- Email: [email protected]
Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats
Pharmacological Reports 66 (2014) 1037–1042
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats Perumal Subramanian a,*, Murugesan Jayakumar a, Jaime Jacqueline Jayapalan b,c, Onn Haji Hashim b,c a b c
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, India University of Malaya Centre for Proteomics Research (UMCPR), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
A R T I C L E I N F O
Article history: Received 4 December 2013 Received in revised form 5 June 2014 Accepted 25 June 2014 Available online 8 July 2014 Keywords: Chronotherapy Fisetin Urea cycle Hyperammonaemia Circadian
A B S T R A C T
Background: Elevated blood ammonia leads to hyperammonaemia that affects vital central nervous system (CNS) functions. Fisetin, a naturally occurring flavonoid, exhibits therapeutic benefits, such as anti-cancer, anti-diabetic, anti-oxidant, anti-angiogenic, neuroprotective and neurotrophic effects. Methods: In this study, the chronotherapeutic effect of fisetin on ammonium chloride (AC)-induced hyperammonaemic rats was investigated, to ascertain the time point at which the maximum drug effect is achieved. The anti-hyperammonaemic potential of fisetin (50 mg/kg b.w. oral) was analysed when administered to AC treated (100 mg/kg b.w. i.p.) rats at 06:00, 12:00, 18:00 and 00:00 h. Amelioration of pathophysiological conditions by fisetin at different time points was measured by analysing the levels of expression of liver urea cycle enzymes (carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS)), nuclear transcription factor kappaB (NF-kB p65), brain glutamine synthetase (GS) and inducible nitric oxide synthase (iNOS) by Western blot analysis. Results: Fisetin increased the expression of CPS-I, OTC, ASS and GS and decreased iNOS and NF-kB p65 in hyperammonaemic rats. Fisetin administration at 00:00 h showed more significant effects on the expression of liver and brain markers, compared with other time points. Conclusions: Fisetin could exhibit anti-hyperammonaemic effect owing to its anti-oxidant and cytoprotective influences. The temporal variation in the effect of fisetin could be due to the (i) chronopharmacological, chronopharmacokinetic properties of fisetin and (ii) modulations in the endogenous circadian rhythms of urea cycle enzymes, brain markers, redox enzymes and renal clearance during hyperammonaemia by fisetin. However, future studies in these lines are necessitated. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction
Abbreviations: AC, ammonium chloride; ASS, argininosuccinate synthetase; CPS-I, carbamoyl phosphate synthetase-I; GPx, glutathione peroxidase; GS, glutamine synthetase; GSH, reduced glutathione; GST, glutathione-S-transferase; HE, hepatic encephalopathy; IkB, inhibitor kappaB; NF-kB p65, nuclear factor kappaB subunit p65; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; OTC, ornithine transcarbamoylase; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-a, tumour necrosis factor-a; UCD, urea cycle disorder. * Corresponding author. E-mail addresses: [email protected], [email protected] (P. Subramanian).
The common feature of urea cycle disorder (UCD) is a defect in ammonia elimination leading to hyperammonaemia and hepatic encephalopathy (HE). The increased level of ammonia in the blood leads to an increase in cerebral uptake across the blood–brain barrier (BBB) [1]. Excess ammonia is toxic to the central nervous system (CNS); elevated cerebral ammonia results in a severe accumulation of glutamine in astrocytes, causing oedema and encephalopathy [2], and leading to a spectrum of neuropsychiatric and neurological symptoms (impaired memory, shortened attention span, sleep–wake inversions, brain oedema, intracranial hypertension, seizures, ataxia and coma) with high mortality [3].
http://dx.doi.org/10.1016/j.pharep.2014.06.018 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
1038
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
Endogenous 24 h rhythmicities of urea cycle enzymes such as carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS) in rat liver and serum have been reported; the enzyme levels are low in light period but exhibit a significant increase in darkness [4,5]. The increase in cerebral ammonia concentration may stimulate glutamine synthetase (GS) activity to metabolise excess ammonia and glutamate into glutamine and thus thwarting neurotoxicity [6]. GS has a robust circadian rhythm in liver and kidney; increased mRNA expression occurs in the dark period when the animal begins activity and feeding coinciding with amino acid metabolism [7]. In humans, the rhythmic pattern is relatively reversed [8]. Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) possesses microbicidal, antiviral, antiparasitic and antitumoural effects. However, abnormal iNOS induction seems to be involved in the pathophysiology of a variety of human diseases. In addition to ammonia and glutamate levels, the NOS and NO system might also be involved in the brain responses to HE [9]. The acrophases of NO and NOS in plasma, brain, kidney, testis and lung occur at midnight, corresponding to the behavioural activity and increased reactive oxygen species (ROS) production [10,11]. NF-kB/Rel proteins, a family of ubiquitous transcription factors, participate in immunological responsiveness, inflammatory processes and cell growth regulation. ROS seem to play a dual role in the NF-kB activation cascade. NF-kB helps to regulate various inflammatory genes in different target cells [12]. The nuclear content of subunit of NF-kB p65 was found to exhibit circadian rhythm in animals [13]. The circadian oscillator components regulate the immune response, and the absence of the core clock component, cryptochrome, caused elevation of NF-kB p65, suggesting a link between circadian rhythm disruption and increased susceptibility to chronic inflammatory diseases [14]. The reappearance of symptoms and serious adverse effects after the discontinuation of treatment are serious drawbacks of many anti-hyperammonaemic agents/therapies. These drugs or therapies are inadequately effective. Therefore, the screening and development of drugs for anti-hyperammonaemic activity are still in progress. This can be achieved by focusing research on the active principles of flavonoids by increasing their efficacy by time dependent administration of drugs at which the effect is maximal. The structural features of flavonoids are the presence of a B-ring catechol group and the presence of a C2–C3 double bond in conjugation with an oxo-group at C4; the first serves to donate a hydrogen/electron to stabilise a radical species, and the second serves to bind transition metal ions such as iron and copper [15]. The anti-oxidant activity of flavonoids and their glycosides has been associated with their capacity to (i) scavenge reactive oxygen and nitrogen species [16], (ii) chelate transition metals that may induce oxidative damage through the Fenton reaction [17], (iii) inhibit pro-oxidant enzymes [18] and (iv) induce anti-oxidant enzymes [19]. Fisetin (3,30 ,40 ,7-tetrahydroxyflavone) is a dietary flavonoid widely distributed in strawberries, apples, persimmons, grapes, onions and cucumbers and displays anti-oxidant [20], antiallergic [21], anti-inflammatory [22], anti-cancer [23,24], neuroprotective [25], neurotrophic [26] and anti-angiogenic [27] activities. Our earlier studies showed preservation of hepatocellular architecture, normalisation of oxidative stress markers, antioxidant enzymes, liver marker enzymes (alanine transaminase, aspartate transaminase and alkaline phosphatase) and astrocytic marker enzyme (soluble guanylyl cyclase) and modulation of their temporal variation towards normalcy in fisetin treated AC-induced rats, although no temporal variations are noticed in AC-alone treated groups [28,29].
Chronotherapy is one of the best therapeutic approaches used to deliver medications in response to endogenous biological rhythms according to the pathophysiology of disease states to optimise treatment outcomes or limit adverse effects. Earlier reports described administration-time differences in the pharmacokinetics of many drugs [8,30]. The maximum clearance of gentamicin, amikacin, and isepamicin was higher when injected during the activity period [31,32]. A higher rate of urinary excretion of ciprofloxacin was found at the beginning of the day [33]. Most laboratory and clinical observations showed that the clearance of drugs was lower during the rest span and higher during the activity span. Circadian variations have been reported in different populations of T total, T helper, and T killer lymphocytes in inflammatory reactions [33]. Further, circadian oscillations in the immune system and the inflammatory response play a major role in the temporal changes in the body’s response to infection and other pathological conditions. Circadian rhythms of activity with gene expression changes have been reported in redox pathway enzymes, including NOS, haem oxygenase (HO), superoxide dismutase (SOD), catalase, reduced glutathione (GSH), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) [34–37]. Modified circadian rhythms of activity have been reported in hyperammonaemic rats [38]. Endogenous 24 h oscillations in gastrointestinal, liver, kidney and other bodily processes are of great importance for therapeutics and for selecting the phase for drugs administration, due to rhythm influences on the pharmacokinetics, effect-duration, efficacy, adverse effects and beneficial outcomes of medications [39]. Investigations on the chronotherapeutic efficacy of anti-hyperammonaemic drugs are completely lacking. However, the time dependent administration of drugs would be helpful in increasing the efficacy and minimising the side effects of drugs. Hence, the present study was designed to assess the chronotherapeutic modulations of liver urea cycle enzymes, NFkB p65, brain GS and iNOS by Western blotting analysis in chronic experimental hyperammonaemic rats. Materials and methods Experimental animals Adult male Wistar rats (180–200 g) obtained from the Central Animal House, Faculty of Medicine, Annamalai University were maintained in air-conditioned room (25 3 8C) with a 12 h light/ 12 h dark cycle. Feed and water were provided ad libitum to the animals. The study protocols were approved by the Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA, Approval No. 737: 2.9.2010), Annamalai University as per the guidelines of Indian Council of Medical Research, New Delhi. Chemicals Fisetin was purchased from Shanxi Jintai Biological (China). Ammonium chloride and other chemicals used in this study were of analytical grade and obtained from Merck and HiMedia, India. The primary polyclonal antibodies (anti-rat CPS-I, anti-rat OTC, anti-rat ASS, anti-rat NF-kB p65, anti-rat GS, anti-rat iNOS and anti-rat b-actin) were purchased from Santa Cruz Biotech, CA, USA. The secondary antibodies were purchased from Bangalore Genei, Bangalore, India. Experimental induction of hyperammonaemia Hyperammonaemia was induced in Wistar rats by intraperitoneal injections of AC (100 mg/kg b.w.) thrice a week for 8 weeks [40,41].
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
1039
Experimental design The animals were divided into seven groups of six animals each. Fisetin (50 mg/kg b.w.) was dissolved in 0.5% DMSO and administered orally along with AC. Group I (control) rats were orally administered with DMSO (0.5%). Group II rats were treated with fisetin alone. Groups III A, II IB, III C and III D were treated with AC at 06:00, 12:00, 18:00 and 00:00 h respectively. Groups IV A, IV B, IV C and IV D were treated with AC and fisetin at 06:00, 12:00, 18:00 and 00:00 h respectively. At the end of the experimental period (8th week), all animals were fasted overnight and sacrificed by cervical dislocation. The liver and brain tissues were excised at 09:00, 15:00, 21:00 and 03:00 h from all subgroups of III and IV, washed with chilled saline and used for further analyses. Western blot analysis Western blotting was performed to analyse the expression patterns of CPS-I, OTC, ASS and NF-kB p65 in liver and iNOS and GS in brain. The liver and brain tissue samples were homogenised in an ice-cold 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)) and centrifuged at 12,000 rpm/min for 15 min at 4 8C. The protein concentration was measured by the method of Lowry et al. [42]. Samples containing 50 mg of total proteins were loaded and separated using 10% SDS polyacrylamide gel electrophoresis. The resolved proteins were blot transferred on to a PVDF membrane (Millipore). The membranes were incubated with blocking buffer containing BSA (5%) for 2 h to reduce non-specific binding sites. The membranes were then incubated with primary antibodies (in Tris-buffered saline and Tween-20 (0.05%, TBST)), recognising CPSI and b-actin (1:1000 dilution), OTC and ASS (1:1000), NF-kB p65 and iNOS (1:500), and GS (1:200) with gentle shaking overnight at 4 8C. Subsequently, the membranes were incubated with their corresponding secondary antibodies (anti-rat IgG conjugated to horseradish peroxidase) for 2 h. The membranes were washed thrice with TBST for 30 min. The protein bands were visualised by an enhanced chemiluminescence method using an ECL kit (GenScript ECL kit, USA). Bands were scanned using a scanner and quantitated by Image J software (Bethesda, USA). Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using SPSS software package 12. Results were expressed as mean S.D. from six rats in each group and p values <0.05 were considered as significant. Results Our earlier studies showed that AC-induced changes on histopathology, oxidative stress markers, anti-oxidants, liver marker enzymes, expression of glial fibrillary acidic protein and soluble guanylyl cyclase do not depend on the time of AC administration and they do not exhibit any circadian rhythm [28,29]. As there are no temporal variations among the AC treated groups in these key indices, we have excluded the subgroups of III from sampling except the AC treated group at 12:00 h as the standard one. Urea cycle enzymes (CPS-I and OTC) expression The liver tissue displayed down-regulated expression of CPS-I and OTC in AC-induced rats compared with controls (Fig. 1A and B).
Fig. 1. Effect of fisetin on CPS-I and OTC protein expression in the liver tissue of control and AC-treated rats (A). Effect of fisetin on CPS-I and OTC protein band intensities scanned using a densitometer (B). The histogram depicts quantitation of three independent experiments (means S.D.). ***p < 0.001, **p < 0.005 and *p < 0.01 compared with control rats; #p < 0.05 compared with AC-control rats.
Administration of fisetin to AC-induced rats caused an increased expression of CPS-I and OTC at all time points. However, the maximum effect (***p < 0.001) was observed at 00:00 h in comparison with other time points. Expression of urea cycle enzyme (ASS) and nuclear transcription factor (NF-kB p65) The representative immunoblot expression of ASS and NF-kB p65 and densitometric analysis (Fig. 2A and B) of liver exhibited down-regulated expression of ASS and up-regulated expression of NF-kB p65 in AC-induced rats (compared with controls). Administration of fisetin to AC-induced rats significantly increased the expression of ASS and down-regulated the expression of NF-kB p65 compared with AC-induced rats. The maximum effect was obtained at 00:00 h (***p < 0.001) when compared with other time points. Astrocytic marker enzyme (GS) and inflammatory marker (iNOS) The brain tissue showed down-regulated expression of GS and up-regulated expressions of GC and iNOS respectively in AC-induced rats (Fig. 3A and B). Administration of fisetin to AC-induced rats significantly upregulated the expression of GS and attenuated iNOS expression and highest effect was observed at 00:00 h (***p < 0.001). Discussion Partial or complete inactivity of any of the urea cycle enzymes can predispose patients to hyperammonaemia [43]. Ammonia is known to be capable of generating free radicals, which could increase superoxide production and decrease the activity of the
1040
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
Fig. 2. Effect of fisetin on ASS and NF-kB p65 protein expression in the liver tissue of control and AC-treated rats (A). Effect of fisetin on ASS and NF-kB p65 protein band intensities scanned using a densitometer (B). The histogram depicts quantitation of three independent experiments (means S.D.). ***p < 0.001, **p < 0.005 and *p < 0.01 compared with control rats; #p < 0.05 compared with AC-control rats.
Fig. 3. Effect of fisetin on GS and iNOS protein expression in the brain tissue of control and AC-treated rats (A). Effect of fisetin on GS and iNOS protein band intensities scanned using a densitometer (B). The histogram depicts quantitation of three independent experiments (means S.D.). ***p < 0.001, **p < 0.005 and *p < 0.01 compared with control rats; #p < 0.05 compared with AC-control rats.
anti-oxidant enzymes, GPx, SOD and catalase [44]. Ammonia induced activation of NMDA (N-methyl-D-aspartate) receptors by glutamate, could lead to an increased level of intracellular Ca2+. The increased Ca2+ triggers the mitochondrial production of ROS, and also induces Ca2+-dependent inhibitor kappa B (IkB) phosphorylation and degradation. Subsequent nuclear translocation of NF-kB and expression of iNOS gene causing increased levels of NO and peroxynitrite; this further increases the nitration and inactivation of proteins. NO could also activate soluble guanylate cyclase, resulting in an increased production of cGMP, leading to cerebral vasodilation [45]. Elevated oxidative/ nitrosative stress altered the mitochondrial permeability transition and dissipated inner membrane potentials, osmotic swelling of the matrix, defective oxidative phosphorylation and cessation of ATP synthesis [46]. The chemical structure, position and degree of hydroxylation are important factors for the biological and pharmacological properties of fisetin [47]. The hydrophobic nature of fisetin, its low molecular weight and size helps it to readily pass through cell membranes and accumulate intracellularly, which could protect hepatocytes/nerve cells from oxidative stress [48]. Numerous reports described that a significant increase in the cerebral concentration of fisetin was involved in preventing or facilitating recovery from neurodegeneration and increasing intracellular glutathione levels [49]. The 3,7-dihydroxyl groups of fisetin can block Ca2+ influx into nerve cells, which is one of the last steps in the cell death cascade of NMDA-receptor mediated oxidative stress [50]. An increase in cerebral concentration, production of intracellular anti-oxidants, and the blockade of Ca2+ ion channels are also considered the most favourable effects of fisetin and could be involved in alleviating hyperammonaemia-mediated toxicity and complications. Hyperammonaemia with alterations in plasma glutamine, alanine, orotic acid and citrulline are the hallmarks of the UCD [51]. The results of the study showed a significantly decreased expression of CPS-I, OTC and ASS in hyperammonaemic rats (Figs. 1A, B and 2A, B) corroborating the previous results [51]. After treatment with fisetin, hyperammonaemic rats showed increased expression of CPS-I, OTC and ASS. The restoration of urea cycle enzyme expression by fisetin could lessen the subsequent hyperammonaemic complications. Glutamine is capable of forming free radicals in astrocytes similar to ammonia. Glutamine has been implicated in brain and astrocyte swelling by creating an osmotic load. Reduced glutamine synthesis might cause an increase in extracellular glutamate in hyperammonaemic rats [52]. The increase in cerebral ammonia levels inhibits the high-affinity uptake of glutamate by inhibiting the glutamate transporter protein and mRNA synthesis [53]. Ammonia-induced nitration of the tyrosine residues of GS (by NO) could lead to a loss of enzyme activity. The increase in GS expression in astroglial cells [54] could activate an endogenous mechanism that could protect neurons from the deleterious effects of excess glutamate and ammonia in the extracellular space caused by hyperammonaemia and/or HE [54]. Our present results showed decreased GS expression in brain of AC-induced hyperammonaemic rats. In contrast, fisetin treatment in hyperammonaemic rats led to a significantly increased expression of GS (Fig. 3A and B). This could be due to the pharmacological anti-hyperammonaemic and free radical quenching potency of fistin and thereby it could limit glutamate-induced neurotoxic injury. Hepatic encephalopathy is a major consequence of hyperammonaemia and is influenced by macrophage-derived cytokines such as interleukins (IL-1 and IL-6) and tumour necrosis factor-a (TNF-a) [55]. Fisetin inhibited TNF-a production by suppressing NF-kB p65 activation and phosphorylation of mitogen-activated protein kinases [22]. Fisetin is also reported to decrease the nuclear
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
levels of NF-kB p65 and its DNA-binding ability and to prevent apoptosis [24]. This multiplicity of effects of fisetin might be due to its interaction with important signalling cascades by selective actions on proteins and lipid kinases, which regulate NF-kB p65 [56]. The earlier data on anti-inflammatory effects of fisetin [22,56] support our present findings of inhibition and/or suppression of NF-kB p65 expression by fisetin (Fig. 2A and B). Hyperammonaemia could be an important factor for abnormal expression of iNOS and elevated production of NO in astroglial cells. Astrocytic induction of iNOS may contribute to neuronal damage in hyperammonaemia [9,57]. Increased NO levels reduce glutamine synthesis, and accompanied by reduced GS activity in hyperammonaemia [9,57]. Our results showed a significantly increased iNOS expression in AC-induced rats suggesting a hyperammonaemia-mediated neuroinflammatory response. After treatment with fisetin, hyperammonaemic rats showed a significantly decreased expression of iNOS (Fig. 3A and B), which could be due to anti-oxidant/anti-inflammatory/neuroprotective effects of fisetin. Biological clock regulates the metabolism in liver and other peripheral tissues, by mediating the expression and/or activity of metabolic enzymes, nuclear receptors and transport systems [4,5,7,14] and impairment of circadian oscillations of biochemical variables and activity was observed under hyperammonaemia [28,38]. Hyperammonaemia and neuroinflammatory responses reduced the feedback modulation of hypothalamic–pituitary– adrenal cortex axis [58] and this could cause modulations of circadian rhythmicities [28,38]. The administration of fisetin at 00:00 h to AC-induced hyperammonaemic rats led to a significant increase in the expression of urea cycle enzymes and decrease in the inflammatory responses than at other time points. This might be due to a temporal variation of effects of fisetin. The anti-oxidant effect of fisetin could prevent the hepatocellular/brain cell damage and thus could maintain the cellular integrity as shown in our previous studies [28,29]. Conclusion The results indicate that fisetin might be a representative agent that could effectively ameliorate hyperammonaemia and its complications and further the chronotherapeutic data showed that there is time dependent variation in the efficacy of fisetin. Future studies are in line in our lab on (i) chronopharmacological properties of fisetin and (ii) modulations in the circadian rhythms of inflammatory markers, redox enzymes and renal clearance during hyperammonaemia would throw light on the mechanism of circadian variability in the efficacy of fisetin. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements Visiting Professorship to PS at Department of Molecular Medicine, University of Malaya, Kuala Lumpur, Malaysia is gratefully acknowledged. Financial support in the form of Research Projects from UGC, New Delhi to PS and to OHH (RP017-13HTM and HIR-MOHE H-20001-00-E000009) is thankfully acknowledged. References [1] Wilkinson DJ, Smeeton NJ, Watt PW. Ammonia metabolism, the brain and fatigue; revisiting the link. Prog Neurobiol 2010;91:200–19. [2] Felipo V, Butterworth RF. Neurobiology of ammonia. Prog Neurobiol 2002;67:259–79.
1041
[3] Bosoi CR, Rose CF. Identifying the direct effects of ammonia on the brain. Metab Brain Dis 2009;24:95–102. [4] Kato H, Mizutani-Funahashi M, Shiosaka S, Nakagawa A. Circadian rhythms of urea formation and argininosuccinate synthetase activity in rat liver. J Nutr 1978;108:1071–7. [5] Moreno LD, Garcıa-Ayala B, Dıaz-Munoz M. Daytime restricted feeding modifies 24 h rhythmicity and subcellular distribution of liver glucocorticoid receptor and the urea cycle in rat liver. Br J Nutr 2012;108:2002–13. [6] Suarez I, Bodega G, Fernandez B. Glutamine synthetase in brain: effect of ammonia. Neurochem Int 2002;41:123–42. [7] Yao Z, DuBois DC, Almon RR, Jusko WJ. Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle. Pharm Res 2006;23:670–9. [8] Rohan SS, Parul KP, Farhatjahan S. Chronotherapeutic approach: circadian rhythm in human and its role in occurrence and severity of diseases. Int J PharmTech Res 2012;4:765–77. [9] Rao VLR. Nitric oxide in hepatic encephalopathy and hyperammonemia. Neurochem Int 2002;41:161–70. [10] Ayers NA, Kapas L, Krueger JM. Circadian variation of nitric oxide synthase activity and cytosolic protein levels in rat brain. Brain Res 1996;707:127–30. [11] Tunctan B, Weigl Y, Dotan A, Peleg L, Zengil H, Ashkenazi I, et al. Circadian variation of nitric oxide synthase activity in mouse tissue. Chronobiol Int 2002;19:393–404. [12] Li X, Stark GR. NF-kB dependent signaling pathways. Exp Hematol 2002;30:285–96. [13] Cecon E, Fernandes PA, Pinato L, Ferreira ZS, Markus RP. Daily variation of constitutively activated nuclear factor kappa B (NFkB) in rat pineal gland. Chronobiol Int 2010;27:52–67. [14] Narasimamurthy R, Hatori M, Nayak SK, Liu F, Panda S, Verma I. Circadian clock protein cryptochrome regulates the expression of proinflammatory cytokines. Proc Natl Acad Sci USA 2012;109:12662–67. [15] Mira L, Fernandez MT, Santos M, Rocha R, Florencio MH, Jennings KR. Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity. Free Radic Res 2002;36:1199–208. [16] Cai Q, Rahn RO, Zhang R. Dietary flavonoids, quercetin, luteolin and genistein, reduce oxidative DNA damage and lipid peroxidation and quench free radicals. Cancer Lett 1997;119:99–107. [17] Cheng IF, Breen K. On the ability of four flavonoids, baicilein, luteolin, naringenin, and quercetin, to suppress the Fenton reaction of the iron–ATP complex. Biometals 2000;13:77–83. [18] Sadik CD, Sies H, Schewe T. Inhibition of 15-lipoxygenases by flavonoids: structure–activity relations and mode of action. Biochem Pharmacol 2003;65:773–81. [19] Lim JH, Park HS, Choi JK, Lee IS, Choi HJ. Isoorientin induces Nrf2 pathwaydriven antioxidant response through phosphatidylinositol 3-kinase signaling. Arch Pharm Res 2007;30:1590–8. [20] Hanneken A, Lin FF, Johnson J, Maher P. Flavonoids protect human retinal pigment epithelial cells from oxidative-stress induced death. Invest Ophthalmol Vis Sci 2006;47:3164–77. [21] Cheong H, Ryu SY, Oak MH, Cheon SH, Yoo GS, Kim KM. Studies of structure activity relationship of flavonoids for the anti-allergic actions. Arch Pharm Res 1998;21:478–80. [22] Gelderblom M, Leypoldt F, Lewerenz J, Birkenmayer G, Orozco D, Ludewig P, et al. The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice. J Cereb Blood Flow Metab 2012;32:835–43. [23] Liao Y, Shih Y, Chao C, Lee X, Chiang T. Involvement of the ERK signaling pathway in fisetin reduces invasion and migration in the human lung cancer cell line A549. J Agric Food Chem 2009;57:8933–41. [24] Sung B, Pandey MK, Aggarwal BB. Fisetin, an inhibitor of cyclin dependent kinase 6, down-regulates nuclear factor-kappa B regulated cell proliferation, anti-apoptotic and metastatic gene products through the suppression of TAK1 and receptor interacting protein-regulated Ikappa B alpha kinase activation. Mol Pharmacol 2007;71:1703–14. [25] Chiruta C, Schubert D, Dargusch R, Maher P. Chemical modification of the multitarget neuroprotective compound fisetin. J Med Chem 2012;55:378–89. [26] Maher P, Akaishi T, Abe K. Flavonoid fisetin promotes ERK dependent longterm potentiation and enhances memory. Proc Natl Acad Sci USA 2006;103:16568–73. [27] Fotsis T, Pepper MS, Montesano R, Aktas E, Breit S, Schweigerer L, et al. Phytoestrogens and inhibition of angiogenesis. Clin Endocrinol Metab 1998;12:649–66. [28] Jayakumar M, Subramanian P. Chronotherapeutic influence of fisetin on ammonium chloride-induced hyperammonemic rats. Biol Rhythm Res 2013;44:577–88. [29] Jayakumar M. Chronopharmacological and chronotherapeutic effects of fisetin in hyperammonemic rats.(Ph.D. thesis) Chidambaram, Tamil Nadu, India: Annamalai University; 2014. [30] Smolensky MH, Lemmer B, Reinberg AE. Chronobiology and chronotherapy of allergic rhinitis and bronchial asthma. Adv Drug Deliv Rev 2007;59:852–82. [31] Choi JS, Kim CK, Lee BJ. Administration-time differences in the pharmacokinetics of gentamicin intravenously delivered to human beings. Chronobiol Int 1999;16:821–9. [32] Yoshiyama Y, Koboyashi T, Ohdo S, Kobayashi T, Shimada H, Tomonaga F, et al. Dosing time-dependent changes of pharmacokinetics of isepamicin in man. J Infect Chemother 1996;2:106–9.
1042
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
[33] Labrecque G, Bureau JP, Reinberg AE. Biological rhythms in the inflammatory response and in the effects of non-steroidal anti-inflammatory agents. Pharmacol Ther 1995;66:285–300. [34] Munoz DM, Munoz HR, Suarez J, Chagoya SV. Day–night cycle of lipid peroxidation in rat cerebral cortex and their relationship to the glutathione cycle and superoxide dismutase activity. Neuroscience 1985;16:859–63. [35] Rubio MF, Agostino PV, Ferreyra GA, Golombek DA. Circadian heme oxygenase activity in the hamster suprachiasmatic nuclei. Neurosci Lett 2003;353:9–12. [36] Sani M, Sebai H, Gadacha W, Boughattas NA, Reinberg A, Mossadok BA. Catalase activity and rhythmic patterns in mouse brain, kidney and liver. Comp Biochem Physiol B: Biochem Mol Biol 2006;145:331–7. [37] Tunon MJ, Gonzalez P, Lopez P, Salido GM, Madrid JA. Circadian rhythms in glutathione and glutathione-S transferase activity of rat liver. Arch Int Physiol Biochim Biophys 1992;100:83–7. [38] Llansola M, Ahabrach H, Errami M, Cabrera-Pastor A, Addaoudi K, Felipo V. Impaired release of cortecosterone from adrenals contribute to impairment of circadian rhythms of activity in hyperammonemic rats. Arch Biochem Biophys 2013;536:164–70. [39] Khan Z, Pillay V, Choonara YE, Toit LC. Drug delivery technologies for chronotherapeutic applications. Pharm Dev Technol 2009;14:602–12. [40] Essa MM, Subramanian P. Hibiscus sabdariffa affects ammonium chlorideinduced hyperammonemic rats. Evid Based Complement Alternat Med 2007;4:321–5. [41] Subash S, Subramanian P. Morin, a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study. Mol Cell Biochem 2009;327:153–61. [42] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:265–75. [43] Enns GM, Chb MB. Neurologic damage and neurocognitive dysfunction in urea cycle disorders. Semin Pediatr Neurol 2008;15:132–9. [44] Kosenko E, Venediktova N, Kaminsky Y, Montoliu C, Felipo V. Sources of oxygen radicals in brain in acute ammonia intoxication in vivo. Brain Res 2003;981:193–200. [45] Haussinger D, Schliess F. Astrocyte swelling and protein tyrosine nitration in hepatic encephalopathy. Neurochem Int 2005;47:64–70.
[46] Bai G, Rao KV, Murthy RK, Panickar KS, Jayakumar AR, Norenberg MD. Ammonia induces the mitochondrial permeability transition in primary cultures of rat astrocytes. J Neurosci Res 2001;66:981–91. [47] Hu JP, Calomme M, Lasure A, Bruyne DT, Pieters L, Vlietinck A, et al. Structure– activity relationship of flavonoids with superoxide scavenging activity. Biol Trace Elem Res 1995;47:327–31. [48] Maher P. Modulation of multiple pathways involved in the maintenance of neuronal function during aging by fisetin. Genes Nutr 2009;4:297–307. [49] Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Radic Biol Med 2001;30:433–46. [50] Rivera F, Urbanavicius J, Gervaz E, Morquio A, Dajas F. Some aspects of the in vivo neuroprotective capacity of flavonoids: bioavailability and structure– activity relationship. Neurotox Res 2004;6:543–53. [51] Beaudet AL, O’Brien WE, Bock HG, Freytag SO, Su TS. The human argininosuccinate synthetase locus and citrullinemia. Adv Hum Genet 1986;15:161–96. [52] Butterworth RF, Le O, Lavoie J, Szerb JC. Effect of portacaval anastomosis on electrically-stimulated release of glutamate from rat hippocampus slices. J Neurochem 1991;56:1481–4. [53] Chan H, Hazell AS, Desjardins P, Butterworth RF. Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes. Neurochem Int 2000;37:243–8. [54] Chan H, Butterworth RF. Evidence for an astrocytic glutamate transporter deficit in hepatic encephalopathy. Neurochem Res 1999;24:1397–401. [55] Braissant O, McLin VA, Cudalbu C. Ammonia toxicity to the brain. J Inherit Metab Dis 2013;36:595–612. [56] Leotoing L, Wauquier F, Guicheux J, Miot-Noirault E, Wittrant Y, Coxam V. The polyphenol fisetin protects bone by repressing NF-kB and MKP-1-dependent signaling pathways in osteoclasts. PLOS ONE 2013;8:e68388. [57] Rao KV, Jayakumar AR, Tong X, Alvarez VM, Norenberg MD. Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes. J Neuroinflam 2010;7:66. [58] Rodrigo R, Cauli O, Gomez-Pinedo U, Agusti A, Hernandez-Rabaza V, GarciaVerdugo JM, et al. Hyperammonemia induces neuroinflammation that contributes to cognitive impairment in rats with hepatic encephalopathy. Gastroenterology 2010;139:675–84.
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats Perumal Subramanian a,*, Murugesan Jayakumar a, Jaime Jacqueline Jayapalan b,c, Onn Haji Hashim b,c a b c
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, India University of Malaya Centre for Proteomics Research (UMCPR), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
A R T I C L E I N F O
Article history: Received 4 December 2013 Received in revised form 5 June 2014 Accepted 25 June 2014 Available online 8 July 2014 Keywords: Chronotherapy Fisetin Urea cycle Hyperammonaemia Circadian
A B S T R A C T
Background: Elevated blood ammonia leads to hyperammonaemia that affects vital central nervous system (CNS) functions. Fisetin, a naturally occurring flavonoid, exhibits therapeutic benefits, such as anti-cancer, anti-diabetic, anti-oxidant, anti-angiogenic, neuroprotective and neurotrophic effects. Methods: In this study, the chronotherapeutic effect of fisetin on ammonium chloride (AC)-induced hyperammonaemic rats was investigated, to ascertain the time point at which the maximum drug effect is achieved. The anti-hyperammonaemic potential of fisetin (50 mg/kg b.w. oral) was analysed when administered to AC treated (100 mg/kg b.w. i.p.) rats at 06:00, 12:00, 18:00 and 00:00 h. Amelioration of pathophysiological conditions by fisetin at different time points was measured by analysing the levels of expression of liver urea cycle enzymes (carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS)), nuclear transcription factor kappaB (NF-kB p65), brain glutamine synthetase (GS) and inducible nitric oxide synthase (iNOS) by Western blot analysis. Results: Fisetin increased the expression of CPS-I, OTC, ASS and GS and decreased iNOS and NF-kB p65 in hyperammonaemic rats. Fisetin administration at 00:00 h showed more significant effects on the expression of liver and brain markers, compared with other time points. Conclusions: Fisetin could exhibit anti-hyperammonaemic effect owing to its anti-oxidant and cytoprotective influences. The temporal variation in the effect of fisetin could be due to the (i) chronopharmacological, chronopharmacokinetic properties of fisetin and (ii) modulations in the endogenous circadian rhythms of urea cycle enzymes, brain markers, redox enzymes and renal clearance during hyperammonaemia by fisetin. However, future studies in these lines are necessitated. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction
Abbreviations: AC, ammonium chloride; ASS, argininosuccinate synthetase; CPS-I, carbamoyl phosphate synthetase-I; GPx, glutathione peroxidase; GS, glutamine synthetase; GSH, reduced glutathione; GST, glutathione-S-transferase; HE, hepatic encephalopathy; IkB, inhibitor kappaB; NF-kB p65, nuclear factor kappaB subunit p65; NMDA, N-methyl-D-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; OTC, ornithine transcarbamoylase; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF-a, tumour necrosis factor-a; UCD, urea cycle disorder. * Corresponding author. E-mail addresses: [email protected], [email protected] (P. Subramanian).
The common feature of urea cycle disorder (UCD) is a defect in ammonia elimination leading to hyperammonaemia and hepatic encephalopathy (HE). The increased level of ammonia in the blood leads to an increase in cerebral uptake across the blood–brain barrier (BBB) [1]. Excess ammonia is toxic to the central nervous system (CNS); elevated cerebral ammonia results in a severe accumulation of glutamine in astrocytes, causing oedema and encephalopathy [2], and leading to a spectrum of neuropsychiatric and neurological symptoms (impaired memory, shortened attention span, sleep–wake inversions, brain oedema, intracranial hypertension, seizures, ataxia and coma) with high mortality [3].
http://dx.doi.org/10.1016/j.pharep.2014.06.018 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
1038
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
Endogenous 24 h rhythmicities of urea cycle enzymes such as carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS) in rat liver and serum have been reported; the enzyme levels are low in light period but exhibit a significant increase in darkness [4,5]. The increase in cerebral ammonia concentration may stimulate glutamine synthetase (GS) activity to metabolise excess ammonia and glutamate into glutamine and thus thwarting neurotoxicity [6]. GS has a robust circadian rhythm in liver and kidney; increased mRNA expression occurs in the dark period when the animal begins activity and feeding coinciding with amino acid metabolism [7]. In humans, the rhythmic pattern is relatively reversed [8]. Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) possesses microbicidal, antiviral, antiparasitic and antitumoural effects. However, abnormal iNOS induction seems to be involved in the pathophysiology of a variety of human diseases. In addition to ammonia and glutamate levels, the NOS and NO system might also be involved in the brain responses to HE [9]. The acrophases of NO and NOS in plasma, brain, kidney, testis and lung occur at midnight, corresponding to the behavioural activity and increased reactive oxygen species (ROS) production [10,11]. NF-kB/Rel proteins, a family of ubiquitous transcription factors, participate in immunological responsiveness, inflammatory processes and cell growth regulation. ROS seem to play a dual role in the NF-kB activation cascade. NF-kB helps to regulate various inflammatory genes in different target cells [12]. The nuclear content of subunit of NF-kB p65 was found to exhibit circadian rhythm in animals [13]. The circadian oscillator components regulate the immune response, and the absence of the core clock component, cryptochrome, caused elevation of NF-kB p65, suggesting a link between circadian rhythm disruption and increased susceptibility to chronic inflammatory diseases [14]. The reappearance of symptoms and serious adverse effects after the discontinuation of treatment are serious drawbacks of many anti-hyperammonaemic agents/therapies. These drugs or therapies are inadequately effective. Therefore, the screening and development of drugs for anti-hyperammonaemic activity are still in progress. This can be achieved by focusing research on the active principles of flavonoids by increasing their efficacy by time dependent administration of drugs at which the effect is maximal. The structural features of flavonoids are the presence of a B-ring catechol group and the presence of a C2–C3 double bond in conjugation with an oxo-group at C4; the first serves to donate a hydrogen/electron to stabilise a radical species, and the second serves to bind transition metal ions such as iron and copper [15]. The anti-oxidant activity of flavonoids and their glycosides has been associated with their capacity to (i) scavenge reactive oxygen and nitrogen species [16], (ii) chelate transition metals that may induce oxidative damage through the Fenton reaction [17], (iii) inhibit pro-oxidant enzymes [18] and (iv) induce anti-oxidant enzymes [19]. Fisetin (3,30 ,40 ,7-tetrahydroxyflavone) is a dietary flavonoid widely distributed in strawberries, apples, persimmons, grapes, onions and cucumbers and displays anti-oxidant [20], antiallergic [21], anti-inflammatory [22], anti-cancer [23,24], neuroprotective [25], neurotrophic [26] and anti-angiogenic [27] activities. Our earlier studies showed preservation of hepatocellular architecture, normalisation of oxidative stress markers, antioxidant enzymes, liver marker enzymes (alanine transaminase, aspartate transaminase and alkaline phosphatase) and astrocytic marker enzyme (soluble guanylyl cyclase) and modulation of their temporal variation towards normalcy in fisetin treated AC-induced rats, although no temporal variations are noticed in AC-alone treated groups [28,29].
Chronotherapy is one of the best therapeutic approaches used to deliver medications in response to endogenous biological rhythms according to the pathophysiology of disease states to optimise treatment outcomes or limit adverse effects. Earlier reports described administration-time differences in the pharmacokinetics of many drugs [8,30]. The maximum clearance of gentamicin, amikacin, and isepamicin was higher when injected during the activity period [31,32]. A higher rate of urinary excretion of ciprofloxacin was found at the beginning of the day [33]. Most laboratory and clinical observations showed that the clearance of drugs was lower during the rest span and higher during the activity span. Circadian variations have been reported in different populations of T total, T helper, and T killer lymphocytes in inflammatory reactions [33]. Further, circadian oscillations in the immune system and the inflammatory response play a major role in the temporal changes in the body’s response to infection and other pathological conditions. Circadian rhythms of activity with gene expression changes have been reported in redox pathway enzymes, including NOS, haem oxygenase (HO), superoxide dismutase (SOD), catalase, reduced glutathione (GSH), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) [34–37]. Modified circadian rhythms of activity have been reported in hyperammonaemic rats [38]. Endogenous 24 h oscillations in gastrointestinal, liver, kidney and other bodily processes are of great importance for therapeutics and for selecting the phase for drugs administration, due to rhythm influences on the pharmacokinetics, effect-duration, efficacy, adverse effects and beneficial outcomes of medications [39]. Investigations on the chronotherapeutic efficacy of anti-hyperammonaemic drugs are completely lacking. However, the time dependent administration of drugs would be helpful in increasing the efficacy and minimising the side effects of drugs. Hence, the present study was designed to assess the chronotherapeutic modulations of liver urea cycle enzymes, NFkB p65, brain GS and iNOS by Western blotting analysis in chronic experimental hyperammonaemic rats. Materials and methods Experimental animals Adult male Wistar rats (180–200 g) obtained from the Central Animal House, Faculty of Medicine, Annamalai University were maintained in air-conditioned room (25 3 8C) with a 12 h light/ 12 h dark cycle. Feed and water were provided ad libitum to the animals. The study protocols were approved by the Institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA, Approval No. 737: 2.9.2010), Annamalai University as per the guidelines of Indian Council of Medical Research, New Delhi. Chemicals Fisetin was purchased from Shanxi Jintai Biological (China). Ammonium chloride and other chemicals used in this study were of analytical grade and obtained from Merck and HiMedia, India. The primary polyclonal antibodies (anti-rat CPS-I, anti-rat OTC, anti-rat ASS, anti-rat NF-kB p65, anti-rat GS, anti-rat iNOS and anti-rat b-actin) were purchased from Santa Cruz Biotech, CA, USA. The secondary antibodies were purchased from Bangalore Genei, Bangalore, India. Experimental induction of hyperammonaemia Hyperammonaemia was induced in Wistar rats by intraperitoneal injections of AC (100 mg/kg b.w.) thrice a week for 8 weeks [40,41].
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
1039
Experimental design The animals were divided into seven groups of six animals each. Fisetin (50 mg/kg b.w.) was dissolved in 0.5% DMSO and administered orally along with AC. Group I (control) rats were orally administered with DMSO (0.5%). Group II rats were treated with fisetin alone. Groups III A, II IB, III C and III D were treated with AC at 06:00, 12:00, 18:00 and 00:00 h respectively. Groups IV A, IV B, IV C and IV D were treated with AC and fisetin at 06:00, 12:00, 18:00 and 00:00 h respectively. At the end of the experimental period (8th week), all animals were fasted overnight and sacrificed by cervical dislocation. The liver and brain tissues were excised at 09:00, 15:00, 21:00 and 03:00 h from all subgroups of III and IV, washed with chilled saline and used for further analyses. Western blot analysis Western blotting was performed to analyse the expression patterns of CPS-I, OTC, ASS and NF-kB p65 in liver and iNOS and GS in brain. The liver and brain tissue samples were homogenised in an ice-cold 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)) and centrifuged at 12,000 rpm/min for 15 min at 4 8C. The protein concentration was measured by the method of Lowry et al. [42]. Samples containing 50 mg of total proteins were loaded and separated using 10% SDS polyacrylamide gel electrophoresis. The resolved proteins were blot transferred on to a PVDF membrane (Millipore). The membranes were incubated with blocking buffer containing BSA (5%) for 2 h to reduce non-specific binding sites. The membranes were then incubated with primary antibodies (in Tris-buffered saline and Tween-20 (0.05%, TBST)), recognising CPSI and b-actin (1:1000 dilution), OTC and ASS (1:1000), NF-kB p65 and iNOS (1:500), and GS (1:200) with gentle shaking overnight at 4 8C. Subsequently, the membranes were incubated with their corresponding secondary antibodies (anti-rat IgG conjugated to horseradish peroxidase) for 2 h. The membranes were washed thrice with TBST for 30 min. The protein bands were visualised by an enhanced chemiluminescence method using an ECL kit (GenScript ECL kit, USA). Bands were scanned using a scanner and quantitated by Image J software (Bethesda, USA). Statistical analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using SPSS software package 12. Results were expressed as mean S.D. from six rats in each group and p values <0.05 were considered as significant. Results Our earlier studies showed that AC-induced changes on histopathology, oxidative stress markers, anti-oxidants, liver marker enzymes, expression of glial fibrillary acidic protein and soluble guanylyl cyclase do not depend on the time of AC administration and they do not exhibit any circadian rhythm [28,29]. As there are no temporal variations among the AC treated groups in these key indices, we have excluded the subgroups of III from sampling except the AC treated group at 12:00 h as the standard one. Urea cycle enzymes (CPS-I and OTC) expression The liver tissue displayed down-regulated expression of CPS-I and OTC in AC-induced rats compared with controls (Fig. 1A and B).
Fig. 1. Effect of fisetin on CPS-I and OTC protein expression in the liver tissue of control and AC-treated rats (A). Effect of fisetin on CPS-I and OTC protein band intensities scanned using a densitometer (B). The histogram depicts quantitation of three independent experiments (means S.D.). ***p < 0.001, **p < 0.005 and *p < 0.01 compared with control rats; #p < 0.05 compared with AC-control rats.
Administration of fisetin to AC-induced rats caused an increased expression of CPS-I and OTC at all time points. However, the maximum effect (***p < 0.001) was observed at 00:00 h in comparison with other time points. Expression of urea cycle enzyme (ASS) and nuclear transcription factor (NF-kB p65) The representative immunoblot expression of ASS and NF-kB p65 and densitometric analysis (Fig. 2A and B) of liver exhibited down-regulated expression of ASS and up-regulated expression of NF-kB p65 in AC-induced rats (compared with controls). Administration of fisetin to AC-induced rats significantly increased the expression of ASS and down-regulated the expression of NF-kB p65 compared with AC-induced rats. The maximum effect was obtained at 00:00 h (***p < 0.001) when compared with other time points. Astrocytic marker enzyme (GS) and inflammatory marker (iNOS) The brain tissue showed down-regulated expression of GS and up-regulated expressions of GC and iNOS respectively in AC-induced rats (Fig. 3A and B). Administration of fisetin to AC-induced rats significantly upregulated the expression of GS and attenuated iNOS expression and highest effect was observed at 00:00 h (***p < 0.001). Discussion Partial or complete inactivity of any of the urea cycle enzymes can predispose patients to hyperammonaemia [43]. Ammonia is known to be capable of generating free radicals, which could increase superoxide production and decrease the activity of the
1040
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
Fig. 2. Effect of fisetin on ASS and NF-kB p65 protein expression in the liver tissue of control and AC-treated rats (A). Effect of fisetin on ASS and NF-kB p65 protein band intensities scanned using a densitometer (B). The histogram depicts quantitation of three independent experiments (means S.D.). ***p < 0.001, **p < 0.005 and *p < 0.01 compared with control rats; #p < 0.05 compared with AC-control rats.
Fig. 3. Effect of fisetin on GS and iNOS protein expression in the brain tissue of control and AC-treated rats (A). Effect of fisetin on GS and iNOS protein band intensities scanned using a densitometer (B). The histogram depicts quantitation of three independent experiments (means S.D.). ***p < 0.001, **p < 0.005 and *p < 0.01 compared with control rats; #p < 0.05 compared with AC-control rats.
anti-oxidant enzymes, GPx, SOD and catalase [44]. Ammonia induced activation of NMDA (N-methyl-D-aspartate) receptors by glutamate, could lead to an increased level of intracellular Ca2+. The increased Ca2+ triggers the mitochondrial production of ROS, and also induces Ca2+-dependent inhibitor kappa B (IkB) phosphorylation and degradation. Subsequent nuclear translocation of NF-kB and expression of iNOS gene causing increased levels of NO and peroxynitrite; this further increases the nitration and inactivation of proteins. NO could also activate soluble guanylate cyclase, resulting in an increased production of cGMP, leading to cerebral vasodilation [45]. Elevated oxidative/ nitrosative stress altered the mitochondrial permeability transition and dissipated inner membrane potentials, osmotic swelling of the matrix, defective oxidative phosphorylation and cessation of ATP synthesis [46]. The chemical structure, position and degree of hydroxylation are important factors for the biological and pharmacological properties of fisetin [47]. The hydrophobic nature of fisetin, its low molecular weight and size helps it to readily pass through cell membranes and accumulate intracellularly, which could protect hepatocytes/nerve cells from oxidative stress [48]. Numerous reports described that a significant increase in the cerebral concentration of fisetin was involved in preventing or facilitating recovery from neurodegeneration and increasing intracellular glutathione levels [49]. The 3,7-dihydroxyl groups of fisetin can block Ca2+ influx into nerve cells, which is one of the last steps in the cell death cascade of NMDA-receptor mediated oxidative stress [50]. An increase in cerebral concentration, production of intracellular anti-oxidants, and the blockade of Ca2+ ion channels are also considered the most favourable effects of fisetin and could be involved in alleviating hyperammonaemia-mediated toxicity and complications. Hyperammonaemia with alterations in plasma glutamine, alanine, orotic acid and citrulline are the hallmarks of the UCD [51]. The results of the study showed a significantly decreased expression of CPS-I, OTC and ASS in hyperammonaemic rats (Figs. 1A, B and 2A, B) corroborating the previous results [51]. After treatment with fisetin, hyperammonaemic rats showed increased expression of CPS-I, OTC and ASS. The restoration of urea cycle enzyme expression by fisetin could lessen the subsequent hyperammonaemic complications. Glutamine is capable of forming free radicals in astrocytes similar to ammonia. Glutamine has been implicated in brain and astrocyte swelling by creating an osmotic load. Reduced glutamine synthesis might cause an increase in extracellular glutamate in hyperammonaemic rats [52]. The increase in cerebral ammonia levels inhibits the high-affinity uptake of glutamate by inhibiting the glutamate transporter protein and mRNA synthesis [53]. Ammonia-induced nitration of the tyrosine residues of GS (by NO) could lead to a loss of enzyme activity. The increase in GS expression in astroglial cells [54] could activate an endogenous mechanism that could protect neurons from the deleterious effects of excess glutamate and ammonia in the extracellular space caused by hyperammonaemia and/or HE [54]. Our present results showed decreased GS expression in brain of AC-induced hyperammonaemic rats. In contrast, fisetin treatment in hyperammonaemic rats led to a significantly increased expression of GS (Fig. 3A and B). This could be due to the pharmacological anti-hyperammonaemic and free radical quenching potency of fistin and thereby it could limit glutamate-induced neurotoxic injury. Hepatic encephalopathy is a major consequence of hyperammonaemia and is influenced by macrophage-derived cytokines such as interleukins (IL-1 and IL-6) and tumour necrosis factor-a (TNF-a) [55]. Fisetin inhibited TNF-a production by suppressing NF-kB p65 activation and phosphorylation of mitogen-activated protein kinases [22]. Fisetin is also reported to decrease the nuclear
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
levels of NF-kB p65 and its DNA-binding ability and to prevent apoptosis [24]. This multiplicity of effects of fisetin might be due to its interaction with important signalling cascades by selective actions on proteins and lipid kinases, which regulate NF-kB p65 [56]. The earlier data on anti-inflammatory effects of fisetin [22,56] support our present findings of inhibition and/or suppression of NF-kB p65 expression by fisetin (Fig. 2A and B). Hyperammonaemia could be an important factor for abnormal expression of iNOS and elevated production of NO in astroglial cells. Astrocytic induction of iNOS may contribute to neuronal damage in hyperammonaemia [9,57]. Increased NO levels reduce glutamine synthesis, and accompanied by reduced GS activity in hyperammonaemia [9,57]. Our results showed a significantly increased iNOS expression in AC-induced rats suggesting a hyperammonaemia-mediated neuroinflammatory response. After treatment with fisetin, hyperammonaemic rats showed a significantly decreased expression of iNOS (Fig. 3A and B), which could be due to anti-oxidant/anti-inflammatory/neuroprotective effects of fisetin. Biological clock regulates the metabolism in liver and other peripheral tissues, by mediating the expression and/or activity of metabolic enzymes, nuclear receptors and transport systems [4,5,7,14] and impairment of circadian oscillations of biochemical variables and activity was observed under hyperammonaemia [28,38]. Hyperammonaemia and neuroinflammatory responses reduced the feedback modulation of hypothalamic–pituitary– adrenal cortex axis [58] and this could cause modulations of circadian rhythmicities [28,38]. The administration of fisetin at 00:00 h to AC-induced hyperammonaemic rats led to a significant increase in the expression of urea cycle enzymes and decrease in the inflammatory responses than at other time points. This might be due to a temporal variation of effects of fisetin. The anti-oxidant effect of fisetin could prevent the hepatocellular/brain cell damage and thus could maintain the cellular integrity as shown in our previous studies [28,29]. Conclusion The results indicate that fisetin might be a representative agent that could effectively ameliorate hyperammonaemia and its complications and further the chronotherapeutic data showed that there is time dependent variation in the efficacy of fisetin. Future studies are in line in our lab on (i) chronopharmacological properties of fisetin and (ii) modulations in the circadian rhythms of inflammatory markers, redox enzymes and renal clearance during hyperammonaemia would throw light on the mechanism of circadian variability in the efficacy of fisetin. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements Visiting Professorship to PS at Department of Molecular Medicine, University of Malaya, Kuala Lumpur, Malaysia is gratefully acknowledged. Financial support in the form of Research Projects from UGC, New Delhi to PS and to OHH (RP017-13HTM and HIR-MOHE H-20001-00-E000009) is thankfully acknowledged. References [1] Wilkinson DJ, Smeeton NJ, Watt PW. Ammonia metabolism, the brain and fatigue; revisiting the link. Prog Neurobiol 2010;91:200–19. [2] Felipo V, Butterworth RF. Neurobiology of ammonia. Prog Neurobiol 2002;67:259–79.
1041
[3] Bosoi CR, Rose CF. Identifying the direct effects of ammonia on the brain. Metab Brain Dis 2009;24:95–102. [4] Kato H, Mizutani-Funahashi M, Shiosaka S, Nakagawa A. Circadian rhythms of urea formation and argininosuccinate synthetase activity in rat liver. J Nutr 1978;108:1071–7. [5] Moreno LD, Garcıa-Ayala B, Dıaz-Munoz M. Daytime restricted feeding modifies 24 h rhythmicity and subcellular distribution of liver glucocorticoid receptor and the urea cycle in rat liver. Br J Nutr 2012;108:2002–13. [6] Suarez I, Bodega G, Fernandez B. Glutamine synthetase in brain: effect of ammonia. Neurochem Int 2002;41:123–42. [7] Yao Z, DuBois DC, Almon RR, Jusko WJ. Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle. Pharm Res 2006;23:670–9. [8] Rohan SS, Parul KP, Farhatjahan S. Chronotherapeutic approach: circadian rhythm in human and its role in occurrence and severity of diseases. Int J PharmTech Res 2012;4:765–77. [9] Rao VLR. Nitric oxide in hepatic encephalopathy and hyperammonemia. Neurochem Int 2002;41:161–70. [10] Ayers NA, Kapas L, Krueger JM. Circadian variation of nitric oxide synthase activity and cytosolic protein levels in rat brain. Brain Res 1996;707:127–30. [11] Tunctan B, Weigl Y, Dotan A, Peleg L, Zengil H, Ashkenazi I, et al. Circadian variation of nitric oxide synthase activity in mouse tissue. Chronobiol Int 2002;19:393–404. [12] Li X, Stark GR. NF-kB dependent signaling pathways. Exp Hematol 2002;30:285–96. [13] Cecon E, Fernandes PA, Pinato L, Ferreira ZS, Markus RP. Daily variation of constitutively activated nuclear factor kappa B (NFkB) in rat pineal gland. Chronobiol Int 2010;27:52–67. [14] Narasimamurthy R, Hatori M, Nayak SK, Liu F, Panda S, Verma I. Circadian clock protein cryptochrome regulates the expression of proinflammatory cytokines. Proc Natl Acad Sci USA 2012;109:12662–67. [15] Mira L, Fernandez MT, Santos M, Rocha R, Florencio MH, Jennings KR. Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity. Free Radic Res 2002;36:1199–208. [16] Cai Q, Rahn RO, Zhang R. Dietary flavonoids, quercetin, luteolin and genistein, reduce oxidative DNA damage and lipid peroxidation and quench free radicals. Cancer Lett 1997;119:99–107. [17] Cheng IF, Breen K. On the ability of four flavonoids, baicilein, luteolin, naringenin, and quercetin, to suppress the Fenton reaction of the iron–ATP complex. Biometals 2000;13:77–83. [18] Sadik CD, Sies H, Schewe T. Inhibition of 15-lipoxygenases by flavonoids: structure–activity relations and mode of action. Biochem Pharmacol 2003;65:773–81. [19] Lim JH, Park HS, Choi JK, Lee IS, Choi HJ. Isoorientin induces Nrf2 pathwaydriven antioxidant response through phosphatidylinositol 3-kinase signaling. Arch Pharm Res 2007;30:1590–8. [20] Hanneken A, Lin FF, Johnson J, Maher P. Flavonoids protect human retinal pigment epithelial cells from oxidative-stress induced death. Invest Ophthalmol Vis Sci 2006;47:3164–77. [21] Cheong H, Ryu SY, Oak MH, Cheon SH, Yoo GS, Kim KM. Studies of structure activity relationship of flavonoids for the anti-allergic actions. Arch Pharm Res 1998;21:478–80. [22] Gelderblom M, Leypoldt F, Lewerenz J, Birkenmayer G, Orozco D, Ludewig P, et al. The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice. J Cereb Blood Flow Metab 2012;32:835–43. [23] Liao Y, Shih Y, Chao C, Lee X, Chiang T. Involvement of the ERK signaling pathway in fisetin reduces invasion and migration in the human lung cancer cell line A549. J Agric Food Chem 2009;57:8933–41. [24] Sung B, Pandey MK, Aggarwal BB. Fisetin, an inhibitor of cyclin dependent kinase 6, down-regulates nuclear factor-kappa B regulated cell proliferation, anti-apoptotic and metastatic gene products through the suppression of TAK1 and receptor interacting protein-regulated Ikappa B alpha kinase activation. Mol Pharmacol 2007;71:1703–14. [25] Chiruta C, Schubert D, Dargusch R, Maher P. Chemical modification of the multitarget neuroprotective compound fisetin. J Med Chem 2012;55:378–89. [26] Maher P, Akaishi T, Abe K. Flavonoid fisetin promotes ERK dependent longterm potentiation and enhances memory. Proc Natl Acad Sci USA 2006;103:16568–73. [27] Fotsis T, Pepper MS, Montesano R, Aktas E, Breit S, Schweigerer L, et al. Phytoestrogens and inhibition of angiogenesis. Clin Endocrinol Metab 1998;12:649–66. [28] Jayakumar M, Subramanian P. Chronotherapeutic influence of fisetin on ammonium chloride-induced hyperammonemic rats. Biol Rhythm Res 2013;44:577–88. [29] Jayakumar M. Chronopharmacological and chronotherapeutic effects of fisetin in hyperammonemic rats.(Ph.D. thesis) Chidambaram, Tamil Nadu, India: Annamalai University; 2014. [30] Smolensky MH, Lemmer B, Reinberg AE. Chronobiology and chronotherapy of allergic rhinitis and bronchial asthma. Adv Drug Deliv Rev 2007;59:852–82. [31] Choi JS, Kim CK, Lee BJ. Administration-time differences in the pharmacokinetics of gentamicin intravenously delivered to human beings. Chronobiol Int 1999;16:821–9. [32] Yoshiyama Y, Koboyashi T, Ohdo S, Kobayashi T, Shimada H, Tomonaga F, et al. Dosing time-dependent changes of pharmacokinetics of isepamicin in man. J Infect Chemother 1996;2:106–9.
1042
P. Subramanian et al. / Pharmacological Reports 66 (2014) 1037–1042
[33] Labrecque G, Bureau JP, Reinberg AE. Biological rhythms in the inflammatory response and in the effects of non-steroidal anti-inflammatory agents. Pharmacol Ther 1995;66:285–300. [34] Munoz DM, Munoz HR, Suarez J, Chagoya SV. Day–night cycle of lipid peroxidation in rat cerebral cortex and their relationship to the glutathione cycle and superoxide dismutase activity. Neuroscience 1985;16:859–63. [35] Rubio MF, Agostino PV, Ferreyra GA, Golombek DA. Circadian heme oxygenase activity in the hamster suprachiasmatic nuclei. Neurosci Lett 2003;353:9–12. [36] Sani M, Sebai H, Gadacha W, Boughattas NA, Reinberg A, Mossadok BA. Catalase activity and rhythmic patterns in mouse brain, kidney and liver. Comp Biochem Physiol B: Biochem Mol Biol 2006;145:331–7. [37] Tunon MJ, Gonzalez P, Lopez P, Salido GM, Madrid JA. Circadian rhythms in glutathione and glutathione-S transferase activity of rat liver. Arch Int Physiol Biochim Biophys 1992;100:83–7. [38] Llansola M, Ahabrach H, Errami M, Cabrera-Pastor A, Addaoudi K, Felipo V. Impaired release of cortecosterone from adrenals contribute to impairment of circadian rhythms of activity in hyperammonemic rats. Arch Biochem Biophys 2013;536:164–70. [39] Khan Z, Pillay V, Choonara YE, Toit LC. Drug delivery technologies for chronotherapeutic applications. Pharm Dev Technol 2009;14:602–12. [40] Essa MM, Subramanian P. Hibiscus sabdariffa affects ammonium chlorideinduced hyperammonemic rats. Evid Based Complement Alternat Med 2007;4:321–5. [41] Subash S, Subramanian P. Morin, a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study. Mol Cell Biochem 2009;327:153–61. [42] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951;193:265–75. [43] Enns GM, Chb MB. Neurologic damage and neurocognitive dysfunction in urea cycle disorders. Semin Pediatr Neurol 2008;15:132–9. [44] Kosenko E, Venediktova N, Kaminsky Y, Montoliu C, Felipo V. Sources of oxygen radicals in brain in acute ammonia intoxication in vivo. Brain Res 2003;981:193–200. [45] Haussinger D, Schliess F. Astrocyte swelling and protein tyrosine nitration in hepatic encephalopathy. Neurochem Int 2005;47:64–70.
[46] Bai G, Rao KV, Murthy RK, Panickar KS, Jayakumar AR, Norenberg MD. Ammonia induces the mitochondrial permeability transition in primary cultures of rat astrocytes. J Neurosci Res 2001;66:981–91. [47] Hu JP, Calomme M, Lasure A, Bruyne DT, Pieters L, Vlietinck A, et al. Structure– activity relationship of flavonoids with superoxide scavenging activity. Biol Trace Elem Res 1995;47:327–31. [48] Maher P. Modulation of multiple pathways involved in the maintenance of neuronal function during aging by fisetin. Genes Nutr 2009;4:297–307. [49] Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Radic Biol Med 2001;30:433–46. [50] Rivera F, Urbanavicius J, Gervaz E, Morquio A, Dajas F. Some aspects of the in vivo neuroprotective capacity of flavonoids: bioavailability and structure– activity relationship. Neurotox Res 2004;6:543–53. [51] Beaudet AL, O’Brien WE, Bock HG, Freytag SO, Su TS. The human argininosuccinate synthetase locus and citrullinemia. Adv Hum Genet 1986;15:161–96. [52] Butterworth RF, Le O, Lavoie J, Szerb JC. Effect of portacaval anastomosis on electrically-stimulated release of glutamate from rat hippocampus slices. J Neurochem 1991;56:1481–4. [53] Chan H, Hazell AS, Desjardins P, Butterworth RF. Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes. Neurochem Int 2000;37:243–8. [54] Chan H, Butterworth RF. Evidence for an astrocytic glutamate transporter deficit in hepatic encephalopathy. Neurochem Res 1999;24:1397–401. [55] Braissant O, McLin VA, Cudalbu C. Ammonia toxicity to the brain. J Inherit Metab Dis 2013;36:595–612. [56] Leotoing L, Wauquier F, Guicheux J, Miot-Noirault E, Wittrant Y, Coxam V. The polyphenol fisetin protects bone by repressing NF-kB and MKP-1-dependent signaling pathways in osteoclasts. PLOS ONE 2013;8:e68388. [57] Rao KV, Jayakumar AR, Tong X, Alvarez VM, Norenberg MD. Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes. J Neuroinflam 2010;7:66. [58] Rodrigo R, Cauli O, Gomez-Pinedo U, Agusti A, Hernandez-Rabaza V, GarciaVerdugo JM, et al. Hyperammonemia induces neuroinflammation that contributes to cognitive impairment in rats with hepatic encephalopathy. Gastroenterology 2010;139:675–84.