The Protective Effect And Mechanism Of Gentiopicroside On Hydrogen Peroxide-induced Oxidative Damage In HepG2 Cells

Background:Oxidative stress(OS)is an important mechanism of various liver injury diseases,which participates in the process of inflammation and liver fibrosis,plays an important role in the pathogenesis of non-alcoholic fatty liver disease and alcoholic liver disease,and is also closely related to the liver injury caused by viral hepatitis.The prevalence of liver disease in Chinese residents is high,but there is a lack of Western medicine in anti-oxidative liver injury,which urgently needs to be developed.Traditional Chinese medicine has unique advantages in the prevention and treatment of liver injury.Clinical application of heat-clearing and dampness-removing Chinese herbal medicines has significant effect in the prevention and treatment of liver injury.In addition,Chinese herbal monomers extracted from natural plants have attracted more and more attention because of their clear structure,wide sources,small toxic and side effects.Many previous studies have shown that some effective components of traditional Chinese medicine have antioxidant effects.Therefore,choosing traditional Chinese medicine monomers as the starting point of research is of great significance for realizing the modernization and internationalization of traditional Chinese medicine.Gentiopicroside is a kind of iridoid terpenoid extracted from Longdancao and Qinjiao,which has anti-inflammatory,antipyretic,antiviral,hepatoprotective,gallbladder-promoting and stomach-protecting effects,but its specific mechanism of action has not been elucidated.Objective:The aim of our study is to investigate the effect of gentiopicroside on hydrogen peroxide(H2O2)induced oxidative damage in HepG2 cells and its molecular mechanism,and to provide experimental basis for the treatment of liver inj ury with traditional Chinese medicine monomers.Methods:The oxidative stress model was established by inducing HepG2 cells with H2O2 for 6 h,and the effect of gentiopicroside on the viability of HepG2 was determined by CCK8 method.Then the experimental groups were divided into blank control group,H2O2 model group,low dose,medium dose and high dose gentiopicroside protection group(10?mol/L,20 ?mol/L,40?mol/L).The content of malondialdehyde(MDA)produced in cells was detected by biological kit.The level of reactive oxygen species(ROS)in cells was detected by fluorescence confocal,the expression level of Nrf2 protein in cells was detected by Western blot and the molecular mechanism of gentiopicroside on Nrf2 activation in HepG2 cells was detected.Results:1.Using different concentration gradient of H2O2 to induce HepG2 cells to establish oxidative stress model for 6 h,the concentration of H2O2 increased exponentially.The results showed that the low concentration group of H2O2 had less effect on the survival rate of HepG2,but with the increase of the concentration of H2O2,the cell survival rate decreased significantly.The IC50 was 798.3?mol/L by statistical analysis,so 800?mol/L was chosen as the modeling concentration in this experiment.2.Gentiopicroside at three concentrations of 10,20 and 40?mol/L was used successively to act on HepG2 cells for 12 h.CCK8 assay showed that all three concentrations of Gentiopicroside had protective effects on oxidative damage caused by H2O2.3.The intracellular MDA level in HepG2 cells in the model group was significantly higher than that in the blank group(0.8767±0.03786 nmol/104 vs 0.5533±0.06028 nmol/104,P<0.01),while the intracellular MDA level(0.7367±0.05508 nmol/104)in the pretreated group was significantly lower than that in the model group(P<0.05).4.The average percentage of intracellular ROS in the model group was significantly higher than that in the blank group(92.67±2.08%vs 50.33±2.08%,P<0.05),and the average percentage of intracellular ROS in the gentiopicroside treatment groups of 10 ?mol/L,20?mol/L,and 40 ?mol/L was(88.33±2.08)%,(87.00±2.00)%,(62.67±5.69)%,which was significantly lower than that in the model group,and gentiopicroside could dose-dependently reduce the oxidative damage caused by H2O2.5.Gentiopicroside can increase the protein amount of Nrf2 in the nucleus,increase AKT phosphorylation,and activate the PI3K/AKT signaling pathway.Conclusions:1.Gentiopicroside has a protective effect against oxidative damage induced by H2O2 in a dose-dependent manner.2.Gentiopicroside can play an antioxidant role by activating the PI3K/AKT signaling pathway and regulating the molecular mechanism of Nrf2 intranuclear metastasis.