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

International Journal of Complementary & Alternative Medicine

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 condition the brain in during 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 must mechanisms 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 molecular biology kits and reagents were used: min. After centrifugation at 15,000X g for 15 min at 4 °C, the

supernatants were neutralized with 2 M KHCO3, centrifuged - -freeTRI reagentTM (Ambion and RNA DEPC by (SigmaLife Technologies Aldrich, RevertTM, USA) Aid Maxima ﬁrst strand SYBR cDNA synthesis kit (Thermo Scientific, Lithuania), TURBO DNA glutamateand used catalyzed for ammonia by glutamate assay. The dehydrogenase quantification in the of ammoniapresence - ofinvolved ammonia. measuring The decrease the rate in O.D.of conversion at 340 nm ofwas α-ketoglutarate recorded. to Green/ ROX qPCR master mix 2X (Thermo Scientific). The primers The 1 mL reaction mixture, contained 50 µL samples, 3.4 (Forwardused in the 5’- present GATCATGAAGACAGGGGCCTT-3’; study were synthesized by ReverseEurofins 5’-CCTGGenomi- GATGAAACCCTGTAGCA-3’);cs India Pvt. Ltd. The rat gene Bcl specific(Forward primers 5’-ACACCAGAATCAAG used were: Bax- 2 mixture was incubated for 5 min at 18-35 °C and reaction was mM α-ketoglutarate and 0.23 mM reduced NADPH in water. The (Forward 5’TCATCAGTTCCATGGCCCAGAC-3’; Reverse 5’-GGTTT- (at 5 min) O.D. at 340 nm was used to calculate concentration of TGTTCGT-3’; Reverse 5’-TGCATTCTTGGATGAAGGGGT-3’); TNF-α- ammoniastarted by in the mM. addition of suitably diluted GDH. Initial and final AGTGCGAGG-3’; Reverse 5’-CCCCAGGAATACTGCCTGCAGAG-3’). TheGCTACGACGTGGGCTA-3’); primers were designed NFκB using (Forward the Fast 5’-PCR GCGTTTCCGTTACA 6.1 software. Western blotting: 100 µg of cytosolic protein was loaded in each lane and subjected to 10% denaturing polyacrylamide Animals gel electrophoresisBriefly, at constant voltage of 100 volts for 2 hours The adult male albino rats weighing 130-160 g, used in this followed by transferring the protein bands on nitrocellulose study, were fed the recommended diet and maintained on 12:12 h light: dark cycle, in an animal house, as per recommendations transfer was checked by Ponceau S staining. The membrane was from the institutional animal ethical committee (IAEC) of Banaras thenmembrane placed overin a blocking night at solution 50 mA at(5% 4 °C.non Efficiency fat dried milk of Protein in 1X Hindu University, for the care and use of the laboratory animals. PBS) for 2h. The membrane was processed for immunodetection of

Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 3/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

Bcl2 antibody (1:1000) and Bax antibody (1:500). HRP-conjugated proteins using ECL western blotting detection kit. For loading gently(20U/μL), and 2incubated μL of 10 mMfor 5 dNTP min atmix, 25 1μL°C followed of reverse by transcriptasecDNA strand control,secondary monoclonal antibody wasanti- used-actin for peroxidasefinal detection antibody of the (1:10,000)respective synthesisto make afor final 60 volumemin at of42 20°C. μL.The The reaction components was terminated were mixed by was used. The normalized densitometric values of each protein heating at 72 °C for 5 min. The cDNA was stored at -80 °C. vs -actin, was recorded 훽using gel densitometry software Alpha Real time (qPCR): The real time qPCR was done with an ABI Imager 2200. 훽 Prism 7500 Sequence Detection System (PE Applied Biosystems, RNA isolation and cDNA synthesis: Total RNA from cortex and cerebellar extracts was isolated using TRI reagent following the CA, USA). The PCR reaction mixture of (20 μL) consisted of 1μL of TRI reagent was centrifuged at 12,000g for 10 min at 4 °C and sample cDNA (diluted 1:10), 1μL of 10 pM of forward and reverse supernatantmanufacture’s was protocol. collected. Briefly, Chloroform the homogenate was added prepared (0.2 mL) in to 1mL the primers and 10 μL of 2X Thermo Scientific SYBR Green/ROX qPCR PCR condition was: 95 °C for 30 sec, followed by 40 cycles at 95 supernatant and vortexed. After 3 min, the tube was centrifuged Master Mix and brought to final volume with RNase free water. °C for 5 sec, and 60 °C for 20 sec. Real-time PCR data was analyzed at 12,000 X g for 10 min, at 4 °C. The centrifugation separated the method, normalizing the Ct values of the indicated mixture into 3 phases, out of which the colorless upper aqueous T gene to the Ct values of GAPDH relative to a control sample. phase containing RNA was collected and 0.5 mL isopropanol using the ΔΔC was added to it. After 5 min, the tube was again centrifuged at Statistical analysis: The statistical analysis of experimental data 12,000X g for 10 min, at 4 °C. The RNA precipitated out in the was done by applying Student’s t test, expressed as mean ± SD and form of a pellet at bottom of the tube and was washed in 1 mL 75% ethanol. After air drying the RNA pellet, it was dissolved in and experimental sets. 60 µL DEPC treated water by repeated pipetting. The dissolved P<0.05 was taken as the level of signiﬁcance between the control RNA pellet was subjected to DNase treatment (DNA free-Ambion) Results to remove any sort of DNA contamination. The total RNA yield was Development of chronic liver failure/ determined and quality assessed by agarose formaldehyde RNA hyperammonemia gel electrophoresis. The HA condition due to TAA induced CLF was evident from The RNA samples with an A /A ratio of 1.8-2.0 were used 260 280 a ~2 x increase (P<0.01) in serum ammonia level of CLF/HA in the cDNA synthesis reaction. rats (Figure 1a). Also, as depicted in Figure 1b&c, as compared synthesis kit was used for cDNA synthesis. The reaction mixture consisted of 4µg RNA, 1 µL random RevertAid hexamer primer, first strand made cDNAup to increased (P<0.05) in the cortex and in the cerebellum of CLF/HA 12 µL with DEPC treated water. The mixture was gently mixed, to the control group rats, the level of ammonia was significantly led HA condition, both in the blood and in the susceptible brain regions.rats. These findings confirm the induction of chronic liver failure centrifuged briefly and the following components were added; 4 µL of 5X reaction buffer, 1μL of RiboLock™ Ribonuclease Inhibitor

Figure 1: Effect of TAA treatment on ammonia level in serum (a), cortex (b) and cerebellum(c). Values are represented as mean ± SD where n=4; * P<0.05; **P<0.01; (control versus CLF), CLF: chronic liver failure.

Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 4/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

Effect of resveratrol on inflammatory factors: TNF-α of Bcl2 and NF-κB the cerebellum (P<0.01), as compared to that observed in the control is group significantly rats. The declined expression in both, level theof Bax cortex (Figure (P<0.001) 5a&c) wasand

and in the cerebellum (P<0.01) of the HA rats, as compared to the Development of neuroinflammation is considered an important also found to be significantly enhanced in the cortex (P<0.001) 2/ marker for HA in the brain. Neuroinflammation was monitored Bax ratio presented in Figure 6a for the cortex (P<0.001) and in by measuring the transcript level of neuroinflammatory markers Figurecontrol 6bgroup for ofthe rats; cerebellum resulting (P<0.001). in a significant However, decline on in treatment the Bcl transcriptTNF-α and in NF-κB. cortex According and cerebellum to Figure of CLF/ 2a&b, HA as rats compared was observed. to the with RSV, the Bcl2/Bax ratio was found to be restored to normal control group rats, a significant increase (P<0.01) in the TNF-α levels in both the brain regions of HA rats (Figure 6a&b). inA similarboth the pattern brain regions was observed of the HA for rats. NF-κB However, transcript the enhanced (Figure

3a&3b), which showed significantly enhanced levels (P<0.001) qPCR analysis of Bcl2 and Bax. According to Figure 7a&b, the These findings were further confirmed by conducting the were restored back to their normal levels in the HA rats treated transcript level of Bcl2 withlevels RSV. of both the neuroinflammatory markers, TNF-α and NF-κB, both the cortex and the cerebellum of the HA rats, as compared to the control group counterparts. was significantly This could decreased be normalized (P<0.01) in both in

Effect of resveratrol on Bcl2 and Bax expression the brain regions of the HA rats with the resveratrol treatment. Similarly, the transcript level of Bax, as shown in Figure 8a&b, Looking at the ratio of Bcl2/Bax is the most effective way to determine the susceptibility of cells for undergoing apoptosis. According to Figure 4a&c, it is evident that the expression level cerebellum of the HA rats, however, due to the RSV treatment, its levelwas significantly recovered to increased the normal (P<0.001) values. in both the cortex and the

Figure 2 mRNA level was normalized with the cycle threshold value of Gapdh for each sample to give a relative quantity of the mRNA expression by 2-ddCt . Values are: Expression represented of as TNF-α mean in ± cerebralSD from cortex4 repeats; (a) andGapdh cerebellum was taken (b) as of the control, internal HA control. and HA+RSV **P<0.01; treated (control rats. versus Cycle thresholdHA) #P<0.05; value ## ofP TNF-α<0.01; (HA versus HA+RSV), HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol.

Figure 3 level was normalized with the cycle threshold value of Gapdh for each sample to give a relative quantity of the mRNA expression by 2-ddCt. Values are represented: Expression as mean ±of SD NF-κB from gene 4 repeats; in cerebral Gapdh cortex was taken(a) and as cerebellum the internal (b) control. of control, ***P <0.001;HA and HA+RSV(control versusrats. Cycle HA) threshold ##P<0.01; value ### ofP<0.001; NF-κB RNA(HA versus HA+RSV), HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol.

Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 5/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

Figure 4 2 in cerebral cortex (a) and cerebellum (c) of the HA and RSV treated HA rats. Western blot analysis of Bcl2 was performed as described in the text of methods. The level of -actin was taken as the loading control. In panels (b) and (d) normalized densitometric : Expression profile of Bcl values of Bcl2/ -actin for cortex and cerebellum respectively, have been presented as mean ± SD from three western blot repeats. **P < 0.01; ***P<0.001; (control versus HA rats), ## < 0.01; ###P<0.001훽 (HA versus HA+ RSV rats). HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol. 훽 훽

Figure 5 performed as described in the text of methods. The level of -actin was taken as the loading control. In panels (b) and (d) normalized densitometric values of: Bax/Expression-actin profile for cortex of Bax and in cerebellumcerebral cortex respectively, (a) and cerebellum have been (c)presented of the HA as andmean RSV ± treatedSD from HA three rats. western Western blot blot repeats. analysis **Pof Bax < 0.01; was ***P<0.001; (control versus HA rats), ###P<0.001 (HA versus훽 HA+ RSV rats). HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol. 훽

Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 6/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

Figure 6: Ratio of Bcl2/Bax in cerebral cortex (a) and cerebellum (b) presented as mean ±SD from three western blot repeats. ***P<0.001; (control versus HA) ###P<0.001; (HA versus HA+ RSV rats). HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol.

Figure 7: Expression of Bcl2 gene in cerebral cortex (a) and cerebellum (b) of control, HA and HA+RSV rats. Cycle threshold value of Bcl2 RNA level was normalized with the cycle threshold value of Gapdh for each sample to give a relative quantity of the mRNA expression by 2-ddCt. Values are represented as mean ± SD from 4 repeats; Gapdh was taken as the internal control. **P<0.01; (control versus HA) ###P<0.001; (HA versus HA+ RSV rats). HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol.

Figure 8: Expression of Bax gene in cerebral cortex (a) and cerebellum (b) of control, HA and HA+RSV rats. Cycle threshold value of Bax RNA level was normalized with the cycle threshold value of Gapdh for each sample to give a relative quantity of the mRNA expression by 2-ddCt. Values are represented as mean ± SD from 4 repeats; Gapdh was taken as the internal control. ***P<0.001; (control versus HA) ###P<0.001; (HA versus HA+ RSV rats). HA: Hyperammonemia, HA+RSV: Hyperammonemia + Resveratrol.

Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 7/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

Discussion

Resveratrol is known to exhibit anti-pathogenic activity (FigureTNF-α amongst 3), it was the interesting most prominent to observe ones a similar [12,52,53]. recovery Moreover, in the against a wide range of diseases mainly by acting as a potent concordant to the RSV dependent decline in TNF-α expression with RSV. Thus, RSV dependent recovery in the expression of are now evident to go hand to hand in a number of pathological level of NFκB mRNA in both the brain regions of HA rats treated ROS scavenger. Moreover, inflammation and ROS generation transcriptionTNF-α and NFκB at gene together, level. in The both argument the brain gets regions support of HA from rats, a andmanifestations, excitotoxicity wherein, have been TNF-α found has to been be responsible found to play for a regulatory triggering advocates that RSV could modulate TNF-α level by altering its neuro-excitotoxicrole [35,36]. In case disorders/diseases of brain disorders like; also, traumatic neuroinflammation brain injury, Ischemia, Alzheimers disease, Parkinson’s disease, etc [17,37- recent report describing modulation of TNF-α mRNA by another 47]. Recent experimental evidences also suggest that natural naturalIn case product, of resveratrol, fisetin, in caseas a of neuroprotectant, HCC pathogenesis another [48]. issue remaining unsolved is how it prevents possible neurodegeneration cytokines while protecting diseased cells from oxidative insult. antioxidants may modulate the expression of inflammatory describedunder the to excitotoxic inhibit glutamate conditions transport involving to the TNF-α glial cells as the from main the examinedFor example, this fisetin, hypothesis a natural for RSV, product a natural has polyphenol,been demonstrated against anto synapticculprit. Thiscleft and aspect thereby is equally allowing important over activation because of glutamate- TNF-α is decrease TNF-α mRNA in HCC cells in vivo [48]. In this article, we AMPA and NMDA receptors while decreasing GABA receptor persistent HA in CLF rats. activity of the post synaptic neurons [17]. Such a situation causes inflammation driven excitotoxic brain disorder developed due to enhanced Ca2+ entry into the neurons and thereby inducing The enhanced levels of ammonia (Figure 1), in blood and in the neuronal death [17]. Such neuronal deaths then must implicate two brain regions selected (cortex and cerebellum), represent a apoptotic mechanisms to execute safe removal of the dying moderate grade HA condition exposing brain cells with increased neurons. The most plausible determinant of intrinsic pathway of ammonia concentration. And as described earlier [33,49,50], apoptosis is the mitochondria damage induced interplay of Bcl2/ these rats were considered to representative of a true CLF led Bax ratio under neuropathological conditions [30-32]. RSV is known to prevent such neurodegeneration by eliminating the ROS insult [12,54,55]. However, ROS generation and mitochondrial HA model of neuroinflammation/excitotoxicity. Using these HA apoptotic factors in the cortex and cerebellum. This is because, damage also go hand to hand by inducing neurodegenration rats, we examined the expression profiles of TNF-α, NFκB and these two brain regions have been demonstrated to undergo in acute HA condition [3,50]. Thus, it is likely that brain cells, particularly, post synaptic neurons, start undergoing apoptosis by ammonia toxicity and HE pathogenesis [33,50,51]. involving classical Bcl -Bax pathways [30-32]. significant neuro-chemical and neuroanatomical changes during 2 The “TNF theory” of HA/HE pathogenesis [8] emphasizes

2/Bax the extent that these cytokines could be used as markers for ratioMoreover, attributed in mainly the present to the declineing context, the Bcl findings (an antiapoptotic in Figure significant roles of TNF-α and other cytokines in HE up to 2 factor)4,5& 6 level clearly with demonstrate the enhanced a significant Bax (pro-apoptotic decline in Bcl factor) be considered a suitable target for therapeutic intervention, level in cerebral cortex and cerebellum of the HA rats and thus encephalopathy grading [4,9-13]. Obviously, TNF-α could however, with limited information on its responsiveness towards suggesting induction of apoptosis in both these brain regions of the HA rats. However, when these HA rats were treated with of the expression of this cytokine by natural product is an RSV, the Bcl /Bax ratio was found to recover back to normal RSV in neuroinflammation and neuroexcitotoxicity. Modulation 2 upcoming strategy [48]. In this respect, results in Figure 2 clearly levels. This further suggests that resveratrol is able to modulate

Bcl2 and Bax expression and thereby, allow the recovery of brain mRNA level in cerebral cortex and cerebellum of the HA rats and cells towards an anti-apoptotic condition from a HA induced pro- demonstrate a cause and effect relationship between HA & TNF-α apototic microenvironment. This argument gets support from a /Bax ratio in the cerebellum thereby support the TNF-α theory of HA pathogenesis. However, it 2 to normal levels due to the in vivo treatment with RSV. Various of CLF rats with even mildest HA condition due to the in vivo was interesting to record recovery of TNF-α expression reaching treatmentsimilar finding with onBacopa recovery extract of Bcl[51]. Indeed, Resveratrol could level by blocking its synthesis and enhancing its degradation as also diminish memory impairment in the vascular dementia strategies have been employed to decrease enhanced TNF-α therapeutic mechanisms in excitotoxic neurological disorders model by attenuating increased expression of Bax and declining [17]. However, examples are limited with regards to RSV and the Bcl expression [32]. modulative expression of this cytokine. In this respect, results 2 Conclusion in Figure 2 are the first of their kind to demonstrate Resveratrol dependent decline in TNF-α mRNA particularly in the cortex and cerebellumThe argument of the rats gets with support HA induced from neuroinflammation.the results in Figure 3 gradeIn conclusion,HA but also these demonstrate findings notthat only it does provide so byevidence modulating for a significant neuroprotective role of Resveratrol during moderate

regulating expression of Bax and Bcl2 in the cortex and cerebellum transcriptiondescribing HA factor dependent that similaris translocated enhancement to the in NFκBnucleus mRNA for ofenhanced the HA rats. expressions Unravelling of TNFα such anda mechanistic NFκB and aspect by reciprocally of in vivo level. This is because, NFκB is known to be a constitutive type neuroprotective action of resveratrol, under a neuro-excitotxic/ enhancing expression of the inflammatory cytokines including

Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 8/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

15. Beste C, Baune BT, Falkenstein M, Konrad C (2010) Variations in the further examination of this mechanism in other brain disorders TNF- gene (TNF- neuro-inflammation condition, is likely to open up the research for mechanisms in a dissociated fashion. Journal of Neurophysiology 104(5):α 2523-2531.α -308G→A) affect attention and action selection Acknowledgementarising out of neuroinflammation and excitotoxicity. 16. Santello M, Bezzi P, Volterra A (2011) TNFalpha controls glutamatergic gliotransmission in the hippocampal dentate gyrus. Neuron 69(5): 988-1001. (54/38/CFPGER/2011/NCD-II) to SKT. A. Khanna acknowledges theThis Lady workTata Memorial was financially Trust- JRF/SRF’ supported awarded by an ICMRto her projectduring 17. Olmos G, Llad the tenure of this work. The authors are thankful to the DST FIST, CAS COSIST, DST Purse and DBT-BHU ISLS programmes in the 2014: 1-12. ό J (2014) Tumor Necrosis Factor Alpha: A link between neuroinflammation and excitotoxicity Mediators of Inflammation Department & Institute of Science for providing instrumentation 18. Tanabe K, Matsushima-Nishiwaki R, Yamaguchi S, Iida H, Dohi S, facilities. et al. (2010) Mechanisms of tumor necrosis factor-alpha-induced

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Citation: Brain. Int J Complement Alt Med 4(2): 00115. DOI: 10.15406/ijcam.2016.04.00115 Khanna A, Trigun SK (2016) Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro-Apoptotic Conditions in Rat Resveratrol Normalizes Hyperammonemia Induced Pro-Inflammatory and Pro- Copyright: 9/9 Apoptotic Conditions in Rat Brain ©2016 Khanna et al.

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