| In China,excessive Acetaminophen(APAP)is a common cause of acute liver failure(ALF).Previous studies have reported that APAP-induced liver injury is related to the accumulation of lipid peroxide caused by the disorder of cellular antioxidant system.Ferroptosis is a recently discovered emerging mode of cell death that is highly associated with intracellular iron overload,oxidative stress,and lipid peroxidation.Regarding the ways of APAP-induced hepatocyte death,it is generally believed that there are cell apoptosis,cell necrosis and programmed necrosis.However,multiple recent studies have shown that ferroptosis is significantly involved in APAP-induced cell death and has the potential to be a new therapeutic target.S-allylcysteine?is a water-soluble compound extracted from ripe garlic.According to relevant research reports,SAC has strong antioxidant,anticancer and anti-inflammatory properties in various diseases,but its effect on ferroptosis and APAP hepatotoxicity is rarely reported.In this experiment,we mainly explored the protective effect of SAC on APAP-induced hepatocyte death,whether ferroptosis is involved,and its possible mechanism.Objective:The study will investigate the protective effect of SAC on APAP-induced hepatocyte injury and its possible mechanism.Methods:1.MTT assay will be used to detect the toxic effects of SAC,ferroptosis inhibitor Fer-1 and Nrf2 pathway inhibitor ML385 on L-02 cells;to detect the toxic effects of different concentrations of APAP on L-02 cells;and to detect the effect of SAC on APAP-induced liver Protective effect in cell damage,and using Fer-1 as a positive control,set up a reversion experiment with ML385.2.In order to explore the antioxidant effect of SAC,the active oxygen detection kit will be used to detect intracellular active oxygen to evaluate the state of cellular oxidative stress.3.Intracellular GSH and MDA will be detected to check the content of lipid peroxide in LO2 cells in this model,and to observe the protective effect of SAC and Fer-1 on lipid peroxidation in L-02 cells.4.In order to prove the occurrence of ferroptosis,RT-qPCR will be used to detect the mRNA expression of four important regulatory molecules of ferroptosis,xCT,GPX4,Tf R1 and PTGS2,and reflect the regulation of these molecules by SAC.5.RT-qPCR and WB will be used to detect the changes of Nrf2/NQO1/HO-1molecules in each group to verify whether SAC achieves protective effect through Nrf2 pathway.Results:1.In the MTT experiment,the results showed that when the concentration of Fer-1 was 1u M,the concentration of ML385 was 2.5u M and the concentration of SAC was 200 u M,it had no obvious effect on the proliferation activity of L-02;the cytotoxicity of APAP on L-02 was concentration-dependent,at a concentration of10 m M,it was close to the median lethal dose;compared with the APAP group,the cell survival rate after SAC pretreatment was significantly increased,and it was concentration-dependent,and the protective effect tended to be the largest at a concentration of 100 u M;after Fer-1 Cell viability increased after pretreatment;the protective effect of SAC could be partially abolished by the pathway inhibitor ML385.2.The results of ROS detection showed that the ROS level in the APAP group was significantly increased,indicating that the intracellular oxidative stress was highly activated.After pretreatment with SAC and Fer-1,the ROS level was significantly lower than that of the APAP group,which suggested that the two could reduce oxidative stress damage by reducing the production of ROS.In addition,compared with the SAC group,the ROS level in the ML385 group rose,indicating that the protective effect of SAC was partially eliminated by ML385.3.The results of GSH and MDA detection showed that after APAP treatment,the concentration of MDA in L-02 cells was significantly increased,and GSH was decreased,and SAC and Fer-1 significantly reduced the level of MDA in cells and increased the level of GSH,and this effect can be reversed by ML385.4.The results of RT-qPCR detection of xCT,GPX4,Tf R1 and PTGS2: APAP treatment can reduce the expression of xCT and GPX4 in L-02 cells,and increase the expression of Tf R1 and PTGS2,while SAC and Fer-1 pretreatment reversed these changes.5.The results of RT-qPCR and WB detection of Nrf2/NQO1/HO-1: WB results showed that APAP treatment could reduce the expression of NQO1 and HO-1 protein,but had no significant effect on the expression of Nrf2 protein;SAC treatment could activate and increase Nrf2,The expression of NQO1 and HO-1 reversed the inhibition of APAP,and the activation of SAC could be eliminated by ML385.Fer-1 can also activate the expression of the above three proteins.The results of RT-qPCR were similar to those of WB.Conclusions:1.SAC pretreatment reduced APAP-induced L-02 cell damage,which may be related to the reduction of ROS generation,GSH depletion,and MDA accumulation.2.Fer-1 can alleviate APAP-induced L-02 cell damage,suggesting that ferroptosis may be involved in APAP-induced liver cell death.3.APAP treatment caused lipid peroxidation in L-02 cells and decreased the expression of ferroptosis markers xCT and GPX4 mRNA,while increasing the expression of ferroptosis markers Tf R1 and PTGS2 mRNA.SAC reversed these changes,indicating that SAC may achieve its protective effect by inhibiting ferroptosis.4.The protective effect of SAC on APAP-induced L-02 cell damage is achieved by activating Nrf2 and downstream proteins NQO1 and HO-1,and the activation of this pathway may be the mechanism of its inhibition of ferroptosis... |
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