INTIMP-03442; No of Pages 9 International Immunopharmacology xxx (2014) xxx–xxx

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NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats Guruvaiah Ponmari a, Arunachalam Annamalai b,⁎, Velliyur Kanniappan Gopalakrishnan c, P.T.V. Lakshmi d, C. Guruvayoorappan b a

Department of Bioinformatics, School of Biotechnology and Health Sciences, Karunya Institute of Technology and Sciences, Karunya University, Coimbatore 641114, Tamil Nadu, India Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya Institute of Technology and Sciences, Karunya University, Coimbatore 641114, Tamil Nadu, India Department of Bioinformatics and Biochemistry, Karpagam University, Coimbatore 641021, Tamil Nadu, India d Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India b c

a r t i c l e

i n f o

Article history: Received 25 June 2014 Received in revised form 8 October 2014 Accepted 21 October 2014 Available online xxxx Keywords: Indigofera caerulea Roxb. Hepatoprotective Nuclear factor κB Proinflammatory cytokines Ferulic acid Liver damage

a b s t r a c t Indigofera caerulea Roxb. is a well known shrub among native medical practitioners in folk medicine used for the treatment of jaundice, epilepsy, night blindness and snake bites. It is also reported to have antioxidant and antimicrobial properties. However its actual efficacy and hepatoprotective mechanism in particular is uncertain. Thus the present study investigates the hepatoprotective effect of the methanolic extract of I. caerulea Roxb. leaves (MIL) and elucidation of its mode of action against carbon tetrachloride (CCl4) induced liver injury in rats. HPLC analysis of MIL when carried out showed peaks close to standard ferulic acid and quercetin. Intragastric administration of MIL up to 2000 mg/kg bw, didn't show any toxicity and mortality in acute toxicity studies. During “in-vivo” study, hepatic injury was established by intraperitoneal administration of CCl4 3 ml/kg bw (30% CCl4 in olive oil; v/v) twice a week for 4 weeks in Sprague–Dawley rats. Further, hepatoprotective activity of MIL assessed using two different doses (100 and 200 mg/kg bw) showed that intra-gastric administration of MIL (200 mg/kg bw) significantly attenuates liver injury. Investigation of the underlying mechanism revealed that MIL treatment was capable of reducing inflammation by an antioxidant defense mechanism that blocks the activation of NF-κB as well as inhibits the release of proinflammatory cytokine TNF-α and IL-1β. The results suggest that MIL has a significant hepatoprotective activity which might be due to the presence of phytochemicals namely analogues of ferulic acid and other phytochemicals which together may suppress the inflammatory signaling pathways and promote hepatoprotective activity against CCl4 intoxicated liver damage. © 2014 Published by Elsevier B.V.

1. Introduction Indigofera (family Fabaceae) is a large genus distributed throughout the tropical and subtropical regions of the world, with a few species reaching the temperate zone in eastern Asia [1]. The plant Indigofera caerulea Roxb. is a shrub that has been known to cure jaundice [2]. The leaf juice is administered orally to cure night blindness, while the root extract is used as a cure for epilepsy [3]. Native medical practitioners and tribal people use it to treat various human ailments such as jaundice, epilepsy and liver diseases [4]. In addition, recently it has been reported that various solvent extracts from I. caerulea Roxb. have

Abbreviations: MIL, methanolic extract of Indigofera caerulea Roxb. leaves; HPLC, High Performance Liquid Chromatography; AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; GPx, glutathione reductase; GSH, reduced glutathione; NFκB, nuclear factor-κB; ROS, Reactive Oxygen Species; TNF-α, Tumor Necrosis Factor-alpha; VLDL, very low density lipoproteins; HE, hematoxylin and eosin. ⁎ Corresponding author. Tel.: +91 9486412961 (mobile); fax: +91 422 2615615. E-mail addresses: [email protected], [email protected] (A. Annamalai).

highly significant antimicrobial and in vitro antioxidant activities and pharmacognostical properties [5,6]. Exposure to toxic chemicals, drugs and environmental pollutants causes damage to biological molecules such as proteins, DNA, lipids and cell membrane structure and its function leads to the metabolic activation of Reactive Oxygen Species (ROS) with the sequential alteration in normal metabolic processes [7]. Free radicals induce oxidative damage capable of generating excessive reactive oxygen species. ROS plays an important role in the pathogenesis of various human degenerative diseases such as liver disorders, aging, atherosclerosis, lungs and kidney damage [8]. The liver being the major site of xenobiotic metabolism is particularly susceptible to oxidative stress. Carbon tetrachloride (CCl4) is a highly toxic chemical and a well known hepatotoxin used extensively to investigate the hepatotoxicity in animal models by initiating lipid peroxidation [9,10]. In reduction reaction CCl4 metabolites biologically activate the hepatic microsomal phase I cytochrome P4502E1 system to form free radicals such as the trichloromethyl radical (CCl3•) that later reacts with oxygen to form its derivative trichloromethyl peroxy radical (CCl3OO•) of various reactive intermediates which incites an inflammatory response [11].

http://dx.doi.org/10.1016/j.intimp.2014.10.021 1567-5769/© 2014 Published by Elsevier B.V.

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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These reactive intermediates covalently bind to cellular molecules and damage the membranes of hepatic cells and organelles. Liver damage induced by CCl4 increases the concentration of the highly reactive lipoperoxide radical which causes peroxidation of lipids and ultimately leads to hepatotoxicity. Increasing concentration of free peroxide radical can induce membrane disintegration of liver hepatocytes, which in turn increases the release of cytosolic enzymes such as aspartate amino-transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transferase (γ-GT) into the circulating blood stream [12]. CCl4 administration triggers inflammation, which turns on the inflammatory molecular events followed by the activation of nuclear factor (NF)-κB and also upregulates the production of inflammatory cytokines Tumor Necrosis Factor-alpha (TNF-α) and Interleukin 1β (IL-1β). Transcription and expression of genes encoding cytokines were regulated by NF-κB [13,14]. Recently there has been an increasing interest over developing potential therapeutic medications and drugs from natural resources. Natural products have provided valuable novel leads or bioactive agents to the pharmaceutical industry that play a vital role in drug discovery. Highly significant results from our previous studies on estimation of total phenolic content and in vitro antioxidant activity of I. caerulea Roxb. encouraged us to carry out this work to determine the hepatoprotective effect of MIL. To the best of our knowledge and in lieu with the existing literature, studies on the protective effect of I. caerulea Roxb. remain unexplored. The present study thus focuses on investigating the role of the methanolic extract of I. caerulea Roxb. against CCl4 induced hepatotoxicity in experimental rats.

Kyoto, Japan) consisting of LC-10ATVp pump, SCL 10A system controller and SPD-M 10ATVp pump SCL 10A system controller and a variable Shimadzu SPD-10ATVp UV VIS detector and a loop injector with a loop size of 20 μl. The peak area was calculated using CLASS-VP software. Reverse-phase chromatographic analysis was carried out in isocratic conditions using a C-18 reverse phase column (250 × 4.6 mm i.d., particle size 5 μm, Luna 5 μ C-18; Phenomenex, Torrance, CA, USA) at 25 °C. The gradient elution of solvent A [water–acetic acid (25:1 v/ v)] and solvent B (methanol) had a significant effect on the resolution of compounds. Detection was carried out at 280 nm. As a result, solvent gradient was formed using dual pumping system by varying the proportion of solvent. Solvent B was increased to 50% in 4 min and subsequently increased to 80% in 10 min at a flow rate of 1.0 ml/min. Gallic acid (GA), caffeic acid (CA), rutin (RU), ferulic acid (FA) and quercetin (QU) were used as internal and external standards. Standards present in MIL were identified by comparing chromatographic peaks with the retention time (Rt) of individual standards [15].

2. Materials and methods

2.6. Acute toxicity studies

2.1. Chemicals

The animals were kept in fasting for overnight providing only water, after which the extract was administered intragastrically with an initial dose of 300 mg/kg bw and the rats were remained under observation for 14 days to check for mortality. Since, toxicity was not observed, the procedure was repeated for the next higher doses of 600, 1000, 1500 and 2000 mg/kg bw. Acute oral toxicity study was performed as per the Organization for Economic Co-operation and Development (OECD) guidelines to test chemicals, Test No. 423 (OECD, 2001; acute oral toxicity–acute toxic class method) [16].

CCl4, olive oil and thiobarbituric acid (TBA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (H2O2) was obtained from Merck (Darmstadt, Germany). Immunohistochemistry (IHC) antibodies were purchased from Santa Cruz Biotech (Santa Cruz, USA). Tumor Necrosis Factor-alpha (TNF-α) and IL-1 beta ELISA kits were purchased from USCN Life Science Inc., Houston, USA and KOMA Biotech Inc., Korea respectively. All other chemicals used in this study were of analytical grade.

2.5. Animals Adult healthy Sprague–Dawley rats of either sex, aged six to eight weeks and weighing 180 ± 20 g were maintained at a controlled temperature of 25 °C to 28 °C on a 12 h light–dark cycle, during which they had free access to standard diet and water ad libitum. Animal studies were conducted according to the regulations of the Institute Animal Ethics Committee and the protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (Reg. No. KU/IAEC/PhD/100 dated 26.07.2012).

2.7. Hepatoprotective experimental studies 2.2. Plant material Fresh leaves of I. caerulea Roxb. were collected from the foothills of Pachaimalai Hills, a part of Eastern Ghats of Tamil Nadu, India. Plant material was identified and authenticated by Botanical Survey of India, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India (Ref No. BSI/SRC/5/23/2010-11/Tech.-1729). 2.3. Extract preparation The leaves were collected, washed thoroughly with tap water, shade dried, homogenized to a fine powder and subjected to cold maceration in 70% methanol. This procedure was based on the basis of previously reported high phenolic content and antioxidant activity in methanolic extract of I. caerulea Roxb. The resulting extract was filtered and the methanolic solution was evaporated in a rotary evaporator under vacuum at 40 °C. This was further freeze dried, ground to fine powder and stored at 4 °C for further studies. 2.4. Compositional analysis of MIL by RP-HPLC The MIL extract was analyzed for phenolic phytoconstituents using a reverse phase High Performance Liquid Chromatography was performed using Shimadzu Liquid Chromatography (Shimadzu Corp.,

Rats were randomly divided into five groups of six animals each (n =6) were used for the study. Group (I) control rats received only vehicles; [olive oil (3 ml/kg bw) and DMSO (3 ml/kg bw)] and were fed with a normal diet. Group (II) rats received intraperitoneal administration of 3 ml CCl4/kg bw (30% CCl4 in olive oil; v/v) twice a week for 4 weeks to cause liver damage. Group (III) rats received Silymarin (50 mg/kg bw) intragastrically through a feeding tube twice a week for 4 weeks. Group (IV) animals were treated with MIL (100 mg/kg bw) intragastrically through a feeding tube twice a week for 4 weeks. Group (V) rats were treated with MIL (200 mg/kg bw) intragastrically through a feeding tube twice a week for 4 weeks. The animals of groups III, IV and V were also administered with intraperitoneal injection of 3 ml CCl4/kg bw (30% CCl4 in olive oil; v/v) twice a week for 4 weeks [17]. At the end of the experiment, 24 h after the CCl4 treatment, the animals were anesthetized with ethyl ether and blood samples were collected through their carotid arteries and serum was obtained by blood centrifugation at 3000 rpm (956 g — Eppendorf 5804 R) for 10 min at

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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Fig. 1. HPLC phenolic profile of MIL.

4 °C. The liver was removed after perfusion with ice cold saline at 4 °C. A part of the liver was immediately transferred to 10% buffered formalin for histopathological examination.

2.7.1. Biochemical determinations Serum analysis of various liver marker enzymes such as glutamatealanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), total cholesterol, triglycerides (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), very low-density lipoprotein (VLDL-C), urea, creatinine, bilirubin, Blood Urea Nitrogen (BUN) and uric acid was assessed spectrophotometrically according to the standard procedure using commercially available diagnostic kits (Agapee Diagnostics India Pvt Ltd, Ernakulum, Kerala, India). Liver tissue homogenates were prepared using ice-cold physiological saline and the resulting suspension was centrifuged at 12,000 rpm (15,294 g — Eppendorf 5804 R) for 20 min at 4 °C. Supernatant was used for the determination of total protein, nitrite (NO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and lipid peroxidation (TBARS) [18].

2.7.2. Histopathology analysis The liver samples of normal and treated animals were fixed with 10% formalin in phosphate buffered saline for 24 h and embedded in paraffin. Sections of 5–6 μm thickness were cut using a microtome and

stained with hematoxylin–eosin before they were examined under the microscope for histopathological changes in the liver. 2.7.3. Effect of MIL on cytokines level (TNF-α and IL-1β) Serum and liver homogenate samples were used for the measurement of TNF-α (USCN Life Science Inc., Houston, USA) and IL-1β (Koma Biotech Inc., Korea) using standard sandwich Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for murine cytokines according to the manufacturer's instruction. 2.7.4. Immunohistochemistry Immunostaining was performed according to standard methods with few modifications [19]. The liver sections were fixed overnight at 56 °C. The sections were de-paraffinized in fresh xylene and rehydrated using graded ethanol solutions. The specimens were dipped in freshly prepared solution of 1% H2O2 in ice cold methanol for 20 min to quench endogenous peroxidase. Specimens were rinsed in phosphate-buffered saline (PBS) and incubated for 1 h in blocking solution (3% BSA, 0.1% Tween-20 in PBS) at room temperature (RT). Sections were incubated with anti-NF-κB antibody (rabbit polyclonal) (1:500) in blocking solution for 12 h at 4 °C, re-equilibrated to RT and washed with PBS, incubated with horse radish peroxidase (HRP) antibody conjugates (1:2500) in blocking solution without Tween-20 for 2 h at RT. Specimens were washed with PBS and incubated with 0.2% solution of 3,3′diaminobenzidine (DAB) until desired stain intensity develops at RT followed by washing in distilled water. Sections were counterstained

Table 1 Effect of MIL and Silymarin on serum biochemical parameters in CCl4 intoxicated rats. Groupa

AST (U/l)

I II III IV V

31.99 451.47 191.26 314.42 218.00

± ± ± ± ±

ALT (U/l) 3.42 43.98# 15.03⁎ 13.12⁎ 23.90⁎

23.04 187.00 86.36 134 95.52

± ± ± ± ±

ALP (U/l) 2.36 20.47# 5.76⁎ 12.95⁎ 6.13⁎

248.08 905.48 293.33 562.00 352.98

± ± ± ± ±

27.17 18.19# 13.41⁎ 18.06⁎ 29.54⁎

γ-GT (U/l)

LDH (U/l)

37.00 208.90 76.16 102.00 114.66

327.00 507.83 347.94 432.00 385.21

± ± ± ± ±

5.76 14.15# 6.47⁎ 5.59⁎ 10.44⁎

± ± ± ± ±

24.76 20.68# 27.98⁎ 26.11⁎ 22.76⁎

Group I: Control (olive oil + DMSO); II: CCl4 (3 ml/kg bw); III: Silymarin (50 mg/kg bw) + CCl4 (3 ml/kg bw); IV: CCl4 (3 ml/kg bw) + MIL (100 mg/kg bw); V: CCl4 (3 ml/kg bw) + MIL (200 mg/kg bw). a Values are expressed mean ± SEM (06 numbers). # Significant difference from the control group (p b 0.05). ⁎ Significant difference from the CCl4 group (p b 0.05).

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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Table 2 Effect of MIL and Silymarin on liver function test in rat. Groupa

Bilirubin (mg/dl)

Urea (mg/dl)

Creatinine (mg/dl)

Uric acid (mg/dl)

BUN (mg/dl)

I II III IV IV

0.65 1.78 1.10 1.36 1.21

18.30 59.16 38.58 46.29 40.00

0.65 1.78 1.10 1.46 1.21

3.26 6.23 4.61 5.55 5.00

12.83 22.16 17.33 21.00 20.50

± ± ± ± ±

0.05 0.07# 0.11⁎ 0.05⁎ 0.11⁎

± ± ± ± ±

1.57 1.76# 1.59⁎ 2.21⁎ 3.31⁎

± ± ± ± ±

0.05 0.07# 0.08⁎ 0.05⁎ 0.07⁎

± ± ± ± ±

0.31 0.32# 0.18⁎ 0.16⁎ 0.18⁎

± ± ± ± ±

0.98 0.98# 0.81⁎ 1.30⁎ 0.54⁎

Group I: control (olive oil + DMSO); II: CCl4 (3 ml/kg bw); III: Silymarin (50 mg/kg bw) + CCl4 (3 ml/kg bw); IV: CCl4 (3 ml/kg bw) + MIL (100 mg/kg bw); V: CCl4 (3 ml/kg bw) + MIL (200 mg/kg bw). a Values are expressed as mean ± SEM (06 numbers). # Significant difference from the control group (p b 0.05). ⁎ Significant difference from the CCl4 group (p b 0.05).

with hematoxylin and mounted with di-n-butylphthalate-polystyrenexylene (DPX). Immunoreactivity was quantitated in a blinded manner by three independent observers and the total number of positively stained cells was quantitated. 2.7.5. Statistical analysis All experimental data comparison between multiple groups was analyzed by one way analysis of variance (ANOVA) using SPSS 18.0 software. Statistically significant data were further analyzed and their means were compared using Duncan's multiple range test. The data was expressed as mean ± SEM and p b 0.05 was considered as statistically significant. 3. Results 3.1. Acute toxicity studies Oral acute toxicity estimation of methanolic extract of I. caerulea Roxb. showed that there was no mortality up to 2000 mg/kg dose. Intragastric administration of methanolic extract of I. caerulea Roxb. neither caused behavioral change nor death of the animals. One tenth (200 mg/kg bw) of maximum dose of the extract tested (2000 mg/ kg bw) which did not indicate mortality was selected for evaluation of hepatoprotective activity. Hence 100 and 200 mg/kg bw consecutive doses were chosen for hepatoprotective studies. 3.2. RP-HPLC analysis of MIL The chromatographic pattern obtained from RP-HPLC profile analysis of phenolic phytoconstituents in the methanolic extract of I. caerulea Roxb. leaves (MIL) chromatogram revealed a peak which was consistent with the chromatographic pattern of the standards such as gallic acid, caffeic acid, rutin, quercetin, and ferulic acid. Quantitative HPLC analysis showed the presence of five standard compounds in MIL (Fig. 1). The relative amount of the five phenolic phytoconstituents found in MIL was in the order of gallic acid (1 μg/g) b caffeic acid (1 μg/g) b rutin (2 μg/g) b quercetin (15 μg/g) b ferulic acid (22 μg/g) respectively.

3.3. Hepatoprotective studies 3.3.1. Effects of MIL on serum marker enzymes Administration of CCl4 increased the levels of serum biochemical parameters such as AST, ALT, ALP, γ-GT and LDH (Table 1). Results showed that the CCl4 treated negative control group had a significant increase (p b 0.05) in the level of enzymatic activity when compared to control group.In contrast the co-treatment of MIL and Silymarin notably decreased (p b0.05) the levels of AST, ALT, ALP, γ-GT and LDH in CCl4 intoxicated group of animals. The level of specific liver marker enzymes in the serum was measured to determine the extent of liver damage in all experimental groups. The liver damage induced by CCl4 significantly elevated (p b 0.05) the range of specific enzymes (Bilirubin, Urea, Creatinine, Uric acid and BUN) in the CCl4 treated negative control group than the other groups. The level of marker enzymes in the Silymarin and MIL (200 mg/kg bw) treated groups was significantly restored toward normal compared to those of normal vehicle control (Table 2). 3.4. Effects of MIL on lipids profile CCl4 intoxication significantly increased (p b 0.05) the level of triglycerides (TGL), total cholesterol (CHOL), LDL and VLDL (Table 3) whereas, the level of HDL was reduced when compared to the vehicle control group of animals. Levels of triglycerides (TGL), total cholesterol (CHOL), LDL and VLDL concentration in MIL (200 mg/kg bw) and Silymarin co-treated groups were significantly restored (p b 0.05) when compared to CCl4 treated negative control group. Whereas, the level of HDL was highly enhanced to compensate the CCl4 mediated toxicity. 3.5. Effect of MIL on enzymatic and non-enzymatic antioxidant status The CCl4 intoxicated negative control group of animals exhibited a significant decrease (p b 0.05) in the level of both enzymatic antioxidants (SOD and CAT) as well as non-enzymatic antioxidants (GPx and GSH) (Fig. 2). Whereas the levels of LPO (TBARS) and nitrite (Table 4)

Table 3 Effect of MIL on lipid profile in rat. Groupa

CHOL (mg/dl)

TGL (mg/dl)

HDL (mg/dl)

LDL (mg/dl)

VLDL (mg/dl)

I II III IV V

87.82 134.00 94.66 118.38 102.66

80.83 99.33 66.99 89.99 82.99

42.38 34.49 41.49 37.00 40.33

27.00 61.00 40.99 56.65 48.66

16.00 19.33 12.99 16.06 15.00

± ± ± ± ±

8.13 11.29# 7.59⁎ 15.86⁎ 9.67⁎

± ± ± ± ±

5.34 7.42# 5.69⁎ 7.85⁎ 8.96⁎

± ± ± ± ±

4.07 2.41# 2.77⁎ 3.98⁎ 1.98⁎

± ± ± ± ±

2.99 6.63# 4.31⁎ 3.41⁎ 7.19⁎

± ± ± ± ±

1.65 1.56# 1.10⁎ 2.57⁎ 1.77⁎

Group I: control (olive oil + DMSO); II: CCl4 (3 ml/kg bw); III: Silymarin (50 mg/kg bw) + CCl4 (3 ml/kg bw); IV: CCl4 (3 ml/kg bw) + MIL (100 mg/kg bw); V: CCl4 (3 ml/kg bw) + MIL (200 mg/kg bw). a Values are expressed as mean ± SEM (06 numbers). # Significant difference from the control group (p b 0.05). ⁎ Significant difference from the CCl4 group (p b 0.05).

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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Fig. 2. Effect of MIL on hepatic antioxidant profile. Each column is mean ± standard error for six rats in each group; columns not sharing a common symbol (#,*) differ significantly from each other (p b 0.05 Duncan's multiple range test). *Units: SOD—Superoxide dismutase: U/mg; Superoxide dismutase one unit is defined as the enzyme concentration required to inhibit the optical density at 569 nm of chromogen production by 50% in 1 min, CAT—Catalase: μM of hydrogen peroxide consumed/min/mg protein, GPx—glutathione peroxidase: μg of reduced glutathione consumed/min/mg protein, GSH-reduced glutathione: μM/mg protein.

were seen to have increased when compared to the normal vehicle control group. These changes were significantly restored (p b 0.05) in the CCl4 + Silymarin and CCl4 + MIL (200 mg/kg bw) treated groups of animals. Oral administration of vehicle control (DMSO + olive oil) did not show any changes in the level of enzymatic and non enzymatic antioxidants. 3.6. Histopathological observations The gross appearances of the liver samples and microscopic assessment of their sections in the experimental groups (A–E) are shown (Fig. 3). Histopathological analysis of liver tissue sections from the normal vehicle control groups exhibited regular cellular architecture with distinct hepatic cells (Fig. 3A). In contrast, the CCl4 intoxicated negative control group showed liver sections with severe structural damage, pseudolobules formation, moderate fatty change and possible loss of hepatocytes (Fig. 3B). Partially preserved hepatocytes with mild inflammation were observed in the liver of rats treated with low dose MIL (100 mg/kg bw) (Fig. 3D). The liver sections of the rat treated with Silymarin and MIL (200 mg/kg bw) showed a mild degree of inflammation and no signs of necrosis (Fig. 3C and Fig. 3E). 3.7. Effect of MIL on cytokines level (TNF-α and IL-1β) CCl4 administration not only causes damage to tissues, but also initiates inflammation, which activates Kupffer cells and which mediates the action of cytokines TNF-α and IL-1β. The level of inflammatory cytokines TNF-α and IL-1β is presented in Fig. 4. Effect of MIL on inflammatory cytokines TNF-α and IL-1β level in CCl4 intoxicated group of animals in serum and liver homogenates (240.29 ± 23.69, 219 ± 20.60 and 1057 ± 74.75, 1016.20 ± 90.19) was enhanced significantly (p b 0.05) than normal control (175.47 ± 16.34, 177 ± 16.56 and 887.48 ± 57.80 and 781.78 ± 37.58) group of animals. MIL (200 mg/kg bw) treatment remarkably (p b 0.05) suppressed the level of inflammatory cytokines in both serum and liver homogenates (185.53 ± 17.89, 185.89 ± 17.75 and 945.95 ± 39.97, 854.46 ± 39.97) comparable with Silymarin treated group of animals (177.70 ± 16.83, 188 ± 17.77 and 942.95 ± 63.90, 827.77 ± 71.69, 942.95 ± 63.90) respectively. These changes show that MIL is associated with the downregulation of Kupffer cell mediated inflammatory cytokines.

3.8. Effect of MIL on the expression of NF-κB NF-κB plays a critical role in chronic inflammatory diseases and its activation is essential for cytokine production. Therefore we have studied the expression of NF-κB and the level of proinflammatory cytokines TNF-α and IL-1β (Fig. 4). Here we have observed the remarkable activation of NF-κB (Fig. 5B) in CCl4 intoxicated group when compared to the vehicle treated control group. Treatment with Silymarin and MIL (200 mg/kg bw) was found to significantly inhibit the NF-κB activation in groups III and V. A notable difference was observed between CCl4 intoxicated negative control and Silymarin co-treated group, MIL (200 mg/kg bw) co-treated group of animals as for as the activation of NF-κB (Fig. 5E). kg bw)/kg bw). Moderate NF-κB immunopositivity (Fig. 5D) were observed in the liver tissue sections of animals treated with a low dose of MIL (100 mg/kg bw). Treatment with MIL dose dependently reduced NF-κB immunoreactivity.

4. Discussion Oxidative stress is one of the most important stimuli that initiates CCl4 mediated liver damage. Lipid peroxidation as a result of oxidative stress is well known to be involved in liver injury [20]. There are a number of studies that have reported the alkynated halogen CCl4 to be widely used as a hepatotoxin to induce liver toxicity in experimental animal models [13]. Histopathology analysis of liver tissue revealed that intragastric administration of CCl4 causes damage to hepatic architecture, as CCl4 is metabolized by the cytochrome P450 mixed oxygenase Table 4 Effect of MIL on TBARS and nitrite in rats. Groupa

TBARS (nM/min/mg protein)

Nitrite (μM/ml)

I II III IV V

5.32 26.29 9.29 21.50 14.09

9.60 41.25 12.47 30.72 18.35

± ± ± ± ±

0.83 0.96# 0.45⁎ 1.57⁎ 1.01⁎

± ± ± ± ±

0.28 0.54# 0.39⁎ 0.94⁎ 0.66⁎

Group I: control (olive oil + DMSO); II: CCl 4 (3 ml/kg bw); III: Silymarin (50 mg/kg bw) + CCl4 (3 ml/kg bw); IV: CCl4 (3 ml/kg bw) + MIL (100 mg/kg bw); V: CCl4 (3 ml/kg bw) + MIL (200 mg/kg bw). a Values are expressed as mean ± SEM (06 numbers). # Significant difference from the control group (p b 0.05). ⁎ Significant difference from the CCl4 group (p b 0.05).

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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Fig. 3. Histomorphological liver sections of control and experimental rats (hematoxylin–eosin staining, 200× magnification). A: vehicle control group; B: CCl4 negative control group (30% CCl4); C: Silymarin positive control group (30% CCl4 + Silymarin 50 mg/kg bw); D: MIL group (30% CCl4 + MIL 100 mg/kg bw); E: MIL control group (30% CCl4 + MIL 200 mg/kg bw).

system found in the endoplasmic reticulum of liver cells and extra hepatic tissues [21]. CYP2E1 is a major cytochrome found to be involved in carbon–chlorine bond reductive cleavage and biotransformation of CCl4 to trichloromethyl radical (CCl3•). These cytochrome systems catalyze many reactions involved in drug metabolism, synthesis of cholesterol and other lipid metabolites both in endogenous substrates such as ethanol and acetone as well as exogenous substrates which includes carbon tetrachloride [22]. In the presence of oxygen, CCl3• generates highly reactive metabolites such as trichloromethyl peroxy (CCl3OO•) free radicals, which covalently bind to the cellular macromolecules, lipids and polyunsaturated fatty acids in the cellular membrane. Trichloromethyl peroxy (CCl3OO•) free radicals react with suitable substrates to complete its electron pair. CCl3OO• is highly reactive with polyunsaturated fatty acids (PUFA) when compared to CCl3• which extracts hydrogen from PUFA subsequently resulting in lipid peroxidation [23]. This lipid peroxidation reaction occurring in fatty acids and lipids leads to the formation of aldehydes, carbonyls and alkanes, which results in production of proteins and DNA adducts, loss of metabolic activation of enzymes, distortion of membrane integrity and thereby changing the structure of the endoplasmic reticulum. Thereby this process leads to inflammation, necrosis and liver damage [24–27].

Intraperitoneal administration of CCl4 induced liver damage and protective efficacy of MIL was measured by the level of serum marker enzymes, lipids profile, antioxidant enzymes, histology, level of inflammatory mediator's TNF-α and IL-1β and immunostaining of NF-κB. CCl4 a well known hepato-toxicant, which exhibits the toxic effects on the liver and eventual accumulation of liver specific enzymes such as ALT, AST, ALP, ACP, LDH, γ-GT and bilirubin; act as specific serological indicators of liver damage. A significant increase in the level of serum marker enzymes in the CCl4 intoxicated group of animals (intraperitoneal administration of 3 ml CCl4/kg bw (30% CCl4/olive oil) twice a week for four weeks) was previously reported [28]. However, animals treated with MIL and Silymarin show that the level of pathological increase in ALT, AST, ALP, LDH, and γ-GT was significantly restored. These results indicate that MIL has the ability to defend liver injury generated by CCl4 intoxicated liver injury. In the serum, the release of hepatocyte enzymes increased the concentration of triglycerides, LDL and total cholesterol whereas, it decreased the level of HDL which is a sign of CCl4 mediated damage on lipid profiles. Thus MIL co-treatment significantly reverts these lipid concentrations which may be attributed to their ability to chelate the metal ions. These results are in agreement with our previous report on the in vitro antioxidant activity of I. caerulea Roxb. [5].

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

G. Ponmari et al. / International Immunopharmacology xxx (2014) xxx–xxx

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Fig. 4. Effect of MIL on hepatic cytokine profile of TNF-α and IL-1β, each column is mean ± standard error; columns not sharing a common symbol (#,*) differ significantly each other (p b 0.05 Duncan's Multiple Range Test — DMRT).

Antioxidant enzymes such as superoxide dismutase and catalase are responsible for the detoxification of hydrogen peroxide and protect tissue from oxidative damage. Data of the present study shows that CCl4 administration significantly depleted the levels of antioxidant defense mechanism. GPx and GSH are well known reductants that metabolize toxic chemicals, drugs and xenobiotics. In the present study significant reduction in the activity of GPx and GSH was observed. Coadministration of MIL (200 mg/kg bw) protects cells from oxidative damage and ROS, which ameliorates the CCl4 mediated damage thereby elevating the activity of SOD, CAT, GPx, and GSH [29,30]. In the present study significant decrease in the activity of antioxidant enzymes suggested that the balance between the oxidant and pro-oxidant was disturbed by CCl4, and co-administration of MIL effectively reverts this imbalance and restores the level of antioxidant enzymes. Our biochemical findings and histopathological observation of the rat liver tissue show that livers from Silymarin (50 mg/kg bw) and MIL (200 mg/kg bw) treated rats had nearly normal liver architecture. Metabolites of toxic agents such as trichloromethyl radical of CCl4, infiltration of inflammatory cells and membrane damaged hepatocytes are the activators of Kupffer cells. Activated Kupffer cells release a wide range of soluble agents including cytokines (TNF-α and IL-1β), Reactive Oxygen Species (ROS) and other factors [31]. Reactive Oxygen Species (ROS) not only cause direct damage to tissues, but also initiate inflammation. Oxidative stress mediated inflammation upregulates the expression of several genes involved in the inflammatory response. This inflammation shows evidence of the activation of NF-κB in CCl4 intoxicated negative control group of animals. As expected the intraperitoneal administration of CCl4 activated

NF-κB and the levels of proinflammatory cytokines TNF-α and IL1β were significantly increased [32,33]. Phytochemicals present in plant sample were seen to inhibit inflammation by decreasing the macrophage production of proinflammatory factors and also by blocking the downstream signal of inflammatory pathways that release cytokines. Compounds exhibiting antioxidant activity also act on Kupffer cells by reducing LPS (lipopolysaccharide)-stimulated TNF-α release and by enhancing interleukin (IL-1β) secretion, which exerts an opposing action of TNF-α effect. The non-parenchymal cells of liver (Kupffer cells) predominant in the liver tissues are primarily involved in the controlled release of cytokines (TNF-α and IL-1β) mitigated by MIL [34,35]. The in vitro antioxidant activity of successive solvent extracts of I. caerulea Roxb. on various radical scavenging assay systems, we found I. caerulea Roxb. to be an effective scavenger of superoxide and hydroxyl anion radicals. The present data revealed that MIL has a protective effect against chronic CCl4 intoxicated liver injury and further reduced the progression of liver damage. RP-HPLC analysis of phenolic phytoconstituents used to quantify the components of MIL revealed the presence of gallic acid, caffeic acid, rutin, quercetin, and ferulic acid as its ingredients. These results confirm that the in vivo hepatoprotective activity of MIL may be associated with the phenolic phytoconstituents present in the extract which have been known for their antioxidant potential [5]. Ferulic acid was present in larger quantities (22 μg/g) than the other standards tested. Ferulic acid, a phenolic compound is an effective scavenger of free radicals and it has been approved in certain countries as food additive to prevent lipid peroxidation [36]. Chronic pretreatment

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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G. Ponmari et al. / International Immunopharmacology xxx (2014) xxx–xxx

Fig. 5. Immunopositive cells with NF-κB expression which are observed as black/brown dots stained with DAB were indicated with arrow marks in black. The effect of MIL and Silymarin on the expression and specific hepatic distribution of NF-κB immunostaining was performed in liver tissue sections (200×). (A) In control livers, NF-κB expression was negligible. (B) Strong NF-κB immunopositivity in CCl4-intoxicated rats. (C) Group of rats that received Silymarin 50 mg/kg bw was similar to control group. (D) Group of rats that received MIL 100 mg/kg bw was moderate NF-κB immunopositivity. (E) Strong NF-κB immunonegative in rats treated with MIL 200 mg/kg. Hypothesis testing was performed by two-way analysis of variance (ANOVA) followed by post-hoc Tukey's test. Results are given as statistically significant at p b 0.05; compared with (a) group II, (b) group IV and (c) group V.

of quercetin does not inhibit LPS-induced inflammation which leads to immediate release of TNF-α and IL-1β during the development of systemic inflammatory responses [37]. Hence, therapeutic potential of MIL against CCl4 induced liver damage was endorsed by FA. Phenolic compounds not included in HPLC analysis might be the possible ingredients in MIL. Previous investigation carried out in animal experimental models also clearly suggests the well-known protective effect of ferulic acid and other phenolic compounds [38,39]. Based on the experimental results demonstrated here, it would be highly desirable to develop a hypothesis that states MIL is highly effective in preventing CCl4 intoxicated inflammatory liver damage and plays an important role in the scavenging of free radicals, upregulating antioxidant enzyme activities, and blocking the activation of NF-κB, followed by the controlled release of proinflammatory cytokines which may suppress the expression of a subsequent inflammatory cascade during inflammation.

Therefore the significant free radical scavenging activity of I. caerulea Roxb. lowers the oxidative stress associated with CCl4 toxic metabolism. In particular the reduced lipid peroxidation may be attributed to the protective effect of I. caerulea Roxb. against liver damage. 5. Conclusion In conclusion, our result evidently demonstrates that MIL has significant protective effect against CCl4 intoxicated inflammatory liver damage. Presence of ferulic acid along with other phenolic phytoconstituents in MIL effectively restores liver function in CCl4 intoxicated liver damage. This report strongly supports the use of I. caerulea Roxb. in treating liver ailment. Altogether it provides an important biological lead with a prospect to be developed as a potential drug for targeting liver diseases.

Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021

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Acknowledgments The author Mr. Ponmari Guruvaiah would like to thank the University Grants Commission, Government of India for their financial assistance through the UGC-RGNF Scheme F.14-2 (SC)/2010 (SA-III). The authors also wish to thank The Chancellor, Karunya University, Coimbatore, India.

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Please cite this article as: Ponmari G, et al, NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl4 induced liver damage in rats..., Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.10.021