Abstract
Drug-induced liver injury (DILI) is a major clinical problem where natural compounds hold promise for its abrogation. Khaya grandifoliola (Meliaceae) is used in Cameroonian traditional medicine for the treatment of liver related diseases and has been studied for its hepatoprotective properties. Till date, reports showing the hepatoprotective molecular mechanism of the plant are lacking. The aim of this study was therefore to identify compounds from the plant bearing hepatoprotective activity and the related molecular mechanism by assessing their effects against acetaminophen (APAP)-induced hepatotoxicity in normal human liver L-02 cells line. The cells were exposed to APAP (10 mM) or co-treated with phytochemical compounds (40 μM) over a period of 36 h and, biochemical and molecular parameters assessed. Three known limonoids namely 17-epi-methyl-6-hydroxylangolensate, 7-deacetoxy-7-oxogedunin and deacetoxy-7R-hydroxygedunin were identified. The results of cells viability and membrane integrity, reactive oxygen species generation and lipid membrane peroxidation assays, cellular glutathione content determination as well as expression of cytochrome P450 2E1 demonstrated the protective action of the limonoids. Immunoblotting analysis revealed that limonoids inhibited APAP-induced c-Jun N-terminal Kinase phosphorylation (p-JNK), mitochondrial translocation of p-JNK and Bcl2-associated X Protein, and the release of Apoptosis-inducing Factor into the cytosol. Interestingly, limonoids increased the expression of Mitogen-activated Protein Kinase Phosphatase (Mkp)-1, an endogenous inhibitor of JNK phosphorylation and, induced the nuclear translocation of Nuclear Factor Erythroid 2-related Factor-2 (Nrf2) and decreased the expression of Kelch-like ECH-associated Protein-1. The limonoids also reversed the APAP-induced decreased mRNA levels of Catalase, Superoxide Dismutase-1, Glutathione-S-Transferase and Methionine Adenosyltransferase-1A. The obtained results suggest that the isolated limonoids protect L-02 hepatocytes against APAP-induced hepatotoxicity mainly through increase expression of Mkp-1 and nuclear translocation of Nrf2. Thus, these compounds are in part responsible of the hepatoprotective activity of K. grandifoliola and further analysis including in vivo and toxicological studies are needed to select the most potent compound that may be useful as therapeutic agents against DILI.
Highlights
Liver is the main site of detoxification and as such, represents the primary target of drug exposure in the body (Kumari and Kakkar, 2012a)
Acetaminophen was purchased from MedChem Express (Monmouth Junction, NJ 08852, United States); Thiazolyl Blue Tetrazolium Bromide, α-Keto-glutaric Acid, L-Alanine, Thiobarbituric Acid, Trichloroacetic Acid, 2 -7 Dichlorodihydrofluorescein diacetate (H2DCFDA), silymarin, Junc N-terminal kinase (JNK) inhibitor SP600125, MITOISO2 – Mitochondria Isolation Kit were purchased from Sigma–Aldrich
APAP was used at the determined concentration of 10 mM. This concentration was found to be toxic for L-02 hepatocytes after 36 h of incubation leading to about 50% decrease of cell viability and great increase of alanine aminotransferase (ALT) leakage into the incubation medium (Figure 2). These observations could be the consequence of metabolic activation of APAP as proven by the overexpression of Cytochrome P450 2E1 (CYP2E1) (Figures 6A,B), depletion of cellular reduced glutathione (GSH) (Figure 6E), overproduction of reactive oxygen species (ROS) (Figure 7A) and increase formation of MDA (Figure 7B) when L-02 hepatocytes were incubated in presence of APAP alone
Summary
Liver is the main site of detoxification and as such, represents the primary target of drug exposure in the body (Kumari and Kakkar, 2012a). DILI is a major clinical problem and public concern worldwide (Jaeschke et al, 2014). It is the case of APAP (N-acetyl-para-amino-phenol), a commonly used analgesic and antipyretic drug that is safe at therapeutic dose; but can cause severe hepatotoxicity and even death at an overdose (Nourjah et al, 2006). APAP-hepatotoxicity cannot only bind to its biotransformation, essentially by the isoform 2E1 of cytochrome P450 enzymes to form a reactive metabolite; N-acetyl-para benzo-quinone imine (NAPQI), and to the excessive generation of ROS. NAPQI and ROS if excessive can covalently bind and/or oxidize cellular macromolecules, causing hepatocytes death and liver injury (Xie et al, 2014). Several studies have recently demonstrated that oxidant stress activates specific downstream biochemical signaling cascades, which perpetuate APAP-induced hepatotoxicity; including the c-Junc N-terminal kinase (JNK) family (Gunawan et al, 2006; Saito et al, 2010; Xie et al, 2014)
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