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 ... -  《-》

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 ... -  《-》

Elucidating the molecular mechanisms behind the therapeutic impact of median nerve stimulation on cognitive dysfunction post-traumatic brain injury.

Ferroptosis represents a form of regulated cellular death dependent upon iron and lipid peroxidation derivatives, holding considerable implications for cerebral and neurologic pathologies. In the present study, we endeavored to elucidate the molecular mechanisms governing ferroptosis and appraise the therapeutic value of electrical stimulation of median nerve in addressing cognitive impairments following traumatic brain injury (TBI), employing a rodent model. In this study, we established a rat model to investigate the cognitive impairments resulting from TBI, followed by the application of median nerve stimulation (MNS). Initially, rats received an intraperitoneal injection of Erastin (2 mg/kg) prior to undergoing MNS. After 24 h of MNS treatment, the rats were subjected to an open field test to evaluate their cognitive and motor functions. Subsequently, we conducted biochemical assays to measure the serum levels of GSH, MDA and SOD. The structural integrity and cellular morphology of hippocampal tissue were examined through H&E staining, Nissl staining and transmission electron microscopy. Additionally, we assessed the expression levels of proteins crucial for neuronal health and function in the hippocampus, including VEGF, SLC7A11, GPX4, Nrf2, α-syn, NEUN and PSD95. Compared to the control group, rats in the stimulation group demonstrated enhanced mobility, reduced levels of tissue damage, a decrease in MDA concentration, and increased levels of GSH and SOD. Additionally, there was a significant upregulation in the expression of proteins critical for cellular defense and neuronal health, including GPX4, SLC7A11, Nrf2, VEGF, α-syn, NEUN, and PSD95 proteins. Conversely, rats in the Erastin group demonstrated decreased mobility, exacerbated pathological tissue damage, elevated MDA concentration, and decreased levels of GSH and SOD. There was also a notable decrease in the expression of GPX4, SLC7CA11, Nrf2, and VEGF proteins. The expression levels of α-syn, NEUN, and PSD95 were similarly diminished in the Erastin group. Each of these findings was statistically significant, indicating that MNS exerts neuroprotective effect in the hippocampal tissue of rats with TBI by inhibiting the ferroptosis pathway. (1) MNS may enhance the cognitive and behavioral performance of rats after TBI; (2) MNS can play a neuroprotective role by promoting the expression of nerve injury-related proteins, alleviating oxidative stress and ferroptosis process; (3) MNS may inhibit ferroptosis of neuronal cells by activating Nrf2/ GPX4 signaling pathway, thereby improving cognitive impairment in TBI rats.

Zhong YJ ，Liu LL ，Zhao Y ，Feng Z ，Liu Y ... -  《-》

The role and possible mechanism of the ferroptosis-related SLC7A11/GSH/GPX4 pathway in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MI/RI) is an unavoidable risk event for acute myocardial infarction, with ferroptosis showing close involvement. We investigated the mechanism of MI/RI inducing myocardial injury by inhibiting the ferroptosis-related SLC7A11/glutathione (GSH)/glutathione peroxidase 4 (GPX4) pathway and activating mitophagy. A rat MI/RI model was established, with myocardial infarction area and injury assessed by TTC and H&E staining. Rat cardiomyocytes H9C2 were cultured in vitro, followed by hypoxia/reoxygenation (H/R) modeling and the ferroptosis inhibitor lipoxstatin-1 (Lip-1) treatment, or 3-Methyladenine or rapamycin treatment and overexpression plasmid (oe-SLC7A11) transfection during modeling. Cell viability and death were evaluated by CCK-8 and LDH assays. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial membrane potential was detected by fluorescence dye JC-1. Levels of inflammatory factors, reactive oxygen species (ROS), Fe2+, malondialdehyde, lipid peroxidation, GPX4 enzyme activity, glutathione reductase, GSH and glutathione disulfide, and SLC7A11, GPX4, LC3II/I and p62 proteins were determined by ELISA kit, related indicator detection kits and Western blot. The ferroptosis-related SLC7A11/GSH/GPX4 pathway was repressed in MI/RI rat myocardial tissues, inducing myocardial injury. H/R affected GSH synthesis and inhibited GPX4 enzyme activity by down-regulating SLC7A11, thus promoting ferroptosis in cardiomyocytes, which was averted by Lip-1. SLC7A11 overexpression improved H/R-induced cardiomyocyte ferroptosis via the GSH/GPX4 pathway. H/R activated mitophagy in cardiomyocytes. Mitophagy inhibition reversed H/R-induced cellular ferroptosis. Mitophagy activation partially averted SLC7A11 overexpression-improved H/R-induced cardiomyocyte ferroptosis. H/R suppressed the ferroptosis-related SLC7A11/GSH/GPX4 pathway by inducing mitophagy, leading to cardiomyocyte injury. Increased ROS under H/R conditions triggered cardiomyocyte injury by inducing mitophagy to suppress the ferroptosis-related SLC7A11/GSH/GPX4 signaling pathway activation.

Chen B ，Fan P ，Song X ，Duan M ... -  《BMC Cardiovascular Disorders》

Electroacupuncture Downregulating Neuronal Ferroptosis in MCAO/R Rats by Activating Nrf2/SLC7A11/GPX4 Axis.

Ischemic stroke involves various pathological processes, among which ferroptosis is crucial. Previous studies by our group have indicated that electroacupuncture (EA) mitigates ferroptosis after ischemic stroke; however, the precise mechanism underlying this effect remains unclear. In the present study, we developed a rat model of middle cerebral artery occlusion/reperfusion. We chose the main acupoint of the treatment methods of the "Awakening and Opening of the Brain". Rats' neurological function and motor coordination were evaluated by neurological function score and the rotarod test, respectively, and the volume of cerebral infarction was analyzed by 2,3,5-triphenyltetrazolium chloride Staining. The cerebrovascular conditions were visualized by time-of-flight magentic resonance angiography. In addition, we detected changes in lipid peroxidation and endogenous antioxidant activity by measuring the malondialdehyde, glutathione, superoxide dismutase activities, glutathione/oxidized glutathione and reduced nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate ratios. Inductively coupled plasma-mass spectrometry, western blot, reverse transcription-polymerase chain reaction, fluoro-jade B staining, immunofluorescence analysis, and transmission electron microscopy were utilized to examine the influence of EA. The results indicate that EA treatment was effective in reversing neurological impairment, neuronal damage, and protecting mitochondrial morphology and decreasing the cerebral infarct volume in the middle cerebral artery occlusion/reperfusion rat model. EA reduced iron levels, inhibited lipid peroxidation, increased endogenous antioxidant activity, modulated the expression of several ferroptosis-related proteins, and promoted nuclear factor-E2-related factor 2 (Nrf2) nuclear translocation. However, the protective effect of EA was hindered by the Nrf2 inhibitor ML385. These findings suggest that EA can suppress ferroptosis and decrease damage caused by cerebral ischemia/reperfusion by activating Nrf2 and increasing the protein expression of solute carrier family 7 member 11 and glutathione peroxidase 4.

Zhu W ，Dong J ，Han Y 《-》