Effect Of Hyperoside On Nrf2-ARE Pathway And Its Protective Effect On Oxidative Stress-Induced L02Cell Damage

Nuclear factor erythroid-2-related factor2plays the role of resisting oxidative stress-induced damage through acting on the antioxidant response element in the promoter region of the antioxidative proteins. Nrf2-ARE is the most important endogenous antioxidant signaling pathways. Hyperoside, an natural product that is widely present in a variety of plants and fruits, belongs to flavonoids compounds. It has been reported to exhibit extensive pharmacological activities, whose mechanism is mainly antioxidant property. Oxidative stress, which is the main pathological mechanism of liver diseases such as alcoholic liver disease, nonalcoholic fatty liver, liver ischemia-reperfusion injury. The flavonoids have been identified to possess the antioxidant effect on the prevention and treatment of liver diseases.ObjectiveIn this study, hypoxia and H2O2were used to make human normal liver cell (L02) damage model, in order to investigate the effect of Hyp on Nrf2-ARE pathway and its pre-protective effect on oxidative stress-induced L02cell injuryMethodsIn vitro cultured L02cells were divided into control group, hypoxia model group, H2O2model group, three different doses of Hyp preconditioning groups. Cell viability was detected by CCK-8. SOD activity and MDA content were measured by the corresponding kits. Flow cytometry was employed to examine the level of ROS. Then the groups were renewedly divided into control group, hypoxia model group, H2O2model group, Hyp treatment and preconditioning group. qRT-PCR and Western blotting were used to determine the gene and protein expression of antioxidative proteins(HO-1, SOD, CAT, GPx) and relative factors(Nrf2, Keapl, Bach1).ResultsHypoxia and H2O2-caused oxidative stress significantly decreased L02cell activity and SOD activity, remarkably increased MDA content and ROS level. In the two model, Hyp preconditioning notably improved these indicators, namely, it weakened the decrease of cell activity and SOD activity, the increase of MDA content and ROS level in order to attenuate oxidative stress-induced L02cell injury. qRT-PCR and Western blot results showed, hypoxia and H2O2-caused oxidative stress significantly upregulated the expression of antioxidative protein HO-1and transcription factor Nrf2. No matter before oxidative damage or after, Hyp treatment further increased the expression of antioxidative protein HO-1, SOD, CAT, GPx and transcription factor Nrf2evidently, meanwhile down-regulated the Bachl expression level, but had no obvious effect on Keapl.ConclusionHypoxia and H2O2-caused oxidative stress induces L02cell injury, Hyp attenuates this damage and pre-protect L02cell, whose mechanism is probably the up-regulation of antioxidative proteins through Nrf2-ARE signaling pathway. The paper takes Hyp as the research object and explores its mechanism from the point of Nrf2/Keapl-Bachl competitive regulation of antioxidative proteins to promote the restoration of redox homeostasis. It provides the experimental proofs to clarify the pharmacological mechanism of flavonoids. It will develop new strategy to take full advantage of traditional Chinese medicine resources against oxidative liver injury.