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MAPK pathway
Accepted Manuscript Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: Role of NFκB/MAPK pathway Sana Nafees, Summya Rashid, Nemat Ali, Syed Kazim Hasan, Sarwat Sultana PII: DOI: Reference:
S0009-2797(15)00090-3 http://dx.doi.org/10.1016/j.cbi.2015.02.021 CBI 7297
To appear in:
Chemico-Biological Interactions
Received Date: Revised Date: Accepted Date:
15 July 2014 28 January 2015 26 February 2015
Please cite this article as: S. Nafees, S. Rashid, N. Ali, S. Kazim Hasan, S. Sultana, Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: Role of NFκB/MAPK pathway, Chemico-Biological Interactions (2015), doi: http://dx.doi.org/10.1016/j.cbi.2015.02.021
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Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: Role of NFκB/MAPK pathway
Sana Nafees, Summya Rashid, Nemat Ali, Syed Kazim Hasan and Sarwat Sultana*
Molecular Carcinogenesis and Chemoprevention Division, Dept. of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India.
*Corresponding author, mailing address: Dr. Sarwat Sultana Molecular Carcinogenesis and Chemoprevention Division, Dept. of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India. E-mail address: [email protected] 1
Abstract Cyclophosphamide is a potent anticancer agent. However its clinical use is restricted because of its marked organ toxicity associated with increased oxidative stress and inflammation. The present study was designed to demonstrate the protective effects of rutin, a naturally occurring bioflavonoid against the hepatotoxicity induced by CP. Rats were subjected to oral pretreatment of rutin (50 and 100 mg/kg b.wt.) against hepatotoxicity induced by i.p. injection of CP (150 mg/kg b.wt.) and were sacrificed after 24h. Hepatoprotective effects of rutin were associated with upregulation of antioxidant enzyme activities and down regulation of serum toxicity markers. Rutin was able to down regulate the levels of inflammatory markers like TNF-α, IL-6 and expressions of p38-MAPK, NFκB, i-NOS and COX-2. Histopathological changes further confirmed the biochemical and immunohistochemical results showing that CP caused significant structural damage to liver which were reversed by pretreatment of rutin. Therefore, our study revealed that rutin may be a promising modulator in attenuating CP induced oxidative stress, inflammation and hepatotoxicity via targeting NFκB and MAPK pathway.
Key words: Cyclophosphamide, rutin, oxidative stress, inflammation, NFκB, MAPK.
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Abbreviations BSA- bovine serum albumin CAT- catalase CDNB- 1-chloro 2, 4-dinitrobenzene COX-2- Cycloxygenase-2 CP- Cyclophosphamide DAB-3,3-diaminobenzidine DTNB- 5, 5′-dithio bis-[2-nitrobenzoic acid] EDTA- ethylene diamine tetra acetic acid GPx -glutathione peroxidase GR- glutathione reductase GSH- reduced glutathione GSSG- oxidized glutathione IL-6 -Interleukin-6 i-NOS- inducible nitric oxide synthase LPO-lipidperoxidation MAPKs- mitogen-activated protein kinases MDA- malondialdehyde NADPH- reduced nicotinamide adenine dinucleotide phosphate NFκB - Nuclear factor kappa B NO- Nitric oxide NOS- nitric oxide synthase ROS- reactive oxygen species TNF-α -Tumor Necrosis Factor XO- xanthine oxidase
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1. Introduction Chemotherapeutic drugs are basically used for the treatment of an array of malignancies. However their therapeutic usage is inadequate because of their clinical side effects [1]. Cyclophosphamide [CP] is an anticancer drug which is extensively used in the treatment of multiple human malignancies and disorders like breast cancer, lung cancer, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis [2]. On the contrary, CP usage is recurrently restricted due to its adverse side effects and toxicities including nephrotoxicity, hepatotoxicity, cardiotoxicity, immunotoxicity and mutagenicity [3]. Acrolein which is a toxic metabolite of CP is responsible for reactive oxygen species [ROS] production and augmentation of lipid peroxidation [4]. One possible mechanism behind hepatotoxity of CP may be oxidative stress as reported previously [5]. The anti-neoplastic effects of CP are due to phosphoramide mustard, whereas acrolein is correlated to its toxic side effects [6]. Acrolein is a powerful soft electrophile that causes hepatoxicity by inactivating proteins and depleting GSH which results in oxidative stress [7]. It has been reported that acrolein toxicity is mediated by increased oxidants, thereby generating oxidative injury[8], signifying that it is not only direct oxidant but it also generates oxidants by various other pathways [9]. Inflammation plays a key role in the regulation of oxidative stress [10]. ROS leads to the activation of an array of transcription factors like nuclear factor kappa B [NFκB], which results in release of proinflammatory cytokines like Tumor Necrosis Factor [TNF-α] and Interleukin-6 [IL-6] [11] which further activate cycloxygenase-2 [COX-2] [12]. It has been reported that in addition to inflammation various antineoplastic agents have also been shown to activate transcription factor NFκB [13]. NFκB is one of the most important transcription factor which helps in the regulation of genes involved in the process of inflammation, cell proliferation and survival [14]. COX-2 is one of the downstream targets of NFκB.
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Uncontrolled expression of COX-2 has been reported in different premalignant and malignant phases [15]. Nitric oxide [NO] is a free radical which is synthesized by the enzyme nitric oxide synthase [NOS]. Among its three isoforms, inducible-nitric oxide synthase [i-NOS] is responsible for the production of elevated levels of NO. It is often considered to be the key executor of toxicity in oxidative stress [16]. The process of i-NOS expression involves different signal transduction pathways, including nuclear translocation of the transcription factor, NFκB [17]. The mitogen-activated protein kinases [MAPKs] are proline targeted serine-threonine kinases, which are transducers of environmental stimulus to the nucleus [18]. The p38MAPK is a member of the MAPK family which is activated by oxidative stress which leads to accumulation of ROS in cell [19]. In various studies it has been reported that the activation of p38-MAPK has been associated in regulating the expression of NFκB and i-NOS [20]. In order to prevent the toxic side effects of CP and its metabolites, augmentation of antioxidant defence enzymes with natural antioxidants is necessary. Therefore, there is a need for effective agents which protect the normal tissue and cells from the undesirable effects of toxicity induced by chemotherapy. Therefore amalgamation of treatment regimen with effective antioxidants might be the desirable approach to alleviate the toxicity induced by CP [21, 22]. Various studies have reported that plant extracts possessing antioxidant activity protect against hepatotoxicity induced by CP [23, 24]. The flavonoids belong to the family of phenolic compounds and possess biochemical and pharmacological properties like antibacterial, antiviral, anti-inflammatory, antiallergic, antithrombotic, antimutagenic and antineoplastic [25]. They affect the cell growth, differentiation and its function owing to their radical scavenging activity. Rutin is a glycone of quercetin having a flavonol structure. It is present in fruits and fruit peel, predominantly in citrus fruits like oranges, grapefruits, lemons and limes. It is also found in buckwheat seeds 5
[26]. It is a member of bioflavonoids with antioxidant, anti-inflammatory, antiallergenic, antiviral and anticarcinogenic properties [27]. It has been reported to scavenge superoxide radicals. It can chelate metal ions, such as ferrous cations, which are involved in the Fenton reaction, which generates ROS and prevents lipid peroxidation [28]. However to the best of our knowledge, there is no report about the effect of rutin against CP induced hepatic oxidative stress and anti-inflammatory activity. Therefore, the main objective of our study was to check the role of activation of NFκB and MAPK pathway, expression of p38-MAPK, NFκB, COX-2, i-NOS and proinflammatory cytokines like TNF-α and IL-6 in the pathogenesis of oxidative damage caused by CP in rat liver and to investigate the protective effect of rutin implicated in the hepatotoxic insult might be caused by acrolein, one of the metabolites of CP. 2. Materials and Methods 2.1. Chemicals Rutin, CP, GR, GSSG, GSH, DTNB, CDNB, BSA, NADPH etc. were obtained from Sigma– Aldrich, USA. All other chemicals and reagents were of the highest purity grade and commercially available. 2.2. Animals Eight weeks old male Wistar rats (150–200 g) were obtained from the Central Animal House Facility of Hamdard University, New Delhi and were housed in a ventilated room at 25 ± 5 0
C under a 12 h light/dark cycle. The animals were acclimatized for 1 week before the study
and had free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India) and water ad libitum. The study was approved by the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) having Registration number and date of registration: (IAEC No: 173/ CPCSEA, 28th Jan 2000).
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2.3. Treatment regimen Male Wistar rats (30) were divided into five groups containing 6 rats in each group. Group I served as a saline treated control. Group II served as positive control and administered intraperitoneal dose (150 mg/kg b.wt.) of CP freshly dissolved in normal saline. Rutin was orally administered at two doses, 50 and 100 mg/kg b.wt. to groups III and IV respectively for 20 consecutive days. Group V received only a high dose of rutin for 20 consecutive days. Group II, III, and IV were given CP (150 mg/kg b.wt.) on 20th day after rutin pretreatment. After 24 h of CP administration rats were sacrificed by cervical dislocation under mild anaesthesia using i.p. pentobarbital (50 mg/kg b.wt.) and blood was taken for various serological
parameters.
Liver
samples
were
taken
at
the
same
time
for
immunohistochemistry, various biochemical and histological parameters. There was 100% survival of animals in all the groups. 2.4. Post-mitochondrial supernatant preparation [PMS] Tissue processing and preparation of post-mitochondria supernatant (PMS) were done as described by S. Nafees et al. [29]. 2.5. Measurement of liver toxicity markers: serum aspartate aminotransferase (AST) and alanine aminotransferase [ALT] AST and ALT activities were determined by the method of Reitman and Frankel [30]. 2.6. Assay for Lactate dehydrogenase [LDH] activity Lactate dehydrogenase activity has been estimated in serum by the method described by Khan et al. [31]. 2.7. Assay for Reduced glutathione [GSH] estimation Reduced glutathione was determined by the method of Jollow et al. [32]. 2.8. Assay for Glutathione reductase [GR] activity Glutathione reductase activity was measured by the method of Carlberg and Mannervik [33].
7
2.9. Assay for Glutathione peroxidase [GPx] activity The activity of Glutathione peroxidase was calculated by the method of Mohandas et al. [34]. 2.10. Assay for catalase activity The catalase activity was assessed by the method of Claiborne [35]. 2.11. Assay for lipid peroxidation [LPO] The assay for lipid peroxidation was done following the method of Wright et al. [36]. 2.12. Assay for Xanthine oxidase [XO] activity Xanthine oxidase activity was done following the method of S. Nafees et al. [37].
2.13. Estimation of protein concentration The protein concentration in all samples was determined by the method of O.H. Lowry [38]. 2.14. Assay for Tumor Necrosis Factor Alpha [TNF-α] TNF-α levels were determined by rat TNF-α kit [eBioscience, Inc., San Diego., USA]. The method is based on enzyme-linked immuno-sorbent assay [ELISA]. Analysis was performed by Elisa Plate Reader [Benchmark plus, BioRad] according to the manufacturer’s instruction. 2.15. Assay for IL-6 IL-6 levels were determined by rat IL-6 kit [eBioscience, Inc., San Diego., USA]. The method is based on enzyme-linked immuno-sorbent assay [ELISA]. Analysis was performed by Elisa Plate Reader [Benchmark plus, BioRad] according to the manufacturer’s instruction. 2.16. Histopathological examination The livers were quickly removed after sacrifice of rats and were fixed in 10% neutral buffered formalin solution for histopathological processing. Sections were stained with hematoxyline and eosin before being observed under an Olympus microscope at 40x magnification. 2.17. Immunohistochemistry
8
To examine the protective effects of rutin on markers of inflammation in the liver COX-2, iNOS, NFκB and p38-MAPK expression in the liver were assessed by immunohistochemical staining. Liver sections on polylysine coated slides obtained were fixed in neutral buffered formalin, and embedded in paraffin and were treated for COX-2, i-NOS, NFκB and p38MAPK antibodies for immunohistochemical analysis. The procedures were processed according to the manufacturer’s protocol recommended for the COX-2, i-NOS, NFκB and p38-MAPK immunohistochemistry with slight modifications. Following deparaffinization and rehydration, sections were irradiated in 0.1 mol/L sodium citrate buffer [pH 6.0] in a microwave oven [medium low temperature] for 20 min. Then the sections were exposed to 3% H2O2 for 10 min to bleach endogenous per-oxidases, followed by rinsing 3 times in Tris buffer [pH 7.4] for 10 min. Sections were selectively incubated under humid conditions using an anti-NFκB antibody [1:400;Thermo Fisher Scientific, USA], anti-COX-2 antibody [1:200; Santacruz Biotechnology, Inc., USA], anti-phospho-p38 [dilution 1:200, Santa Cruz] and anti-i-NOS antibody (1:200; Thermo Fisher Scientific, USA) for overnight at 4 °C. Next day, slides were washed 3 times in Tris buffer for 10 min each. The specificity of the antibodies was tested by omission of the primary antibodies and a positive control of rat tonsil tissue. After washing in Tris buffer (pH 7.4), tissues were visualized with 3,3’diaminobenzidine [DAB] and counterstained with hematoxyline. Finally, the sections were dehydrated in xylene, mounted with DPX and cover slipped. Slides prepared for each case were examined by light microscopy. Positive and negative controls were conducted in parallel with COX-2, i-NOS, NFκB and p38-MAPK stained sections. Staining of sections with commercially available antibodies served as the positive control. Negative controls included staining tissue sections with omission of the primary antibody. 2.18. Statistical analysis
9
The data from individual groups were presented as the mean ± SD. Differences between groups were analyzed using analysis of variance [ANOVA] followed by Tukey-Kramer multiple comparisons test and minimum criterion for statistical significance was set at p<0.05 for all comparisons. 2.19. Quantitative evaluation of NFκB immunostaining Percentages of NFκB immunostained nuclei were calculated in each selected section for control rats, toxicant treated rats and rats given rutin as a modulator using the following formula: Number of labelled nuclei x 100/total number (labelled + unlabelled) of nuclei [39]. NFκB immunostained nuclei were considered positive regardless of staining intensity. All slides were examined by two independent observers who were unaware of the experimental protocol. The slides with discrepant evaluations were re-evaluated, and a consensus was reached. Measurements were carried out using an Olympus BX51 (Hamburg, Germany) microscope using objectives with 40x magnification. 2.20. Quantitative evaluation of COX-2, i-NOS and p38-MAPK immunostaining According to the diffuseness of the DAB staining, sections were graded as 0 (no staining), 1 (staining, 25%), 2 (staining between 25% and 50%), 3 (staining between 50% and 75%), or 4 (staining >75%). According to staining intensity, sections were graded as follows: 0 (no staining), 1 (weak but detectable staining), 2 (distinct staining) or 3 (intense staining). Immunohistochemical staining scores were obtained by adding the diffuseness and intensity scores. All slides were examined by two independent observers who were unaware of the experimental protocol. The slides with discrepant evaluations were reevaluated, and a consensus was reached. Measurements were carried out using an Olympus BX51 (Hamburg, Germany) microscope at 40× magnification.
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3. Results 3.1.
Rutin
pretreatment
modulates
aspartate
aminotransferase
[AST],
alanine
aminotransferase [ALT] and lactate dehydrogenase [LDH] activity AST, ALT and LDH were significantly elevated in CP-treated rats (Table 3). Prophylactic treatment of rutin at a higher dose prevented CP induced elevation in serum levels of these enzymes (##p < 0.01 and < 0.05,
##
p < 0.01 and
###
p < 0.01) as compared to untreated control. Significant reduction [#p
###
p < 0.001] in these marker enzymes was observed in the rutin
pretreatment groups and found to be effective in the reduction of level of these enzymes when compared to CP administered group. Only rutin treated group showed no significant difference compared with the control group.
Henceforth there was a significant recovery after rutin
supplementation [Table 1]. 3.2. Rutin pretreatment restored the hepatic reduced glutathione [GSH] level GSH level was depleted significantly [***p < 0.001] in CP treated group when compared with the control group. GSH level in the rutin pretreated groups increased significantly [##p < 0.01 and
###
p < 0.001] when compared to CP treated group in a dose dependent manner.
However rutin alone did not show any significant changes in GSH level compared to control group [Table 2]. 3.3. Rutin pretreatment ameliorates the activities of antioxidant enzymes A significant depletion in hepatic antioxidant enzymes viz., GPx, GR [Table 2] and CAT [Table 3] was observed in the CP treated group when compared to the control group [***p < 0.001]. Pretreatment with rutin was found to be significantly effective in restoring the activities of these antioxidant enzymes [#p < 0.05,
11
##
p < 0.01 and
###
p < 0.001] in a dose
dependent manner. There was no significant difference in the activity of these antioxidant enzymes between control and only rutin treated group. 3.4. Rutin pretreatment decreased XO activity and MDA formation A significant [**p < 0.01 and
***
p < 0.001] increase in the XO activity and MDA formation
was found in the CP administered group when compared with the control group. It has been observed that pretreatment with rutin leads to significant reduction in the elevated level of MDA and XO activity significantly [#p < 0.05, ##p < 0.01 and
###
p < 0.001] when compared
to CP treated group in a dose dependent manner. There was no significant difference in the activity of these antioxidant enzymes between control and only rutin treated group [Table 3]. 3.5. Cytokines analysis 3.5.1 Rutin inhibit the TNF-α and IL-6 production Levels of proinflammatory cytokine TNF-α and IL-6 were found elevated significantly in CP treated group as compared to control group [***p < 0.001]. Pretreatment with rutin significantly [#p < 0.05,
##
p < 0.01] decreased TNF-α and IL-6 levels. However no
significance difference was observed in the levels of TNF-α and IL-6 between control and only rutin pretreated group [Figure 1 & 2]. 3.6. Effect of rutin on histological changes The liver architecture of control group was normal, devoid of any signs of inflammatory changes. Nucleolus was clear, the central veins and hepatic lobule were clearly seen. In CP treated group, dilated and congested sinusoidal space with infiltration of lymphocytes and small portal space with inflammation were observed which are indicative of hepatocellular injury. Pretreatment with rutin reverted these changes in liver marked by less dilated and congested sinusoidal space with less inflammatory cell infiltration [Figure 3]. 3.7. Effect of rutin pretreatment on immunohistochemical analysis of i-NOS, COX-2 and P38-MAPK 12
The expressions of i-NOS, COX-2 and P38-MAPK are given in figure fig.4, fig.5 and fig.6. Staining intensity of i-NOS, COX-2 and P38-MAPK was remarkably high in CP treated group as compared to control group [***p < 0.001 and
**
p < 0.01]. Pretreatment with rutin
caused a noticeable reduction in the staining intensity of i-NOS, COX-2 and p-38 dose dependently [#p < 0.05, ##p < 0.01 and ###p < 0.001]. 3.8. Effect of rutin pretreatment on expression of NFκB The expression of NFκB protein is given in fig. 7. The immunohistochemical study revealed that in CP treated group intense and positive stained nuclei were seen as compared to the control group [**p < 0.01]. Rutin pretreatment reduced nuclear staining of NFκB dose dependently [#p < 0.05 and ##p < 0.01]. 4. Discussion Therapeutic effectiveness with anticancer drugs like CP is limited due to its evident toxicity on several target organs in addition to the risk of secondary tumors in patients [40]. Therefore there is an urgent need for potent chemopreventive agents which can diminish toxicity and effectively make the possible use of CP so that there will be improvement in the quality of life of the patients undergoing CP therapy. Findings of our study reveal that rutin exhibits preventive potential against CP-induced oxidative and inflammatory events. Biochemical, molecular and histopathological data demonstrates that alleviated effect of rutin is accredited to its antioxidative and antinflammatory potential. It has been evaluated that antioxidants show protection in the normal cells against the toxicity induced by CP [41, 42]. It has been reported that treatment with CP induces hepatotoxicity due to which there is elevation in the levels of serum marker enzymes [AST, ALT and LDH] which depicts hepatic damage [43]. Similarly in our study CP enhanced serum toxicity markers significantly and pretreatment with rutin showed protection against CP induced hepatotoxicity as shown by reduced level of serum enzymes which were compared to that of control group. It has been 13
evaluated that CP induced hepatic injury which led to the induction of oxidative stress due to excessive formation of ROS which caused lipid peroxidation [LPO] [44, 45]. Our results showed increased LPO in the liver of CP treated rats. Pretreatment with rutin prevented CP induced lipid peroxidation which may possibly be associated with the free radical scavenging activity of rutin, suppressing oxidative stress. GSH plays a crucial role in shielding cells against oxidative insult. CP treatment causes depletion of GSH level which might be attributed to the direct conjugation of acrolein with GSH, thus reducing its cellular level and leads to induction of oxidative stress [46]. Our study demonstrated that the mechanism of hepatoprotection by rutin against CP induced toxicity and hepatic damage is associated with the inhibition of oxidative stress by preventing GSH depletion which is in concurrence with the previous findings [47]. Pretreatment with rutin augmented the levels of GSH which may be because of enhancement of GSH synthesizing enzyme activities. Cellular antioxidant defence enzymes like CAT, GPx and GR help in detoxification of the free radicals and following oxidative stress. Dismutation of the superoxide anion leads to the formation of H2O2 and O2 which is further detoxified to water by CAT and GPx. During this reaction, GSH is oxidized to GSSG which is reduced back to GSH via GR. As a result, the activity of these antioxidant enzymes is essential for protection against oxidative stress. Hepatotoxicity induced by CP is attributed to oxidative stress caused by reduction of the antioxidant enzymes [48]. In our study activity of antioxidant enzymes like CAT, GPx and GR in the hepatic tissue were significantly decreased by CP treatment indicating marked oxidative stress. Our results demonstrated that the antioxidant status in the liver was markedly diminished after CP administration. Previous studies demonstrate that natural antioxidants protect against CP induced hepatic injury [49, 17]. In our study we have demonstrated that rutin showed protection against CP induced hepatotoxicity by preventing
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the generation of oxidative stress and increasing the hepatic antioxidant defense armoury involving GSH-dependent enzymes. Cytokines are low molecular weight proteins which are secreted primarily by the activation of lymphocytes and macrophages and are responsible for the maintenance of homeostasis by lymphoid, inflammatory and hematopoietic cells respectively [50]. TNF-α is a key element for the prevention of inflammatory toxicity induced by chemical compounds [51]. It has been reported in the previous studies that the levels of TNF-α and IL-6 were increased in the rats administered with CP [52]. In our study rutin reduced the levels of TNF-α and IL-6 which is in agreement with the findings of the previous studies [53]. In several inflammatory diseases, NFκB pathway is a therapeutic target because it plays an essential role in the activation of transcriptional factors like TNF-α, IL-6, COX-2 and i-NOS [54]. Dysregulation of NFκB has been associated with different pathological conditions which includes cancer and other inflammatory diseases [55]. In our study we have demonstrated that rutin plays potential role in attenuating ROS generated and TNF-α induced inflammation through downregulation of p38-MAPK, NFκB, COX-2 and i-NOS. ROS are thought to be involved in the activation of NFκB [56]. Various antioxidants are known to exhibit an inhibitory action on the activation of NFκB by modulating the redox status of the cell [57, 58]. In line with this, TNF-α induced ROS generation may possibly be associated with the stimulation of NFκB pathway [56], while inhibition of NFκB signalling could be due to the significant free radical scavenging properties of rutin. Our results suggest the significance of intracellular ROS levels, for rutin-inhibited ROS induced NFκB activation in hepatic tissues. To prevent inflammatory diseases it is an effective approach to target COX-2 and i-NOS [59]. A number of proinflammatory cytokines such as TNF-α and IL-6 are well-known to participate in stimulating i-NOS and COX-2 [60, 61]. Our results demonstrated that rutin 15
remarkably inhibited the activation of TNF- α, IL-6, NFκB, COX-2 and i-NOS induced by CP may be correlated with the inactivation of these proinflammatory cytokines. It has been reported that ROS, proinflammatory cytokines further stimulates redox sensitive transcription factors by activating mitogen-activated protein kinase (MAPK) signalling pathways [62, 63]. p38-MAP kinase, one of the most widely studied cell signalling cascade, plays an important role in inflammation via regulation of activation of COX-2 and NFκB in response to oxidative stress [64]. p38-MAPK pathway regulates production of proinflammatory cytokines which play critical role in inflammation through transcriptional activation of transcription factors like NFκB strongly supports its role in inflammation [65]. Our results demonstrated that there was highest expression of p38, NFκB in CP treated group as compared to control group. However rutin significantly suppressed the activation of p38- MAPK and NFκB which supports the anti-inflammatory effect of rutin. Histopathological studies showed that CP caused destruction of the hepatic tissue which was supported by the swelling and reduction of sinusoidal spaces in liver tissues. This may be due to potency in metabolite of CP which caused destruction of the membrane. The pathological changes due to the CP induced toxicity are correlated with distorted enzyme activities [66] and upregulation of proteins. In our study rutin pretreatment showed a protective effect on liver morphology. 5. Conclusion Our study demonstrated that rutin alleviated ROS induced oxidative stress, inflammation via targeting p38-MAPK, NFκB, COX-2, i-NOS and TNF-α, IL-6 which may be due to the acrolein, metabolite of CP. More research is needed to explain the down-regulation pathways of the modulatory effects of rutin against CP induced hepatotoxicity. In conclusion, rutisn attenuates CP induced hepatic toxicity in Wistar rats and may be clinically useful in the form
16
of a combinational therapy after further confirmatory studies both at pre clinical and clinical levels.
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Figure Legends Fig.1: Results represent mean ± SE of six animals per group. Levels of proinflammatory cytokine
TNF-α was found elevated significantly in CP treated group when compared with the control group [***p < 0.001]. Pretreatment with rutin significantly [#p < 0.05,
##
p < 0.01] decreased
TNF-α level dose dependently. There is no significance difference in the level of TNF-α between control and only rutin treated group. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig.2 Results represent mean ± SE of six animals per group. Levels of proinflammatory cytokine
IL-6 was found elevated significantly in CP treated group when compared with the control group [***p < 0.001]. Pretreatment with rutin significantly [#p < 0.05,
##
p < 0.01] decreased
IL-6 level dose dependently. There is no significance difference in the level of IL-6 between control and only rutin treated group. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt.
Fig. 3 [A-E] Representative photomicrograph of histopathological examination of rat liver 40x [A]
The liver architecture of control group was normal, devoid of any signs of inflammatory changes. Nucleolus was clear, the central veins and hepatic lobule were clearly seen. [B] In CP treated group, dilated and congested sinusoidal space with infiltration of lymphocytes and small portal space with inflammation were observed which are indicative of hepatocellular injury as shown by arrows. [C & D] Pretreatment with rutin reverted these changes in liver marked by less dilated and congested sinusoidal space with less inflammatory cell infiltration as shown by arrows. [E] Liver showed normal histology almost similar to control. Rutin; D1 = 50
mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig. 4 [A-D] Representative photomicrograph of immunohistochemistry of i-NOS of rat liver 40 x
[A] Control group showing negligible staining. [B] Staining intensity of i-NOS, was remarkably high in CP treated group as compared to control group as shown by arrows. 26
[C&D] Pretreatment with rutin caused a noticeable reduction in the staining intensity of iNOS dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.;
D2 = 100
mg/kg/b.wt. Fig. 4.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (##p < 0.01, and
###
p < 0.001). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100
mg/kg/b.wt. Fig. 5 [A-D] Representative photomicrograph of immunohistochemistry of COX-2 of rat liver 40 x.
[A] Control group showing negligible staining. [B] Staining intensity of COX-2 was remarkably high in CP treated group as compared to control group as shown by arrows. [C & D] Pretreatment with rutin caused a noticeable reduction in the staining intensity of COX-2 dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig. 5.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (##p < 0.01 and
###
p < 0.001). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100
mg/kg/b.wt. Fig. 6 [A-D] Representative photomicrograph of immunohistochemistry of p38-MAPK of rat liver 40x. [A] Control group showing negligible staining [B] Staining intensity of p38-MAPK, was
remarkably high in CP treated group as compared to control group as shown by arrows. [C & D] Pretreatment with rutin caused a noticeable reduction in the staining intensity of p38MAPK dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig.6.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (#p < 0.05 and ##p < 0.01). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. 27
Fig. 7 [A-D] Representative photomicrograph of immunohistochemistry of NFκB of rat liver 40x.
[A] Control group showing very less immunopositive cells. [B] Number of immunopositive cells was remarkably high in CP treated group as shown by arrows. [C&D] Pretreatment with rutin caused a noticeable reduction in the number of NFκB immunopositive cells dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig.7.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (#p < 0.05 and ##p < 0.01). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt.
28
Immunohistochemical Score
5
***
4.5 4
#
3.5 3 2.5 2
###
1.5 1 0.5 0
Group I
Group II
Group III
Group IV
Fig.5.1 Quantitative estimation of Cox-2
Immunohistochemical Score
6
**
5 4
## 3
###
2 1 0 Group I
Group II
Group III
Group IV
Fig.4.1 Quantitative estimation of i-NOS
Immunohistochemical Score
6
**
5 4
#
3
##
2 1 0 Group I
Group II
Group III
Group IV
Fig.6.1 Quantitative estimation of P38-MAPK
35
** 30 25 Labelling index %
#
20 15
##
10 5 0 Group I
Group II Group III Fig.7.1 Quantitative estimation of NFҡB
Group IV
Table.1 Results of pretreatment of Rutin on serum markers enzymes like AST, ALT and LDH on cyclophosphamide administration in liver of Wistar rats. Treatment regimen per group
AST(IU/L)
ALT(IU/L)
LDH (n mol NADH oxidised / min/ mg protein) 163.99 ± 40.70
Group I (control)
51.36 ± 0.08
49.01±0.25
Group II (only CP)
98.79 ± 0.39***
100.24±0.87***
507.84 ± 25.26 **
Group III (D1+CP)
71.85 ± 0.54 ###
70.13±0.30###
296.24 ± 40.70 #
Group IV (D2+CP)
53.07 ± 0.22 ###
52.89±0.33 ###
179.86 ± 3.34##
Group V (only D2)
52.98 ± 4.09
51.85±1.72
169.28 ± 12.06
. Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (**P < 0.01,
***
P < 0.001). Results obtained are significantly different from
cyclophosphamide administered group (#P < 0.05, ##P < 0.01 and b.wt.; D2 = 100mg/kg b.wt.
29
###
P < 0.001). Rutin; D1= 50mg/kg
Table.2 Results of pretreatment of Rutin on antioxidant enzymes like GSH, GR and GPx on cyclophosphamide administration in liver of Wistar rats. Treatment regimen per group
Group I (control)
GSH (n mol CDNB Conjugate formed /g tissue) 9.15 ± 0.18
GR (n mol NADPH Oxidized/min/ mg protein) 298.09 ±2.94
Group II (only CP)
2.07 ± 0.31 ***
107.03±2.58***
Group III (D1+CP)
5.50 ± 0.66 ##
205.45±5.64###
Group IV (D2+CP)
8.17 ± 0.16 ###
288.02±1.03 ###
Group V (only D2)
8.63 ± 0.12
291.57±0.43
GPX (n mol NADPH Oxidized/min/ mg protein) 265.28 ± 0.91 99.82 ± 0.97 *** 173.21 ± 7.06### 246.96 ± 13.85### 251.11 ± 1.38
Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (***P < 0.001). Results obtained are significantly different from cyclophosphamide treated group (##P < 0.01 and (###P < 0.001). RutinD1= 50mg/kg/b wt; D2 = 100mg/kg/b wt.
30
Table.3 Results of pretreatment of Rutin on antioxidant enzymes like catalase, XO and LPO on cyclophosphamide administration in liver of Wistar rats. Treatment regimen per group
Group I (control)
Catalase (nmol H2O2 consumed/min/mg protein) 29.28 ± 2.19
XO (µg uric acid formed/min/mg protein) 0.27 ± 0.07
LPO (n mol MDA formed / hr/ g tissue)
Group II (only CP)
10.94 ± 7.84***
0.86 ± 0.05 **
27.71 ± 1.46 ***
Group III (D1+CP)
20.40 ± 8.38#
0.65 ±0.02 #
19.55 ± 0.56 #
Group IV (D2 +CP)
26.14 ± 20.47 ##
0.31 ±0.031##
12.12 ± 1.36 ###
Group V (only D2)
27.08 ± 16.22
0.29 ± 0.05
11.10 ± 0.66
10.89 ± 0.45
Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (**P < 0.01 and #
***
P < 0.001). Results obtained are significantly different from
cyclophosphamide treated group ( P < 0.05,
##
P < 0.01) and (###P<0.001). RutinD1= 50mg/kg/b wt; D2 =
100mg/kg/b wt.
31
Graphical Abstract Short Term study (21 days)
HpH
Rutin
Cyclophosphamide
Oxidative Stress
Hepatotoxicity
Inflammation
Rutin
GSH, GPx, GR, XO, LPO AST, ALT, LDH
Rutin
NFҡB
P38-MAPK
TNF-α, IL-6, i-NOS,Cox-2
Manuscript Highlights
• Role of rutin in alleviating oxidative stress in liver. • Role of rutin in targeting hepatic injury by inhibition of inflammation in liver tissue. • Role of rutin in suppressing histopathological changes induced by cyclophosphamide induced hepatotoxicity.
32
S0009-2797(15)00090-3 http://dx.doi.org/10.1016/j.cbi.2015.02.021 CBI 7297
To appear in:
Chemico-Biological Interactions
Received Date: Revised Date: Accepted Date:
15 July 2014 28 January 2015 26 February 2015
Please cite this article as: S. Nafees, S. Rashid, N. Ali, S. Kazim Hasan, S. Sultana, Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: Role of NFκB/MAPK pathway, Chemico-Biological Interactions (2015), doi: http://dx.doi.org/10.1016/j.cbi.2015.02.021
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Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: Role of NFκB/MAPK pathway
Sana Nafees, Summya Rashid, Nemat Ali, Syed Kazim Hasan and Sarwat Sultana*
Molecular Carcinogenesis and Chemoprevention Division, Dept. of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India.
*Corresponding author, mailing address: Dr. Sarwat Sultana Molecular Carcinogenesis and Chemoprevention Division, Dept. of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India. E-mail address: [email protected] 1
Abstract Cyclophosphamide is a potent anticancer agent. However its clinical use is restricted because of its marked organ toxicity associated with increased oxidative stress and inflammation. The present study was designed to demonstrate the protective effects of rutin, a naturally occurring bioflavonoid against the hepatotoxicity induced by CP. Rats were subjected to oral pretreatment of rutin (50 and 100 mg/kg b.wt.) against hepatotoxicity induced by i.p. injection of CP (150 mg/kg b.wt.) and were sacrificed after 24h. Hepatoprotective effects of rutin were associated with upregulation of antioxidant enzyme activities and down regulation of serum toxicity markers. Rutin was able to down regulate the levels of inflammatory markers like TNF-α, IL-6 and expressions of p38-MAPK, NFκB, i-NOS and COX-2. Histopathological changes further confirmed the biochemical and immunohistochemical results showing that CP caused significant structural damage to liver which were reversed by pretreatment of rutin. Therefore, our study revealed that rutin may be a promising modulator in attenuating CP induced oxidative stress, inflammation and hepatotoxicity via targeting NFκB and MAPK pathway.
Key words: Cyclophosphamide, rutin, oxidative stress, inflammation, NFκB, MAPK.
2
Abbreviations BSA- bovine serum albumin CAT- catalase CDNB- 1-chloro 2, 4-dinitrobenzene COX-2- Cycloxygenase-2 CP- Cyclophosphamide DAB-3,3-diaminobenzidine DTNB- 5, 5′-dithio bis-[2-nitrobenzoic acid] EDTA- ethylene diamine tetra acetic acid GPx -glutathione peroxidase GR- glutathione reductase GSH- reduced glutathione GSSG- oxidized glutathione IL-6 -Interleukin-6 i-NOS- inducible nitric oxide synthase LPO-lipidperoxidation MAPKs- mitogen-activated protein kinases MDA- malondialdehyde NADPH- reduced nicotinamide adenine dinucleotide phosphate NFκB - Nuclear factor kappa B NO- Nitric oxide NOS- nitric oxide synthase ROS- reactive oxygen species TNF-α -Tumor Necrosis Factor XO- xanthine oxidase
3
1. Introduction Chemotherapeutic drugs are basically used for the treatment of an array of malignancies. However their therapeutic usage is inadequate because of their clinical side effects [1]. Cyclophosphamide [CP] is an anticancer drug which is extensively used in the treatment of multiple human malignancies and disorders like breast cancer, lung cancer, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis [2]. On the contrary, CP usage is recurrently restricted due to its adverse side effects and toxicities including nephrotoxicity, hepatotoxicity, cardiotoxicity, immunotoxicity and mutagenicity [3]. Acrolein which is a toxic metabolite of CP is responsible for reactive oxygen species [ROS] production and augmentation of lipid peroxidation [4]. One possible mechanism behind hepatotoxity of CP may be oxidative stress as reported previously [5]. The anti-neoplastic effects of CP are due to phosphoramide mustard, whereas acrolein is correlated to its toxic side effects [6]. Acrolein is a powerful soft electrophile that causes hepatoxicity by inactivating proteins and depleting GSH which results in oxidative stress [7]. It has been reported that acrolein toxicity is mediated by increased oxidants, thereby generating oxidative injury[8], signifying that it is not only direct oxidant but it also generates oxidants by various other pathways [9]. Inflammation plays a key role in the regulation of oxidative stress [10]. ROS leads to the activation of an array of transcription factors like nuclear factor kappa B [NFκB], which results in release of proinflammatory cytokines like Tumor Necrosis Factor [TNF-α] and Interleukin-6 [IL-6] [11] which further activate cycloxygenase-2 [COX-2] [12]. It has been reported that in addition to inflammation various antineoplastic agents have also been shown to activate transcription factor NFκB [13]. NFκB is one of the most important transcription factor which helps in the regulation of genes involved in the process of inflammation, cell proliferation and survival [14]. COX-2 is one of the downstream targets of NFκB.
4
Uncontrolled expression of COX-2 has been reported in different premalignant and malignant phases [15]. Nitric oxide [NO] is a free radical which is synthesized by the enzyme nitric oxide synthase [NOS]. Among its three isoforms, inducible-nitric oxide synthase [i-NOS] is responsible for the production of elevated levels of NO. It is often considered to be the key executor of toxicity in oxidative stress [16]. The process of i-NOS expression involves different signal transduction pathways, including nuclear translocation of the transcription factor, NFκB [17]. The mitogen-activated protein kinases [MAPKs] are proline targeted serine-threonine kinases, which are transducers of environmental stimulus to the nucleus [18]. The p38MAPK is a member of the MAPK family which is activated by oxidative stress which leads to accumulation of ROS in cell [19]. In various studies it has been reported that the activation of p38-MAPK has been associated in regulating the expression of NFκB and i-NOS [20]. In order to prevent the toxic side effects of CP and its metabolites, augmentation of antioxidant defence enzymes with natural antioxidants is necessary. Therefore, there is a need for effective agents which protect the normal tissue and cells from the undesirable effects of toxicity induced by chemotherapy. Therefore amalgamation of treatment regimen with effective antioxidants might be the desirable approach to alleviate the toxicity induced by CP [21, 22]. Various studies have reported that plant extracts possessing antioxidant activity protect against hepatotoxicity induced by CP [23, 24]. The flavonoids belong to the family of phenolic compounds and possess biochemical and pharmacological properties like antibacterial, antiviral, anti-inflammatory, antiallergic, antithrombotic, antimutagenic and antineoplastic [25]. They affect the cell growth, differentiation and its function owing to their radical scavenging activity. Rutin is a glycone of quercetin having a flavonol structure. It is present in fruits and fruit peel, predominantly in citrus fruits like oranges, grapefruits, lemons and limes. It is also found in buckwheat seeds 5
[26]. It is a member of bioflavonoids with antioxidant, anti-inflammatory, antiallergenic, antiviral and anticarcinogenic properties [27]. It has been reported to scavenge superoxide radicals. It can chelate metal ions, such as ferrous cations, which are involved in the Fenton reaction, which generates ROS and prevents lipid peroxidation [28]. However to the best of our knowledge, there is no report about the effect of rutin against CP induced hepatic oxidative stress and anti-inflammatory activity. Therefore, the main objective of our study was to check the role of activation of NFκB and MAPK pathway, expression of p38-MAPK, NFκB, COX-2, i-NOS and proinflammatory cytokines like TNF-α and IL-6 in the pathogenesis of oxidative damage caused by CP in rat liver and to investigate the protective effect of rutin implicated in the hepatotoxic insult might be caused by acrolein, one of the metabolites of CP. 2. Materials and Methods 2.1. Chemicals Rutin, CP, GR, GSSG, GSH, DTNB, CDNB, BSA, NADPH etc. were obtained from Sigma– Aldrich, USA. All other chemicals and reagents were of the highest purity grade and commercially available. 2.2. Animals Eight weeks old male Wistar rats (150–200 g) were obtained from the Central Animal House Facility of Hamdard University, New Delhi and were housed in a ventilated room at 25 ± 5 0
C under a 12 h light/dark cycle. The animals were acclimatized for 1 week before the study
and had free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India) and water ad libitum. The study was approved by the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) having Registration number and date of registration: (IAEC No: 173/ CPCSEA, 28th Jan 2000).
6
2.3. Treatment regimen Male Wistar rats (30) were divided into five groups containing 6 rats in each group. Group I served as a saline treated control. Group II served as positive control and administered intraperitoneal dose (150 mg/kg b.wt.) of CP freshly dissolved in normal saline. Rutin was orally administered at two doses, 50 and 100 mg/kg b.wt. to groups III and IV respectively for 20 consecutive days. Group V received only a high dose of rutin for 20 consecutive days. Group II, III, and IV were given CP (150 mg/kg b.wt.) on 20th day after rutin pretreatment. After 24 h of CP administration rats were sacrificed by cervical dislocation under mild anaesthesia using i.p. pentobarbital (50 mg/kg b.wt.) and blood was taken for various serological
parameters.
Liver
samples
were
taken
at
the
same
time
for
immunohistochemistry, various biochemical and histological parameters. There was 100% survival of animals in all the groups. 2.4. Post-mitochondrial supernatant preparation [PMS] Tissue processing and preparation of post-mitochondria supernatant (PMS) were done as described by S. Nafees et al. [29]. 2.5. Measurement of liver toxicity markers: serum aspartate aminotransferase (AST) and alanine aminotransferase [ALT] AST and ALT activities were determined by the method of Reitman and Frankel [30]. 2.6. Assay for Lactate dehydrogenase [LDH] activity Lactate dehydrogenase activity has been estimated in serum by the method described by Khan et al. [31]. 2.7. Assay for Reduced glutathione [GSH] estimation Reduced glutathione was determined by the method of Jollow et al. [32]. 2.8. Assay for Glutathione reductase [GR] activity Glutathione reductase activity was measured by the method of Carlberg and Mannervik [33].
7
2.9. Assay for Glutathione peroxidase [GPx] activity The activity of Glutathione peroxidase was calculated by the method of Mohandas et al. [34]. 2.10. Assay for catalase activity The catalase activity was assessed by the method of Claiborne [35]. 2.11. Assay for lipid peroxidation [LPO] The assay for lipid peroxidation was done following the method of Wright et al. [36]. 2.12. Assay for Xanthine oxidase [XO] activity Xanthine oxidase activity was done following the method of S. Nafees et al. [37].
2.13. Estimation of protein concentration The protein concentration in all samples was determined by the method of O.H. Lowry [38]. 2.14. Assay for Tumor Necrosis Factor Alpha [TNF-α] TNF-α levels were determined by rat TNF-α kit [eBioscience, Inc., San Diego., USA]. The method is based on enzyme-linked immuno-sorbent assay [ELISA]. Analysis was performed by Elisa Plate Reader [Benchmark plus, BioRad] according to the manufacturer’s instruction. 2.15. Assay for IL-6 IL-6 levels were determined by rat IL-6 kit [eBioscience, Inc., San Diego., USA]. The method is based on enzyme-linked immuno-sorbent assay [ELISA]. Analysis was performed by Elisa Plate Reader [Benchmark plus, BioRad] according to the manufacturer’s instruction. 2.16. Histopathological examination The livers were quickly removed after sacrifice of rats and were fixed in 10% neutral buffered formalin solution for histopathological processing. Sections were stained with hematoxyline and eosin before being observed under an Olympus microscope at 40x magnification. 2.17. Immunohistochemistry
8
To examine the protective effects of rutin on markers of inflammation in the liver COX-2, iNOS, NFκB and p38-MAPK expression in the liver were assessed by immunohistochemical staining. Liver sections on polylysine coated slides obtained were fixed in neutral buffered formalin, and embedded in paraffin and were treated for COX-2, i-NOS, NFκB and p38MAPK antibodies for immunohistochemical analysis. The procedures were processed according to the manufacturer’s protocol recommended for the COX-2, i-NOS, NFκB and p38-MAPK immunohistochemistry with slight modifications. Following deparaffinization and rehydration, sections were irradiated in 0.1 mol/L sodium citrate buffer [pH 6.0] in a microwave oven [medium low temperature] for 20 min. Then the sections were exposed to 3% H2O2 for 10 min to bleach endogenous per-oxidases, followed by rinsing 3 times in Tris buffer [pH 7.4] for 10 min. Sections were selectively incubated under humid conditions using an anti-NFκB antibody [1:400;Thermo Fisher Scientific, USA], anti-COX-2 antibody [1:200; Santacruz Biotechnology, Inc., USA], anti-phospho-p38 [dilution 1:200, Santa Cruz] and anti-i-NOS antibody (1:200; Thermo Fisher Scientific, USA) for overnight at 4 °C. Next day, slides were washed 3 times in Tris buffer for 10 min each. The specificity of the antibodies was tested by omission of the primary antibodies and a positive control of rat tonsil tissue. After washing in Tris buffer (pH 7.4), tissues were visualized with 3,3’diaminobenzidine [DAB] and counterstained with hematoxyline. Finally, the sections were dehydrated in xylene, mounted with DPX and cover slipped. Slides prepared for each case were examined by light microscopy. Positive and negative controls were conducted in parallel with COX-2, i-NOS, NFκB and p38-MAPK stained sections. Staining of sections with commercially available antibodies served as the positive control. Negative controls included staining tissue sections with omission of the primary antibody. 2.18. Statistical analysis
9
The data from individual groups were presented as the mean ± SD. Differences between groups were analyzed using analysis of variance [ANOVA] followed by Tukey-Kramer multiple comparisons test and minimum criterion for statistical significance was set at p<0.05 for all comparisons. 2.19. Quantitative evaluation of NFκB immunostaining Percentages of NFκB immunostained nuclei were calculated in each selected section for control rats, toxicant treated rats and rats given rutin as a modulator using the following formula: Number of labelled nuclei x 100/total number (labelled + unlabelled) of nuclei [39]. NFκB immunostained nuclei were considered positive regardless of staining intensity. All slides were examined by two independent observers who were unaware of the experimental protocol. The slides with discrepant evaluations were re-evaluated, and a consensus was reached. Measurements were carried out using an Olympus BX51 (Hamburg, Germany) microscope using objectives with 40x magnification. 2.20. Quantitative evaluation of COX-2, i-NOS and p38-MAPK immunostaining According to the diffuseness of the DAB staining, sections were graded as 0 (no staining), 1 (staining, 25%), 2 (staining between 25% and 50%), 3 (staining between 50% and 75%), or 4 (staining >75%). According to staining intensity, sections were graded as follows: 0 (no staining), 1 (weak but detectable staining), 2 (distinct staining) or 3 (intense staining). Immunohistochemical staining scores were obtained by adding the diffuseness and intensity scores. All slides were examined by two independent observers who were unaware of the experimental protocol. The slides with discrepant evaluations were reevaluated, and a consensus was reached. Measurements were carried out using an Olympus BX51 (Hamburg, Germany) microscope at 40× magnification.
10
3. Results 3.1.
Rutin
pretreatment
modulates
aspartate
aminotransferase
[AST],
alanine
aminotransferase [ALT] and lactate dehydrogenase [LDH] activity AST, ALT and LDH were significantly elevated in CP-treated rats (Table 3). Prophylactic treatment of rutin at a higher dose prevented CP induced elevation in serum levels of these enzymes (##p < 0.01 and < 0.05,
##
p < 0.01 and
###
p < 0.01) as compared to untreated control. Significant reduction [#p
###
p < 0.001] in these marker enzymes was observed in the rutin
pretreatment groups and found to be effective in the reduction of level of these enzymes when compared to CP administered group. Only rutin treated group showed no significant difference compared with the control group.
Henceforth there was a significant recovery after rutin
supplementation [Table 1]. 3.2. Rutin pretreatment restored the hepatic reduced glutathione [GSH] level GSH level was depleted significantly [***p < 0.001] in CP treated group when compared with the control group. GSH level in the rutin pretreated groups increased significantly [##p < 0.01 and
###
p < 0.001] when compared to CP treated group in a dose dependent manner.
However rutin alone did not show any significant changes in GSH level compared to control group [Table 2]. 3.3. Rutin pretreatment ameliorates the activities of antioxidant enzymes A significant depletion in hepatic antioxidant enzymes viz., GPx, GR [Table 2] and CAT [Table 3] was observed in the CP treated group when compared to the control group [***p < 0.001]. Pretreatment with rutin was found to be significantly effective in restoring the activities of these antioxidant enzymes [#p < 0.05,
11
##
p < 0.01 and
###
p < 0.001] in a dose
dependent manner. There was no significant difference in the activity of these antioxidant enzymes between control and only rutin treated group. 3.4. Rutin pretreatment decreased XO activity and MDA formation A significant [**p < 0.01 and
***
p < 0.001] increase in the XO activity and MDA formation
was found in the CP administered group when compared with the control group. It has been observed that pretreatment with rutin leads to significant reduction in the elevated level of MDA and XO activity significantly [#p < 0.05, ##p < 0.01 and
###
p < 0.001] when compared
to CP treated group in a dose dependent manner. There was no significant difference in the activity of these antioxidant enzymes between control and only rutin treated group [Table 3]. 3.5. Cytokines analysis 3.5.1 Rutin inhibit the TNF-α and IL-6 production Levels of proinflammatory cytokine TNF-α and IL-6 were found elevated significantly in CP treated group as compared to control group [***p < 0.001]. Pretreatment with rutin significantly [#p < 0.05,
##
p < 0.01] decreased TNF-α and IL-6 levels. However no
significance difference was observed in the levels of TNF-α and IL-6 between control and only rutin pretreated group [Figure 1 & 2]. 3.6. Effect of rutin on histological changes The liver architecture of control group was normal, devoid of any signs of inflammatory changes. Nucleolus was clear, the central veins and hepatic lobule were clearly seen. In CP treated group, dilated and congested sinusoidal space with infiltration of lymphocytes and small portal space with inflammation were observed which are indicative of hepatocellular injury. Pretreatment with rutin reverted these changes in liver marked by less dilated and congested sinusoidal space with less inflammatory cell infiltration [Figure 3]. 3.7. Effect of rutin pretreatment on immunohistochemical analysis of i-NOS, COX-2 and P38-MAPK 12
The expressions of i-NOS, COX-2 and P38-MAPK are given in figure fig.4, fig.5 and fig.6. Staining intensity of i-NOS, COX-2 and P38-MAPK was remarkably high in CP treated group as compared to control group [***p < 0.001 and
**
p < 0.01]. Pretreatment with rutin
caused a noticeable reduction in the staining intensity of i-NOS, COX-2 and p-38 dose dependently [#p < 0.05, ##p < 0.01 and ###p < 0.001]. 3.8. Effect of rutin pretreatment on expression of NFκB The expression of NFκB protein is given in fig. 7. The immunohistochemical study revealed that in CP treated group intense and positive stained nuclei were seen as compared to the control group [**p < 0.01]. Rutin pretreatment reduced nuclear staining of NFκB dose dependently [#p < 0.05 and ##p < 0.01]. 4. Discussion Therapeutic effectiveness with anticancer drugs like CP is limited due to its evident toxicity on several target organs in addition to the risk of secondary tumors in patients [40]. Therefore there is an urgent need for potent chemopreventive agents which can diminish toxicity and effectively make the possible use of CP so that there will be improvement in the quality of life of the patients undergoing CP therapy. Findings of our study reveal that rutin exhibits preventive potential against CP-induced oxidative and inflammatory events. Biochemical, molecular and histopathological data demonstrates that alleviated effect of rutin is accredited to its antioxidative and antinflammatory potential. It has been evaluated that antioxidants show protection in the normal cells against the toxicity induced by CP [41, 42]. It has been reported that treatment with CP induces hepatotoxicity due to which there is elevation in the levels of serum marker enzymes [AST, ALT and LDH] which depicts hepatic damage [43]. Similarly in our study CP enhanced serum toxicity markers significantly and pretreatment with rutin showed protection against CP induced hepatotoxicity as shown by reduced level of serum enzymes which were compared to that of control group. It has been 13
evaluated that CP induced hepatic injury which led to the induction of oxidative stress due to excessive formation of ROS which caused lipid peroxidation [LPO] [44, 45]. Our results showed increased LPO in the liver of CP treated rats. Pretreatment with rutin prevented CP induced lipid peroxidation which may possibly be associated with the free radical scavenging activity of rutin, suppressing oxidative stress. GSH plays a crucial role in shielding cells against oxidative insult. CP treatment causes depletion of GSH level which might be attributed to the direct conjugation of acrolein with GSH, thus reducing its cellular level and leads to induction of oxidative stress [46]. Our study demonstrated that the mechanism of hepatoprotection by rutin against CP induced toxicity and hepatic damage is associated with the inhibition of oxidative stress by preventing GSH depletion which is in concurrence with the previous findings [47]. Pretreatment with rutin augmented the levels of GSH which may be because of enhancement of GSH synthesizing enzyme activities. Cellular antioxidant defence enzymes like CAT, GPx and GR help in detoxification of the free radicals and following oxidative stress. Dismutation of the superoxide anion leads to the formation of H2O2 and O2 which is further detoxified to water by CAT and GPx. During this reaction, GSH is oxidized to GSSG which is reduced back to GSH via GR. As a result, the activity of these antioxidant enzymes is essential for protection against oxidative stress. Hepatotoxicity induced by CP is attributed to oxidative stress caused by reduction of the antioxidant enzymes [48]. In our study activity of antioxidant enzymes like CAT, GPx and GR in the hepatic tissue were significantly decreased by CP treatment indicating marked oxidative stress. Our results demonstrated that the antioxidant status in the liver was markedly diminished after CP administration. Previous studies demonstrate that natural antioxidants protect against CP induced hepatic injury [49, 17]. In our study we have demonstrated that rutin showed protection against CP induced hepatotoxicity by preventing
14
the generation of oxidative stress and increasing the hepatic antioxidant defense armoury involving GSH-dependent enzymes. Cytokines are low molecular weight proteins which are secreted primarily by the activation of lymphocytes and macrophages and are responsible for the maintenance of homeostasis by lymphoid, inflammatory and hematopoietic cells respectively [50]. TNF-α is a key element for the prevention of inflammatory toxicity induced by chemical compounds [51]. It has been reported in the previous studies that the levels of TNF-α and IL-6 were increased in the rats administered with CP [52]. In our study rutin reduced the levels of TNF-α and IL-6 which is in agreement with the findings of the previous studies [53]. In several inflammatory diseases, NFκB pathway is a therapeutic target because it plays an essential role in the activation of transcriptional factors like TNF-α, IL-6, COX-2 and i-NOS [54]. Dysregulation of NFκB has been associated with different pathological conditions which includes cancer and other inflammatory diseases [55]. In our study we have demonstrated that rutin plays potential role in attenuating ROS generated and TNF-α induced inflammation through downregulation of p38-MAPK, NFκB, COX-2 and i-NOS. ROS are thought to be involved in the activation of NFκB [56]. Various antioxidants are known to exhibit an inhibitory action on the activation of NFκB by modulating the redox status of the cell [57, 58]. In line with this, TNF-α induced ROS generation may possibly be associated with the stimulation of NFκB pathway [56], while inhibition of NFκB signalling could be due to the significant free radical scavenging properties of rutin. Our results suggest the significance of intracellular ROS levels, for rutin-inhibited ROS induced NFκB activation in hepatic tissues. To prevent inflammatory diseases it is an effective approach to target COX-2 and i-NOS [59]. A number of proinflammatory cytokines such as TNF-α and IL-6 are well-known to participate in stimulating i-NOS and COX-2 [60, 61]. Our results demonstrated that rutin 15
remarkably inhibited the activation of TNF- α, IL-6, NFκB, COX-2 and i-NOS induced by CP may be correlated with the inactivation of these proinflammatory cytokines. It has been reported that ROS, proinflammatory cytokines further stimulates redox sensitive transcription factors by activating mitogen-activated protein kinase (MAPK) signalling pathways [62, 63]. p38-MAP kinase, one of the most widely studied cell signalling cascade, plays an important role in inflammation via regulation of activation of COX-2 and NFκB in response to oxidative stress [64]. p38-MAPK pathway regulates production of proinflammatory cytokines which play critical role in inflammation through transcriptional activation of transcription factors like NFκB strongly supports its role in inflammation [65]. Our results demonstrated that there was highest expression of p38, NFκB in CP treated group as compared to control group. However rutin significantly suppressed the activation of p38- MAPK and NFκB which supports the anti-inflammatory effect of rutin. Histopathological studies showed that CP caused destruction of the hepatic tissue which was supported by the swelling and reduction of sinusoidal spaces in liver tissues. This may be due to potency in metabolite of CP which caused destruction of the membrane. The pathological changes due to the CP induced toxicity are correlated with distorted enzyme activities [66] and upregulation of proteins. In our study rutin pretreatment showed a protective effect on liver morphology. 5. Conclusion Our study demonstrated that rutin alleviated ROS induced oxidative stress, inflammation via targeting p38-MAPK, NFκB, COX-2, i-NOS and TNF-α, IL-6 which may be due to the acrolein, metabolite of CP. More research is needed to explain the down-regulation pathways of the modulatory effects of rutin against CP induced hepatotoxicity. In conclusion, rutisn attenuates CP induced hepatic toxicity in Wistar rats and may be clinically useful in the form
16
of a combinational therapy after further confirmatory studies both at pre clinical and clinical levels.
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Figure Legends Fig.1: Results represent mean ± SE of six animals per group. Levels of proinflammatory cytokine
TNF-α was found elevated significantly in CP treated group when compared with the control group [***p < 0.001]. Pretreatment with rutin significantly [#p < 0.05,
##
p < 0.01] decreased
TNF-α level dose dependently. There is no significance difference in the level of TNF-α between control and only rutin treated group. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig.2 Results represent mean ± SE of six animals per group. Levels of proinflammatory cytokine
IL-6 was found elevated significantly in CP treated group when compared with the control group [***p < 0.001]. Pretreatment with rutin significantly [#p < 0.05,
##
p < 0.01] decreased
IL-6 level dose dependently. There is no significance difference in the level of IL-6 between control and only rutin treated group. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt.
Fig. 3 [A-E] Representative photomicrograph of histopathological examination of rat liver 40x [A]
The liver architecture of control group was normal, devoid of any signs of inflammatory changes. Nucleolus was clear, the central veins and hepatic lobule were clearly seen. [B] In CP treated group, dilated and congested sinusoidal space with infiltration of lymphocytes and small portal space with inflammation were observed which are indicative of hepatocellular injury as shown by arrows. [C & D] Pretreatment with rutin reverted these changes in liver marked by less dilated and congested sinusoidal space with less inflammatory cell infiltration as shown by arrows. [E] Liver showed normal histology almost similar to control. Rutin; D1 = 50
mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig. 4 [A-D] Representative photomicrograph of immunohistochemistry of i-NOS of rat liver 40 x
[A] Control group showing negligible staining. [B] Staining intensity of i-NOS, was remarkably high in CP treated group as compared to control group as shown by arrows. 26
[C&D] Pretreatment with rutin caused a noticeable reduction in the staining intensity of iNOS dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.;
D2 = 100
mg/kg/b.wt. Fig. 4.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (##p < 0.01, and
###
p < 0.001). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100
mg/kg/b.wt. Fig. 5 [A-D] Representative photomicrograph of immunohistochemistry of COX-2 of rat liver 40 x.
[A] Control group showing negligible staining. [B] Staining intensity of COX-2 was remarkably high in CP treated group as compared to control group as shown by arrows. [C & D] Pretreatment with rutin caused a noticeable reduction in the staining intensity of COX-2 dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig. 5.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (##p < 0.01 and
###
p < 0.001). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100
mg/kg/b.wt. Fig. 6 [A-D] Representative photomicrograph of immunohistochemistry of p38-MAPK of rat liver 40x. [A] Control group showing negligible staining [B] Staining intensity of p38-MAPK, was
remarkably high in CP treated group as compared to control group as shown by arrows. [C & D] Pretreatment with rutin caused a noticeable reduction in the staining intensity of p38MAPK dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig.6.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (#p < 0.05 and ##p < 0.01). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. 27
Fig. 7 [A-D] Representative photomicrograph of immunohistochemistry of NFκB of rat liver 40x.
[A] Control group showing very less immunopositive cells. [B] Number of immunopositive cells was remarkably high in CP treated group as shown by arrows. [C&D] Pretreatment with rutin caused a noticeable reduction in the number of NFκB immunopositive cells dose dependently as shown by arrows. Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt. Fig.7.1. Results represent mean±standard error of mean of 6 animals. Results obtained are significantly different from group I (**p < 0.01). Results obtained are significantly different from group II (#p < 0.05 and ##p < 0.01). Rutin; D1 = 50 mg/kg/b.wt.; D2 = 100 mg/kg/b.wt.
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Immunohistochemical Score
5
***
4.5 4
#
3.5 3 2.5 2
###
1.5 1 0.5 0
Group I
Group II
Group III
Group IV
Fig.5.1 Quantitative estimation of Cox-2
Immunohistochemical Score
6
**
5 4
## 3
###
2 1 0 Group I
Group II
Group III
Group IV
Fig.4.1 Quantitative estimation of i-NOS
Immunohistochemical Score
6
**
5 4
#
3
##
2 1 0 Group I
Group II
Group III
Group IV
Fig.6.1 Quantitative estimation of P38-MAPK
35
** 30 25 Labelling index %
#
20 15
##
10 5 0 Group I
Group II Group III Fig.7.1 Quantitative estimation of NFҡB
Group IV
Table.1 Results of pretreatment of Rutin on serum markers enzymes like AST, ALT and LDH on cyclophosphamide administration in liver of Wistar rats. Treatment regimen per group
AST(IU/L)
ALT(IU/L)
LDH (n mol NADH oxidised / min/ mg protein) 163.99 ± 40.70
Group I (control)
51.36 ± 0.08
49.01±0.25
Group II (only CP)
98.79 ± 0.39***
100.24±0.87***
507.84 ± 25.26 **
Group III (D1+CP)
71.85 ± 0.54 ###
70.13±0.30###
296.24 ± 40.70 #
Group IV (D2+CP)
53.07 ± 0.22 ###
52.89±0.33 ###
179.86 ± 3.34##
Group V (only D2)
52.98 ± 4.09
51.85±1.72
169.28 ± 12.06
. Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (**P < 0.01,
***
P < 0.001). Results obtained are significantly different from
cyclophosphamide administered group (#P < 0.05, ##P < 0.01 and b.wt.; D2 = 100mg/kg b.wt.
29
###
P < 0.001). Rutin; D1= 50mg/kg
Table.2 Results of pretreatment of Rutin on antioxidant enzymes like GSH, GR and GPx on cyclophosphamide administration in liver of Wistar rats. Treatment regimen per group
Group I (control)
GSH (n mol CDNB Conjugate formed /g tissue) 9.15 ± 0.18
GR (n mol NADPH Oxidized/min/ mg protein) 298.09 ±2.94
Group II (only CP)
2.07 ± 0.31 ***
107.03±2.58***
Group III (D1+CP)
5.50 ± 0.66 ##
205.45±5.64###
Group IV (D2+CP)
8.17 ± 0.16 ###
288.02±1.03 ###
Group V (only D2)
8.63 ± 0.12
291.57±0.43
GPX (n mol NADPH Oxidized/min/ mg protein) 265.28 ± 0.91 99.82 ± 0.97 *** 173.21 ± 7.06### 246.96 ± 13.85### 251.11 ± 1.38
Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (***P < 0.001). Results obtained are significantly different from cyclophosphamide treated group (##P < 0.01 and (###P < 0.001). RutinD1= 50mg/kg/b wt; D2 = 100mg/kg/b wt.
30
Table.3 Results of pretreatment of Rutin on antioxidant enzymes like catalase, XO and LPO on cyclophosphamide administration in liver of Wistar rats. Treatment regimen per group
Group I (control)
Catalase (nmol H2O2 consumed/min/mg protein) 29.28 ± 2.19
XO (µg uric acid formed/min/mg protein) 0.27 ± 0.07
LPO (n mol MDA formed / hr/ g tissue)
Group II (only CP)
10.94 ± 7.84***
0.86 ± 0.05 **
27.71 ± 1.46 ***
Group III (D1+CP)
20.40 ± 8.38#
0.65 ±0.02 #
19.55 ± 0.56 #
Group IV (D2 +CP)
26.14 ± 20.47 ##
0.31 ±0.031##
12.12 ± 1.36 ###
Group V (only D2)
27.08 ± 16.22
0.29 ± 0.05
11.10 ± 0.66
10.89 ± 0.45
Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (**P < 0.01 and #
***
P < 0.001). Results obtained are significantly different from
cyclophosphamide treated group ( P < 0.05,
##
P < 0.01) and (###P<0.001). RutinD1= 50mg/kg/b wt; D2 =
100mg/kg/b wt.
31
Graphical Abstract Short Term study (21 days)
HpH
Rutin
Cyclophosphamide
Oxidative Stress
Hepatotoxicity
Inflammation
Rutin
GSH, GPx, GR, XO, LPO AST, ALT, LDH
Rutin
NFҡB
P38-MAPK
TNF-α, IL-6, i-NOS,Cox-2
Manuscript Highlights
• Role of rutin in alleviating oxidative stress in liver. • Role of rutin in targeting hepatic injury by inhibition of inflammation in liver tissue. • Role of rutin in suppressing histopathological changes induced by cyclophosphamide induced hepatotoxicity.
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