Quercetin inhibits hydrogen peroxide-induced cleavage of heat shock protein 90 to prevent glutathione peroxidase 4 degradation via chaperone-mediated autophagy

Background: Oxidative stress is caused by the accumulation of Reactive Oxygen Species (ROS) and the depletion of free radical scavengers, which is closely related to Ferroptosis in diseases. Quercetin, as a natural flavonoid compound, has been reported to have multiple pharmacological effects on the basis of its anti-oxidative and anti-ferroptotic activities. This study was designed to explore the specific mechanism of quercetin against Ferroptosis induced by hydrogen peroxide (H₂O₂).

Methods: The HT22 cells (mouse hippocampal neuronal cells) treated with 40 μg·ml^-1 H₂O₂ were used to investigate the role of Ferroptosis in oxidative stress damage and the regulation of quercetin (7.5, 15, 30 μmol·l^-1), as evidenced by assessments of cell viability, morphological damage, Fe²⁺ accumulation, and the expressions of ferroptotic-related proteins. The changes in the expression levels of Glutathione Peroxidase 4 (GPX4), heat shock cognate protein 70 (HSC70), lysosomal-associated membrane protein 2a (LAMP-2a), and heat shock protein (HSP90) were assessed by qPCR, western blotting (WB) and immunofluorescence (IF) assays. Additionally, the interactions of GPX4, HSC70, LAMP-2a, and HSP90 were examined by co-immunoprecipitation (Co-IP) assay to elucidate the impact of quercetin on the degradation pathway of GPX4 and the CMA pathway. To further explore the regulatory mechanism of quercetin, the si-LAMP-2a and HSP90 mutant cells were conducted.

Results: Pretreatment with 30 μmol·l^-1 quercetin for 6 h significantly enhanced the survival rate (p < 0.05), maintained cell morphology, and inhibited Fe²⁺ levels in HT22 cells exposed to H₂O₂ (40 μg·ml^-1). HT22 cells under oxidative stress showed lower expressions of GPX4 and ferritin heavy chain 1 (FTH1), and a higher level of Acyl-CoA synthetase long-chain family member 4 (ACSL4) (p < 0.05). And quercetin significantly reversed the expressions of these ferroptotic proteins (p < 0.05). Moreover, the autophagic lysosomal pathway inhibitor CQ effectively increased the expression of GPX4 in oxidative stress cell model. Further study showed that H₂O₂ increased the activity of macroautophagy and chaperone-mediated Autophagy (CMA), while quercetin notably suppressed the levels of microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ), LAMP-2a, and the activity of lysosomes (p < 0.01). Additionally, quercetin disrupted the interactions of GPX4, HSC70, and LAMP-2a, reduced cellular levels of CMA by decreasing the cleaved HSP90 (c-HSP90), and these effects were reversed in the R347 mutant HT22 cells.

Conclusions: Quercetin has a significantly protective effect on oxidative stress cell model through the inhibition on Ferroptosis, which is related to the degradation of GPX4 via CMA. And quercetin decreases the level of c-HSP90 induced by H₂O₂ to reduce the activity of CMA by binding to R347 of HSP90.

Chaperone-mediated autophagy; Ferroptosis; Heat shock protein 90; Oxidative stress; Quercetin.