Four Active Monomers From Moutan Cortex Play A Protective Role Against Oxidative Stress And Liver Injury By Activating Nrf2/keap1 Signaling Pathway

Objective:The purpose of this study was to extract and isolate the active ingrediants from Moutan Cortex,and was to identify the structures of ingrediants.Additionally,the inhibitory effects and material basis of Moutan Cortex on oxidative stress and liver injury were illustrated.Human liver hepatocellular carcinoma?HepG2?induced by hydrogen peroxide?H₂O₂?was adopted as cell model for the reveal potential anti-oxidative mechanism of ingrediants.Moreover,acute liver injury of mice stimulated by carbon tetrachloride?CCl₄?was used to evaluate their anti-oxidative and hepatoprotective effect.After that,the therapeutic effect of active monomers on acute liver injury by itself or synergistic administration were discussed as well.Methods and results:1.The roots of Moutan Cortex were crushed and ultrasonic extracted with 85%ethanol for 3 times.Extract liquid was firstly condensed and then suspended in water,following different active fractions were obtained by respectively extracting with petroleum ether,ethyl acetate,n-butanol and water.Forthermore,these fractions were isolated and purified by silica gel column and using the crystal to gain four active monomers.The structures were identified as paeonol?Pae?,quercetin?Qct?,?-sitosterol?Sit?and gallic acid?Ga?by ¹³C NMR,¹H NMR and MS.Their purities were determined by HPLC and were 95.38%,95.13%,96.90%,95.24%,respectively.2.In order to verify and compare the in vitro antioxidant capacities of active ingrediants?Pae,Qct,Sit and Ga?from Moutan Cortex.HepG2 induced by H₂O₂ cell was selected as the in vitro evaluation of oxidative stress model.The detection of CCK8 cell proliferation and toxicity was used to confirm the administrated concentrations and times of four monomers and H₂O₂ on HepG2 cell.The Annexin V-FITC/PI kit was employed to analyze the apoptotic rate of HepG2 cell.The fluorescence intensity of reactive oxygen species?ROS?in cells were tested by fluorescence microscopy.ELISA assays were used to measure the level of the total antioxidant capacity?T-AOC?,catalase?CAT?and superoxide dismutase?SOD?of the active ingrediants in the cell supernatant.The expressions of protein linked to oxidative stress and the binding activities of nuclear factor E2-related factor 2-antioxidant response element?Nrf2-ARE?complexes were determined by western blot and EMSA kit.Meanwhile,the expressions of Nrf2 and its downstream proteins were observed after the cell was pretreated by ML385?inhibitor of Nrf2?.The in vitro results indicated that the survival rates of four monomers and H₂O₂ on HepG2 cells apparently reduced with the increase of administrated dosage and time.After the pretreatment of active ingrediants,the apoptosis rates and ROS contents of HepG2cells were inhibited;the levels of T-AOC,CAT and SOD were remarkably up-regulated;the Nrf2/Kelch-like ECH-associated protein 1?Keap1?pathway was activated;the binding activity of Nrf2-ARE and the levels of Nrf2,heme oxygenase-1?HO-1?and quinone oxidoreductase 1?NQO1?expression were also promoted.When HepG2 cell were treated by monomers,the levels of Nrf2,HO-1 and NQO1 were obviously increased,this result was contrary to that stimulated by ML385 inhibitor.It means that these active ingrediants inhibited the oxidative stress were through the activation of Nrf2/Keap1signaling pathway.Among the four ingrediants,the anti-oxidative effects of Pae and Qct exhibited better than that of Sit and Ga,this result provided a theoretical and material basis for deep study on acute liver injury.3.For revealing the independent or synergistic effect of Pae and Qct on anti-oxidative and hepatoprotective activities in vivo experiment,mice were chosen to establish acute liver injury model induced by CCl₄ with intraperitoneal injection of 2%CCl₄.The pathological change in mice livers was explored by hematoxylin and eosin?H&E?staining and immunofluorescence staining.The activities of alanine transaminase?ALT?,aspartate aminotransferase?AST?,glutathione?GSH?and SOD in serum of mice induced by CCl₄ was detected by ELISA kit.The expression of Nrf2 and HO-1 proteins in mice livers were measured by Western blot.Meanwhile,the levels of mRNA of Nrf2,HO-1 and NQO1 in mice livers were analyzed by RT-PCR.The results showed that Pae extremely relieved the symptoms of inflammatory infiltration,central venous hyperemia and hepatocyte necrosis;reduced the content of ALT and AST;enhanced the activities of GSH and SOD in mice serum;augmented the expression of Nrf2 and HO-1 protein and the level of mRNA of Nrf2,HO-1 and NQO1 in mice livers.In addition,other active monomer group as well alleviated the inflammatory symptoms and actived Nrf2/Keap1signaling pathway.The anti-oxidative effect of Pae was significantly better than that of Qct.The synergistic effect of Pae and Qct were not exhibited here.The antioxidant activities of Pae and Qct from Moutan Cortex were independent rather than synergistic effect in this study.Conclusion:To sum up,the active ingrediants extracted from Moutan Cortex were respectively identified as Pae,Qct,Sit and Ga.The in vitro results clearly showed that the four active ingrediants inhibited the oxidative stress by activating Nrf2/Keap1 signaling pathways,scavenging ROS free radicals and enhancing the antioxidative effects.The antioxidant capacities of Pae and Qct were better in vitro than that of Sit and Ga.After that,the Pae and Qct were administered alone or in combination with each other to treat liver damages induced by CCl₄,the in vivo results revealed that Pae and Qct administrated alone or in combination exerted the antioxidative and hepatoprotective abilities,but the effect of Pae administrated alone was stronger than that of the combination of Pae and Qct.Therefore,the antioxidant activity of Pae and Qct from Moutan Cortex was independent rather than synergistic effect.