Hepatoprotective effect of ginsenoside Rg1 from Panax ginseng on carbon tetrachloride‐induced acute liver injury by activating Nrf2 signaling pathway in mice

Oxidative stress and inflammatory response are well known to be involved in the pathogenesis of acute liver injury. This study was performed to examine the hepatoprotective effect of ginsenoside Rg1 (Rg1) against CCl₄‐induced acute liver injury, and further to elucidate the involvement of Nrf2 signaling pathway in vivo and in vitro. Mice were orally administered Rg1 (15, 30, and 60 mg/kg) or sulforaphane (SFN) once daily for 1 week prior to 750 μL/kg CCl₄ injection. The results showed that Rg1 markedly altered relative liver weights, promoted liver repair, increased the serum level of TP and decreased the serum levels of ALT, AST and ALP. Hepatic oxidative stress was inhibited by Rg1, as evidenced by the decrease in MDA, and increases in GSH, SOD, and CAT in the liver. Further research demonstrated that Rg1 suppressed liver inflammation response through repressing the expression levels of inflammation‐related genes including TNF‐α, IL‐1β, IL‐6, COX‐2, and iNOS. In addition, Rg1 enhanced antioxidative stress and liver detoxification abilities by up‐regulating Nrf2 and its target‐genes such as GCLC, GCLM, HO‐1, NQO1, Besp, Mrp2, Mrp3, Mrp4, and down‐regulating Cyp2e1. However, the changes in Nrf2 target‐genes, as well as ameliorative liver histology induced by Rg1 were abrogated by Nrf2 antagonist all‐transretinoic acid in vivo and Nrf2 siRNA in vitro. Overall, the findings indicated that Rg1 might be an effective approach for the prevention against acute liver injury by activating Nrf2 signaling pathway.     

Investigation of the effect of a panel of model hepatotoxins on the Nrf2-Keap1 defence response pathway in CD-1 mice

The Keap1-Nrf2-ARE signalling pathway has emerged as an important regulator of the mammalian defence system to enable detoxification and clearance of foreign chemicals. Recent studies by our group using paracetamol (APAP), diethylmaleate and buthionine sulphoximine have shown that for a given xenobiotic molecule, Nrf2 induction in the murine liver is associated with protein reactivity and glutathione depletion. Here, we have investigated, in vivo, whether the ability of four murine hepatotoxins, paracetamol, bromobenzene (BB), carbon tetrachloride (CCl4) and furosemide (FS) to deplete hepatic glutathione (GSH) is related to induction of hepatic Nrf2 nuclear translocation and Nrf2-dependent gene expression. Additionally, we studied whether hepatic Nrf2 nuclear translocation is a general response during the early stages of acute hepatic chemical stress in vivo. Male CD-1 mice were administered APAP (3.5 mmol/kg), FS (1.21 mmol/kg), BB (4.8 mmol/kg) and CCl4 (1 mmol/kg) for 1, 5 and 24h. Each compound elicited significant serum ALT increases after 24h (ALT U/L: APAP, 3036+/-1462; BB, 5308+/-2210; CCl4, 5089+/-1665; FS, 2301+/-1053), accompanied by centrilobular damage as assessed by histopathology. Treatment with APAP also elicited toxicity at a much earlier time point (5h) than the other hepatotoxins (ALT U/L: APAP, 1780+/-661; BB, 161+/-15; CCl4, 90+/-23; FS, 136+/-27). Significant GSH depletion was seen with APAP (9.6+/-1.7% of control levels) and BB (52.8+/-6.2% of control levels) 1h after administration, but not with FS and CCl4. Western Blot analysis revealed an increase in nuclear Nrf2, 1h after administration of BB (209+/-10% control), CCl4 (146+/-3% control) and FS (254+/-41% control), however this was significantly lower than the levels observed in the APAP-treated mice (462+/-36% control). The levels of Nrf2-dependent gene induction were also analysed by quantitative real-time PCR and Western blotting. Treatment with APAP for 1h caused a significant increase in the levels of haem oxygenase-1 (HO-1; 2.85-fold) and glutamate cysteine ligase (GCLC; 1.62-fold) mRNA. BB and FS did not affect the mRNA levels of either gene after 1h of treatment; however CCl4 significantly increased HO-1 mRNA at this time point. After 24h treatment with the hepatotoxins, there was evidence for the initiation of a late defence response. BB significantly increased both HO-1 and GCLC protein at this time point, CCl4 increased GCLC protein alone, although FS did not alter either of these proteins. In summary, we have demonstrated that the hepatotoxins BB, CCl4 and FS can induce a small but significant increase in Nrf2 accumulation in hepatic nuclei. However, this was associated with modest changes in hepatic GSH, a delayed development of toxicity and was insufficient to activate an early functional adaptive response to these hepatotoxins.     

Role of MRP2 and GSH in intrahepatic cycling of toxins

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. alpha-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR(-) rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 micromol absolute i.v.) to Wistar and Mrp2-deficient TR(-) rats and collected bile. While both arsenite and GSH secretion were absent in TR(-) rats, the total secretion of arsenite into Wistar bile (2.91 micromol) was accompanied by a excess secretion of 24 micromol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR(-) rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.     

α‐Lipoic acid protects against microcystin‐LR induced hepatotoxicity through regeneration of glutathione via activation of Nrf2

Microcystins (MCs), as the most dominant bloom‐forming strains in eutrophic surface water, can induce hepatotoxicity by oxidative stress. Alpha‐lipoic acid (α‐LA) is a super antioxidant that can induce the synthesis of antioxidants, such as glutathione (GSH), by nuclear factor erythroid 2‐related factor 2 (Nrf2). However, the potential molecular mechanism of α‐LA regeneration of GSH remains unclear. The present study aimed to investigate whether α‐LA could reduce the toxicity of MCs induced in human hepatoma (HepG2), Bel7420 cells, and BALB/c mice by activating Nrf2 to regenerate GSH. Results showed that exposure to 10 μM microcystin‐leucine arginine (MC‐LR) reduced viability of HepG2 and Bel7402 cells and promoted the formation of reactive oxygen species (ROS) compared with untreated cells. Moreover, the protection of α‐LA included reducing the level of ROS, increasing superoxide dismutase activity, and decreasing malondialdehyde. Levels of reduced glutathione (rGSH) and rGSH/oxidized glutathione were significantly increased in cells cotreated with α‐LA and MC‐LR compared to those treated with MC‐LR alone, indicating an ability of α‐LA to attenuate oxidative stress and MC‐LR‐induced cytotoxicity by increasing the amount of rGSH. α‐LA can mediate GSH regeneration through the Nrf2 pathway under the action of glutathione reductase in MC‐LR cell lines. Furthermore, the data also showed that α‐LA‐induced cytoprotection against MC‐LR is associated with Nrf2 mediate pathway in vivo. These findings demonstrated the potential of α‐LA to resist MC‐LR‐induced oxidative damage of liver.     

Multidrug resistance-related protein 1 (MRP1) function and localization depend on cortical actin

MRP1 (ABCC1) is known to be localized in lipid rafts. Here we show in two different cell lines that localization of Mrp1/MRP1 (Abcc1/ABCC1) in lipid rafts and its function as an efflux pump are dependent on cortical actin. Latrunculin B disrupts both cortical actin and actin stress fibers. This results in partial loss of actin and Mrp1/MRP1 (Abcc1/ABCC1) from detergent-free lipid raft fractions, partial internalization of Mrp1/MRP1 (Abcc1/ABCC1), and reduction of Mrp1/MRP1 (Abcc1/ABCC1)-mediated efflux. Pretreatment with nocodazole prevents latrunculin B-induced loss of cortical actin and all effects of latrunculin B on Mrp1 (Abcc1) localization and activity. However, pretreatment with tyrphostin A23 does not prevent latrunculin B-induced loss of cortical actin, lipid raft association, and efflux activity, but it does prevent latrunculin B-induced internalization of Mrp1 (Abcc1). Cytochalasin D disrupts actin stress fibers but not cortical actin and this inhibitor much less affects Mrp1/MRP1 (Abcc1/ABCC1) localization in lipid rafts, internalization, and efflux activity. In conclusion, cortical actin disruption results in reduced Mrp1/MRP1 (Abcc1/ABCC1) activity concomitant with a partial shift of Mrp1/MRP1 (Abcc1/ABCC1) out of lipid raft fractions and partial internalization of the ABC transporter. The results suggest that reduced Mrp1 (Abcc1) function is correlated to the loss of lipid raft association but not internalization of Mrp1 (Abcc1).     

Glutathione-dependent interaction of heavy metal compounds with multidrug resistance proteins MRP1 and MRP2

The interactions of three heavy metal-containing compounds, cisplatin (CDDP), arsenic trioxide (As₂O₃), and mercury dichloride (HgCl₂), with the multidrug resistance transporters MRP1 and MRP2 and the involvement of glutathione (GSH)-related processes herein were investigated. In Madin–Darby canine kidney cells stably expressing MRP1 or MRP2, viability, GSH content, calcein efflux and polarized GSH efflux were measured as a function of exposure to CDDP, As₂O₃ and HgCl₂. In isolated Sf9-MRP1 and Sf9-MRP2 membrane vesicles, the interaction with MRP-associated ATPase activity was measured. In the latter model system adduct formation with GSH is not an issue.

The data show that (1) CDDP interacts with both MRP1 and MRP2, and GSH appears to play no major role in this process, (2) As₂O₃ interacts with MRP1 and MRP2 in which process GSH seems to be essential, and (3) HgCl₂ interacts with MRP1 and MRP2, either alone and/or as a metal–GSH complex.     

Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines.

The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and contribute to the current knowledge of drug transport. The aim of the present study was to characterize three rat epithelial cell lines for transporter expression and activity. Transporter expression was assessed in intestinal IEC-6 and renal GERP and NRK-52E cells using RT-PCR and Western blot analysis. Pgp and Mrp transport activity were analyzed by measuring calcein accumulation and glutathione-S-bimane efflux, respectively. The three cell lines showed Pgp expression and Pgp-dependent transport, both decreasing with culture time after reaching confluency. Besides Pgp, cells expressed Mrp1, Mrp3, Mrp4, and Mrp5, while Mrp2 and Mrp6 were absent. In addition, they showed temperature- and Mrp-dependent efflux of glutathione-S-bimane. Exposure to a panel of different inhibitors showed that this efflux was probably mediated by Mrp4. In conclusion, the three rat epithelial cell lines investigated showed Pgp and Mrp expression and transport. Mrp dependent transport was most likely mediated by Mrp4. In future, these cell lines may be used as in vitro models to study drug transport.     

Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l-gamma-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K(m) for glutamate and ATP, and increasing the K(i) for GSH inhibition. To characterize the enzyme structure-function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.