MAPEG Related Mechanisms Of Aristolochic Acid I Induced Hepatotoxicity In Canines

Part Ⅰ. MAPEG mediated inflammation of aristolochic acid I induced hepatotoxicity in canines.Objective:To evaluate hepatotoxicity of aristolochic acid I (AAI) in canines, and the influence of AAI on hepatic inflammatory mediator cysteinyl leukotrienes (cysLTs) and prostaglandin E2(PGE2), which is mediated by membrane associated proteins ineicosanoid and glutathione metabolism (MAPEG) superfamily. Methods:Canines were treated with AAI capsules of3mg/kg/day orally, while control group were given empty capsules (n=4). All canines were administrated for10days continuously, and sacrificed for collection of hepatic samples2days after drug withdrawal. Blood samples of canines were collected for liver damage evaluation after0,1,5,9,11days of AAI treatment. Hematoxylin and eosin staining was used to assess hepatic pathological status. The modification of cysLTs was detected by EIA kit. Western blot and immunohistochemistry (IHC) were applied to evaluate the expression and distribution of5-lipoxygenase activating protein (FLAP), microsomal glutathione transferase2(mGST2), mGST3, leukotriene C4synthase (LTC4S) and microsomal prostaglandin E synthase (mPGES-1) of canines liver.Results:Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were up-regulated significantly after10days of AAI administration. Hepatic sections were characterized by extravasated blood in hepatic sinusoid and central veins, infiltrated inflammatory cells around central veins, and disarranged hepatic lobule. Which indicate AAI may induce hepatic inflammation of canines. Synthesis of cysLTs was increased through FLAP and mGST2overexpression, while mGST3and LTC4S had no significant changes. FLAP was located in hepatic kupffer cells, while mGST2was expressed in cytoplasm of hepatocytes. Suggesting that AAI probably induces hepatic inflammation of canines, through FLAP and mGST2mediated up-regulation of inflammatory mediator cysLTs. Although total expression of hepatic mPGES-1was down-regulated, the expression was up-regulated significantly in regions around central veins, which indicates AAI may damage regions of central veins through activation of mPGES-1in these areas.Conclusions:After exposed to3mg/kg/day of AAI capsules for10days, AAI induces hepatic inflammation of canines mainly through (1) FLAP and mGST2mediated elevation of cysLTs,(2) regional increase of mPGES-1around central veins. Part Ⅱ. Mechanisms of aristolochic acid I induced hepatotoxicity in canines.Objective:To evaluate whether inflammation signaling transduction pathways of interleukin-6(IL-6) and epidermal growth factor receptor (EGFR) are involved in AAI induced hepatotoxicity in canines, and its related molecular mechanisms.Methods:Canines were treated with AAI capsules of3mg/kg/day orally, while control group were given empty capsules (n=4). All canines were given continuous administration for10days, and sacrificed for collection of hepatic samples2days after drug withdrawal. Nucleus nuclear factor-KB (NF-κB) and phosphorylation of cytoplasm inhibitor of κB (IκB) were detected by western blot. Production of hepatic IL-6was measured by ELISA. Expression and location of IL-6Ra and EGFR were determined by western blot and IHC analysis. Mitochondrial dependent apoptosis pathway related proteins including Bax, Bcl-2, caspase3and cytochrome C; mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38were performed with western blot analysis.Results:Nuclear translocation of NF-κB was activated as well as enhancement of cytoplasm IκB phosphorylation. Reflecting NF-κB translocation participates in AAI induced hepatic inflammation of canines. Both content of IL-6and expression of IL-6Ra were increased, IL-6Ra was located in cytomembrane of hepatocytes, but p-EGFR expression was decreased. Suggesting AAI probably induces hepatic inflammation via IL-6related pathways selectively. ERK and JNK, but not p38, were activated by AAI treatment. Suggesting AAI may induces generation of cysLTs through activation of ERK, while JNK probably participates in mitochondrial dependent apoptosis. Enhanced Bax/Bcl-2ratio and release of cytochrome C, as well as cleaved caspase3were observed in liver of canines after AAI treatment, which indicates JNK induced mitochondrial dependent apoptosis pathway may participate in AAI induced hepatotoxicity.Conclusions:AAI induces hepatic inflammation of canines probably via (1) inflammatory mediator cysLTs induced NF-κB translocation, and sequentially increased secretion of IL-6,(2) JNK activated mitochondrial dependent apoptosis. Meanwhile, AAI could induce hepatic generation of cysLTs through activation of ERK.