Mol Cell Biochem (2014) 396:269–280 DOI 10.1007/s11010-014-2162-8

Rutin modulates ASC expression in NLRP3 inflammasome: a study in alcohol and cerulein-induced rat model of pancreatitis Ravikumar Aruna • Arumugam Geetha Periyanayagam Suguna

•

Received: 10 April 2014 / Accepted: 14 July 2014 / Published online: 25 July 2014 Ó Springer Science+Business Media New York 2014

Abstract Inflammasomes are protein complexes formed in response to tissue injury and inflammation to regulate the formation of proinflammatory cytokines. Nod-like receptor pyrin domain containing 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation. Caspase activation recruitment domain (CARD) is an interaction motif found in all the major components of NLRP3 inflammasome such as apoptosis associated speck-like CARD containing protein (ASC) and procaspase-1. NLRP3 activates procaspase-1 with the concerted action of CARD domain of ASC. In the present study, the effect of rutin, a natural flavonoid on the expression of ASC of NLRP3, was investigated in rats treated with ethanol (EtOH) and cerulein (Cer). Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats were fed normal diet, whereas groups 3 and 4 rats were fed EtOH (36 % of total calories) containing diet for a total period of 5 weeks and also administered Cer (20 lg/kg body weight i.p.) thrice weekly for the last 3 weeks. In addition, groups 2 and 4 rats received daily 100 mg/kg body weight of rutin from third week. Rutin co-administration significantly decreased the level of pancreatic marker enzymes, oxidative stress markers, inflammatory markers, mRNA expression of caspase-1, cytokines, ASC–NLRP3, and protein expression of caspase-1 and ASC in rats received EtOH–Cer. The results of the study revealed that rutin can reduce inflammation in pancreas probably by influencing the down regulation of ASC–NLRP3 which might result in the

R. Aruna  A. Geetha (&)  P. Suguna Department of Biochemistry, Bharathi Women’s College, Affiliated to University of Madras, Broadway, Chennai 600 108, Tamil Nadu, India e-mail: [email protected]

reduced activation of caspase-1 and controlled cytokine production. Keywords Caspase-1  ASC–NLRP3  EtOH–cerulein  Pancreatitis  Proinflammatory cytokines  Rutin Abbreviations NLRP3 Nod-like receptor pyrin domain containing 3 CARD Caspase activation recruitment domain ASC Apoptosis associated speck-like CARD containing protein

Introduction Pancreatitis is a painful, life-threatening disorder of the pancreas, associated with alcohol abuse, gall stone diseases, trauma, medications, and metabolic disturbances. Prolonged ingestion of large amount of alcohol is a major risk factor for the development of chronic pancreatitis [1]. Alcohol may promote chronic pancreatitis through toxic effect of its metabolites on acinar cells, oxidant stress, and also through the activation of pancreatic stellate cells, the key fibrogenic cells of pancreas [2]. Although alcohol consumption is associated with chronic pancreatitis in developed countries, evidences suggest that alcohol alone cannot cause pancreatitis. Cerulein (Cer) administration has been shown to potentiate the toxic effects of ethanol (EtOH). Cer can stimulate the acinar cells to excrete a large amount of digestive enzyme resulting in pancreatitis characterized by a higher serum enzyme level, interstitial edema, and the vacuolation of acinar cells [3]. Administration of Cer, a cholecystokinin (CCK) analog, stimulates the pancreatic acinar cells via CCK receptors, which leads to prematuration of proteolytic enzyme [4].

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Fig. 1 Activation of caspase-1 by NLRP3 inflammasome

Pancreatic inflammation is initiated by local production of mediators such as IL-1b and IL-18 [5]. The levels of these proinflammatory cytokines are shown to be elevated in the Cer-induced model of pancreatitis [6]. Generation of mature IL-1b and IL-18 is dependent on the proteolytic activity of caspase-1, which itself becomes activated in molecular platforms, referred to as inflammasomes. Inflammasomes are multi protein complexes that link recognition of damage-associated molecular patterns (DAMPs) by members of the nod-like receptor (NLR) family of cytosolic pattern recognition receptors to the activation of caspase-1 and processing and release of the proinflammatory cytokines IL-1b and IL-18 [7]. The inflammasome-initiating event is recognition of intracellular DAMPs derived from host (danger or stress signals) by a cytosolic NLR. An inflammasome is comprised of an NLR receptor, the sensor which is thought to detect the alarm signal to induce the formation of protein platform for the activation of caspase-1. The caspase activation recruitment domain (CARD) present in the adaptor protein apoptosis associated speck-like CARD containing protein (ASC) interacts with the CARD of procaspase-1 and the pyrin domain (PYD) of ASC interacts with PYD of NLR as shown in the diagrammatic representation (Fig. 1). The resulting assembly of different domains ultimately causes cleavage of procaspase-1 and its subsequent activation [8]. A prominent example of an NLR protein-linked autoinflammatory disease is systemic AA amyloidosis [9]. NLR PYD containing 3 (NLRP3) has been shown to play a central role in the induction of obesity and insulin resistance [10].

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The current treatment for pancreatitis is only supportive therapy for pain management. Hence the multiple pathogenicity of pancreatitis demands the identification of novel plant products with multiple mode of therapeutic action by modulating enzyme activities, metabolism, receptor functioning, signal transduction machinery, scavenging free radicals, etc., at various levels. Polyphenolic compounds such as flavonoids and coumarins have interesting medicinal properties, exerting antilipo peroxidant, anti-inflammatory, antiallergic, antiviral, antibacterial, and anticancer effects [11]. Rutin is a multifunctional anti-inflammatory [12] flavonoid, richly present in Emblica officinalis and Fagopyrum esculentum and chemically known as 5,7,30 ,40 , tetrahydroxy flavonol-3-rhamnoglucoside. Rutin has been shown to possess antihepatotoxic, antiulcer, antiallergic and antiviral properties [13]. Rutin also possesses antioxidant activity [14] and reduces LDL oxidation. The aim of the present study is focused on evaluating the anti-inflammatory activity of rutin. The study also evaluated the modulating action of rutin on ASC of NLRP3 inflammasome in rat pancreatitis model-induced by EtOH and Cer administration.

Materials and methods Chemicals and reagents Rutin was purchased from Santa Cruz Biotechnology, Inc. (Canada) and caspase-1 assay kit was obtained from Abcam. AxyPrep multisource total RNA miniprep kit was

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purchased from Axygen Biosciences, USA and cDNA reverse transcription kit was purchased from Applied Biosystems, USA. ELISA kit for IL-1b was purchased from Abcam and IL-18 from Invitrogen. All other chemicals and reagents used were of analytical grade. Experimental protocol Studies were performed in male albino Wistar rats weighing 175–200 g. Animals were housed in polycarbonate cages (four rats/cage) with wood chip bedding and fed standard laboratory chow and tap water. They were maintained in a climate-controlled animal room (temperature 22 ± 3 °C, relative humidity 60 ± 5 %) with a 12/12 h light/dark cycle. The animals were randomly allocated to four groups.

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homogenate prepared was used for the estimations within 2 h after sacrifice. For all the estimations involving proteins and enzymes, the reagents were prepared in proteasefree water and stored at 4 °C. The fecal materials weighing 4.5–5.5 g was dissolved in 0.5 % sodium bicarbonate (1/10), and this suspension was again diluted with 0.5 % sodium bicarbonate (1/100). Determination of serum lipase activity The activity of lipase (L) in serum was measured by the method of Lowry and Tinsley [16]. Olive oil/triton X 100 emulsion was used and the liberated free fatty acid was assayed spectrophotometrically at 715 nm and the activity was expressed as IU/l. Determination of serum amylase activity

Induction of pancreatitis After the acclimatization period, all the animals, except those in the normal and drug control group (groups 1 and 2), received isocalorically adjusted diet containing EtOH (36 % of total calories) for a total period of 5 weeks and i.p. injections of Cer at a dose of 20 lg/kg body weight thrice weekly for the last 3 weeks of the experimental period [15]. Animals in the drug control (group 2) and treatment group (group 4) received 100 mg/kg body weight of rutin from third week till the experimental period. A dose–response study was conducted with different concentrations (25, 50, 100, and 200 mg/kg body weight) of rutin co-administered to EtOH–Cer-treated rats. A dose of 100 mg/kg body weight of rutin was selected which gave the optimal pancreato-protective activity against EtOH– Cer-induced pancreatitis measured in terms of serum enzymes and histopathological alterations. This study was conducted according to the guidelines given by the Institutional Animal Ethics Committee. At the end of experimental period, rats were administered ketamine hydrochloride (30 mg/kg body weight) and killed by cervical decapitation; immediately, the blood was collected and the plasma/serum separated was stored at 4 °C until analyses.

The method of Gomori [17] was used to determine the activity of amylase (A). The method was based on the activity of enzyme on starch and the measurement of maltose was liberated using Lugol’s iodine solution. Assay of fecal trypsin Fecal trypsin was measured according to the method of McGowan and Wills [18]. Assay of IL-1b The assay was performed according to manufacturer’s instructions (ab100767). The antibody precoated wells were added with standard or serum sample. IL-1b present in a sample is bounded to the wells by the immobilized antibody. Biotinylated secondary antibody was added after washing the wells thoroughly. The unbound biotinylated antibody was washed and HRP-conjugated streptavidin was added to the wells. TMB substrate solution was added to all the wells after repeated washing. The stop solution changes the color from blue to yellow, and the intensity of the color was measured at 450 nm. The activity was expressed as pg/ml.

Biochemical investigations

Assay of IL-18

Preparation of tissue homogenate and fecal suspension

The assay was carried out as per the instruction of kit manual (KRC2341). The serum sample in duplicate or aliquots standard was pipetted into antibody immobilized wells. After the incubation, biotinylated secondary antibody was added. Streptavidin peroxidase was added after removal of excess secondary antibody. Then, the substrate solution was added to react with the bound enzyme to produce color. The intensity of this color was measured

Pancreas was removed immediately, washed in ice cold saline, and kept at -20 °C. It was homogenized in 0.1 M Tris–HCl buffer, pH 7.4, and centrifuged at low speed to remove any cell debris. The supernatant was used for the determination of caspase-1, total collagen, lipid peroxides, reduced glutathione (GSH), and antioxidant enzymes. The

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spectrophotometrically at 450 nm. The activity of IL-18 was expressed as pg/ml. Assay of caspase-1 Caspase-1 activity was measured according to the method of Thornberry et al. [19]. Briefly, the pancreas was homogenized in a lysis buffer [25 mM HEPES (pH 7.5), 1 mM EDTA, 10 lg of aprotinin/ml, 10 lg of leupeptin/ ml, 2 mM dithiothreitol] at 5 ml/100 mg of pancreas tissue. Extracts were centrifuged at 15,0009g for 30 min at 4 °C, and the supernatant was centrifuged again at 200,0009g for 1 h at 4 °C. The cytosol was used for caspase-1 activity measurements. According to the kit manufacturer instruction (AB39470), the assay in undiluted serum or pancreas extract was performed. Reactions with enzyme preparation alone, with enzyme mixed with caspase-1 substrate (Ac-YVAD-pNA) or inhibitor (AcYVAD-CHO), and with substrate alone were also run as controls. A recombinant caspase-1 enzyme was used as a positive control. The activity was measured by proteolytic cleavage of Ac-YVAD-pNA for 4 h at 37 °C. The plates were read at 405 nm.

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method of Kakkar et al. [26]. The enzyme activity was expressed as U/mg protein. The decomposition of H2O2 was kinetically measured at 240 nm to determine the activity of catalase (CAT) (Aebi [27]) and expressed as lM of H2O2 consumed/min/mg protein.

RT-PCR analysis RT-PCR was performed to measure mRNA transcript levels in the rat pancreatic tissue. Total RNA was isolated from frozen tissues (AxyPrep multisource Total RNA miniprep) and quantified. It was subjected to reverse transcription using cDNA reverse transcription kit. For real-time PCR, ABI PRISM Sequence Detection System 7700 (Applied Biosystems, USA) was performed. For each sample, triplicate test reactions and a control reaction without reverse transcriptase were analyzed for expression of the gene of interest and the results were normalized. The sequences of forward and reverse oligonucleotide primers for real-time PCR are tabulated in Table 1. PCR conditions were as follows: 40 cycles of 95 °C for 20 s (denaturation), 55 °C for 30 s (annealing), and 60 °C for 30 s (extension). Ct values obtained were used to quantify mRNA expression.

Determination of myeloperoxidase activity Western blot analysis Myeloperoxidase (MPO) activity in the pancreatic tissue was measured according to the method of Bradley et al. [20]. The enzyme activity was expressed as U/mg protein. Estimation of lipid peroxides and oxidative stress index The level of lipid peroxides in plasma was determined by measuring thiobarbituric acid-reacting substances (TBARS) [21]. The value was expressed as nmol/ml plasma and nmol/ 100 mg tissue protein. FOX 2 method with minor modifications was used to measure the peroxide content in plasma [22]. According to the method of Miller et al. [23] total antioxidant capacity (TAC) was determined. The decolorization of the assay mixture containing 2,20 -azino bis 3-ethyl benzo-thiazoline-6-sulfonate and the sample was monitored by measuring the absorbance at 734 nm and the % inhibition was calculated by using trolox as positive control. The ratio of total peroxides to TAC was calculated as oxidative stress index (OSI). Estimation of GSH and antioxidant enzymes GSH level was determined by the method of Moron et al. [24]. GSH peroxidase (GPx) was assayed by the method of Flohe and Gunzler [25]. The activity of GPx was expressed as nM of GSH oxidized/min/mg protein. Superoxide dismutase (SOD) activity was measured according to the

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Pancreatic tissue was homogenized in a buffer containing 250 mM sucrose, 20 mM HEPES–KOH (pH 7.0), 10 mM KCl, 1 mM EGTA, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and the protease inhibitor mixture. The homogenate was centrifuged at 1,0009g for 10 min at 4 °C and then supernatant was centrifuged for 1 h at 100,0009g. Then, the supernatant was used for western blotting. Protein concentration in the cell lysate was estimated by using Bradford method [28]. 20 lg of protein was resolved on denaturing 15 % polyacrylamide gels with a 6 % stacking gel and then transferred to nitrocellulose membrane electrophoretically. The membrane was blocked with 5 % skim milk powder in Tris-buffered saline (TBS; 20 mM Tris– HCl, pH 7.4, and 0.5 M NaCl). Blot was washed three times for 5 min with 0.1 % T-TBS (Tween 20 in TBS) for 1 h at room temperature and then incubated independently with primary antibodies selectively against ASC (1:500; Novus Biologicals, CA, USA) and caspase-1 (1:500; Abcam, Cambridge, USA) for 1 h. After washing, each blot was incubated with HRP-conjugated goat anti-rabbit IgG as the secondary antibody (Genei, Bangalore, India, 1:1,000) for 1 h and then washed three times in T-TBS solution for 5 min followed by incubation with DAB solution as substrate and dried. The brown-colored bands developed were subjected to densitometric tracing using DeGelDAS.

Mol Cell Biochem (2014) 396:269–280 Table 1 Primer sequences for RT-PCR analysis

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Genes

Forward primer

b-Actin

50 -GAGAAGATTTGGCACCACAC-30

Caspase-1 IL-1b

Reverse primer

0

50 -CAGCTGATGGACCTGACTGA-30

0

0

50 -AAAGAAGGTGCTTGGGTCCT-30

0

0

50 -ATCCCCATTTTCATCCTTCC-30

5 -TATGGAAAAGGCACGAGACC-3 5 -CAGGAAGGCAGTGTCACTCA-3

IL-18

5 -GAGGACTGGCTGTGACCCTA-3

TNF-a

50 -CTCGAGTGACAAGCCCGTAG-30 0

ASC

50 -CATCACAATGCCAGTGGTAC-30

0

0

5 -TTATGGAAGAGTCTGGAGCT-3

50 -TTGACCTCAGCGCTGAGCAG-30 50 -AATGAGTGCTTGCCTGTGTT-30

Table 2 Levels of serum lipase/amylase ratio, fecal trypsin, and pancreatic total collagen

Effect of rutin on the inflammatory markers

Groups

Table 3 shows the activity of MPO, caspase-1, IL-1b, and IL-18. MPO activity in the pancreas was found elevated by 2-fold in EtOH–Cer-administered rats when compared to normal rats (p = 0.000). Rutin co-administration was found to decrease the activity of MPO significantly (p = 0.000). The decrease was found to be 1.8-fold when compared to EtOH–Cer-administered rats. The activity levels of caspase-1 in serum and pancreatic tissue showed 7.6- and 9-fold increase, respectively, in EtOH–Cer control rats when compared to normal control. Caspase-1 was significantly decreased in rutin co-administered rats by 4.4-fold in serum and 6.7-fold in pancreas. Serum cytokines IL-1b and IL-18 levels showed significant elevation in EtOH–Cer-administered rats (p = 0.000). We found that IL-1b and IL-18 were increased by 2- and 1.5-fold, respectively, when compared to normal control rats. A significant fall in the levels of IL1b (1.9-fold) and IL-18 (1.3-fold) was observed in rutin coadministered rats. The cytokines level did not show much difference when normal control (group 1) and rutin control groups were compared (p = 1.000). The mRNA and protein expression of ASC and caspase1 are presented in Figs. 2 and 3. Figures 4, 5 and 6 show the mRNA expression of IL-1b, IL-18, and TNF-a, respectively. As illustrated in the above-mentioned figures, the mRNA expressions of ASC, caspase-1, IL-1b, IL-18, and TNF-a were up regulated significantly (p = 0.000) in EtOH–Cer-administered rats when compared to normal control. The protein expressions of ASC and caspase-1 were also up regulated by 1.6- and 2.9-fold, respectively, in EtOH–Cer-administered rats. The above mentioned inflammatory markers and ASC were found to be down regulated significantly (p = 0.000) by rutin coadministration.

Lipase/amylase ratio

Trypsin (mg/min/100 mg feces)

Normal control

1.24 ± 0.16

12.7 ± 1.89

Rutin control

1.27 ± 0.14NS

12.4 ± 1.53NS

EtOH ? Cer

5.8 ± 0.82*

8.2 ± 0.87*

EtOH ? Cer ? rutin

2.5 ± 0.29*

12.1 ± 1.39*

Values are expressed as mean ± SD for six animals in each group. Control versus rutin control, control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer NS non significant * p = 0.000

Statistical analysis Data were analyzed by using a commercially available statistics software package (SPSS for window V. 10). The statistical significance of mean values between different groups was determined by applying one-way ANOVA with post hoc Bonferroni test and the p value\0.05 was considered as significant.

Results Effect of rutin on pancreatic marker enzyme levels The level of L/A ratio and fecal trypsin activity is furnished in Table 2. The serum L/A ratio showed 4.7-fold (p = 0.000) elevation in EtOH–Cer control rats (group 3) when compared to normal control (group 1). The coadministration of rutin (group 4) was found to reduce the L/A ratio by 2-fold (p = 0.000). The excretion of fecal trypsin was found decreased by 1.5-fold in EtOH–Cer-treated rats (group 3) when compared to normal control rats. Rutin co-administration was found to increase the fecal excretion of trypsin by 1.5-fold (p = 0.000) when compared to EtOH–Cer control. The level of serum L/A ratio and fecal trypsin activity showed minimal difference between normal control and rutin control groups.

Effect of rutin on oxidative stress markers The levels of oxidative stress markers are represented in Table 4. The levels of TBARS, LHP, and OSI were found elevated significantly in EtOH–Cer-administered rats with

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Table 3 Levels of serum IL-1b, IL-18, caspase-1 and pancreatic MPO, caspase-1 in experimental animals Groups

IL-1b (pg/ml)

IL-18 (pg/ml)

Caspase-1

MPO (U/mg protein)

(pg/ml)

(pmol/mg protein)

10.5 ± 1.41

10.2 ± 1.21

Normal control

12.3 ± 1.59

170.1 ± 17.18

Rutin control

11.4 ± 1.33NS

167.9 ± 24.85NS

9.9 ± 1.27NS

9.7 ± 1.02NS

1.93 ± 0.28 1.71 ± 0.23NS

EtOH ? Cer

24.5 ± 2.52*

250.5 ± 31.31*

80.1 ± 11.77*

92.8 ± 12.34*

3.6 ± 0.41*

EtOH ? Cer ? rutin

13.1 ± 1.91*

191.3 ± 25.44*

18.3 ± 1.99*

13.9 ± 3.04*

2.02 ± 0.21*

Values are expressed as mean ± SD for six animals in each group. Control versus rutin control, control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer NS non significant * p = 0.000

Fig. 2 The mRNA and protein expression of ASC in experimental animals. a mRNA expression of ASC, b western blot analyses of ASC and c histogram of ASC protein expression. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni. Values are

mean ± SD of six rats. *p = 0.000 for control versus EtOH ? Cer, $ p = 0.045 EtOH ? Cer ? rutin versus EtOH ? Cer, p = 1.000 for control versus rutin control (not significant)

the fold change of 1.7, 1.6, and 2.7, respectively, when compared to normal control. Rutin co-administered rats showed significant reduction in the levels of these oxidative stress markers (p = 0.000). It was observed that the OSI was found decreased by 2.5-fold when compared to EtOH–Cer control groups. The TAC level was found to be decreased by 1.7-fold in EtOH–Cer control rats when compared to that of normal control rats. The co-administration of rutin was found to reduce this alteration by 1.6fold (p = 0.000).

Effect of rutin on GSH and antioxidants

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As indicated in Table 5, the activity of antioxidant enzymes GPx, SOD, and CAT was significantly decreased (p = 0.000) in EtOH–Cer-administered rats when compared to normal control rats. The decrease was found to be at the level of 3-, 1.7-, and 1.5-fold, respectively. In rutin co-administered rats, the activity of these enzymes was found to be increased (p = 0.000) than in EtOH–Cer control rats. We found that there was 2.5-fold increase in

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Fig. 3 The mRNA and protein expression of caspase-1 in experimental animals. a mRNA expression of caspase-1, b western blot analyses of caspase-1 and c histogram of caspase-1 protein expression. Data were analyzed by one-way ANOVA followed by post hoc

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Bonferroni. Values are mean ± SD of six rats. *p = 0.000 for control versus EtOH ? Cer, @p = 0.003 EtOH ? Cer ? rutin versus EtOH ? Cer, p = 1.000 for control versus rutin control (not significant)

Fig. 4 mRNA expression of IL-1b in pancreas of experimental rats. Data were analyzed by one way ANOVA followed by post hoc Bonferroni. Values are mean ± SD of six rats. *p = 0.000 for control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer, p = 1.000 for control versus rutin control (not significant)

the activity of GPx in rutin co-administered rats. The enzyme activity levels were not significantly different in normal control and rutin control groups.

Discussion The main events occurring in the pancreatic acinar cells that initiate and propagate pancreatitis include inhibition of secretion, activation of intracellular proteases, and generation of inflammatory mediators [29]. Alcohol abuse is a key etiologic factor in the mechanism of

pancreatitis. Chronic EtOH exposure increases the content of digestive and lysosomal enzymes within the acinar cell and decreases the stability of the organelles that contain digestive enzymes and lysosomes. Leakage of digestive enzymes to the interstitium generates selfdigestion and pathological inflammation of pancreatic tissues. These events have been correlated with the acinar morphological changes which are observed in the well-established experimental model of Cer-induced pancreatitis, as well as in human pancreatitis [30]. Hence this model of EtOH and Cer-induced pancreatitis was applied for the study.

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Fig. 5 mRNA expression of IL-18 in pancreas of experimental rats. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni. Values are mean ± SD of six rats. *p = 0.000 for control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer, p = 1.000 for control versus rutin control (not significant)

Fig. 6 mRNA expression of TNF-a in pancreas of experimental rats. Data were analyzed by one-way ANOVA followed by post hoc Bonferroni. Values are mean ± SD of six rats. *p = 0.000 for control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer, p = 1.000 for control versus rutin control (not significant)

The serum levels of both A and L have substantial sensitivity and specificity for the diagnosis of pancreatitis [31]. Serum A activity could reflect the exocrine pancreatic insufficiency. The serum L level, another widely accepted

marker of pancreatitis, rises after the onset of pancreatitis in parallel with the A level [32]. In this study, the level of L/A ratio rose markedly in the EtOH–Cer-treated group showing the severity of pancreatitis. In rutin co-administered group, the elevations are significantly minimized. This observation is the prime evidence for the preventive effect of rutin against EtOH–Cer-induced pancreatitis. Excess or abnormal activation of trypsin by the proteolytic cleavage of trypsinogen in the pancreas can lead to a series of events that cause pancreatic self-digestion, resulting in pancreatitis. Subnormal fecal trypsin indicates pancreatic dysfunction [33]. Due to acute necrosis or malignant growth in the pancreas, a fall in fecal trypsin may occur because of duct obstruction or reduced secretion of enzymes. When the pancreas does not produce enough trypsin and chymotrypsin, decreased level of trypsin was excreted in the feces. Active trypsin appears in the blood stream during pancreatitis but becomes rapidly bound to protein inhibitors such as alpha1-antitrypsin and alpha2macroglobulin and this might have resulted in reduced excretion of fecal trypsin. Our result shows that rutin coadministration significantly increase the level of fecal excretion of trypsin. This shows that rutin might inhibit the auto activation of trypsin and hence from activating the

Table 4 Activity levels of plasma peroxide content, total antioxidant capacity, and oxidative stress index in experimental animals Groups

TBARS (nmol/100 mg protein)

Peroxides (mmol/ml)

TAC (mmol trolex eq./l)

OSI

Normal control

3.03 ± 0.32

179.9 ± 25.73

343.2 ± 47.7

0.53 ± 0.06

177.8 ± 20.8NS

336.4 ± 49.7NS

0.52 ± 0.07NS

Rutin control EtOH ? Cer EtOH ? Cer ? rutin

2.8 ± 0.34NS 5.2 ± 0.74*

280.1 ± 38.4*

200.9 ± 23.5*

1.42 ± 0.2*

3.05 ± 0.41*

186.3 ± 23.0*

330.1 ± 35.7*

0.57 ± 0.06*

Values are expressed as mean ± SD for six animals in each group. Control versus rutin control, control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer NS non significant * p = 0.000

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Table 5 Activity levels of antioxidant enzymes and glutathione in pancreatic tissue of the experimental animals Groups

GSH (mg/g protein)

GPx (nM of GSH oxidized/min/mg protein)

SOD (U/mg protein)

CAT (lmol H2O2 consumed/min/mg protein)

Normal control

12.9 ± 1.5

1.52 ± 0.16

13.9 ± 2.0

107.6 ± 14.2

Rutin control EtOH ? Cer EtOH ? Cer ? rutin

12.3 ± 1.8

NS

1.48 ± 0.2

NS

13.6 ± 1.4

NS

102.9 ± 12.1NS

6.7 ± 0.9*

0.5 ± 0.06*

8.4 ± 0.1*

70.3 ± 10.2*

12.1 ± 1.6*

1.27 ± 0.18*

12.8 ± 1.5*

98.7 ± 12.5*

Values are expressed as mean ± SD for six animals in each group. Control versus rutin control, control versus EtOH ? Cer, EtOH ? Cer ? rutin versus EtOH ? Cer NS non significant * p = 0.000

enzyme cascade which otherwise leads to autodigestion, acinar cell death, and pancreatitis. Thus, rutin proves that it is a potent pancreato-protective agent. MPO has been implicated in promoting tissue damage during inflammation. The predominant physiological activity of MPO is to convert hydrogen peroxide and chloride to hypochlorous acid [34]. Under the influence of hypochlorite, cell membrane chlorination occurs with the subsequent disturbances leading to cell death [35]. Hypochlorous acid converts alpha-amino acids to aldehyde in neutrophils [36] which is cytotoxic in nature. MPO in excess promotes the formation of reactive oxygen metabolites that can damage normal cells with the liberation of intracellular proteinases. In the present study, EtOH–Certreated rats showed elevated level of MPO which might have caused neutrophil activation and liberated biologically active substances, cytokines, and reactive oxygen species (ROS). The products, formed under the influence of MPO, primarily have protective properties; however, its uncontrolled increase leads to damage of the organism’s own cells. Animals co-administered with rutin showed decreased level of MPO, an evidence for its anti-inflammatory action. The discovery of NLRs as an essential component of the immune system triggered significant interest in the study of their role in the pathogenesis of inflammatory diseases. NLRs comprise a large family of intracellular proteins that are believed to be primarily involved in the innate immune response to microbial pathogens through the recognition of conserved pathogen-associated molecular patterns [37]. However, NLRs also contribute to inflammation by sensing ‘‘danger signals’’ (i.e., endogenous molecules that are produced during tissue damage or inflammation) [38]. The bipartite adaptor protein ASC bridges the interaction between NLRP3 and the caspase-1 by means of homotypic interactions involving its PYD and CARD motifs, making it essential for activation of the inflammasome [39]. Activated caspase-1 processes the cytosolic precursors of the related cytokines IL-1b and IL-18, thus allowing secretion

of biologically active cytokines. It has been proved that mice lacking caspase-1 are defective in the maturation and secretion of IL-1b and IL-18. IL-1b participates in the generation of systemic and local responses to infection, injury, and immunological challenges by inducing the ‘‘acute phase response’’ characterized by elevated formation of acute phase proteins and leukocytosis. IL-18 lacks the pyrogenic activity as that of IL-1b, but involved in the induction of several secondary proinflammatory cytokines, chemokines, and cell-adhesion molecules. Cytosolic pathogen receptors and their downstream molecules have recently garnered attention in the field of inflammation. One such molecule is ASC, an adaptor molecule consisting of a C-terminal CARD and N-terminal PYD. ASC of inflammasome has been established to activate procaspase-1 through its oligomerization and process pro-IL-1b and pro-IL-18 into IL-1b and IL-18, respectively [40]. ASC and PYD family proteins seem to interact via their PYD domain, while the CARD domain of ASC has been shown to bind to the CARD domain of procaspase-1. Thus, PYD functions as the oligomerization domain and CARD as the effector domain of ASC in the caspase-1 activation pathway [41]. In EtOH–Cer treatment, the ASC was found to be up regulated. The up-regulated ASC might have interacted with procaspase-1 attributed by PYD (NLR)– PYD (ASC) interactions. This might enhance the oligomerization of ASC thereby induces caspase-1 activation. This might have led to processing of pro IL-1b and pro-IL18 to their matured forms. The anti-inflammatory activity of rutin might have been due to the down regulation of ASC expression in pancreas. Cytokines are a group of low molecular weight proteins that play crucial role in induction and progression of inflammatory reactions in pancreatitis. In normal cells, caspase-1 is present in a catalytically inactive proform. The formation of inflammasome initiates autocatalytic activation of caspase-1, resulting in the cleavage of proenzyme into a 20 kDa (p20) and 10 kDa (p10) subunits [19]. The

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active enzyme then assembles into two heterodimers of p20 and p10 subunits, containing two active sites. The elevated level of caspase-1 was observed in EtOH–Cer-administered rats than in rats co-administered with rutin. Caspase-1, proteolytically convert pro IL-1b into the bioactive cytokine IL-1b. IL-1b is a proinflammatory cytokine that lacks a signal peptide and needs cleavage in order to be activated and released. Mature IL-1b is a potent pyrogen with pleiotropic functions including the activation of lymphocytes and endothelial cells and the initiation of the acute phase response [42]. Caspase-1 can also cleave other members of the IL-1 family. Caspase-1-mediated cleavage has also been demonstrated for the activation of pro-IL-18. IL-18 is initially formed as a larger 22.3 kDa inactive protein that is cleaved by caspase-1 to a 17.3 kDa active form. Unlike IL-1b, IL-18 is constitutively expressed in its precursor form in many cells and requires prior activation by caspase-1. IL-18 in turn can induce IFN-c and other proinflammatory cytokines secretion and also activates NK cells. The activation of inflammatory cells that release cytokines such as TNF-a and IL-1b is an important cascade in the pathogenesis of pancreatitis [43]. TNF-a and IL-1b are derived predominantly from activated macrophages and act via specific cell membrane bound receptors. The proinflammatory cytokine, TNF-a has an important role in various biological functions, including cell proliferation, cell differentiation, survival, apoptosis and necrosis, and in inflammatory disorders. Secretion of TNF-a by several stressful stimuli has been demonstrated in many cell types, including pancreatic acinar cells. It is well established that pancreatic acinar cells produce TNF-a [44]. The present study shows that TNF-a expression in pancreas is significantly reduced in rutin co-administered rats than in EtOH– Cer control rats. The anti-inflammatory activity of rutin might be attributed to the inhibiting effect on caspase activation. Oxidative damage is considered to be a common factor in the pathogenesis of pancreatitis. This is due to increase in the generation of ROS. Superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals (OH) are important ROS causing tissue damage, and lipid peroxides level is an indicator of the generation of ROS in tissues. The higher lipid peroxidation levels indicated increased production of ROS within the tissue. These radicals function in concert to induce cell degeneration via peroxidation of membrane lipids, breaking of DNA strands, and denaturing cellular proteins [45]. In the present study, we have observed a significant increase in TBARS, LHP, OSI and decrease in TAC levels in EtOH–Cer treatment. These alterations are significantly minimized by co-administering rutin, along with EtOH–Cer.

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Superoxide ions are potentially harmful and they are removed by a number of scavenger mechanisms in vivo. SOD preferentially scavenges superoxide [46] and CAT catalyzes the decomposition of hydrogen peroxide which otherwise leads to oxidative stress in cells [47]. Decreased activity of SOD, CAT, and GPx and depletion of GSH in EtOH–Cer-treated rats showed that oxidative stress plays an augmenting role in inflammation induced by EtOH and Cer. The decreased SOD activity could be due to the oxidative inactivation of the enzyme by ROS generation. GSH is a major non-protein thiol that plays a central role in coordinating the antioxidant defense process. It is involved in the maintenance of normal cell structure through its redox and detoxification reactions [48]. GSH in association with GPx detoxify H2O2 and protect the cells against oxidative damage. Interestingly, activities of all these defense enzymes and level of GSH in pancreas were markedly greater in rutin co-administered rats than in EtOH–Cer control rats. These findings imply that the oral administration of rutin has a protective effect against pancreatitis through the free-radical scavenging activity.

Conclusion The results of the study revealed that rutin inhibits the activation of caspase-1, the key enzyme involved in the maturation of cytokines probably by down regulating the ASC domain of NLRP3 in pancreas to protect the gland from the hazardous effects of cytokines. Acknowledgments We thank Mr. Pazanimuthu Annamalai, Principal Scientist, Department of Biomedical Sciences, Sri Ramachandra University, for his expert technical support in RT-PCR study. This work was supported by Indian Council of Medical Research (ICMR) [Senior Research Fellow, file no.: 45/51/2012/BMS/TRM], New Delhi, India.

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