NF-κB activation and proinflammatory cytokines mediated protective effect of Indigofera caerulea Roxb. on CCl 4 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 b Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya Institute of Technology and Sciences, Karunya University, Coimbatore 641114, Tamil Nadu, India c Department of Bioinformatics and Biochemistry, Karpagam University, Coimbatore 641021, Tamil Nadu, India d Centre for Bioinformatics, Pondicherry University, Puducherry 605014, India abstract article info Article history: Received 25 June 2014 Received in revised form 8 October 2014 Accepted 21 October 2014 Available online 30 October 2014 Keywords: Indigofera caerulea Roxb. Hepatoprotective Nuclear factor κB Proinflammatory cytokines Ferulic acid Liver damage 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 anti- microbial 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 (CCl 4 ) induced liver injury in rats. HPLC analysis of MIL when carried out showed peaks close to standard ferulic acid and quercetin. Intragastric ad- ministration 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 CCl 4 3 ml/kg bw (30% CCl 4 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 acti- vation 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 name- ly analogues of ferulic acid and other phytochemicals which together may suppress the inflammatory signaling pathways and promote hepatoprotective activity against CCl 4 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 practi- tioners 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 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 ac- tivation of Reactive Oxygen Species (ROS) with the sequential alteration in normal metabolic processes [7]. Free radicals induce oxidative dam- age 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 (CCl 4 ) is a highly toxic chemical and a well known hepatotoxin used extensive- ly to investigate the hepatotoxicity in animal models by initiating lipid peroxidation [9,10]. In reduction reaction CCl 4 metabolites biologically activate the hepatic microsomal phase I cytochrome P4502E1 system to form free radicals such as the trichloromethyl radical (CCl 3 •) that later reacts with oxygen to form its derivative trichloromethyl peroxy radical (CCl 3 OO•) of various reactive intermediates which incites an inflammatory response [11]. International Immunopharmacology 23 (2014) 672–680 Abbreviations: MIL, methanolic extract of Indigofera caerulea Roxb. leaves; HPLC, High Performance Liquid Chromatography; AST, aspartate transaminase; ALT, alanine transami- nase; ALP, alkaline phosphatase; GPx, glutathione reductase; GSH, reduced glutathione; NF- κB, 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: aannamalai2001@yahoo.com, drannamalai@karunya.edu (A. Annamalai). http://dx.doi.org/10.1016/j.intimp.2014.10.021 1567-5769/© 2014 Published by Elsevier B.V. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp