rats of Sprague-Dawley strain were grouped into four; control, alcohol (4 g/kg), Ksheerabala (15 μL/1 ml milk/100 g) and Ksheerabala (15 μL/1 ml milk/100 g) + alcohol (4 g/kg). After the experimental period (90 days), the animals were sacrificed and the effect of Ksheerabala (101) was studied on oxidative stress, inflammatory markers, and induction of transcription factor in brain. Results were statistically analyzed by one-way ANOVA.
RESULTS: The activities of antioxidant enzymes and reduced glutathione which were decreased in alcohol-treated rats, increased significantly in co-administered groups. The lipid peroxidation products and protein carbonyls which were increased significantly in alcohol-treated rats decreased significantly in co-administered groups. The expression of gamma-glutamyl cysteine synthase decreased significantly in alcohol-treated rats and increased significantly in co-administered groups. The transcription factor nuclear factor-κB (NFκB) which was up-regulated in alcohol-treated rats was down-regulated in co-administered rats. The histopathology reinforced these results.
CONCLUSION: Ksheerabala (101) attenuates alcohol-induced oxidative stress and down-regulates the expression of NFκB in rat brain.

Introduction

Alcohol is the world's most widely used psychoactive drug, but chronic alcohol consumption leads to permanent organ damage or death. Alcohol abuse can result in brain damage and neurodegeneration.[1] Alcohol may also injure the brain by increasing oxidative stress.[2] Although the mechanisms behind oxidative stress is not well-understood, numerous studies have demonstrated that chronic ethanol consumption is accompanied by both oxidative damage to cellular proteins, lipids, and DNA[34] and reduced levels of the endogenous antioxidants.[5] Excessive production of reactive oxygen species has been proposed as a potential mechanism for ethanol-induced neuronal damage.[6] There is evidence suggesting the involvement of oxidative stress in neurodegenerative diseases.[7]

Growing evidence indicates the role that inflammation plays as a potential pathogenic factor in many central nervous system (CNS) diseases, including neurodegenerative diseases.[89] The hallmark of the neuroinflammation is the activation of glial cells and the production of cytokines and inflammatory mediators that trigger neural damage.[9] Alcohol not only stimulates glial cells, but may also induce a proinflammatory response in the brain.[10] Chronic alcohol intake upregulates inflammatory mediators in both brain and astroglial cells, activating signaling events associated with inflammation.[11]

Many drugs in Ayurveda possess neuroprotective effect. Ksheerabala (101) is an Ayurvedic drug, which is also used as a nerve tonic. The textual reference of Ksheerabala (101) is found in Ashtangahridaya.[12] It is used in Ayurveda to treat arthritis, CNS disorders and insomnia. It also enhances the functioning of sense organs. The main contents of Ksheerabala (101) are Bala (Sida Cordifolia Linn., belongs to Malvaceae family), Ksheera (cow's milk) and Tila Taila (Sesamum oil). Previous study showed that Ksheerabala reduces the oxidative stress induced by quinolinic acid.[13] Hence, the main focus of the present study was to evaluate the antioxidant and neuroprotective properties of Ksheerabala (101) against alcohol-induced neurotoxicity.

Materials and Methods

Animals

Male albino rats (Sprague-Dawley strain) weighing between 100 and 140 g bred and reared in our animal house were used for the experiment.

A total of 24 rats were divided into 4 groups of 6 rats each.

Group I: Control

Group II: Alcohol (4 g/kg b.wt)

Group III: Ksheerabala (15 μl/100 g b.wt/day)

Group IV: Alcohol (4 g/kg body weight + Ksheerabala (15 μl/100 g b.wt/day).

Animals were housed in polypropylene cages. Cages were kept in a room that was maintained between 28°C and 32°C. The light cycle was 12 h light and dark. Animals were handled using the laboratory animal welfare guidelines.[14] Rats were fed with rat feed (Ashirvad Private Ltd., India). Food and water were given ad libitum. The study protocol was approved by the institutional animal ethics committee [IAEC–KU-14/2009-2010-BC-MI (22)].

The dose of alcohol[15] and Ksheerabala (101)[13] were selected from the previous studies. Alcohol (4 g/kg body weight, 1:1 dilution) and Ksheerabala (101) (15 μl dissolved in 1 ml milk/100 g b.wt) were given orally by gastric intubation. Alcohol was purchased from Merck India and Ksheerabala (101) was procured from Kottakkal Arya Vaidyasala, Kottakkal, Kerala, India (Batch No. 126165).

Alcohol and Ksheerabala (101) were given separately every morning for 90 days to the co-administered group. At the end of the experimental period, the animals were sacrificed. The brain was dissected out and cleaned with ice-cold phosphate buffer saline, blotted dry and immediately transferred to ice-cold containers for various biochemical evaluations. Blood was collected in clean, dry test tubes and allowed to clot for 30 min at room temperature. The clear serum was separated after centrifugation at 2000 g for 10 min and used immediately for the assay of various parameters.

Biochemical analysis

The tissue was extracted[16] and superoxide dismutase (SOD),[17] catalase,[18] glutathione reductase (GR) activity,[19] glutathione peroxidase (GPx) activity,[2021] malondialdehyde (MDA) estimation,[22] hydroperoxides (HP) estimation,[23] conjugated dienes, (CD)[24] protein carbonyls estimation,[25] tissue protein estimation,[26] glutathione (GSH),[27] isolation of monocytes,[28] cyclooxygenase (COX) activity,[29] and lipooxygenase (LOX)[30] were assayed.

Total RNA isolation

Total RNA was isolated from the brain using TRI Reagent (Sigma-Aldrich).[31]

Reverse transcription polymerase chain reaction

The isolated RNA was used for reverse transcription- polymerase chain reaction (RT-PCR) to study the expression of nuclear factor-κB (NFκB), gamma- glutamyl cysteine synthase (γGCS) and glyceraldehyde-3-phosphate dehydrogenase. Primer sequences are given in Table 1. Total tissue RNA (2 mg) was primed with 0.05 mg oligo dT and reverse-transcribed by omniscript RT using a cDNA synthesis kit (Qiagen). PCR was carried out using an Eppendorf thermocycler (model 5332). The PCR mixture contained 10 mM-Tris (pH 8.3), 50 mM-KCl, 1.5 mM-MgCl2, deoxynucleoside triphosphate (20 mM each), gene-specific primers (0.5 mM each) and Taq polymerase (0.025 units/ml). After an initial denaturation step at 94°C, 35 amplification cycles were performed. A final extension step of 5 min at 72°C was performed in order to complete the PCR. The amplified product was analyzed by electrophoresis on 2% agarose gel containing ethidium bromide. Then, the gels were subjected to densitometric scanning (Bio-Rad Gel Doc) to determine the optical density of each, and then normalized.

Table 1

Primer sequences used for RT-PCR analysis

Statistical analysis

The results were analyzed using a statistical program SPSS/PC+, Version 17.0 (SPSS Inc., Chicago, IL, USA). A one-way ANOVA was employed for comparison among the six groups. Duncan's post-hoc multiple comparison tests of significant differences among groups were determined, P < 0.05 was considered to be significant.

Results

The activities of catalase, SOD, GPx, GR [Table 2] were significantly decreased in the brain of the alcohol-treated group compared to the control. There was also a significant increase in the activities of these enzymes and the concentration of GSH in group administered Ksheerabala (101) along with alcohol compared with alcohol group.

Table 2

Activities of antioxidant enzymes in the brain

The level of lipid peroxidation products MDA, HP, and CD in brain [Table 3] was increased significantly in alcohol-treated groups compared to control group and the concentration of these reduced significantly in co-administered group when compared with alcohol-treated group. The level of GSH was significantly decreased in the brain of the alcohol-treated group compared to the control. The concentration of protein carbonyls increased significantly in alcohol-treated group compared to the control group and the concentration of these reduced significantly in co-administered group when compared with alcohol-treated group [Table 4].

Table 3

Concentration of lipid peroxidation products in the brain

Table 4

Concentration of protein carbonyls and reduced glutathione in brain

The activities of inflammatory markers COX and LOX in monocytes [Table 5] were increased significantly in alcohol-treated rats compared to control group and their activities were decreased significantly in co-administered group compared to alcohol-treated rats.

Table 5

Activities of inflammatory markers in monocytes

The mRNA expressions of NFκB and γGCS were evaluated by RT-PCR. In alcohol-treated rat brain, PCR products of NFκB had a marked increase compared to control rats. Treatment with Ksheerabala reduced the levels of expression of NFκB [Figure 1] gene. There was no significant change in the expression of these genes in Ksheerabala treated rats. The mRNA expression of γGCS [Figure 2] was markedly decreased in alcoholic rats, and the changes were reversed by treatment with Ksheerabala.

Figure 1

(a) Expression of nuclear factor kB (NFkB) at mRNA level, (b) Intensity of NFkB mRNA using gel doc. C: Control; A: Alcohol; K: Ksheerabala; KA: Ksheerabala; K + A: Ksheerabala + Alcohol. The mean intensity was measured and expressed as INT/mm2. Results are expressed as the average of quadruplicate experiments ± standard deviation. Different letter indicates values statistically significant at P < 0.05

Figure 2

(a) Expression of gamma-glutamyl cysteine synthetase (gamma-glutamylcysteine synthetase) at mRNA level; (b) Intensity of γGCS mRNA using gel doc. C: Control; A: Alcohol; K: Ksheerabala; KA: Ksheerabala; K + A: Ksheerabala + Alcohol. The mean intensity was measured and expressed as INT/mm2. Results are expressed as the average of quadruplicate experiments ± standard deviation. Different letter indicates values statistically significant at P < 0.05

Discussion

The brain is one of the major target organs for the actions of alcohol, and heavy alcohol consumption has long been associated with brain damage. The brain processes large amounts of oxygen in a relatively small mass, and has a high content of substrates available for oxidation (that is, polyunsaturated fatty acids and catecholamines) in conjunction with low antioxidant activities; making it extremely susceptible to oxidative damage.[32] Experiments with cells of rat brains have shown that a single dose of alcohol results in increases in lipid HP levels.[33] Our results are in line with the findings that alcohol causes oxidative damage in the brain. This was evidenced by higher levels of lipid peroxidation products and lower activities of scavenging enzymes. In addition to the attenuation of antioxidant enzyme activity, the antioxidant GSH was also shown to be decreased by ethanol exposure. This is in line with the observations of previous study.[34] The reduced level of GSH can be correlated with decreased expression of γGCS, the rate-limiting enzyme of GSH biosynthesis.[35] In the present study, it was observed an increase in activities of these enzymes, decreased lipid peroxidation products and increased GSH content in the brain of rats administered Ksheerabala (101) along with alcohol indicating that Ksheerabala (101) possess significant protective effect against alcohol-induced oxidative damage. This is in agreement with earlier studies in which Ksheerabala reduced quinolinic acid-induced neurotoxicities.[13]

Oxidative damage to proteins generates increased carbonyl groups due to oxidation of sensitive amino acids, such as histidine, proline, arginine, and lysine. These oxidatively modified proteins are functionally inactive and are degraded by the enzyme alkaline protease.[36] In consistent with this fact, there was an increase in the level of protein carbonyls in alcohol-treated rats. The co-administration of Ksheerabala (101) along with alcohol reduced the peroxidation of proteins in the brain.

Oxidative stress and inflammation are likely linked and may be difficult to tease apart. Several animal studies suggest that chronic alcohol intake alters CNS immune and inflammatory responses through interference with NFκB and expression of NFκB controlled genes.[37] NFκB is classically activated through dissociation from IkB in the cytoplasm.[38] DNA-binding and transactivation functions are also regulated through posttranslational modifications of NFκB proteins in the cell nucleus. Thus, chronic alcohol administration up-regulates inflammatory mediators in the animal brain, and isolated astrocytes followed by activation of NFκB and the upregulation of inducible NO synthase[39] and COX-2 expression. In inflammation, the prostaglandins are synthesized as a result of various cytokines signaling COX-2 transcription. It was found that 5-LOX pathway is crucially involved in brain disorders, and 5-LOX inhibition could act as a neuroprotective.[40] Prostaglandin E2, the main prostaglandin synthesized during inflammation, is formed via COX-mediated catalysis. The down-regulation in the expression of NFκB in the co-administered rats indicates that Ksheerabala (101) can down-regulate the alcohol induced upregulation of these genes, and thus COX and LOX mediated inflammation. The reduced activities of COX and LOX in co-administered group in comparison with alcohol also support this.

The antioxidant and anti-inflammatory effect of Ksheerabala (101) may due to the additive effect of its individual components. The main contents of Ksheerabala (101) are Bala (S. cordifolia), Ksheera (cow's milk) and Tila Taila (Sesamum oil). There are reports that milk caseins possess significant antioxidant activity.[41] The studies have shown that sesame oil protects against lipopolysaccharide-stimulated oxidative stress in rats.[42] The consumption of this oil influences beneficially the blood glucose, glycosylated hemoglobin, lipid peroxidation, and antioxidant levels in diabetic rats.[43] The plant S. cordifolia belongs to the family Malvaceae is a widely used herb for a number of neurodegenerative disorders. The previous studies showed that it had a protective effect on quinolinic acid-induced neurotoxicity,[44] and it had a potent action against alcohol-induced hepatotoxicity.[15]

Conclusion

The results confirmed that alcohol-induced toxic effects may be due to free radical mechanisms and provide evidence that Ksheerabala (101) significantly protects brain cells and reduces the severity of damage caused by alcohol intoxication. The mechanism of action may be by suppression of the activation of a transcription factor, that is, NFκB and concomitant reduction in the neuroinflammation. However, further clinical trials are needed to determine whether Ksheerabala (101) that inhibit the activation of NFκB will be effective in preventing and possibly treating alcohol-induced brain injury in humans.

Financial support and sponsorship

Council of Scientific and industrial Research.

Conflicts of interest

There are no conflicts of interest.