Background/Aims: Cerebral ischemia-reperfusion (I/R) injury involves multiple independently fatal terminal pathways. CK2α/NADPH oxidase is an important signaling pathway associated with ischemia-reperfusion injury, and miR-125b can regulate oxidative stress-related injury. In this study, we investigated whether the effect of miR-125b in rat brain I/R injury occurs through its modulation of the CK2α/NADPH oxidase pathway. Methods: Rats were subjected to 2 h of cerebral ischemia followed by 24 h of reperfusion to establish an I/R injury model. Neurological deficit was evaluated using a five-point score. Infarct volume was evaluated with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and RT-PCR was used to detect expressions of miR125b and CK2α. We then examined the association between miR-125b expression and the CK2α/NADPH oxidative signaling pathway in a PC-12 cell oxygen-glucose deprivation and reoxygenation (OGD/R) injury model. Transfection with miR-125b mimics, an miR-125b inhibitor, and luciferase reporter gene plasmid was accomplished using commercial kits. In these cells, Western blots were used to detect the levels of expression of CK2α, cleaved caspase-3, NOX2, and NOX4. RT-PCR was used to detect the expressions of CK2α, miR125b, NOX2, and NOX4. We evaluated Lactate Dehydrogenase (LDH) level, NADPH oxidase activity, and caspase-3 activity using commercial kits. Mitochondrial reactive oxygen species (ROS) were measured by fluorescence microscopy. For both PC-12 cells and rat brains, histological analyses were conducted to observe morphological changes, and apoptosis was measured using a commercial kit. Results: I/R rats exhibited an increase in neurological deficit score, infarct volume, and cellular apoptosis, along with miR-125b elevation and CK2α downregulation. OGD/R treatment increased PC-12 cells’ injuries, cellular apoptosis, and ROS levels. These changes were associated with miR-125b elevation, CK2α downregulation and activations of NOX2 and NOX4, mimicking our in vivo findings. All of these effects were reversed by the inhibition of miR-125b, confirming a strong correlation between miR-125b activity and the CK2α/NADPH oxidase signaling pathway. Conclusions: Based on these observations, we conclude that inhibition of miR-125b protects the rat brain from I/R injury by regulating the CK2α/NADPH oxidative signaling pathway.

Traumatic brain injury (TBI) is a major source of mortality and long-term disability worldwide. The mechanisms associated with TBI development are poorly understood, and little progress has been made in the treatment of TBI. Tanshinone IIA is an effective agent to treat a variety of disorders; however, the mechanisms of Tanshinone IIA on TBI remain unclear. The aim of the present study was to investigate the therapeutic potential of Tanshinone IIA on TBI and its underlying molecular mechanisms. Changes in microvascular permeability were examined to determine the extent of TBI with Evans blue dye. Brain edema was assessed by measuring the wet weight to dry weight ratio. The expression levels of CD11, interleukin- (IL-) 1β, and tumor necrosis factor- (TNF-) α mRNA were determined by reverse transcription-quantitative PCR. Aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), and p47phox protein expression levels were detected by western blotting. Superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-PX) activities, and malondialdehyde (MDA) content were determined using commercial kits. Cell apoptosis was detected by western blotting and TUNEL staining. Tanshinone IIA (10 mg/kg/day, intraperitoneal administration) significantly reduced brain water content and vascular permeability at 12, 24, 48, and 72 h after TBI. Tanshinone IIA downregulated the mRNA expression levels of various factors induced by TBI, including CD11, IL-1β, and TNF-α. Notably, CD11 mRNA downregulation suggested that Tanshinone IIA inhibited microglia activation. Further results showed that Tanshinone IIA treatment significantly downregulated AQP4 and GFAP expression. TBI-induced oxidative stress and apoptosis were markedly reversed by Tanshinone IIA, with an increase in SOD and GSH-PX activities and a decrease in the MDA content. Moreover, Tanshinone IIA decreased TBI-induced NADPH oxidase activation via the inhibition of p47phox. Tanshinone IIA attenuated TBI, and its mechanism of action may involve the inhibition of oxidative stress and apoptosis.

Oxymatrine (OMT) is the major quinolizidine alkaloid extracted from the root of Sophora flavescens Ait and has been shown to exhibit a diverse range of pharmacological properties. The aim of the present study was to investigate the role of OMT in diabetic brain injury in vivo and in vitro. Diabetic rats were induced by intraperitoneal injection of a single dose of 65mg/kg streptozotocin (STZ) and fed a high-fat and high-cholesterol diet. Memory function was assessed using a Morris water maze test. A SH-SY5Y cell injury model was induced by incubation with glucose (30mM/l) to simulate damage in vitro. The serum fasting blood glucose, insulin, serum S100B, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were analyzed using commercial kits. Morphological changes were observed using Nissl staining and electron microscopy. Cell apoptosis was assessed using Hoechst staining and TUNEL staining. NADPH oxidase (NOX) and caspase-3 activities were determined. The effects of NOX2 and NOX4 knockdown were assessed using small interfering RNA. The expression levels of NOX1, NOX2, and NOX4 were detected using reverse transcription-quantitative PCR and western blotting, and the levels of caspase-3 were detected using western blotting. The diabetic rats exhibited significantly increased plasma glucose, insulin, reactive oxygen species (ROS), S-100B, and MDA levels and decreased SOD levels. Memory function was determined by assessing the percentage of time spent in the target quadrant, the number of times the platform was crossed, escape latency, and mean path length and was found to be significantly reduced in the diabetic rats. Hyperglycemia resulted in notable brain injury, including histological changes and apoptosis in the cortex and hippocampus. The expression levels of NOX2 and NOX4 were significantly upregulated at the protein and mRNA levels, and NOX1 expression was not altered in the diabetic rats. NOX and caspase-3 activities were increased, and caspase-3 expression was upregulated in the brain tissue of diabetic rats. OMT treatment dose-dependently reversed behavioral, biochemical, and molecular changes in the diabetic rats. In vitro, high glucose resulted in increases in reactive oxygen species (ROS), MDA levels, apoptosis, and the expressions of NOX2, NOX4, and caspase-3. siRNA-mediated knockdown of NOX2 and NOX4 decreased NOX2 and NOX4 expression levels, respectively, and reduced ROS levels and apoptosis. The results of the present study suggest that OMT alleviates diabetes-associated cognitive decline, oxidative stress, and apoptosis via NOX2 and NOX4 inhibition.

Copper is an important mineral, and moderate copper is required to maintain physiological processes in nervous system including cerebral ischemia/reperfusion (I/R) injury. Over the past few decades, copper induced cell death, named cuprotosis, has attracted increasing attention. Several lines of evidence have confirmed cuprotosis exerts pivotal role in diverse of pathological processes, such as cancer, neurodegenerative diseases, and I/R injury. Therefore, an in-depth understanding of the interaction mechanism between copper-mediated cell death and I/R injury may reveal the significant alterations about cellular copper-mediated homeostasis in physiological and pathophysiological conditions, as well as therapeutic strategies deciphering copper-induced cell death in cerebral I/R injury.

Ferroptosis, an iron-dependent cell death pathway driven by lipid peroxidation, critically contributes to cerebral ischemia/reperfusion (I/R) injury. In ischemic stroke, neuronal ferroptosis arises from iron overload and antioxidant system failure. Excessive Fe²⁺ fuels hydroxyl radical production via the Fenton reaction, accelerating lipid peroxidation in PUFA-rich membranes. Concurrently, cysteine depletion and GPX4 inactivation disrupt the GPX4-glutathione antioxidant axis, allowing toxic lipid peroxide accumulation. Enzymes like ACSL4 and LPCAT3 esterify PUFAs into phospholipids, enabling lipoxygenases (LOXs) to generate peroxides, while impaired repair pathways (eg, FSP1-CoQ10) exacerbate damage. Therapeutic strategies target iron chelators (eg, deferoxamine), lipid peroxidation inhibitors (liproxstatin-1), and GPX4 activators to restore redox balance. Targeting propagation mediators (eg, Galectin-13, which reduces SLC7A11 membrane localization) may limit damage spread. Combinatorial therapies addressing iron regulation and lipid peroxidation pathways offer promising neuroprotection against I/R-related neurodegeneration, positioning ferroptosis as a key druggable target.