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

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). 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. 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. 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.

Peng C ，Li H ，Mao Q ，Tang K ，Sun M ，Ai Q ，Yang Y ，Liu F ... -  《-》

Emodin protects against severe acute pancreatitis-associated acute lung injury by activating Nrf2/HO-1/GPX4 signal and inhibiting ferroptosis in vivo and in vitro.

Severe acute pancreatitis (SAP) has high morbidity, a complicated and dangerous course, and many complications, including severe pulmonary complications. SAP-associated acute lung injury (SAP-ALI) is still a significant challenge for surgeons because of its high mortality. Therefore, more effective treatment methods are urgently needed. Emodin (EMO) has shown tremendous potential in treating many refractory diseases. However, its protection mechanism in SAP-ALI needs to be further clarified. This study was undertaken to investigate the protective effects of EMO against lung injury in SAP rats and alveolar epithelial cells, with a particular focus on the classical ferroptosis pathway. In an in vivo study, forty SD rats were evenly split into five groups: sham operation (SO) group, the biliopancreatic duct was retrogradely injected with 5% sodium taurocholate (STC) to create the SAP group, SAP + EMO group was administered EMO via gavage to the rats following the modeling, SAP + ML385 group (a given inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2)), SAP + ML385 + EMO group. In an in vitro study, alveolar epithelial A549 cell lines were exposed to lipopolysaccharide (LPS) and treated with EMO. ML385 was also used to inhibit the expression of Nrf2. Pancreatic and lung tissue damage was evaluated using histological examination and molecular experiments. Enzyme-linked immunosorbent assays (ELISA) were used to assess the levels of pro-inflammatory cytokines, Fe2+, and associated oxidative stress indicators in the serum and cell supernatant. Real-time polymerase chain reaction (PCR), Western blot (WB), and immunofluorescence were used to find the expressions of related mRNAs and proteins in the lung tissue or A549 cells. The findings demonstrated that suppressing Nrf2 expression exacerbated the inflammatory response brought on by SAP and the pathological alterations of SAP-ALI. Emodin treatment reversed this pathological change by activating the Nrf2/Heme Oxygenase-1 (HO-1)/glutathione peroxidase 4 (GPX4) signal path. Moreover, these results also showed that EMO, contrary to the effects of ML385, suppressed the ferroptosis response, which manifested as up-regulated glutathione (GSH) and GPX4 levels in vivo and in vitro and down-regulated malondialdehyde (MDA), superoxide dismutase (SOD), Fe2+, and reactive oxygen species (ROS) levels. Our results demonstrated that EMO effectively inhibited ferroptosis both in vivo and in vitro, while also modulating the Nrf2/HO-1/GPX4 signaling pathway to provide protection against SAP-ALI.

Shen G ，Wen H ，Li H ，Zhang X ，Lan B ，Dong X ，Ge P ，Luo Y ，Chen H ... -  《BMC GASTROENTEROLOGY》

BRIP1 Induced Ferroptosis to Inhibit Glioma Cells and was Associated with Increased Oxidative Stress.

Chen C ，Wu ZH ，Lu XJ ，Shi JL ... -  《-》

Amentoflavone attenuates homocysteine-induced neuronal ferroptosis-mediated inflammatory response: Involvement of the SLC7A11/GPX4 axis activation.

Elevated homocysteine (Hcy) levels, referred to hyperhomocysteinemia, are associated with an increased risk of several neurological disorders. Ferroptosis and inflammation play a vital role in Hcy-induced neuronal dysfunction. Amentoflavone (AMF), an active natural biflavone compound, exhibits antioxidative, anti-inflammatory, and neuroprotective activities. This study aimed to explore the potential effects of AMF on Hcy-induced neuronal injury, with a particular focus on the underlying mechanisms involving ferroptosis and inflammation. We assessed neuronal damage in HT22 cells by measuring cell viability, lactate dehydrogenase (LDH) release, and proliferation rates. Additionally, we evaluated oxidative stress markers including the levels of reactive oxygen species (ROS), MitoSOX, mitochondrial membrane potential (MMP), malondialdehyde (MDA), and glutathione (GSH). Iron metabolism and ferroptosis-related gene expressions (Ptgs2, Tfr1, and Fth1) were quantified. TheSLC7A11/GPX4 axis was also detected. Our results showed that AMF treatment dramatically mitigated Hcy-induced neuronal injury by increasing cell viability, decreasing LDH release, and promoting cell proliferation. AMF treatment also reduced Hcy-induced oxidative stress and lipid peroxidation, as evidenced by reduced ROS, MitoSOX, MMP, and MDA levels, along with an increased GSH content in HT22 cells. In addition, AMF treatment reduced iron content and ferroptosis-related gene mRNA levels. However, Erastin, a ferroptosis inducer, blocked these neuroprotective effects of AMF. Ferroptosis inhibitor Ferrostatin-1 also attenuated Hcy-induced ferroptosis. Moreover, both AMF and Ferrostatin-1 effectively mitigated Hcy-induced inflammation, which was again antagonized by Erastin. Mechanistically, AMF treatment enhanced SLC7A11/GPX4 axis in Hcy-treated HT22 cells. In conclusion, these findings suggest that AMF possesses neuroprotection against Hcy-induced injury primarily by inhibiting ferroptosis-mediated inflammation, partly through the activation of SLC7A11/GPX4 axis.

Wang Z ，Wang B ，Jin X 《-》

Vaccarin Ameliorates Renal Fibrosis by Inhibiting Ferroptosis via Nrf2/SLC7A11/GPX4 Signaling Pathway.

Vaccarin is a natural flavonoid glycoside with anti-inflammatory, antioxidant and nephroprotective effects. However, the effects of vaccarin on renal fibrosis (RF) and its molecular mechanisms remain unclear. This study aimed to investigate the effects of vaccarin on RF and its molecular mechanisms. Network pharmacology was used to analyze the effect of vaccarin on RF, and molecular docking and molecular dynamics simulations were performed to assess the binding of nuclear factor erythroid 2-related factor 2 (Nrf2) to vaccarin. A mouse model of unilateral ureteral obstruction (UUO) was established in vivo, and human renal tubular epithelial (HK2) cells were induced with transforming growth factor-β (TGF-β) and RSL3, respectively, as an in vitro model. The anti-fibrotic effect of vaccarin was observed by histopathological staining and determination of fibrous markers. Changes in oxidative stress and ferroptosis-related markers were detected by kits, Western blot (WB), qRT-PCR and immunofluorescence (IF). Finally, Nrf2 inhibitors were added to the in vitro model to observe the effects on fibrosis and ferroptosis. Vaccarin and RF cross genes are enriched for oxidative stress. Nrf2 binds stably to vaccarin. Both in vivo and in vitro experiments showed that vaccarin treatment reduced the expression of fibrosis markers, decreased the levels of reactive oxygen species (ROS), malondialdehyde (MDA), lipid peroxidation (LPO) and Fe2+, and increased glutathione (GSH) secretion. In addition, vaccarin down-regulated the expression of Long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2) and NADPH oxidase 1 (NOX1), and up-regulated Nrf2 and its downstream solute transport family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression. Mechanistic studies indicated that vaccarin activated the Nrf2/SLC7A11/GPX4 pathway to inhibit ferroptosis, and this inhibition was effectively reversed by the Nrf2 inhibitor. Vaccarin ameliorates RF by inhibiting ferroptosis via Nrf2/SLC7A11/GPX4 pathway.

Cui M ，Xu Q ，Duan L ，Lu J ，Hu J ... -  《Drug Design Development and Therapy》