GSH Depletion Sensitizes T-BHP-Induced Ferroptosis In Hepatocytes: Protective Effects Of Caffeic Acid Phenethyl Ester As Well As Baicalein

Objective of the study:The current global trend of increasing incidence of liver diseases year by year has caused heavy mental and economic burden to patients and society.Recent studies have found that liver diseases are closely related to ferroptosis,and many liver diseases exhibit ferroptosis characterized by increased lipid peroxidation and iron ions and inhibition of antioxidant system during the development of liver diseases,and screening for compounds that can inhibit ferroptosis has the potential to achieve the goal of treating liver diseases.Both enhanced lipid peroxidation and inhibition of the antioxidant system can lead to ferroptosis in cells.In constructing an experimental model of ferroptosis,a model that enhances lipid peroxidation and inhibits the antioxidant system is more consistent with the development of liver disease than a single inducing factor,and this model will be more accurate and comprehensive in assessing the therapeutic potential and mechanism of action of compounds on liver disease.Flavonoids are widely found in many plants and fruits.Baicalein（BA）,one of the flavonoids,has been shown to have a neuroprotective effect in a disease model of post-traumatic seizures by inhibiting ferroptosis,and thus BA has the potential to inhibit ferroptosis in the treatment of liver diseases.Caffeic acid phenethyl ester（CAPE）has anti-inflammatory and antioxidant activity and chelates iron ions,but whether it can inhibit ferroptosis in hepatocytes has not been studied.Therefore,we constructed a model of hepatocyte ferroptosis that enhances lipid peroxidation while inhibiting the antioxidant system,and on this basis,we investigated the inhibitory effects of BA and CAPE on ferroptosis and evaluated the application prospects of BA and CAPE in the treatment of liver diseases.Study content:L02 liver cells were treated（BSO/t-BHP）with a combination of tert-butyl hydroperoxide（t-BHP）,which enhances cellular lipid peroxidation,and L-buthionine-sulfoximine（L-BSO）,which inhibits intracellular antioxidant glutathione synthesis.An ferroptosis model was constructed to examine the inhibitory effects of CAPE and BA on ferroptosis in hepatocytes by detecting cell viability,cell morphology,cell membrane integrity,oxidative stress levels,Fe²⁺and glutathione content.Results:（1）BSO/t-BHP induced ferroptosis in hepatocytes:compared to the BSO and t-BHP groups,BSO/t-BHP significantly exacerbated L02 cell damage,resulting in decreased cell viability,impaired cell membrane integrity,increased levels of oxidative stress,and increased Fe²⁺content,and ferroptosis inhibitors（desferrioxamine,Ferrostatin-1,and Liproxstatin-1）can effectively inhibit the cell damage caused by BSO/t-BHP.（2）Both BA and CAPE effectively inhibited BSO/t-BHP-induced ferroptosis:increased cell viability,protected damaged cell membranes,reduced oxidative stress levels and Fe²⁺content,but did not increase glutathione content.Conclusion:CAPE and BA can effectively inhibit BSO/t-BHP-induced ferroptosis in hepatocytes by reducing iron overload and have potential for application in the treatment of liver diseases.