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Resveratrol Normalizes Hyperammonemia Induced Pro- Inflammatory and Pro-Apoptotic Conditions in Rat Brain

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Resveratrol Normalizes Hyperammonemia Induced Pro- Inflammatory and Pro-Apoptotic Conditions in Rat Brain International Journal of Complementary & Alternative Medicine Resveratrol Normalizes Hyperammonemia Induced Pro- Inflammatory and Pro-Apoptotic Conditions in Rat Brain Research Article Abstract Chronic liver failure (CLF) led hyperammonemia (HA) is known to develop a Volume 4 Issue 2 - 2016 considered responsible for mounting neurological complications associated metabolic brain disorder known as hepatic encephalopathy (HE). TNF-α is now with HA/HE. However, the mechanism that connects inflammation and Department of Zoology, Banaras Hindu University, India neuroexcitotoxicity is not yet clear. Resveratrol (RSV) is a natural antioxidant and known to mediate its therapeutic actions mainly by scavenging Reactive *Corresponding author: Surendra K Trigun, Department oxygen species (ROS). RSV is predicted to modulate many cellular targets of Zoology, Institute of Science, Banaras Hindu University, as well; however, there is little information on its neuroprotective roles. This Varanasi-221005, India, Tel: +91-542-6702523; Email: NFkB and apoptotic factors; Bcl2 & Bax in cerebral cortex and cerebellum of the CLFarticle rats describes (induced the by effect administration of RSV treatment of 100 onmg the thioacetamide expression profiles i.p. for of10 TNF-α, days) Received: July 31, 2016 | Published: August 26, 2016 (p<0.001) in both of the brain regions (cerebral cortex and cerebellum) of HA ratsconfirming was observed. moderate This grade was HA.consistent A significant with a increasesimilar enhancement in mRNA level in ofthe TNF-α level declineof NFkB, in a Bcl2transcription level (p<0.001) factor forwith TNF-α a significantly synthesis, enhanced and thus, levelsuggests of Bax induction further suggestedof TNF-α leda neurodegenerative inflammation in conditionthe brain induring those persistentbrain regions HA. of A the significant HA rats. Moreover, post treatment of those HA rats, with 10 mg/Kg b.w. RSV for a week, was found to bring all the factors towards their normal level in both the brain regions. The finding suggest that RSV is able to ameliorate moderate HA induced in the rat brain primarily by modulating expression of the inflammatory and apoptoticTNF-α led factors.inflammatory cascade and proapoptotic neurodegenerative condition Keywords: Apoptosis; Cortex; Cerebellum Hyperammonemia; Neuro inflammation; Moderate HE; TNF-α; Introduction During recent past, “TNF theory” (Tumor necrosis factor) of HE pathogenesis could draw much attention due to a close Ammonia neurotoxicity is considered responsible for development of a serious nervous system disorder called hepatic of CLF patients with HE [8]. Later studies have also emphasized encephalopathy (HE). This situation arises mainly due to liver association between the level of TNF-α and ammonia in the brain dysfunction led hyperammonemia (HA). HE is characterized by the extent that these cytokines could be used as markers for the neuropsychiatric manifestations related to motor dysfunction, encephalopathysignificant involvement grading [4,9-13].of TNF-α and other cytokines in HE to memory impairment, and deranged sleep-awake cycle [1,2]. Since HE affects the quality of life of the patients, it is important to It is now evident that the microglia and astroglia cells in develop effective therapeutic strategy against HE. This necessitates understanding the neuro-chemistry of HA pathogenesis. and astrocyte induced synaptic strengthening [14-17]. However, whenbrain synthesizeproduced TNF-αin higher to regulate amounts higher during order neuropathology, brain functions Since ammonia crosses the blood brain barrier easily, they are known to potentiate glutamate induced cytotoxicity by prolonged HA condition, a common situation during chronic inhibiting glutamate transport to the glial cells from the synaptic liver failure (CLF), is likely to maintain increased ammonia cleft [12,16-19]. Even development of MHE has been described to concentration in brain [3,4]. The information, mainly derived from the cell culture system and from animal models with acute severity of CLF or HA in the CLF patients [20]. Moreover, some HE, suggest that the increased brain ammonia level mainly drives be dependent more on enhanced inflammatory markers than the could prevent ammonia toxicity in astroglial cells by normalizing and also over activates glutamate-NMDA receptor (N-Methyl- findings from in vitro studies suggest that resveratrol treatment astrocytes to undergo significant morpho-pathological changes D-Aspartate Receptor) pathway in the post-synaptic neurons of resveratrol in ameliorating ammonia toxicity. However, the [1,5-7]. Moreover, NMDAR inhibition could not be translated mechanismROS/RNS level by which[12,21,22]. resveratrol Thus, hinting does so towards is unclear. a significant role into prevention of ammonia neurotoxicity, mainly because threshold glutamate-NMDAR activation is necessary for normal Resveratrol (3, 3, 4’-trihydroxy-Trans stilbene), a natural neurological functions including higher order brain functions and polyphenol, is found in a number of dietary sources such as grapes, normal synaptic activity. berries, and red wine [23-25]. It acts as an effective cardioprotector, Submit Manuscript | http://medcraveonline.com Int J Complement Alt Med 2016, 4(2): 00115 Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 2/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al. anti-aging, and anti-carcinogenic agent in vivo [12,26-28]. Development of chronic liver failure (CLF)/HA and Some reports also suggest that this natural polyphenol exerts treatment schedule neuroprotection during certain neurodegenerative challenges [12,26-28] mainly due to its ROS scavenging properties and/or by activating some antioxidant enzymes. Some recent observations, rats was developed by the administration of thioacetamide (TAA) mainly derived from in vitro studies, suggest that ammonia as standardized The CLF/HA modelin our oflab neuroinflammation [33]. The rats were in divided adult male into albino three induced impaired glutamatergic communication in astroglial cells groups with six rats in each. Group A: control (C), administered with could be recovered with resveratrol treatment [29]. Since astroglia 0.9% NaCl i.p, once daily for 10 days; Group B: Hyperammonemia (CLF/HA), administered with 100 mg/Kgbw TAA (prepared in 0.9 % NaCl) i.p once daily for 10 days; Group C: CLF/HA + Resveratrol implicatedcells are the in main impaired source glutamateof inflammatory transport cytokines, during it ammoniais argued (CLF/HA + RSV) administered with 10 mg/Kgbw Resveratrol i.p th neurotoxicity.that resveratrol And could if it does also so,prevent then this inflammatory polyphenol mustmechanisms be able once daily, dissolved in 1% DMSO, starting from 8 day onwards th to down regulate apoptotic mechanism which otherwise becomes till 14 day. The Resveratrol was administered 4h after the TAA upregulated during such challenges [30-32]. treatment. The selected dose of Resveratrol was able to recover Thus, to investigate whether resveratrol is able to modulate th theTAA hyperammonemic induced neurobehavioural condition, deficit ammonia in the assay rats. Afterwas performed24 h of the inlast the dose pooled given, serum all the of ratsall the were six sacrificedrats from controlon 15 day.and HATo confirmgroups. inflammation-apoptosis pathway in the HA brain, the expression The cortex and cerebellum was dissected out and stored at -80 °C profiles of TNF-α (a proinflammatory marker), NFκB (a apoptotic factors (Bcl2/Bax) were examined in the cortex and for further studies. cerebellumtranscriptional (the regulator two susceptible of inflammatory brain regions) cytokines) of theand CLFintrinsic rats with moderate grate HA. Preparation of cortex and cerebellum extracts Material and methods Cerebral cortex and cerebellum extracts were prepared in 0.02 M Tris-Cl (pH 7.4) containing protease inhibitors. Extracts Chemicals were centrifuged at 35,000 g for 45 min at 4°C. The supernatant collected was used for the ammonia assay. Protein content was All chemicals used were of analytical grade purchased determined by the method of Lowry et al [34]. from E-Merck and Sisco research Laboratory, Mumbai (India) except N,N’-methylenebisacrylamide, Acrylamide, N,N,N’N’- Mitochondria-free tissue extract was prepared in an extraction tetramethylethylenediamine (TEMED), phenylmethylsulphonyl medium consisting of 400 mM sucrose, 0.2 mM benzamidine, 1 purchased from Sigma-Aldrich, USA. Primary antibodies used were 7.4), and 0.02% heparin. Initially, the extracts were centrifuged at fluoride (PMSF), Ponceau S and bromophenol blue, which were mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) (pH supplied from the following companies: rabbit polyclonal Bcl2 and Bax from Cell Signaling Technology and rabbit monoclonal -actin obtain cytosolic fractions. The protein content in the extract was from Sigma Aldrich. Goat anti-Rabbit horseradish peroxidase estimated12,000×g forby Lowry15 min method and finally as mentioned at 19,000×g earlier. for 40 min at 4 °C to (HRP) conjugated secondary antibodies were supplied훽 from Ammonia assay: The ammonia assay was performed using a Genei. ECL western blotting detection kit was procured from kit supplied by Sigma-Aldrich, USA. The serum as well as cortex and cerebellum extract were deproteinized in 1/5 volume of Cayman chemicals company. Thermo Scientific. The trans-Resveratrol was supplied from the ice-cold 100 g/ L Trichloroacetic acid and kept on ice for 15 The following
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