Mangiferin exerts hepatoprotective activity against D-galactosamine induced acute toxicity and oxidative/nitrosative stress via Nrf2–NFκB pathways
Graphical abstract
Highlights
►Galactosamine induces hepatocytes death via oxidative and nitrosative stress. ►Mangiferin exerts hepatoprotective effect/antioxidant defense via Nrf2 pathway. ►Mangiferin exerts anti-inflammatory responses by inhibiting NF-κB. ►Mangiferin suppresses galactosamine-induced repression of IL-10 mRNA.
Introduction
Nowadays, liver disease is a serious global health problem. Liver injury occurs because of its exposure to different agents, like chemicals, alcohols, viruses and auto-immune diseases (Sugiyama et al., 1999). Although extensive progress in the treatment of liver disease by several oral hepatoprotective agents has been carried out, the existing synthetic drugs have several limitations. Therefore, search for newer drugs (with minimum side effects) obtained from traditional medicines continues. d-Galactosamine (GAL) is a well known experimental hepatotoxin usually used to produce acute toxicity in rat liver (Manna et al., 2007, Sinha et al., 2007a, Muntane et al., 1998, El Mofty et al., 1975, Nagaki et al., 1994). This model has been shown to be closely related to human viral hepatitis and acute self-limiting hepatitis with inflammation, necrosis and regeneration, resembling a drug induced liver disease in human (Wills and Asha, 2006, Jonker et al., 1992, Decker and Keppler, 1972). Both oxidative and nitrosative stress play a key role in the pathogenesis of GAL-induced hepatic injury (Kucera et al., 2006, Siendones et al., 2003). Several investigators also showed that GAL-induced hepatocyte death was mediated by NFκB dependent iNOS over expression (Ranchal et al., 2006, Siendones et al., 2003, Siendones et al., 2004), TNFα and other pro-inflammatory cytokines (IL-1β, IL-6 etc.) (Lozano et al., 2003). It has been reported earlier that most of the genes overexpressed in inflammation (e.g. those encoding proinflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes) are controlled predominantly by the nuclear factor kappa B (NF-κB) (Christman et al., 2000, Aggarwal et al., 2006). The induction of oxidative stress is due to increased production of ROS and/or depletion of antioxidants. The intracellular antioxidant defense mechanism consists of a high cellular level of antioxidants, such as glutathione (GSH), and a family of phase II detoxification enzymes, including glutathione S-transferases (GST) and NADPH:quinine oxidoreductase (NQO1). However, these genes encoding antioxidant proteins and phase II detoxifying enzymes are controlled by a transcription factor nuclear erythroid 2-related factor 2 (Nrf2). In response to oxidative stress, Nrf2 is translocated to the nucleus from cytosol, where it sequentially binds to the antioxidant response element (ARE), resulting in a cytoprotective response characterized by an upregulation of antioxidant enzymes and decreased sensitivity to oxidative stress damage (Dhakshinamoorthy and Jaiswal, 2001, Jaiswal, 2004). Therefore, any agent that can modulate these genes and exert antioxidant power would be beneficial at this present pathophysiological condition.
Mangiferin (2-C-β-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) has attracted considerable interest due to its potent antioxidant (Moreira et al., 2001, Ghosh et al., 2011), antitumor, antiviral (Guha et al., 1996, Yoshimi et al., 2001), antidiabetic (Ichiki et al., 1998, Miura et al., 2001) and immunomodulatory activities (Leiro et al., 2004). This naturally occurring polyphenol is widely distributed in the plants belonging to Anacardiaceae and Gentianaceae families (e.g. Mangifera indica, mango), especially in the leaves, bark and bark root (Yoshimi et al., 2001). In Philippines, mango leaves are used as tea and the juice of the leaf is useful in bleeding dysentery (Quisumbing, 1978). In Cuba, extract of mango plant is manufactured in industrial scale to be used as nutritional supplement, cosmetic and phytomedicine. The bark and seeds of this plant act as astringents (Nunez-Selles et al., 2002). In traditional Indian medicine, mangiferin is also used for melancholia and nervous debility (Bhattacharya et al., 1972). Mangiferin has been reported to inhibit NFκB activation and a series of pro-inflammatory cytokines (Leiro et al., 2004) an immunomodulatory agent. In addition, it increased the cellular GSH, inhibit lipid peroxidation and quench ROS because of its inherent antioxidant property. All these properties indicate that besides being a ROS scavenger, this xanthone also possesses the ability to modulate the expression of a number of genes playing very important roles in regulating apoptosis and inflammation. However, there is no previous experimental evidence in the literature describing the protective role of mangiferin in GAL-induced hepatotoxicity in rats.
The present study has been undertaken to evaluate whether the antioxidant properties of mangiferin was mediated via Nrf2 dependent pathways against GAL-induced hepatic pathophysiology. Besides, we also assessed whether the regulation of NFκB, iNOS and pro-inflammatory cytokines by mangiferin accounted for its hepatoprotective role in GAL intoxicated animals.
Section snippets
Chemicals
Anti caspase-3, anti iNOS, anti NFκB, anti HO-1, anti GSTα and anti NQO1 antibodies were purchased from Sigma-Aldrich Chemical Company (St. Louis, USA). Anti Nrf2 and Lamin B2 antibodies were purchased from Santa Cruz Biotechnology (CA, USA). d-galactosamine and all other reagents were bought from Sisco Research Laboratory (Mumbai, India).
Mangiferin extraction
Mangiferin was extracted from the bark of M. indica following the method of Singh et al. (2009). Bose Institute Experimental Farm has its own mango trees and
Characterization of mangiferin
Fig. S1A represents the structure of mangiferin.
Fig. S1B represents the reverse phase HPLC chromatogram with a single peak at retention time 10 min.
Fig. S1C represents the mass spectrum of mangiferin. HRMS (ESI) analysis: calculated m/z 445.0747 [M + Na]+, observed m/z 445.0748 [M + Na]+.
Fig. S1D represents the 1H NMR spectrum of mangiferin. 1H NMR (500 MHz, DMSO-d6, δ ppm): 13.81 (1 H, s, 1-OH), 10.3–10.8 (3 H, m,3,6,7-OH), 7.38 (1 H, s, 8-H), 6.85 (1 H, s, 5-H), 5.36 (1 H, s, 4-H), 4.59 (1 H, d, J =
Discussion
The present study has been conducted to find out whether a reduction in free radical generation and/or gene regulation was relevant to the cytoprotective properties of mangiferin administered during GAL-induced hepatic pathophysiology. Mangiferin exerted its cytoprotective role via both antioxidant actions as well as by reducing the expressions of NFkB, iNOS and cytokines induced by GAL.
GAL is an extensively used hepatotoxicant. It distinctly reduces hepatic UDP-glucuronic acid level and
Conflict of interest
The authors have declared that no conflict of interest exists.
Acknowledgments
The authors are grateful to Mr. Prasanta Pal for the technical assistance of the study.
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