Protective Effects And Mechanisms Of Picroside Ⅱ On Hydrogen Peroxide-Induced L02Cell Damage

Objective:With the model of H2O2damaged L02cells, this research would clear out the liver protective effects of Picroside Ⅱ, and investigate the regulation of Keapl-Nrf2-ARE antioxidant signaling pathway.Methods:1. The protective effects of Pic Ⅱ on the H2O2damaged L02cellsExcept control group,L02cells were pretreated by different concentrations of Pic Ⅱ for3h, and treated by0.6mM H2O2for1h, which induced oxidative damage,then the cells vitality, the scavenging activity of ROS, the LDH content of the cell culture supernatant and mitochondrial membrane potential(MMP) and MDA content were measured to analyze the liver protective effects of Pic Ⅱ.2. The effects of Pic Ⅱ on the Ⅱ-phase detoxification enzymes expression of H2O2damaged L02cells.Except control group, L02cells were pretreated by different concentrations of Pic Ⅱ for3h, and then treated by0.6mM H2O2for1h, which induced oxidative damage. Real-time RT-PCR wes used to test the mRNA expressions of GST、NQO1、Gclc and Gclm.3. The activation effects of Pic II on the signaling pathway Keapl-Nrf2-ARE expression of H2O2damaged L02cell.Except control group, L02cells were pretreated by different concentrations of Pic Ⅱ for3h, and treated by0.6mM H2O2for lh, which induced oxidative damage. Then Real-time RT-PCR and Western Blotting were used to test the mRNA and protein expressions of Keapl, Nrf2and HO-1at1h,24h and48h.Result:1. Compared with the control group, H2O2treatment resulted in markedly decrease of cell viability,, Picroside Ⅱ pretreatment could inhibite the decrease of L02cell viability elevated LDH content and increased level of MMP,MDAand ROS, which were induced by H2O2treatment. The results showed in dose-dependent to some extents.2. Compared with the control group,the mRNA expressions of GST,NQO1,Gclc and Gclm decreased in model group, increased in pic Ⅱ pretreatment groups. And the results showed in dose-dependent to some extents.3. After Pic Ⅱ pretreatment for3h and H2O2treatment for1h, the mRNA and protein expressions of HO-1markedly increased, and the mRNA and protein expressions of Nrf2and Keapl were no significant change. Continuing for24h, the mRNA and protein expressions of HO-1in high concentration PicⅡ pretreatment group and model group are higher than it in control group, the mRNA and protein expressions of Nrf2in the high concentration Pic Ⅱ pretreatment group are higher than other groups, The different treatments had no effects on the mRNA and protein expression of Keapl. Culture for48h, the protein expression of HO-1in this group shows a increasing tendency comparing with the control group and model group, the mRNA and protein expressions of Nrf2in the high concentration PicⅡ pretreatment group were higher than them in the model group, but the mRNA and protein of Nrf2in each treatment groups were lower than them in the control group. PicⅡ had the up-regulation effect on the mRNA expression and the down-regulation effect on the protein expression of Nrf2.Conclusion:1.PicⅡ attenuates H2O2-induced L02cell damage, and the effect is related to direct antioxidant of ROS scavenging.2.Pic Ⅱ increases the antioxidative ability of L02cell to protect the cell from injury by up-regulating the mRNA expression of Ⅱ phase antioxidase.3.PicⅡ could induce the mRNA and protein expression of Nrf2,and increasethe keap1degradation after translation leve,.Up-regulate the expression of antioxidant components ARE’s downstream of the antioxidase to protect the cell form injury. Activating Keap1-Nrf2-ARE signaling pathway is the one of the liver cell protection mechanism of Pic Ⅱ.