Study On Cytoprotective Effective Of Paeonol Against Alcohol-Induced Acute Liver Injury

In China,as people’s living standards have greatly increased,the consumption of alcoholic beverages has continued to increase,and the incidence of alcoholic liver disease(ALD)has also increased year by year.At present,the relevant treatment of ALD has attracted widespread attention.The pathogenesis of ALD has also been widely studied.However,there is no breakthrough in the treatment of alcoholic liver disease.The precise pathogenesis needs further elucidation.Paeonol is an active ingredient extracted from the dry root barks of the peony family Ranunculaceae.Its scientific name is 2’-hydroxy-4’-methoxyacetophenone,which is reported to have analgesic,anti-inflammatory,antipyretic and inhibit allergy and other pharmacological activities.In this thesis,HepG2 cells and C57BL/6 mice were used as in vitro and in vivo models to explore the protective effect and preliminary molecular mechanism of paeonol on acute alcoholic liver injury.The content and results of this study are as follows:1.Paeonol pretreatment had protective effects on alcohol-induced HepG2 cell injury.The results of MTT assay showed that 300 mM alcohol did not significantly inhibit the survival rate of HepG2 cells,but the subsequent experimental results showed that 300 mM alcohol significantly increased the levels of ROS and inflammatory factors in HepG2 cells,indicating that 300 mM alcohol can not kill HepG2 in large quantities.However,300 mM alcohol have destroyed the redox balance within the cells and caused an inflammatory reaction.Pretreatment with paeonol(2,6 and 18 μM)inhibited the increase of ROS and inflammatory cytokines in HepG2 cells induced by alcohol in a concentration-dependent manner.Cell morphology experiments also showed that paeonol pretreatment had protective effects on alcohol-induced HepG2 cell injury.2.Paeonol protects HepG2 cells against alcohol via SIRT1/Nrf2/NF-κB signaling pathway.The cells were pretreated with medium containing paeonol(2,6 and 18 μM)or physiological saline instead of paeonol for 24 hours and then treated with alcohol(300 mM)for 24 hours.Paeonol pretreatment promoted the nuclear transfer of Nrf2 and inhibited NF-κB,and reduced the expression of alcohol-induced anti-oxidation proteins NQO-1 and HO-1.SIRT1 plays an important role in regulating the expression of Nrf2.Pretreatment with paeonol inhibits alcohol-induced reduction of HO-1,NQO-1,SIRT1,and nuclear Nrf2,while the treatment of paeonol with SIRT1 inhibitor NAM attenuates the ability of paeonol to maintain these proteins.3.The results of serum markers and histopathological observations all confirmed the protective effect of paeonol on alcohol-fed C57BL/6 mice.Alcohol treatment significantly increased the serum ALT and AST levels in C57BL/6 mice,while the ALT and AST levels in the paeonol pretreatment group were closer to the normal group.Paeonol pretreatment significantly inhibited alcohol-induced reduction in serum GSH consumption and TNF-α expression.HE staining showed that the liver sections from the normal group and paeonol single group had complete central vein and radioactive heparin.Alcohol treatment causes marked edema,balloon degeneration and irregular hepatic cords.However,pretreatment with paeonol significantly inhibited alcohol-induced liver injury.Slices of alcohol+ paeonol(25 mg/kg)showed limited edema,ballooning degeneration,and irregular hepatic cords,there was no obvious pathological changes in hepatocytes in the alcohol + paeonol(25 mg/kg).4.Paeonol inhibited alcohol-induced liver injury in C57BL/6 mice by regulating nuclear transfer of Nrf2 and NF-κB.Western blotting showed that compared with the control group,paeonol treatment significantly promoted SIRT1 expression,promoted Nrf2 nuclear translocation,and promoted NF-κB p65 cytoplasmic metastasis.For SIRT1,compared with the control group,SIRT1 expression in the alcohol-treated group did not change significantly,but the SIRT1 expression level in the paeonol-treated group was higher than that in the corresponding paeonol+alcohol-treated group.Compared with the control group,the expression of Nrf2 in the cytoplasm of the alcohol treatment group increased,and the expression of cytoplasmic Nrf2 was decreased in the paeonol +alcohol group,and the expression of nuclear Nrf2 was increased.Compared with the control group,the expression of NF-κB in the alcohol treatment group increased,the expression of NF-κB in the paeonol + alcohol group decreased,and the expression of the cytoplasmic NF-κB increased.In summary,paeonol pretreatment has protective effects on acute alcoholic liver injury.By inhibiting the expression of SIRT1,Nrf2,HO-1,NQO-1 and other proteins against alcohol-induced oxidative stress,it reduces inflammation induced by alcohol,such as TNF-a,IL-6 and IL-1β,by inhibiting the expression of NF-κB.Factors release,antagonize the excessive inflammatory response.