Liver Protective Effects And Molecular Pharmacological Mechanisms Of Ginsenoside Rg1

Part Ⅰ The protective effect of Rg1 on acetaminophen induced liver injuryObjective:To investigate the liver protective effects and molecular pharmacological mechanisms of ginsenoside Rg1（Rg1）on acetaminophen induced liver injury.Methods:Drug induced liver injury mice model was induced by intraperitoneal injection of APAP.The activities of serum lactate dehydrogenase（LDH）,alanine aminotransferase（ALT）,aspartate transaminase（AST）and the expression of malondialdehyde（MDA）,reduced glutathione（GSH）,superoxide dismutase（SOD）and catalase（CAT）in the liver tissue homogenates as well as hematoxylin-eosin（H&E）staining were used to evaluate the protective effect of Rg1 on APAP-induced liver injury.The protein expression of Nrf2 in the nuclear and cytoplasmic,epoxy chloropropane kelch sample related protein-1（Keap1）,glutamate-cysteine ligase catalytic subunit（GCLC）,glutamate-cysteine ligase modifier subunit（GCLM）,NAD（P）H:quinine oxidoreductase 1（NQO1）,heme oxygenase-1（HO-1）,multidrug resistance-related protein 2（Mrp2）,Mrp3 and Mrp4 were detected by western blot.The mRNA levels of UDP-glucuronyl transferase1a1（Ugt1a1）,Ugt1a6,Ugt2b1,hydroxysteroid sulfotransferase2a1（Sult2a1）,cytochrome P450 2E1（Cyp2e1）,Cyp3a11 and Cyp1a2 were detected by RT-PCR.Nrf2 antagonist all-transretinoic acid（ATRA）and Nrf2 gene silencing test was performed in vivo and in vitro to determine whether the liver protective effects of Rg1 were dependent on Nrf2.Results:Rg1 enhanced antioxidant and detoxification capacity by up-regulating of Nrf2 and its downstream genes GCLC,GCLM,HO-1,NQO1,Ugt1a1,Ugt1a6,Ugt2b1 and Sult2a1.Rg1 enhanced the excretion of reactive or conjugated metabolites by repressing the activities of Cyp2e1,Cyp3a11,Cyp1a2 and up-regulating the expression of Mrp2,Mrp3 and Mrp4.In vivo and in vitro studies further demonstrated that the hepatoprotective effect of Rg1 on liver injury induced by APAP was due to the Nrf2-mediated regulation of above genes after activation of Nrf2.Conclusion:Rg1 was involved in the regulation of hepatic efflux transporters and enzymes,effectively reducing the toxic metabolites accumulation in the liver,enhancing the antioxidant capacity and promoting glutathione synthesis to protect against APAP induced liver injury.Part Ⅱ The protective effect of Rg1 on carbon tetrachloride induced liver injuryObjective:To investigate the liver protective effects and molecular pharmacological mechanisms of Rg1 on carbon tetrachloride induced liver injury.Methods:The protective effects of Rg1 on acute liver injury induced by CCl₄ in mice were evaluated by H&E staining and serum biochemical indicators.The expression of inflammatory factors tumor necrosis factor-α（TNF-α）,cyclooxygenase-2（COX-2）,and inducible nitric oxidesynthase（iNOS）were determined by western blot to investigate the anti-inflammatory mechanism of Rg1 on CCl₄ induced inflammatory response.The number of BrdU-positive hepatocytes was used as an index to reveal the effect of Rg1 on hepatic repair.The protein expression of Nrf2,NQO1,GCLM,bile salt export pump（Bsep）,Mrp2,Mrp3,Mrp4 and the mRNA levels of GCLC,HO-1 and Cyp2e1 were detected to evaluate the anti-oxidative stress and detoxification effects of Rg1 on CCl₄ induced liver injury.Nrf2 antagonist ATRA and Nrf2 gene silencing test was performed in vivo and in vitro to determine whether the liver protective effects of Rg1 were dependent on Nrf2.Results:Rg1 relieved inflammatory response induced by CCl₄ via a down-regulating effect on the expression of inflammatory cytokines（TNF-α,IL-1β,IL-6,COX-2 and iNOS）.Rg1 enhanced antioxidant capacity by up-regulating of Nrf2and its downstream genes GCLC,GCLM,HO-1 and NQO1.Rg1 also enhanced the excretion of reactive or conjugated metabolites by down-regulating the activity of Cyp2e1 and up-regulating the expression of Bsep,Mrp2,Mrp3 and Mrp4.Meanwhile,Rg1 promoted hepatocyte proliferation.The regulatory effect of Rg1 on these factors was eliminated by Nrf2 antagonist ATRA.In vitro gene silencing experiment directly demonstrate that Nrf2 was involved in the hepatoprotective effect of Rg1 in CCl₄induced liver injury mice.Conclusion:Rg1 was involved in the regulation of hepatic efflux transporters and enzymes,effectively reducing the toxic metabolites accumulation in the liver,enhancing the anti-oxidative stress ability,reducing inflammatory reaction and promoting the proliferation of hepatocytes to protect against CCl₄ induced liver injury.Part Ⅲ The protective effect of Rg1 on LPS/D-GalN induced liver injuryObjective:To investigate the liver protective effects and molecular pharmacological mechanisms of Rg1 on lipopolysaccharide/D-galactosamine induced liver injury.Methods:Acute liver injury mice model was induced by intraperitoneal injection of LPS/D-GalN.The severity of liver damage induced by LPS/D-GalN and the protective effect of Rg1 were evaluated by the survival rate,liver/body weight ratio and the gross examination of the livers.Serum ALT,AST,the expression of MDA,myeloperoxidase（MPO）,SOD,GSH,and reactive oxygen species（ROS）in the liver tissue homogenates were determined as indicators of protective effects of Rg1 on liver injury induced by LPS/D-GalN in mice.The mRNA levels of inflammatory factors TNF-α,IL-1β,IL-6,monocyte chemotactic protein-1（Mcp-1）,macrophage inflammatory protein-2（Mip-2）,iNOS and anti-inflammatory factor IL-10 were determined by RT-PCR to investigate the anti-inflammatory mechanism of Rg1.Meanwhile,the expression of TNF-αin mouse liver was determined by immunohistochemistry to further evaluate the anti-inflammatory effect of Rg1.The protein expression of toll-like receptor 4（TLR4）downstream genes nuclear factor-κB p65（NF-κB p65）and mitogen-activated protein kinase（MAPK）were determined by western blot.The mRNA levels of Nrf2 downstream gene were determined by RT-PCR.TLR4 specific inhibitor TAK-242 was performed in vitro to verify the role of TLR4 in anti-oxidative stress and anti-inflammatory response effects of Rg1 on LPS/D-GalN induced liver injury.Results:In the experiment of LPS/D-GalN induced liver injury,Rg1 relieved inflammatory response induced by LPS/D-GalN via a down-regulating effect on the expression of inflammatory cytokines（TNF-α,IL-1β,IL-6,Mcp-1,Mip-2 and iNOS）.Rg1 inhibited the protein expression of TLR4 and its downstream genes including NF-κB and MAPKs,which are involved in inflammatory response.Rg1 dramatically reduced oxidative stress by regulating the expression of efflux transporter Mrp2 and various enzymes including GCLC,GCLM,HO-1 and NQO1.The changes in these genes and protein induced by Rg1 were abrogated by TLR4 antagonist TAK-242 in vitro.Conclusion:Through the inhibition of TLR4,Rg1 reduced the inflammatory reaction and enhanced antioxidative stress ability to protect against LPS/D-GalN induced liver injury.