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 (HO).

METHODS

The HT22 cells (mouse hippocampal neuronal cells) treated with 40 μg·ml HO were used to investigate the role of ferroptosis in oxidative stress damage and the regulation of quercetin (7.5, 15, 30 μmol·l), as evidenced by assessments of cell viability, morphological damage, Fe 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 quercetin for 6 h significantly enhanced the survival rate (p < 0.05), maintained cell morphology, and inhibited Fe levels in HT22 cells exposed to HO (40 μg·ml). 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 HO 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 HO to reduce the activity of CMA by binding to R347 of HSP90.