Protective Effects And Mechanisms Of Hyperoside On Hydrogen Peroxide-Induced L02 Cell Damage

At present, hepatitis, alcohol-induced liver injury, nonalcoholic fatty liver, ischemia-reperfusion liver injury, drug-induced liver disease and hepatoma have become the public health problems. It has been recently recognized that oxidative stress participates in the pathophysiologic changes of various liver disorders. Vitamin A,β-carotene, vitamin E, vitamin C and GSH are usually used for scavenging excess ROS. However, this effect may interfere with the normal physiological function of free radicals, such as NO and H2O2. Therefore, searching for antioxidants which enhance cellular defense system and maintain the redox homeostasis may be of crucial importance for intervention of oxidative liver disorders.Recent studies have demonstrated that the cellular antioxidant system is tightly regulated by the Keap1-Nrf2-ARE signaling pathway, which is known to up-regulate the expression of antioxidant genes. Thus, inducers of this signaling pathway may represent a promising strategy against oxidative liver disorders.Flavonoids have been identified to exhibit a broad variety of beneficial effects on various liver disease. Most of these effects are associated with their antioxidant properties. Flavonoids can exhibit indirect antioxidant protection against oxidative stress through inducing antioxidant enzymes by modulating the Keap1-Nrf2-ARE signaling pathway. However, the precise role of the Keap1-Nrf2-ARE signaling pathway in flavonoids-mediated cytoprotective effects remains to be elucidated. Hyperoside(Hyp) is a glycoside of flavonol, has been reported to possess cytoprotective effects against ischemic brain damage, myocardial injury, liver damage and kidney damage. In addition to its properties of direct free radical scavenging and metal-chelation, hyperoside may be able to modulate the activities of antioxidant enzymes. However, very few studies to date have focused on the gene expressions of these antioxidants, even though this mechanism may account for the increased antioxidant enzymatic activity associated with hyperoside treatment. To this end, the present study was designed to investigate the protective effect of hyperoside against H2O2-induced cell damage and to assess its capability to activate the Keap1-Nrf2-ARE signaling pathway.Methods1. Development and validation of a HPLC method for the determination of Hyp in L02 cells after 0, 3, 6, 12, 24 h of incubation.2. The cytoprotective effects of Hyp were evaluated by cell viability assay, LDH leakage study, measurement of intracellular ROS scavenging activity and MMP. The activities of SOD and HO-1 were also determined by spectrophotometry.3. After hyperoside treatment, RT-PCR and real time RT-PCR were used for analysis of HO-1, Nrf2 and Keap1, respectively. HO-1 transcriptional activity was evaluated by luciferase reporter assay. The HO-1, Nrf2, Keap1 and Bach1 expression and the phosphorylation of MAPK were analyzed by western blot.Results1. A HPLC method for determination of Hyp in L02 cells was developed. The stability, repetition, and line relation of this HPLC method is very well. Intracellular concentration of Hyp was increased with extended incubation time. It peaked at 24 h of hyperoside administration (11.88%).2. H2O2 treatment resulted in markedly decrease of cell viability, increase of LDH leakage rate, decrease of MMP and ROS scavenging activity, which were dose-dependently (10-800μM) inhibited by hyperoside pretreatment. The cell viability was increased in a time-dependent manner after 6, 12 and 24 h of Hyp administration. Interestingly, the ROS scavenging activity in L02 cells was transiently decreased after 1h of hyperoside pretreatment while the HO-1 activity was increased with the same incubation time. Both activities were time-dependently increased with prolonged periods of incubation.3. Hyperoside treatment was also associated with increased HO-1 expression at both mRNA and protein levels; furthermore, these up-regulations were dose- and time-dependent. Hyperoside treatment enhanced Nrf2 mRNA and protein expressions in dose- and time-dependent manners, but did not significantly affect Keap1 mRNA expression. The nuclear translocation of Nrf2 was also facilitated by hyperoside. Keap1 protein expression in the cytoplasm was significantly decreased after 6 or 12 h of hyperoside treatment (200μM). Extending the treatment period to 24 h did not result in further significant changes to Keap1 concentration. Keap1 was not detected in the nucleus under the same experimental conditions.4. Western blot analysis revealed that hyperoside treatment (200μM) was associated with increased phosphorylation of ERK and p38; however, phosphorylation of JNK was unaffected by the presence of hyperoside. In addition, treatment with specific inhibitor of ERK (PD98059) or p38 (SB203580) significantly suppressed hyperoside-mediated Nrf2 nuclear translocation. In contrast, the activation of Nrf2 nuclear translocation was not inhibited by the inhibitor of JNK (SP600125).5. The protein level of Bach1 in the nucleolar compartment was significantly decreased after 3 h of hyperoside treatment (200μM), which was inhibited by LMB.Conclusions1. Hyp is able to penetrate L02 cell membrane and the intracelllular concentration of Hyp was increased with the incubation time, and peaked at 24 h.2. Hyp attenuates H2O2-induced L02 cell damage.3. The increase in ROS scavenging activity may contribute to Hyp's cytoprotective effect.4. Hyp attenuates H2O2-induced L02 cell damage by up-regulating HO-1 expression via the MAPK-dependent Keap1-Nrf2-ARE signaling pathway.5. Facilitation the translocation of Bach1 from nuclear to cytoplasm is another important mechanism of Hyp's cytoprotective effect.