Total extracts from Abelmoschus manihot (L.) alleviate radiation-induced cardiomyocyte ferroptosis via regulating redox imbalances mediated by the NOX4/xCT/GPX4 axis.

Radiation-induced heart disease (RIHD) is one of the most serious complications in patients receiving chest radiotherapy, partially offsetting its benefits. At present, there is a lack of effective treatments for RIHD. Ferroptosis is a newly discovered type of cell death that results from iron-dependent lipid peroxide accumulation. It was recently shown that irradiation generates severe ferroptosis, providing new insights for the treatment of RIHD. Abelmoschus manihot (L.) possesses excellent pharmacological properties and is widely used in treating various ischemic heart and brain diseases; however, its efficacy and mechanism in treating RIHD are unknown. This study aimed to investigate the efficacy and mechanism of total extracts from A. manihot (L.) (TEA) in treating RIHD. C57BL/6 mice and H9C2 cells were exposed to irradiation to induce RIHD in vivo and in vitro, respectively. In vivo, we evaluated the protective effects of TEA (150 and 300 mg/kg) on RIHD. Body and heart weight changes of mice were calculated in each group, and malondialdehyde (MDA) level, glutathione/oxidized glutathione (GSH/GSSH) and nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) ratios, western blot, heart histology, and immunohistochemistry were used to evaluate TEA effectiveness. We identified the potential mechanism of radiation-induced cardiomyocyte injury in H9C2 cells treated with small interfering RNA. We determined the effective dose of TEA (0.6 mg/mL) using a Cell Counting Kit-8 assay. Intracellular Fe2+ and lipid peroxidation levels were detected by Phen Green™ SK diacetate probe, BODIPY 581/591 C11 staining, and MDA, GSH, and NADPH kits, and the level of target protein was evaluated by immunofluorescence and western blot. Radiation inhibited system Xc-cystine (xCT)/glutathione peroxidase 4 (GPX4) expression and activity in cardiomyocytes in a time and dose-dependent manner. After silencing xCT/GPX4, MDA significantly increased and GSH/GSSH and NADPH/NADP+ ratios were reduced. xCT/GPX4 inhibition drove ferroptosis in radiation-induced H9C2 injury. Oxidative stress in H9C2 was significantly enhanced by irradiation, which also significantly increased NADPH oxidase 4 (NOX4) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression in vivo and in vitro. Inhibition of xCT/GPX4 drove ferroptosis in radiation-induced H9C2 injury, which was aggravated by inactivation of Nrf2 and alleviated by inhibition of NOX4. Compared with the ionizing radiation-only group, TEA improved body weight loss, MDA levels, and histological changes induced by irradiation in mice hearts, and increased the ratio of GSH/GSSH and NADPH/NADP+in vivo; it also reduced lipid peroxidation and intracellular Fe2+ accumulation, restored MDA levels, and elevated the ratios of GSH/GSSH and NADPH/NADP+ in irradiation-injured H9C2 cells. TEA up-regulated Nrf2, xCT, and GPX4 expression and inhibited NOX4 expression in vivo and in vitro. Ferroptosis induced by redox imbalance mediated through the NOX4/xCT/GPX4 axis is a potential mechanism behind radiation-induced cardiomyocyte injury, and can be prevented by TEA.

Xu Z ，Wang Y ，Yang W ，Han W ，Ma B ，Zhao Y ，Bao T ，Zhang Q ，Lin X ... -  《-》

[Ultrafiltration of Angelicae Sinensis Radix and Astragali Radix inhibits ferroptosis and improves pulmonary fibrosis in rats by regulating Nrf2/xCT/GPX4 signaling pathway].

Wang CL ，Ren CZ ，Wang XY ，Chen QL ，Lyu XF ，Zhi XD ，Wu X ，Jiang HG ，Zhao XK ，Li YD ... -  《-》

Naringenin alleviates myocardial ischemia/reperfusion injury by regulating the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) /System xc-/ glutathione peroxidase 4 (GPX4) axis to inhibit ferroptosis.

Xu S ，Wu B ，Zhong B ，Lin L ，Ding Y ，Jin X ，Huang Z ，Lin M ，Wu H ，Xu D ... -  《-》

Salvia miltiorrhiza suppresses cardiomyocyte ferroptosis after myocardial infarction by activating Nrf2 signaling.

Ferroptosis, a recently identified non-apoptotic form of cell death reliant on iron, is distinguished by an escalation in lipid reactive oxygen species (ROS) that are iron-dependent. This phenomenon has a strong correlation with irregularities in iron metabolism and lipid peroxidation. Salvia miltiorrhiza Bunge (DS), a medicinal herb frequently utilized in China, is highly esteemed for its therapeutic effectiveness in enhancing blood circulation and ameliorating blood stasis, particularly during the treatment of cardiovascular diseases (CVDs). Numerous pharmacological studies have identified that DS manifests antioxidative stress effects as well as inhibits lipid peroxidation. However, ambiguity persists regarding the potential of DS to impede ferroptosis in cardiomyocytes and subsequently improve myocardial damage post-myocardial infarction (MI). The present work focused on investigating whether DS could be used to prevent the ferroptosis of cardiomyocytes and improve post-MI myocardial damage. In vivo experiments: Through ligation of the left anterior descending coronary artery, we constructed both a wild-type (WT) and NF-E2 p45-related factor 2 knockout (Nrf2-/-) mouse model of MI. Effects of DS and ferrostatin-1 (Fer-1) on post-MI cardiomyocyte ferroptosis were examined through detecting ferroptosis and myocardial damage-related indicators as well as Nrf2 signaling-associated protein levels. In vitro experiments: Erastin was used for stimulating H9C2 cardiomyocytes to construct an in vitro ferroptosis cardiomyocyte model. Effects of DS and Fer-1 on cardiomyocyte ferroptosis were determined based on ferroptosis-related indicators and Nrf2 signaling-associated protein levels. Additionally, inhibitor and activator of Nrf2 were used for confirming the impact of Nrf2 signaling on DS's effect on cardiomyocyte ferroptosis. In vivo: In comparison to the model group, DS suppressed ferroptosis in cardiomyocytes post-MI and ameliorated myocardial damage by inducing Nrf2 signaling-related proteins (Nrf2, xCT, GPX4), diminishing tissue ferrous iron and malondialdehyde (MDA) content. Additionally, it enhanced glutathione (GSH) levels and total superoxide dismutase (SOD) activity, effects that are aligned with those of Fer-1. Moreover, the effect of DS on alleviating cardiomyocyte ferroptosis after MI could be partly inhibited through Nrf2 knockdown. In vitro: Compared with the erastin group, DS inhibited cardiomyocyte ferroptosis by promoting the expression of Nrf2 signaling-related proteins, reducing ferrous iron, ROS, and MDA levels, but increasing GSH content and SOD activity, consistent with the effect of Fer-1. Additionally, Nrf2 inhibition increased erastin-mediated ferroptosis of cardiomyocytes through decreasing Nrf2 signaling-related protein expressions. Co-treatment with DS and Nrf2 activator failed to further enhance the anti-ferroptosis effect of DS. MI is accompanied by cardiomyocyte ferroptosis, whose underlying mechanism is probably associated with Nrf2 signaling inhibition. DS possibly suppresses ferroptosis of cardiomyocytes and improves myocardial damage after MI through activating Nrf2 signaling.

Wu YT ，Zhang GY ，Li L ，Liu B ，Wang RY ，Song RQ ，Hua Y ，Bi YM ，Han X ，Zhang F ，Wang D ，Xie LP ，Zhou YC ... -  《-》

Electroacupuncture Inhibits Neural Ferroptosis in Rat Model of Traumatic Brain Injury via Activating System Xc(-)/GSH/GPX4 Axis.

Ferroptosis is an iron-dependent regulating programmed cell death discovered recently that has been receiving much attention in traumatic brain injury (TBI). xCT, a major functional subunit of Cystine/glutamic acid reverse transporter (System Xc-), promotes cystine intake and glutathione biosynthesis, thereby protecting against oxidative stress and ferroptosis. The intention of this research was to verify the hypothesis that electroacupuncture (EA) exerted an anti-ferroptosis effect via an increase in the expression of xCT and activation of the System Xc-/GSH/GPX4 axis in cortical neurons of TBI rats. After the TBI rat model was prepared, animals received EA treatment at GV20, GV26, ST36 and PC6, for 15 min. The xCT inhibitor Sulfasalazine (SSZ) was administered 2h prior to model being prepared. The degree of neurological impairment was evaluated by means of TUNEL staining and the modified neurological severity score (mNSS). Specific indicators of ferroptosis (Ultrastructure of mitochondria, Iron and ROS) were detected by transmission electron microscopy (TEM), Prussian blue staining (Perls stain) and flow cytometry (FCM), respectively. GSH synthesis and metabolism-related factors in the content of the cerebral cortex were detected by an assay kit. Real-time quantitative PCR (RT-QPCR), Western blot (WB), and immunofluorescence (IF) were used for detecting the expression of System Xc-/GSH/GPX4 axisrelated proteins in injured cerebral cortex tissues. EA successfully relieved nerve damage within 7 days after TBI, significantly inhibited neuronal ferroptosis, upregulated the expression of xCT and System Xc-/GSH/GPX4 axis forward protein and promoted glutathione (GSH) synthesis and metabolism in the injured area of the cerebral cortex. However, aggravation of nerve damage and increased ferroptosis effect were found in TBI rats injected with xCT inhibitors. EA inhibits neuronal ferroptosis by up-regulated xCT expression and by activating System Xc-/GSH/GPX4 axis after TBI, confirming the relevant theories regarding the EA effect in treating TBI and providing theoretical support for clinical practice.

Li N ，Wang R ，Ai X ，Guo J ，Bai Y ，Guo X ，Zhang R ，Du X ，Chen J ，Li H ... -  《-》