Genistein modulates the expression of NF-κB and MAPK (p-38 and ERK1/2), thereby attenuating d-Galactosamine induced fulminant hepatic failure in Wistar rats

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Highlights

  • First study to evaluate hepatoprotective effect of Genistein against d-GalN

  • Genistein prevents oxidative damage induced by d-GalN.

  • Genistein blunts iNOS, COX-2, NF-ĸB, IKKα/β and MAPK expression.

  • Genistein prevents d-GalN induced apoptosis and necrosis.

Abstract

Genistein is an isoflavanoid abundantly found in soy. It has been found to play an important role in the prevention of various chronic diseases including cancer. In this study, we evaluated potential therapeutic properties of Genistein against d-Galactosamine (d-GalN) induced inflammation and hepatotoxicity in male Wistar rats. Fulminant hepatic failure (FHF) was induced in rats by intraperitoneal injection of d-GalN (700 mg/kgBW). Genistein (5 mg/kgBW/day) was given as pre-treatment for 30 days via intra-gastric route followed by d-GalN (700 mg/kgBW) injection. The hepatoprotective and curative effects of Genistein were evident from a significant decrease in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels as well as prevention of histological damage by pre-treatment of Genistein. Genistein pre-treatment significantly inhibited the increased protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing nitric oxide (NO) and prostaglandin-E2 (PGE) levels, respectively. In addition Genistein significantly suppressed the production of d-GalN-induced proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. These inhibitory effects were associated with the suppression of nuclear factor-kappa B (NF-ĸB) activation, IKKα/β and Mitogen activated protein kinase (MAPK) phosphorylation by Genistein in d-GalN-treated animals. In conclusion, our results suggest that Genistein may serve as a potential supplement in the prevention of hepatic and inflammatory diseases. Furthermore Genistein is able to maintain the redox potential and strengthens the antioxidant defense system of a cell.

Introduction

Inflammation is a complex biological process in living organisms that reflects the response of a cell to harmful stimuli such as infection, cellular damage, and tissue injury. It is considered to be an innate immune response beneficial to host survival (Krakauer, 2004). The inflammatory reactions result from considerable changes in a number of cellular and biochemical processes involving the downstream regulation of proinflammatory protein expression and the upregulation of anti-inflammatory protein expression that facilitate the recruitment of immune cells, whereas pro-inflammatory cytokines facilitate this process (Pasparakis, 2009, Kim and Park, 2012). However, inappropriate control and a prolonged inflammatory response have been identified as crucial risk factors that may lead to the development of various chronic diseases such as autoimmune disorders, cancer, and vascular diseases (Chen and Nunez, 2010). iNOS and COX-2 are the two important mediators of inflammation which regulate the inflammatory process by producing NO and PGE2, respectively (Jeong et al., 2011). Therefore, a compound possessing inhibitory effects on iNOS and COX-2 expression could have great potential to improve the treatment of chronic inflammation. d-GalN is one of the major factors that stimulate the inflammatory response by stimulating various proinflammatory mediator cytokines such as interferon-γ (IFN-γ), IL-1β, interleukin-6 (IL-6), and TNF-α. It has been associated with the activation of MAPK also (Ganai et al., 2014). It induces FHF that closely resembles human viral hepatitis (Wills and Asha, 2006). d-GalN is a specific hepatotoxicant that depletes uridine triphosphate thereby inhibiting macromolecule synthesis (Decker and Keppler, 1974). As a transcriptional inhibitor, d-GalN potentiates the toxic effects of lipopolysaccharide in liver (Xiong et al., 1999).

NF-κB plays an important role in mediating the inflammatory response, cellular proliferation, and cell adhesion. Its activation is controlled by the IkB kinase (IKK) complex, which is linked with the induction of IκB phosphorylation at two specific serine residues (Ser32 and Ser36), resulting in IκB degradation through the ubiquitin–proteasome system (Beauparlant and Hiscott, 1996, Pasparakis, 2009). The free NF-κB then translocates to the nucleus and is bound to its binding sites on the target genes, such as iNOS and COX-2 thereby stimulating the gene expression (Appleby et al., 1994, Lin et al., 1996). Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK constitute the MAPK family whose persistent activation has been associated with an increase in the development of human inflammatory diseases due to the induction of iNOS expression (Chan and Riches, 2001). Hence, the two targets i.e., NF-κB and MAPK signaling pathways are considered to be an attractive therapeutic strategy for the development of anti-inflammatory drugs.

FHF is a life-threatening clinical syndrome that results from severe impairment of liver function. Mortality without supportive management and/or liver transplantation is in excess of 70–90%. Without liver transplantation (LT), the overall prognosis for patients with FHF is quite poor, with survival rates usually reported between 10% and 30% (Dhiman et al., 1998). FHF caused by alcohol, viral infection or non-alcoholic steatohepatitis are highly associated with acute/chronic inflammation (Crispe, 2009, Tacke et al., 2009). A wide variety of inflammatory cells such as natural killer cells, T cells, dendritic cells and macrophages are recruited during liver inflammation (Crispe, 2009). The involvement of hepatic resident macrophage has been found to be of crucial importance in exciting the liver injury because of great production of inflammatory cytokines including TNF-α, IL-1β and IL-6 and reactive oxygen species in response to inflammatory stimuli (Crispe, 2009).

Genistein is a soy derived bioactive polyphenol that has been a subject of numerous studies (Sarkar and Li, 2003). Various epidemiological, in vitro, and animal studies have evaluated the anti-cancer, cardioprotective, anti-osteoporotic, antioxidant, anti-inflammatory activities of Genistein. It has been also associated with the inhibition of tyrosine-specific protein kinase in malignant cells (Akiyama et al., 1987, Goldwyn et al., 2000, Wang, 2000, Caldarelli et al., 2005, Zhang et al., 2008, Park et al., 2010, Rusin et al., 2010). These properties of Genistein are attributed to its strong anti-proliferative and anti-apoptotic potential (Messina and Loprinzi, 2001). Early studies have evaluated the potential of Genistein to blunt NFĸ-B, MAPK (p-38 and ERK1/2) and TNF-α thus ameliorating muscle function and morphology in mdx mice (Messina et al., 2011). It acts as a potential anti-inflammatory and anti-necrotic agent in experimental liver damage caused by d-GalN (Kuzu et al., 2007) and it has been revealed that Genistein activates the antioxidant profile, decreases IL-6 and TNF-α concentrations in insulin resistant rats, thereby preventing oxidative damage and ameliorating the fatty liver (Mohamed Salih et al., 2009). In addition, Genistein has been shown to prevent and protect against acetaminophen (APAP) induced liver toxicity due to the inhibition of APAP biotransformation and the resistance to oxidative stress via the modulation of the activities of the metabolism and the antioxidant enzyme. These properties of Genistein make it a popular compound for drug development and to be used as a neutraceutical.

In the present study, we evaluated the anti-inflammatory and hepatoprotective effect of Genistein in d-GalN treated male Wistar rats. These results provide evidence that Genistein may be a potential anti-inflammatory and hepatoprotective supplement.

Section snippets

Chemicals and reagents

All the chemicals and reagents were procured from Sigma Aldrich Pvt. Ltd. India.

Preparation of doses

Genistein was dissolved in 1.25% aqueous DMSO. All the doses were prepared as a suspension.

Acute oral toxicity —acute toxic class method

Acute oral toxicity study (Ecobichon, 1997) was performed as per OECD-423 guidelines. Wistar rats (n = 6) selected by random sampling technique were used for the study. The animals were fasted overnight and provided full access to water after which Genistein was administered at the dose level of 5 mg/kg body weight by

Lethality and histological analysis

In d-GalN group, rats began to die at 8 h of d-GalN injection. The mortality was 40% at 10 h and stabilized at 87% at 24 h of d-GalN treatment. However, pretreatment with Genistein (5 mg/kg/day) significantly reduced the mortality and attenuated the damage. The ratio of animals which died to the number of animals used was found to be 5:6. The median survival time was found to be 14 h after d-GalN (alone) injection. Genistein pre-treatment significantly reduced the mortality and increased the median

Discussion

FHF is a clinical syndrome of divergent etiology. The consequences of FHF may be hepatic encephalopathy and renal, cardiac, or pulmonary failure and evokes a rapid hepatic dysfunction.

Our current study examined the protective effects of Genistein against inflammation and FHF induced by d-GalN. We found that Genistein is able to reduce oxidative stress induced by d-GalN and inhibits proinflammatory and apoptotic pathways.

d-GalN has been widely used as a model hepatotoxicant that induces FHF

Conflict of interest

The authors declare no conflict of interest with this manuscript.

Acknowledgment

The financial support by University Grants Commission (F.No-7-357/2011(BSR)) is highly acknowledged.

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