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BACKGROUND: Studies have found that activation of nuclear factor-erythroid 2-related factor 2/heme oxidase-1 (Nrf2/HO-1) pathway can alleviate oxidative stress caused by cerebral ischemia-reperfusion injury, but whether human bone marrow mesenchymal stem cells (hBMSC) can activate Nrf2/HO-1 pathway to alleviate cerebral ischemia-reperfusion injury is still lacking relevant studies. OBJECTIVE: To investigate whether intracranial transplantation of hBMSC alleviates oxidative stress injury in cerebral ischemia-reperfusion animal models by activating Nrf2/HO-1 pathway. METHODS: Totally 40 male SPF SD rats were randomly divided into sham operation group, model group, hBMSC transplantation group, hBMSC+solvent group and hBMSC+Nrf2 inhibitor group. Each group consisted of eight animals. In the model group and the hBMSC transplantation group, middle cerebral artery occlusion model was prepared by thread embolization method. The thread embolization was removed 1 hour later, and 30 μL PBS or hBMSC cultured to at least passage 5 was injected into the right cortex and striatum of rats. In the hBMSC+Nrf2 inhibitor group and hBMSC+solvent group, the left ventricle was injected with Nrf2 inhibitor Brusatol and its solvent dimethyl sulfoxide respectively 24 hours before model establishment, then the middle cerebral artery occlusion model was prepared, and hBMSC was injected. Relevant indexes were detected 3 days after transplantation. RESULTS AND CONCLUSION: (1) CT and TTC staining showed the same area and volume of cerebral infarction: model group > hBMSC+Nrf2 inhibitor group > hBMSC+solvent group > hBMSC transplantation group > sham operation group. (2) Hematoxylin-eosin staining and Nissl’s staining showed that the ischemic brain tissue was intact and the neurons were normal in the sham operation group. Compared with the model group, the pathological morphology and neuronal injury of the hBMSC transplantation group and the hBMSC+solvent group were significantly improved. Compared with the hBMSC+solvent group, the hBMSC+Nrf2 inhibitor group had more serious pathological morphology and neuronal damage. (3) Western blot assay and oxidative stress index detection results showed that compared with the sham operation group, Nrf2 and HO-1 proteins were decreased (all P < 0.05), malondialdehyde was increased and superoxide dismutase was decreased (all P < 0.05) in the model group. Compared with the model group, the expression levels of Nrf2 and HO-1 proteins were increased (all P < 0.05), malondialdehyde was decreased and superoxide dismutase was increased (all P < 0.05) in the hBMSC transplantation group and the hBMSC+solvent group. Compared with the hBMSC+solvent group, the expression levels of Nrf2 and HO-1 proteins were simultaneously decreased (all P < 0.05), and malondialdehyde was increased and superoxide dismutase was decreased (all P < 0.05) in the hBMSC+Nrf2 inhibitor group. (4) These results indicate that hBMSC can alleviate cerebral ischemia-reperfusion injury possibly by activating Nrf2/HO-1 pathway. [ABSTRACT FROM AUTHOR]

Background/Objectives: Ischemic stroke is a leading cause of disability and mortality worldwide, with current therapies limited in addressing its complex pathophysiological mechanisms, such as inflammation, oxidative stress, apoptosis, and impaired autophagy. Glycyrrhizic acid (GA), a bioactive compound from licorice (Glycyrrhiza glabra L.), has demonstrated neuroprotective properties in preclinical studies. This review consolidates current evidence on GA's pharmacological mechanisms and assesses its potential as a therapeutic agent for ischemic stroke. Methods: This review examines findings from recent preclinical studies and reviews on GA's neuroprotective effects, focusing on its modulation of inflammation, oxidative stress, apoptosis, and autophagy. Studies were identified from major scientific databases, including PubMed, Web of Science, and Embase, covering research from January 2000 to August 2024. Results: GA has demonstrated significant neuroprotective effects through the modulation of key pathways, including HMGB1/TLR4/NF-κB and Keap1/Nrf2, thereby reducing neuroinflammation, oxidative stress, and apoptosis. Additionally, GA promotes autophagy and modulates immune responses, suggesting it could serve as an adjunct therapy to enhance the efficacy and safety of existing treatments, such as thrombolysis. Conclusions: Current findings underscore GA's potential as a multi-targeted neuroprotective agent in ischemic stroke, highlighting its anti-inflammatory, antioxidant, and anti-apoptotic properties. However, while preclinical data are promising, further clinical trials are necessary to validate GA's therapeutic potential in humans. This review provides a comprehensive overview of GA's mechanisms of action, proposing directions for future research to explore its role in ischemic stroke management. [ABSTRACT FROM AUTHOR]

Cerebral ischemia-reperfusion injury (CI/RI) is a complex process leading to neuronal damage and death, with mitophagy implicated in its pathogenesis. However, the significance of mitophagy in CI/RI remains debated. We hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitophagy via the PINK1/Parkin pathway, and aggravating CI/RI. Rat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively. To evaluate the success of the CI/R modeling, TTC and HE staining were employed. The determination of serum biochemical indexes was carried out using relative assay kits. The Western Blot analysis was employed to assess the expression of ATAD3A, TRIM25, as well as mitophagy-related proteins (PINK1, Parkin, P62, and LC3II/LC3I). The mRNA levels were detected using QRT-PCR. Mitochondrial membrane potential was assessed through JC-1 staining. Mitosox Red Assay Kit was utilized to measure mitochondrial reactive oxygen species levels in PC12 cells. Additionally, characterization of the mitophagy structure was performed using transmission electron microscopy (TEM). Our findings showed down-regulation of ATAD3A and up-regulation of TRIM25 in both in vivo and in vitro CI/RI models. Various experimental techniques such as Western Blot, JC-1 staining, Mitosox assay, Immunofluorescence assay, and TEM observation supported the occurrence of PINK1/Parkin signaling pathway-mediated mitophagy in both models. ATAD3A suppressed mitophagy, while TRIM25 promoted it during CI/RI injury. Additionally, the results indicated that TRIM25 interacted with and ubiquitinated ATAD3A via the proteasome pathway, affecting ATAD3A protein stability and expression. TRIM25 promoted Pink1/Parkin-dependent excessive mitophagy by destabilizing ATAD3A, exacerbating CI/RI. Targeting TRIM25 and ATAD3A may offer therapeutic strategies for mitigating CI/RI and associated neurological damage. [Display omitted] • We introduced a previously unidentified interaction between TRIM25 and ATAD3A. • This research highlighted the role of TRIM25 in ubiquitinating ATAD3A. • TRIM25 enhanced Pink1/Parkin-dependent mitophagy by destabilizing ATAD3A. • Ubiquitination of ATAD3A by TRIM25 worsens cerebral ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

[Display omitted] • HACE1 overexpression alleviated cerebral ischemia–reperfusion injury. • HACE1 overexpression inhibited neuronal apoptosis in rats with cerebral ischemia. • HACE1 overexpression repressed oxidative stress in rats with cerebral ischemia. • HACE1 overexpression activated the PI3K/AKT/Nrf2 pathway. • HACE1-induced neuroprotection was reversed by blocking the PI3K/AKT pathway. HECT domain and Ankyrin repeat-containing E3 ubiquitin protein ligase 1 (HACE1) is an E3 ubiquitin ligase involving oxidative stress, an important contributor in cerebral ischemia–reperfusion injury (CIRI). It was proposed to be associated with the PI3K/AKT pathway and Nrf2 nuclear translocation, which are important players of oxidative stress. Therefore, we supposed that HACE1 might affect CIRI by regulating the PI3K/AKT/Nrf2 pathway. Here, we used the transient middle cerebral artery occlusion-reperfusion (tMCAO/R) model to induce CIRI in rats and found lower HACE1 expression in ischemic rats compared with the control. To explore the exact role of HACE1, the lentivirus vector carrying the HACE1 sequence was administrated to rats by intracerebroventricular injection (1 × 109 TU/mL, 9 μL) one week before tMCAO/R operation. HACE1 overexpression alleviated tMCAO/R-induced brain damage in rats. Further studies revealed that it reduced oxidative stress via activating the PI3K/AKT/Nrf2 pathway, thereby inhibiting neuronal apoptosis in the ischemic penumbra of rats with CIRI. Then, differentiated PC12 cells were cultured in oxygen-glucose deprivation-reoxygenation (OGD/R) conditions (OGD: 1 % O 2 , 94 % N2, and 5 % CO 2 ; R: normal atmosphere) to simulate CIRI in vitro. Similarly, HACE1 overexpression inhibited neuronal apoptosis caused by OGD/R treatment. The PI3K inhibitor LY294002 reversed the inhibitory effects of HACE1 overexpression on oxidative stress in OGD/R-injured cells, accompanied by the inactivated AKT/Nrf2 pathway. Altogether, our results suggest that HACE1 protects against oxidative stress-induced neuronal apoptosis in CIRI by activating the PI3K/AKT/Nrf2 pathway, providing a new insight into the CIRI treatment. [ABSTRACT FROM AUTHOR]

Cerebral ischemia reperfusion injury is a severe neurological impairment that occurs after blood flow reconstruction in stroke, and microglia cell pyroptosis is one of its important mechanisms. Electroacupuncture has been shown to be effective in mitigating and alleviating cerebral ischemia reperfusion injury by inhibiting neuroinflammation, reducing cellular pyroptosis, and improving neurological function. In this experiment, we divided the rats into three groups, including the sham operation (Sham) group, the middle cerebral artery occlusion/reperfusion (MCAO/R) group, and the pre-electroacupuncture (EAC) group. Pre-electroacupuncture group was stimulated with electroacupuncture of a certain intensity on the Baihui (GV 20) and Dazhui (GV 14) of the rat once a day from the 7th day to the 1st day before the MCAO/R operation. The extent of cerebral infarction was detected by TTC staining. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. The number of neurons and morphological changes were accessed by Nissl staining and HE staining. The cellular damage was detected by TUNEL staining. In addition, the expression levels of RhoA, pyrin, GSDMD, Caspase1, cleaved-Caspase1, Iba-1, CD206, and ROCK2 were examined by western blotting and immunofluorescence. The results found that pre-electroacupuncture significantly attenuated neurological impairment and cerebral infarction compared to the post-MCAO/R rats. In addition, pre-electroacupuncture therapy promoted polarization of microglia to the neuroprotective (M2) phenotype. In addition, pre-electroacupuncture inhibited microglia pyroptosis by inhibiting RhoA/pyrin/GSDMD signaling pathway, thereby reducing neuronal injury and increasing neuronal survival in the MCAO/R rats. Taken together, these results demonstrated that pre-acupuncture could attenuate cerebral ischemia-reperfusion injury by inhibiting microglial pyroptosis. Therefore, pre-electroacupuncture might be a potential preventive strategy for ischemic stroke patients. Hightlights: 1. Pre-electroacupuncture can decrease cerebral infarct volume after stroke. 2. Pre-electroacupuncture can effectively reduce neuroinflammation and inhibit microglia pyroptosis. 3. Pre-electroacupuncture can inhibit pyroptosis by RhoA/pyrin/GSDMD pathway. [ABSTRACT FROM AUTHOR]

Cerebral ischemia–reperfusion injury significantly contributes to global morbidity and mortality. Loganin is a natural product with various neuroprotective effects; however, it lacks targeted specificity for particular cells or receptors, which may result in reduced therapeutic efficacy and an increased risk of side effects. To address the limitations of loganin, we developed LA-1, a novel compound incorporating an Arg-Gly-Asp (RGD) peptide to target integrin receptor αvβ3, enhancing brain-targeting efficacy. LA-1 exhibited optimal nanoscale properties, significantly improved cell viability, reduced ROS production, and enhanced survival rates in vitro. In vivo, LA-1 decreased infarct sizes, improved neurological function, and reduced oxidative stress and neuroinflammation. Proteomic analysis showed LA-1 modulates PI3K/Akt and Nrf2/HO-1 pathways, providing targeted neuroprotection. These findings suggest LA-1's potential for clinical applications in treating cerebral ischemia–reperfusion injury. [ABSTRACT FROM AUTHOR]

Objective To analyze the effects of agatroban combined with mitochondrial transplantation on endothelial function and hemorheology in mice with cerebral ischemia-reperfusion injury. Methods 50 SPF grade Kunming mice, with 10 as the normal group, 38 mices were successfully modeled and divided into a model group of 9, a mitochondrial transplantation group of 9, an Agatroban group of 10, and a combination group of 10. The mitochondrial transplantation group was given exogenous mitochondrial extract, the agatroban group was given agatroban, the combined group was given agatroban combined with mitochondrial transplantation, and the blank group and model group were injected with normal saline. Observation of changes in cerebral infarction volume, pathological morphology of brain tissue, endothelial function, hemorheology, and relative expression of apoptosis related proteins in mice after 7 days of intervention. Results Compared with normal group, cerebral infarction volume ratio, ET-1, whole blood viscosity, plasma viscosity, Caspase-3 and Bax protein expression levels were increased in model group, mitochondrial transplantation group, agatroban group and combined group, while NO level and Bcl-2 protein expression levels were decreased (P < 0.05). Compared with the model group, the cerebral infarction volume ratio, ET-1, whole blood viscosity, plasma viscosity, Caspase-3 and Bax protein expressions in the mitochondrial transplantation group, agatroban group and combined group were decreased, and the combined group was lower than the mitochondrial transplantation group and agatroban group, and the NO level and Bcl-2 protein expression were increased. The combined group was higher than that of mitochondrial transplantation group and agatriban group (P < 0.05). Conclusion In mice with cerebral ischemia-reperfusion injury, endothelial function injury was improved, hemorheology was regulated, and brain injury was alleviated after agatroban combined with mitochondrial transplantation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Oxidative injury is considered to be one of the important factors of cerebral ischemia-reperfusion injury. Superoxide dismutase 2 (SOD2) is a key mitochondrial antioxidant molecule, and fenofibrate can regulate the expression of SOD2 by activating peroxisome proliferator-activated receptor α.OBJECTIVE: To explore whether the mechanism of fenofibrate in the treatment of cerebral ischemia-reperfusion injury depends on the expression of SOD2. METHODS: The TALENs system was used to construct SOD2 transgenic mice. The transgenic mice were genotyped by PCR and DNA sequencing techniques. The expression of SOD2 protein in transgenic mice was detected by western blot assay. Wild-type and SOD2 transgenic mice were randomly divided into four groups: wild-type control group (n=6), wild-type fenofibrate group (n=6), SOD2 transgenic control group (n=5) and SOD2 transgenic fenofibrate group (n=5). A mouse model of middle cerebral artery occlusion was prepared using the suture-occlusion method. After 90 minutes of ischemia, the thread was removed to reperfuse cerebral blood flow for 30 minutes. A cerebral blood flow monitor was used to monitor local cerebral blood flow. Brain tissue slices were taken for 2,3,5-triphenyltetrazolium chloride staining to analyze the situation of cerebral infarction in each group. RESULTS AND CONCLUSION: After PCR and DNA sequencing analysis, nine SOD2+/+ transgenic mice were successfully constructed. After cerebral ischemiareperfusion, the wild-type fenofibrate group showed partial recovery of cerebral blood flow and significantly reduced cerebral infarction volume compared with the wild-type control group (P < 0.001). There was no significant difference in cerebral blood flow and cerebral infarction volume between the SOD2 transgenic fenofibrate group and the SOD2 transgenic control group. The SOD2 transgenic control was superior to the wild-type control group in terms of improving cerebral blood flow and cerebral infarction (P < 0.001). There were also no significant differences in cerebral blood flow and cerebral infarction volume between the wild-type fenofibrate group and the SOD2 transgenic control group and between the wild-type fenofibrate group and the SOD2 transgenic fenofibrate group. To conclude, the expression of SOD2 is one of the mechanisms of fenofibrate in the treatment of cerebral ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Background: Cerebral ischemic disease is a common cerebrovascular disease, especially ischemic stroke. Exercise has protective functions on brain tissues following cerebral ischemia‐reperfusion injury (CIRI), but its preventive effects and mechanisms in CIRI remain unclear. We aimed to investigate the effects and mechanisms of exercise preconditioning on CIRI. Methods: The middle cerebral artery occlusion (MCAO) operation was prepared to establish CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE‐TIMP1, exercise preconditioning, MCAO operation plus intraventricular injection of OE‐TIMP1) groups. Results: The results indicated that exercise preconditioning suppressed approximately 66.67% of neurological deficit scores and 73.79% of TIMP1 mRNA expression in MCAO rats, which were partially offset by OE‐TIMP1. The protective effects of exercise against neuron death status and cerebral infarction size in MCAO rats were reversed by OE‐TIMP1. It also confirmed that exercise weakened apoptosis and oxidative stress damage, with notable increases of B‐cell lymphoma‐2, superoxide dismutase, and glutathione peroxidase production, and evident decreases of BCL2‐associated X, caspase 3, and malondialdehyde in MCAO rats, while these effects were partially reversed by OE‐TIMP1. Additionally, the inhibitory effects of exercise on the protein levels of TIMP1, hypoxia‐inducible factor‐alpha, vascular endothelial growth factor receptor 2, vascular endothelial growth factor, and neurogenic locus notch homolog protein 1 in MCAO rats were partially reversed by OE‐TIMP1. Conclusion: Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1, which provided new therapeutic strategies for preventing CIRI. [ABSTRACT FROM AUTHOR]

Objective To investigate the neuroprotective effect and mechanism of salvia polyphenolic acid for injection (SAFI) on cerebral ischemia-reperfusion injury in rats. Methods A total of 100 SD rats were randomly divided into sham surgery group, model group, low, medium and high dose (5, 10, 20 mg·kg-1) salvia polyphenolic acid groups, with 20 rats in each group. After being continuously administrated by intraperitoneal injection of SAFI once daily for three days, the rat model of middle cerebral artery occlusion/reperfusion (MCAO/ R) was established using the thread embolization method at 1 hour after the last administration. The neurological deficit of rats was evaluated by Zea Longa score. The cerebral infarction volume was detected by 2, 3, 5- triphenyltetrazolium chloride (TTC) staining. The levels of serum NADPH oxidase (NOX), 4-hydroxynonanal (4- HNE), 8-hydroxydeoxyguanosine (8-OHdG), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-18 (IL-18), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) were detected by ELISA kits. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of brain tissue and the morphology of neurons. The apoptosis of neuronal cells in brain tissue was detected by TUNEL. Immunofluorescence staining was used to detect the expression level of glial fibrillary acidic protein (GFAP) in brain tissue. Western Blot was used to detect the protein expression of NLRP3 and Caspasel in brain tissue. Results Compared with the sham surgery group, neurological deficit scores in model group increased remarkably (P<0.01). The cerebral infarction volume increased significantly (P<0.01). Serious pathological damage of brain was observed, and neuronal density decreased significantly (P<0.01). The apoptosis rate of cortical cells increased obviously (P<0.01). The levels of serum NOX, 4-HNE, 8-OHJC, MCP-1, TNF-α, IL-1β, IL-1β, IL-6 and ICAM-1 increased significantly (P<0.05, P<0.01). The protein expression of GFAP, NLRP3 and Caspasel in brain significantly upregulated (P<0.01). Compared with the model group, neurological deficit scores in medium and high dose SAFI groups decreased remarkably (P<0.01). The cerebral infarction volume decreased significantly (P<0.01). Neuronal damage was ameliorated to varying degrees, and neuronal density increased significantly (P<0.05, P<0.01). The apoptosis rate of cortical cells decreased obviously (P<0.01). The levels of serum NOX, 4-HNE, 8-OHJC, MCP-1, TNF-α, IL-1β, IL-18, IL-6 and ICAM-1 decreased significantly (P<0.05, P<0.01). The protein expression of GFAP, NLRP3 and Caspasel in brain significantly downregulated (P<0.01). Conclusion SAFI has a protective effect on MCAO/R rats, which can significantly reduce oxidative stress and inflammatory responses, thereby reducing pathological damage and apoptosis of brain tissue. Its mechanism may be related to the inhibition of NLRP3/Caspase-1 signaling pathway and astrocyte activation. [ABSTRACT FROM AUTHOR]

Transient receptor potential melastatin (TRPM) channels have emerged as potential therapeutic targets for cerebral ischemia–reperfusion (I/R) injury. We highlight recent findings on the involvement of TRPM channels in oxidative stress, mitochondrial dysfunction, inflammation, and calcium overload. We also discuss the challenges and future directions in targeting TRPM channels for cerebral I/R injury. [ABSTRACT FROM AUTHOR]

This study aimed to observe the effects of acupuncture on iron metabolism in the hippocampus of rats with cerebral ischemia-reperfusion injury (CIRI) and to explore the mechanism of acupuncture on neural repair in CIRI rats. After general feeding for 3 days, 16 of 48 healthy Sprague–Dawley rats were selected according to a random number table to form a sham-occlusion group, and the models were prepared in the remaining rats. After successful modeling, the rats were randomized into model and acupuncture groups, with 16 rats in each group. The CIRI models were prepared using the modified Zea Longa suture-occlusion method. In the acupuncture group, the rats were fixed and acupuncture was delivered at "Shuigou" (GV26), "Dazhui" (GV14), and "Baihui" (GV20), and the needles were manipulated once every 15 min. In the sham-occlusion and model groups, rats were fixed without acupuncture. The intervention was performed for 30 min in each group, once every 12 h, seven times. The degree of neurological impairment was assessed by using the modified Garcia score. Cerebral blood flow (CBF) was monitored using a laser speckle blood flow imaging system. The cerebral infarction area ratio was measured using triphenyltetrazolium chloride staining. Ferric ion aggregation in brain tissue was noted using Prussian blue staining. The ferric ion and glutathione (GSH) contents of the hippocampal tissue on the ischemic side were detected using ferric ion and GSH kits. The protein expression of transferrin receptor 1 (TFR1), iron regulatory protein 2 (IRP2), and ferroportin (FPN) was detected via western blotting. (1) Before intervention, compared with the sham-occlusion group, the Garcia neurological score was significantly reduced in the model and acupuncture groups (both P < 0.01). After the intervention, compared with the sham-occlusion group, the Garcia neurological score in the model group decreased significantly (P < 0.01), and the score in the acupuncture group was elevated compared with the model group (P < 0.05). Compared to before the intervention, the Garcia neurological score increased after the intervention in the acupuncture group (P < 0.05). (2) Compared with the sham-occlusion group, in the model group, CBF was reduced, the infarct area was significantly enlarged, ferric ion aggregation increased in the hippocampus, the content of ferric ion increased, and that of GSH decreased significantly (both P < 0.01), while the protein expression of TFR1 and IRP2 was upregulated and that of FPN was downregulated (both P < 0.05). When compared with the model group, in the acupuncture group, CBF was increased, the infarct area was reduced (P < 0.01), the ferric ion aggregation was reduced in the hippocampus, the content of ferric ion decreased, and that of GSH increased significantly (P < 0.05, P < 0.01); the protein expression of TFR1 and IRP2 was downregulated and that of FPN was upregulated (both P < 0.05). Acupuncture can alleviate CIRI, which may be related to the regulation of iron homeostasis-associated transferrin in the hippocampus, attenuation of ferric ion aggregation in hippocampal tissue, reduction of lipid peroxidation, and inhibition of ferroptosis. [ABSTRACT FROM AUTHOR]

Cerebral ischemia–reperfusion injury (CIRI) leads to malignant brain edema, blood–brain barrier destruction, and neuronal apoptosis. N6-methyladenosine (m6A) RNA modification in CIRI was still limited explored. In this study, MeRIP- and RNA-sequencing were performed of middle cerebral artery occlusion and reperfusion (MCAO/R) rats to find novel potential molecular targets. Transcription factor TFAP2B stood out of which its m6A abundance decreased associated with a marked reduction of its mRNA based on cojoint interactive bioinformatics analysis of the MeRIP- and RNA-sequencing data. It was suggested TFAP2B could have a role in CIRI. Functionally, overexpression of TFAP2B in cultured primary neurons could effectively improve the cell survival and pro-survival autophagy in parallel with reduced cell apoptosis during OGD/R in vitro. Through the RNA-sequencing of TFAP2B overexpressed primary neurons and subsequent validation experiments, it was found that mitophagy receptor BNIP3 was one of the important targets of TFAP2B in OGD/R neurons through which TFAP2B could bind to its promoter region for transcriptional activation of BNIP3, thereby enhancing BNIP3-mediated mitophagy to protect against OGD/R injury of neurons. Lastly, TFAP2B was demonstrated to alleviate the MCAO/R damage to a certain extent in vivo. Although it failed to confirm TFAP2B dysregulation was m6A dependent in current research, this is the first research of TFAP2B in CIRI field with important guiding significance. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (LncRNAs) play a substantial role in the process of cerebral ischemia-reperfusion injury (CIRI). The present work aimed to determine the probable mechanism by which LncRNA TUG1 exacerbates CIRI via the miR-340-5p/phosphatase and tensin homolog (PTEN) pathway. After developing a middle cerebral artery occlusion/reperfusion (MCAO/R) model, pcDNA-TUG1 together with miR-340-5p agomir were administrated in vivo. Furthermore, the neurologic defects in rats were assessed by a modified neurological severity score. Moreover, 2,3,5-Triphenyl-2 H-tetrazolium chloride stain-step was performed to determine the brain's infarct size. In addition, western blotting, immunohistochemistry, and qRT-PCR experiments were utilized for gauging the proteomic/genomic expression-profiles. Luciferase reporter assay validated correlations across TUG1, miR-340-5p, together with PTEN. The results indicated relatively reduced miR-340-5p levels in MCAO/R models, while upregulated TUG1 levels. The pcDNA-TUG1-treated rats indicated increasing neurological dysfunction, whereas the miR-340-5p agomir-treated rats showed improvement. Furthermore, miR-340-5p was determined to be the expected and confirmed TUG1 target. All things considered, the findings suggested that PTEN can serve as the target of miR-340-5p. In addition, TUG1 served as a miR-340-5p ceRNA, which promotes PTEN modulation. Furthermore, TUG1 overexpression decreased miR-340-5p's capacity to fend against CIRI. Conclusively, this work proved that in CIRI, targeting the TUG1/miR-340-5p/PTEN regulatory axis is a viable approach for the treatment of ischemic stroke. [ABSTRACT FROM AUTHOR]

Ischemic preconditioning (IPC) provides ischemic tolerance and neuroprotection during cerebral ischemia reperfusion (CI/R) injury. Diabetes abolishes the beneficial effects of conditioning phenomenon during CI/R. The study investigates the role of T-type calcium ion channel in IPC mediated protection during diabetes mellitus. The study employed Swiss Albino mice. Animals were divided into 3 normoglycaemic groups (Sham, CI/R, and IPC) and 4 hyperglycaemic groups (Sham, CI/R, IPC, and ML218 + IPC). CI/R injury was induced in Swiss Albino mice by occlusion of common carotid arteries followed by reperfusion. IPC was given prior to CI/R injury and diabetes was induced using streptozotocin (STZ). Animals were assessed for learning, memory, motor coordination, neurological function, cerebral infarction, edema, and histopathological alterations. Biochemical assessments were performed for calcium binding proteins (Calmodulin (CaM), calcium/calmodulin-dependent protein kinase II (CaMKII), and S100B), oxidative stress (4-hydroxy-2-nonenal (4-HNE)), glutathione (GSH), inflammation (nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), tumor necrosis factor (TNF-α), interleukin (IL-10)), inducible nitric oxide synthase (iNOS) levels, and acetylcholinesterase activity (AChE) in brain supernatants. NF-kB, iNOS, and S100B serum levels were also assessed. CI/R animals (normoglycemic and hyperglycaemic) showed impairment in learning, memory, motor coordination, and neurological function along with increase in cerebral infarction, edema, and histopathological alterations. Furthermore, increase in brain calcium-binding proteins, oxidative stress, inflammation, and AChE along with serum NF-kB, iNOS, and S100B levels were recorded in CI/R animals. IPC ameliorated CI/R induced behavioral, biochemical, and histopathological impairment, however no beneficial effects were observed in IPC (diabetic) mice. Administration of ML218 (10 mg/kg; i.p.), a selective T-type calcium channel re-established the IPC mediated neuroprotection in CI/R diabetic animals. In conclusion, IPC-mediated neuroprotection was abolished in diabetic mice. T-type calcium ion channel antagonism plays an important role in the IPC-mediated neuroprotection during hyperglycaemia. [ABSTRACT FROM AUTHOR]

Cerebral ischemia‒reperfusion (I/R) injury seriously threatens the lives of patients. Astragalus polysaccharide (APS) is the main active ingredient of Astragalus membranaceus and has a wide range of pharmacological activities. Here, we aimed to explore the impacts of APS on cerebral I/R injury and its specific mechanisms. We established a cerebral I/R injury model using middle cerebral artery occlusion (MCAO)-treated rats and oxygen glucose deprivation/reoxygenation (OGD/R)-treated BV2 cells. The interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin (IL-10) levels were determined using corresponding ELISA kits and RT‒qPCR. The levels of M1 microglial markers (INOS and CD16) and M2 microglial markers (Arg-1 and CD206) were measured by RT‒qPCR. The O-linked N-acetylglucosamine modification (O-GlcNAcylation), O-GlcNAc transfer (OGT) and O-GlcNAc glycosidase (OGA) protein levels were measured by Western blot. Our results showed that APS treatment decreased IL-1β (179.72 ± 9.08 vs. 81.33 ± 6.30) and IL-6 (445.56 ± 33.09 vs. 234.75 ± 27.62) levels and increased IL-10 (41.95 ± 4.18 vs. 86.40 ± 7.16) levels in OGD/R-treated BV2 cells (p < 0.001). In addition, APS promoted the M2 polarization of OGD/R-treated BV2 cells, manifested by an increase in Arg-1 (0.43 ± 0.04 vs. 0.76 ± 0.03) and CD206 (0.36 ± 0.03 vs. 0.65 ± 0.06) and a decrease in INOS (2.84 ± 0.39 vs. 1.56 ± 0.19) and CD16 (4.04 ± 0.36 vs. 1.88 ± 0.09) in OGD/R-treated BV2 cells (p < 0.001). Additionally, APS treatment increased the O-GlcNAcylation and OGT levels in OGD/R-treated BV2 cells, while OGT knockdown reversed the effect of APS in OGD/R-treated BV2 cells and MCAO-treated rats (p < 0.05). Our study demonstrated that APS alleviated cerebral I/R injury by promoting the M2 polarization of microglia by enhancing OGT-mediated O-GlcNAcylation. [ABSTRACT FROM AUTHOR]

Chang-Sheng Ma,1,2,&ast; Ya-Ping Ma,1,3,&ast; Bo Han,1,2,&ast; Wan-Li Duan,1 Shu-Chen Meng,1 Min Bai,1 Hao Dong,1 Li-Ying Zhang,1 Meng-Yuan Duan,1,2 Jing Liu,1 Ai-Jun Deng,2 Mao-Tao He1,2 1Department of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong, People’s Republic of China; 2Department of Ophthalmology, Affiliated Hospital of Shandong Second Medical University, Weifang, People’s Republic of China; 3Department of Pathology, The 942Hospital of the People’s Liberation Army Joint Logistic Support Force, Yinchuan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Mao-Tao He, Department of Basic Medical Sciences, Shandong Second Medical University, Weifang, Shandong, People’s Republic of China, Tel +86-18209617186, Fax +86-0536-3081201, Email hemaotao@sdsmu.edu.cn Ai-Jun Deng, Department of Ophthalmology, Affiliated Hospital of Shandong Second Medical University, Weifang, People’s Republic of China, Email dengaijun@hotmail.comPurpose: Ischemic stroke is a refractory disease wherein the reperfusion injury caused by sudden restoration of blood supply is the main cause of increased mortality and disability. However, current therapeutic strategies for the inflammatory response induced by cerebral ischemia-reperfusion (I/R) injury are unsatisfactory. This study aimed to develop a functional nanoparticle (MM/ANPs) comprising apelin-13 (APNs) encapsulated in macrophage membranes (MM) modified with distearoyl phosphatidylethanolamine-polyethylene glycol-RVG29 (DSPE-PEG-RVG29) to achieve targeted therapy against ischemic stroke.Methods: MM were extracted from RAW264.7. PLGA was dissolved in dichloromethane, while Apelin-13 was dissolved in water, and CY5.5 was dissolved in dichloromethane. The precipitate was washed twice with ultrapure water and then resuspended in 10 mL to obtain an aqueous solution of PLGA nanoparticles. Subsequently, the cell membrane was evenly dispersed homogeneously and mixed with PLGA-COOH at a mass ratio of 1:1 for the hybrid ultrasound. DSPE-PEG-RVG29 was added and incubated for 1 h to obtain MM/ANPs.Results: In this study, we developed a functional nanoparticle delivery system (MM/ANPs) that utilizes macrophage membranes coated with DSPE-PEG-RVG29 peptide to efficiently deliver Apelin-13 to inflammatory areas using ischemic stroke therapy. MM/ANPs effectively cross the blood-brain barrier and selectively accumulate in ischemic and inflamed areas. In a mouse I/R injury model, these nanoparticles significantly improved neurological scores and reduced infarct volume. Apelin-13 is gradually released from the MM/ANPs, inhibiting NLRP3 inflammasome assembly by enhancing sirtuin 3 (SIRT3) activity, which suppresses the inflammatory response and pyroptosis. The positive regulation of SIRT3 further inhibits the NLRP3-mediated inflammation, showing the clinical potential of these nanoparticles for ischemic stroke treatment. The biocompatibility and safety of MM/ANPs were confirmed through in vitro cytotoxicity tests, blood-brain barrier permeability tests, biosafety evaluations, and blood compatibility studies.Conclusion: MM/ANPs offer a highly promising approach to achieve ischemic stroke-targeted therapy inhibiting NLRP3 inflammasome-mediated pyroptosis. Keywords: apelin-13, cerebral ischemia-reperfusion injury, macrophage membrane, ischemic stroke therapy, pyroptosis

Peijie Zhou,&ast; Shangshang Yu,&ast; Xuan Wang,&ast; Xiaofei Zhang, Dongyan Guo, Chongbo Zhao, Jiangxue Cheng, Jing Wang, Jing Sun Department of Pharmaceutics, College of Pharmacy, The Key Laboratory of Basic and New Drug Research of Traditional Chinese Medicine, Shaanxi Provincial Engineering Technology Research Center for Traditional Chinese Medicine Decoction Pieces, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jing Sun, Email ph.175@163.comBackground: Cerebral ischaemia-reperfusion injury (CIRI) could worsen the inflammatory response and oxidative stress in brain tissue. According to previous studies, ferulic acid methyl ester (FAME), as the extract with the strongest comprehensive activity in the traditional Chinese medicine Huang Hua oil dot herb, has significant anti-oxidative stress and neuroprotective functions, and can effectively alleviate CIRI, but its mechanism of action is still unclear.Methods: Firstly, the pharmacological effects of FAME were investigated by in vitro oxidative stress and inflammatory experiments. Secondly, evaluate the therapeutic effects of FAME in the treatment of CIRI by brain histopathological staining and cerebral infarct area by replicating the in vivo MACO model. Thirdly, RNA-Seq and network pharmacology were utilized to predict the possible targets and mechanisms of FAME for CIRI at the molecular level. Finally, the expression of key target proteins, as well as the key regulatory relationships were verified by molecular docking visualization, Western Blotting and immunohistochemistry.Results: The results of in vitro experiments concluded that FAME could significantly reduce the content of TNF-α, IL-1β and ROS, inhibiting COX-2 and iNOS protein expression in cells(p< 0.01). FAME was demonstrated to have anti-oxidative stress and anti-inflammatory effects. The results of in vivo experiments showed that after the administration of FAME, the area of cerebral infarction in rats with CIRI was reduced, the content of Bcl-2 and VEGF was increased(p< 0.05). Network pharmacology and RNA-Seq showed that the alleviation of CIRI by FAME may be through PI3K-AKT and HIF-1 signaling pathway. Enhanced expression of HIF-1α, VEGF, p-PI3K, p-AKT proteins in the brain tissues of rats in the FAME group was verified by molecular docking and Western Blotting.Conclusion: FAME possesses significant anti-inflammatory and anti-oxidative stress activities and alleviates CIRI through the PI3K/HIF-1α/VEGF signaling pathway. Keywords: ferulic acid methyl ester, cerebral ischemia-reperfusion injury, WGCNA, RNA-Seq, network pharmacology, molecular docking techniques

Abstract Background Stroke is a globally dangerous disease capable of causing irreversible neuronal damage with limited therapeutic options. Meldonium, an inhibitor of carnitine-dependent metabolism, is considered an anti-ischemic drug. However, the mechanisms through which meldonium improves ischemic injury and its potential to protect neurons remain largely unknown. Methods A rat model with middle cerebral artery occlusion (MCAO) was used to investigate meldonium’s neuroprotective efficacy in vivo. Infarct volume, neurological deficit score, histopathology, neuronal apoptosis, motor function, morphological alteration and antioxidant capacity were explored via 2,3,5-Triphenyltetrazolium chloride staining, Longa scoring method, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, rotarod test, transmission electron microscopy and Oxidative stress index related kit. A primary rat hippocampal neuron model subjected to oxygen–glucose deprivation reperfusion was used to study meldonium’s protective ability in vitro. Neuronal viability, mitochondrial membrane potential, mitochondrial morphology, respiratory function, ATP production, and its potential mechanism were assayed by MTT cell proliferation and cytotoxicity assay kit, cell-permeant MitoTracker® probes, mitochondrial stress, real-time ATP rate and western blotting. Results Meldonium markedly reduced the infarct size, improved neurological function and motor ability, and inhibited neuronal apoptosis in vivo. Meldonium enhanced the morphology, antioxidant capacity, and ATP production of mitochondria and inhibited the opening of the mitochondrial permeability transition pore in the cerebral cortex and hippocampus during cerebral ischemia–reperfusion injury (CIRI) in rats. Additionally, meldonium improved the damaged fusion process and respiratory function of neuronal mitochondria in vitro. Further investigation revealed that meldonium activated the Akt/GSK-3β signaling pathway to inhibit mitochondria-dependent neuronal apoptosis. Conclusion Our study demonstrated that meldonium shows a neuroprotective function during CIRI by preserving the mitochondrial function, thus prevented neurons from apoptosis.

To explore the protective effects of 6-shogaol (6-SH) against cerebral is chemia-reperfusion injury (CIRI) in rats, molecular docking was utilized to investigate the spontaneous binding ability of 6-SH with microRNA-26a-5p (miRNA-26a-5P). Dual luciferase reporter gene assays were employed to validate the targeted relationship between miRNA-26a-5p and death associated protein kinase 1 (DAPK1). The CIRI rat model was induced using the suture method, with male SD rats randomly assigned to sham surgery, model, and 6-shogaol groups. Improved neurological function scoring was conducted 72 hours post-CIRI, accompanied by Triphenyltetrazolium chloride (TTC) staining. Post-scoring, brain tissue specimens were collected for hematoxylin-eosin (HE) staining to observe neuronal pathological damage. Transmission electron microscopy was utilized to examine neuronal ultrastructure and autophagy. Immunofluorescence was employed for the detection of autophagic markers, and real-time fluorescence quantification, along with protein immunoblotting, were performed to assess proteins and genes related to autophagy and calcium overload. The results indicate that 6-shogaol can upregulate miRNA-26a-5p expression, inhibit DAPK1 expression, and alleviate excessive autophagy and calcium overload after CIRI in rats, showcasing its neuroprotective properties. [ABSTRACT FROM AUTHOR]

Reactive astrocyte activation in the context of cerebral ischemia/reperfusion (I/R) injury gives rise to two distinct subtypes: the neurotoxic A1 type and the neuroprotective A2 type. DJ‐1 (Parkinson disease protein 7, PARK7), originally identified as a Parkinson's disease‐associated protein, is a multifunctional anti‐oxidative stress protein with molecular chaperone and signaling functions. SHP‐1 (Src homology 2 domain‐containing phosphatase‐1) is a protein tyrosine phosphatase closely associated with cellular signal transduction. miR‐155 is a microRNA that participates in cellular functions by regulating gene expression. Recent studies have uncovered the relationship between DJ‐1 and astrocyte‐mediated neuroprotection, which may be related to its antioxidant properties and regulation of signaling molecules such as SHP‐1. Furthermore, miR‐155 may exert its effects by influencing SHP‐1, providing a potential perspective for understanding the molecular mechanisms of stroke. A middle cerebral artery occlusion/reperfusion (MCAO/R) model and an oxygen–glucose deprivation/reperfusion (OGD/R) model were established to simulate focal cerebral I/R injury in vivo and in vitro, respectively. The in vivo interaction between DJ‐1 and SHP‐1 has been experimentally validated through immunoprecipitation. Overexpression of DJ‐1 attenuates I/R injury and suppresses miR‐155 expression. In addition, inhibition of miR‐155 upregulates SHP‐1 expression and modulates astrocyte activation phenotype. These findings suggest that DJ‐1 mediates astrocyte activation via the miR‐155/SHP‐1 pathway, playing a pivotal role in the pathogenesis of cerebral ischemia–reperfusion injury. Our results provide a potential way for exploring the pathogenesis of ischemic stroke and present promising targets for pharmacological intervention. [ABSTRACT FROM AUTHOR]

Excessive reactive oxygen species (ROS) generated during cerebral ischemic reperfusion (CIRI) are crucial for subsequent tissue damage. However, despite the potential benefits of antioxidants reported in clinical applications, few have proven effective in treating CIRI, particularly in the elderly. Epicatechin (EC) is a catechol flavonoid monomer derived from natural tea plants. Multiple phenolic hydroxyl groups give it strong antioxidant properties, which can not only degrade ROS through chemical reactions between hydroxyl and ROS but also enhance the activity of antioxidant enzymes in cells, and it is easy to penetrate the blood–brain barrier. But its antagonistic effect on age‐related CIRI and potential medicinal value are still unknown. Nuclear factor erythroid 2‐related factor 2 (Nrf2) is the most important transcription factor regulating the expression of antioxidant proteins in the body. This study first compared the pathological differences of the Nrf2 system in CIRI between 2‐month‐old and 12‐month‐old Sprague–Dawley (SD) rats. Subsequently, EC was administered to 12‐month‐old rat models of middle cerebral artery occlusion and reperfusion (MCAO/R) and senescent SH‐SY5Y cell models subjected to oxygen glucose deprivation/reoxygenation (OGD/R). EC treatment improved cerebral morphology and function; increased p‐Nrf2, heme oxygenase‐1 (HO‐1), superoxide dismutase (SOD), and glutathione (GSH) expression; reduced infarct volume; and neuronal apoptosis in senescent rats. Moreover, EC enhanced cellular activity and the expression of p‐Nrf2, HO‐1, and quinone oxidoreductase‐1 (NQO‐1) while decreasing ROS and malondialdehyde (MDA) levels and mitigating apoptosis in senescent SH‐SY5Y cells. These effects were reversed upon si‐Nrf2. In sum, we confirm that EC exerts neuroprotective effects by upregulating Nrf2/ARE and reducing oxidative stress, suggesting that EC may be a promising drug for the treatment of senile cerebral apoplexy. This study also provides a scientific basis for the development and selection of new drugs for ischemic stroke in elderly patients. [ABSTRACT FROM AUTHOR]

Pyroptosis has been regarded as caspase-1-mediated monocyte death that induces inflammation, showing a critical and detrimental role in the development of cerebral ischemia-reperfusion injury (IRI). MARCH1 is an E3 ubiquitin ligase that exerts potential anti-inflammatory functions. Therefore, the study probed into the significance of MARCH1 in inflammation and pyroptosis elicited by cerebral IRI. Middle cerebral artery occlusion/reperfusion (MCAO/R)-treated mice and oxygen glucose deprivation/reoxygenation (OGD/R)-treated hippocampal neurons were established to simulate cerebral IRI in vivo and in vitro. MARCH1 and PCSK9 expression was tested in MCAO/R-operated mice, and their interaction was identified by means of the cycloheximide assay and co-immunoprecipitation. The functional roles of MARCH1 and PCSK9 in cerebral IRI were subsequently determined by examining the neurological function, brain tissue changes, neuronal viability, inflammation, and pyroptosis through ectopic expression and knockdown experiments. PCSK9 expression was increased in the brain tissues of MCAO/R mice, while PCSK9 knockdown reduced brain damage and neurological deficits. Additionally, inflammation and pyroptosis were inhibited in OGD/R-exposed hippocampal neurons upon PCSK9 knockdown, accompanied by LDLR upregulation and NLRP3 inflammasome inactivation. Mechanistic experiments revealed that MARCH1 mediated ubiquitination and degradation of PCSK9, lowering PCSK9 protein expression. Furthermore, it was demonstrated that MARCH1 suppressed inflammation and pyroptosis after cerebral IRI by downregulating PCSK9 both in vivo and in vitro. Taken together, the present study demonstrate the protective effect of MARCH1 against cerebral IRI through PCSK9 downregulation, which might contribute to the discovery of new therapies for improving cerebral IRI. [ABSTRACT FROM AUTHOR]

Cerebral ischemia–reperfusion injury (CIRI) is a significant pathological process in stroke, characterized by neuronal cell death and neurological dysfunction. Metformin, commonly used for diabetes management, has been noted for its neuroprotective properties, though its effects on CIRI and the mechanisms involved remain unclear. This study explored the neuroprotective impact of metformin on CIRI, focusing on its potential to modulate the c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) signaling pathways. Using in vitro models of oxygen–glucose deprivation/reperfusion (OGD/R) in neuronal cells and in vivo mouse models of middle cerebral artery occlusion (MCAO), the effects of metformin were assessed. Cell viability was measured with Cell Counting Kit-8 (CCK-8), protein expression via Western Blot (WB), and apoptosis through flow cytometry. The extent of brain injury in mice was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining, while JNK and p38 activation statuses were detected through WB and phospho-JNK (p-JNK) immunofluorescence staining. Results showed that metformin significantly improved the viability of HT22 cells post-OGD/R, reduced apoptosis, and decreased OGD/R-induced phosphorylation of JNK and p38 in vitro. In vivo, metformin treatment notably reduced brain infarct volume in MCAO mice, inhibited p-p38 and p-JNK expression, and enhanced neurological function. These findings suggest that metformin exerts neuroprotective effects against CIRI by modulating the JNK/p38 signaling pathway, highlighting its potential therapeutic value in treating cerebral ischemia–reperfusion injury and paving the way for clinical applications. [ABSTRACT FROM AUTHOR]

Background: Stroke is a globally dangerous disease capable of causing irreversible neuronal damage with limited therapeutic options. Meldonium, an inhibitor of carnitine-dependent metabolism, is considered an anti-ischemic drug. However, the mechanisms through which meldonium improves ischemic injury and its potential to protect neurons remain largely unknown. Methods: A rat model with middle cerebral artery occlusion (MCAO) was used to investigate meldonium's neuroprotective efficacy in vivo. Infarct volume, neurological deficit score, histopathology, neuronal apoptosis, motor function, morphological alteration and antioxidant capacity were explored via 2,3,5-Triphenyltetrazolium chloride staining, Longa scoring method, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, rotarod test, transmission electron microscopy and Oxidative stress index related kit. A primary rat hippocampal neuron model subjected to oxygen–glucose deprivation reperfusion was used to study meldonium's protective ability in vitro. Neuronal viability, mitochondrial membrane potential, mitochondrial morphology, respiratory function, ATP production, and its potential mechanism were assayed by MTT cell proliferation and cytotoxicity assay kit, cell-permeant MitoTracker® probes, mitochondrial stress, real-time ATP rate and western blotting. Results: Meldonium markedly reduced the infarct size, improved neurological function and motor ability, and inhibited neuronal apoptosis in vivo. Meldonium enhanced the morphology, antioxidant capacity, and ATP production of mitochondria and inhibited the opening of the mitochondrial permeability transition pore in the cerebral cortex and hippocampus during cerebral ischemia–reperfusion injury (CIRI) in rats. Additionally, meldonium improved the damaged fusion process and respiratory function of neuronal mitochondria in vitro. Further investigation revealed that meldonium activated the Akt/GSK-3β signaling pathway to inhibit mitochondria-dependent neuronal apoptosis. Conclusion: Our study demonstrated that meldonium shows a neuroprotective function during CIRI by preserving the mitochondrial function, thus prevented neurons from apoptosis. [ABSTRACT FROM AUTHOR]

The newly identified estrogen receptor, G protein-coupled receptor 30 (GPR30), is prevalent in the brain and has been shown to provide significant neuroprotection. Recent studies have linked ferroptosis, a newly characterized form of programmed cell death, closely with cerebral ischemia–reperfusion injury (CIRI), highlighting it as a major contributing factor. Consequently, our research aimed to explore the potential of GPR30 targeting in controlling neuronal ferroptosis and lessening CIRI impacts. Results indicated that GPR30 activation not only improved neurological outcomes and decreased infarct size in a mouse model but also lessened iron accumulation and malondialdehyde formation post-middle cerebral artery occlusion (MCAO). This protective effect extended to increased levels of Nrf2 and GPX4 proteins. Similar protective results were replicated in PC12 cells subjected to Oxygen Glucose Deprivation and Reoxygenation (OGD/R) using the GPR30-specific agonist G1. Importantly, inhibition of Nrf2 with ML385 curtailed the neuroprotective effects of GPR30 activation, suggesting that GPR30 mitigates CIRI primarily through inhibition of neuronal ferroptosis via upregulation of Nrf2 and GPX4. [ABSTRACT FROM AUTHOR]

AIM: To investigate the impact of aquaporin 4 (AQP4) expression inhibition on autophagy and apoptosis in a mouse model of cerebral ischemia-reperfusion (I/R) injury, and to elucidate its underlying mechanism. METHODS: Cerebral I/R injury was induced in mice via transient middle cerebral artery occlusion (tMCAO). Totally 60 mice were randomly divided into sham group, I/R group, AQP4 inhibition group, and 3-methyladenine (3-MA) group, with 15 mice in each group. Among them, the mice in sham and I/R groups received intraperitoneal injections of normal saline, while those in AQP4 inhibition group and 3-MA group received intraperitoneal injections of AER-271 (2 mg·kg−1·d−1) and AER-271+3-MA (2 mg·kg−1 ·d−1) for 3 d, respectively, once per day. Longa score was adopted to assess the neurological function, and to record changes in body weight. Cerebral infarction volume and histopathological alterations were evaluated using hematoxylin-eosin staining. Western blot analysis was performed to determine the levels of AQP4, LC3- II, P62 and cleaved caspase-3, while the LC3-II, P62, cleaved caspase-3 and NeuN (neuronal marker) colocalization and expression assessment were conducted with immunofluorescence. RESULTS: The mice in I/R and AQP4 inhibition groups exhibited extensive cerebral infarction, cerebral edema, and elevated Longa scores. However, in comparision to I/ R group, the mice in AQP4 inhibition group showed significantly reduced cerebral infarct volume, cerebral edema volume, and Longa score (P<0.05). Additionally, in contrast to sham group, the mice in I/R group displayed increased ex⁃ pression of AQP4, LC3-II and cleaved caspase-3 (P<0.01), accompanied by decreased body weight and P62 expression (P<0.05 or P<0.01). Furthermore, compared with I/R group, the mice in both AQP4 inhibition group and 3-MA group demonstrated a decrease in the expression levels of AQP4, LC3-II and cleaved caspase-3 (P<0.05 or P<0.01), along with increased body weight and P62 expression (P<0.05 or P<0.01). Nonetheless, no significant differences were ob⁃ served between AQP4 inhibition group and 3-MA group regarding Longa score, cerebral infarct volume, body weight, and the expression of AQP4, LC3-II, cleaved caspase-3 and P62. CONCLUSION: Inhibition of AQP4 expression significantly reduces cerebral infarction area and nerve injury severity in tMCAO mice. Moreover, AQP4 expression inhibition decelerates autophagy and apoptosis after cerebral infarction, with the additional autophagy inhibitor showing no notable impact on the protective effect of AQP4 inhibition. [ABSTRACT FROM AUTHOR]

Cerebral ischemia-reperfusion injury (IRI), occurring after blood supply restoration, contributes significantly to stroke-related deaths. This study explored the combined impact and mechanisms of astragaloside IV (AS-IV), hydroxysafflor yellow A (HSYA), and their combination in mitigating IRI. Male Sprague–Dawley (SD) rats were randomized to the Sham, MCAO, MCAO+AS-IV, MCAO+HSYA, and MCAO+AS-IV+HSYA groups. Neurological deficits and cerebral infarction were examined after restoring the blood supply to the brain. Pathomorphological changes in the cerebral cortex were observed via HE staining. IL-1β and IL-18 were quantified using ELISA. The expression of NF-κB and GSDMD in the ischemic cerebrum was analyzed using immunohistochemistry. The expression levels of NLRP3, ASC, IL-1β, Caspase-1, and GSDMD in the ischemic cerebrum were evaluated using Western blot. The MCAO+AS-IV, MCAO+HSYA, and MCAO+AS-IV+HSYA groups exhibited notably better neurological function and cerebral infarction compared with the MCAO group. The combined treatment demonstrated superior brain tissue injury alleviation. Reductions in NF-κB, GSDMD positive cells, and NLRP3/ASC/IL-1β/Caspase-1/GSDMD protein expression in the ischemic brain were significantly more pronounced with the combined therapy, indicating a synergistic effect in countering cerebral IRI via the NF-κB/NLRP3/Caspase-1/GSDMD pathway inhibition of cell pyroptosis-induced injury. [ABSTRACT FROM AUTHOR]

Cerebral ischemia–reperfusion injury (CIRI) is the second leading cause of death worldwide, posing a huge risk to human life and health. Therefore, investigating the pathogenesis underlying CIRI and developing effective treatments are essential. Ferroptosis is an iron-dependent mode of cell death, which is caused by disorders in iron metabolism and lipid peroxidation. Previous studies demonstrated that ferroptosis is also a form of autophagic cell death, and nuclear receptor coactivator 4(NCOA4) mediated ferritinophagy was found to regulate ferroptosis by interfering with iron metabolism. Ferritinophagy and ferroptosis are important pathogenic mechanisms in CIRI. This review mainly summarizes the link and regulation between ferritinophagy and ferroptosis and further discusses their mechanisms in CIRI. In addition, the potential treatment methods targeting ferritinophagy and ferroptosis for CIRI are presented, providing new ideas for the prevention and treatment of clinical CIRI in the future. [ABSTRACT FROM AUTHOR]

Introduction: Cerebral ischemia‐reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. Methods: We utilized high‐throughput transcriptome sequencing and weighted gene co‐expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK‐N‐SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. Results: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. Conclusion: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments. [ABSTRACT FROM AUTHOR]

Objective To observe the ameliorating effect of intragastric administration of Klotho protein on cerebral ischemia-reperfusion injury (CIRI) in rats with acute cerebral infarction (ACI) and to explore its mechanism. Methods Fifty SD male rats were divided into the sham operation group, model group, low-dose Klotho group, medium-dose Klotho group and high-dose Klotho group, with 10 rats in each group. CIRI models after ACI were established in the model group, low-dose Klotho group, medium-dose Klotho group and high-dose Klotho group. In the sham operation group, only carotid artery vessel separation and wound suture were performed, and carotid artery clamping was not performed. After being fed for 24 h, the rats in the low-dose, medium-dose and high-dose Klotho groups were treated with 25，50 and 100 mg/kg Klotho protein, respectively, and the rats in the model group and sham operation group were treated with equal volume of normal saline. All rats in the five groups were treated once a day, for 14 days. Longa scoring method and balance beam scoring method were used to evaluate the neurological scores of rats in each group. Cerebral tissues of rats in each group were taken after decapitation. Hematoxylin staining method was used to measure the apoptosis rate of cerebral cortex neurons of rats, and receptor-interacting protein kinase 1（RIP1) and RIP3 proteins in cerebral cortex tissues of rats were detected by Western blotting. The inflammatory mediators interleukin (IL) -1β, tumor necrosis factor (TNF) -α and IL-6 were detected by enzyme-linked immunoassay, MDA was detected by TBA, SOD was detected by enzyme-linked immunoassay, and GSH-Px was detected by ultraviolet colorimetry. Results The the neurological score and cerebral cortical neuron apoptosis rate of rats in the sham operation group were lower than those in the other groups (all P<0. 05), and those in the model group were higher than those in the other groups (all P<0. 05), and those in the high-dose Klotho group were higher than those in the low-dose Klotho group and medium-dose Klotho group (all P<0. 05), and those in the mediumdose Klotho group were higher than those in the low-dose Klotho group (all P<0. 05) . The relative expression levels of RIP1 and RIP3 proteins and the expression levels of IL-1β, TNF-α, IL-6 and MDA in the cerebral cortex of rats in the sham operation group were lower than those in the other groups, while the expression levels of SOD and GSH-Px were higher than those in the other groups (all P<0. 05) . The relative expression levels of RIP1 and RIP3 proteins and the expression levels of IL-1β, TNF-α, IL-6 and MDA in cerebral cortex of rats in the model group were higher than those in the other groups, while the expression levels of SOD and GSH-Px were lower than those in the other groups (all P<0. 05) . The relative expression levels of RIP1 and RIP3 proteins and the expression levels of IL-1β, TNF-α, IL-6 and MDA in the cerebral cortex of rats in the high-dose Klotho group were lower than those in the low-dose Klotho group and medium-dose Klotho group, while the expression levels of SOD and GSH-Px were higher than those of the low-dose and medium-dose Klotho groups (all P<0. 05) . The relative expression levels of RIP1 and RIP3 protein and the expression levels of IL-1β, TNF-α, IL-6 and MDA in cerebral cortex of rats in the medium-dose Klotho group were lower than those in the low-dose Klotho group, while the expression levels of SOD and GSH-Px were higher than those in the low-dose Klotho group (all P< 0. 05) . Conclusion Gastric administration of 100 mg/kg Klotho protein can alleviate CIRI in ACI rats, and its mechanism may be related to inhibiting the expression of RIP1 and RIP3 proteins in cerebral cortex and inhibiting the inflammatory response and oxidative stress response at the site of brain injury. [ABSTRACT FROM AUTHOR]

The precise and targeted delivery of therapeutic agents to the lesion sites remains a major challenge in treating brain diseases represented by ischemic stroke. Herein, we modified liposomes with mesenchymal stem cells (MSC) membrane to construct biomimetic liposomes, termed MSCsome. MSCsome (115.99 ± 4.03 nm) exhibited concentrated accumulation in the cerebral infarcted hemisphere of mice with cerebral ischemia-reperfusion injury, while showing uniform distribution in the two cerebral hemispheres of normal mice. Moreover, MSCsome exhibited high colocalization with damaged nerve cells in the infarcted hemisphere, highlighting its advantageous precise targeting capabilities over liposomes at both the tissue and cellular levels. Leveraging its superior targeting properties, MSCsome effectively delivered Dl-3-n-butylphthalide (NBP) to the injured hemisphere, making a single-dose (15 mg/kg) intravenous injection of NBP-encapsulated MSCsome facilitate the recovery of motor functions in model mice by improving the damaged microenvironment and suppressing neuroinflammation. This study underscores that the modification of the MSC membrane notably enhances the capacity of liposomes for precisely targeting the injured hemisphere, which is particularly crucial in treating cerebral ischemia-reperfusion injury. We constructed biomimetic liposomes based on mesenchymal stem cell membrane, possessing precise brain-targeting properties at both tissue and cellular levels, which can efficiently target the infarcted hemisphere and are further internalized by damaged nerve cells. [Display omitted] • MSCsome incorporated cellular properties onto the inherent characteristics of liposomes. • MSCsome accumulated in the cerebral infarcted hemisphere, while showing non-regionally selective distribution in normal mice. • A single-dose intravenous injection of NBP-MSCsome showed superior efficacy in treating cerebral ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

Backgrounds: Mature angiogenesis plays a critical role in improving cerebral ischemia-reperfusion injury (CIRI). Glycolysis serves as the primary energy source for brain microvascular endothelial cells (BMECs), whereas other vascular cells rely on aerobic respiration. Therefore, intercellular variations in energy metabolism could influence mature angiogenesis. Taohong Siwu Decoction (THSWD) has demonstrated efficacy in treating ischemic stroke (IS), yet its potential to promote mature angiogenesis through glycolysis activation remains unclear. Methods: In this study, we established a middle cerebral artery occlusion/reperfusion (MCAO/R) model in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. We assessed neuroprotective effects using neurobehavioral scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin-eosin (HE) staining, and Nissl staining in MCAO/R rats. Additionally, we evaluated mature angiogenesis and glycolysis levels through immunofluorescence, immunohistochemistry, and glycolysis assays. Finally, we investigated THSWD's mechanism in linking glycolysis to mature angiogenesis in OGD/R-induced BMECs. Results: In vivo experiments demonstrated that THSWD effectively mitigated cerebral damage and restored neurological function in MCAO/R rats. THSWD significantly enhanced CD31, Ang1, PDGFB, and PDGFR-ß expression levels, likely associated with improved glucose, pyruvate, and ATP levels, along with reduced lactate and lactate/pyruvate ratios. In vitro findings suggested that THSWD may boost the expression of mature angiogenesis factors (VEGFA, Ang1, and PDGFB) by activating glycolysis, increasing glucose uptake and augmenting lactate, pyruvate, and ATP content, thus accelerating mature angiogenesis. Conclusion: THSWD could alleviate CIRI by activating the glycolysis pathway to promote mature angiogenesis. Targeting the glycolysis-mediated mature angiogenesis alongside THSWD therapy holds promise for IS treatment. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the protective effect and underlying mechanism of orexin A on cerebral ischemia-reperfusion injury, specifically through vasodilation mediated by the hypoxia inducible factor-1α (HIF-1α)-Endothelin-1(ET-1)/endothelial nitric oxide synthase (eNOS) pathway. A model of middle cerebral artery occlusion was established in both wild-type SD rats with exogenous orexin A intervention and in orexin A transgenic rats. Neurological deficit scores and cerebral infarction areas were assessed, and ischemic cortical blood flow was monitored. Gene and protein expression levels of HIF-1α, HIF-2α, ET-1, and three types of NOS were detected using real-time RT-qPCR and Western blot analysis, respectively. Additionally, nitric oxide (NO) levels in the cortex were analyzed through biochemical detection methods. Orexin A demonstrated a protective effect by reducing cerebral infarction and improving neurological deficits, which was achieved by increasing cortical blood flow during reperfusion. This protective mechanism was associated with upregulated HIF-1α expression, downregulated ET-1 expression, upregulated eNOS expression, and increased NO production. This study demonstrates the protective effect of orexin A on cerebral ischemia-reperfusion injury, achieved by regulating the release of vasomotor substances to enhance cortical blood flow during reperfusion. These findings suggest that orexin A may represent a potential therapeutic strategy for ischemic stroke.

Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that occurs when blood supply is restored to ischemic brain tissue and is one of the leading causes of adult disability and mortality. Multiple pathological mechanisms are involved in the progression of CIRI, including neuronal oxidative stress and mitochondrial dysfunction. Isoliquiritigenin (ISL) has been preliminarily reported to have potential neuroprotective effects on rats subjected to cerebral ischemic insult. However, the protective mechanisms of ISL have not been elucidated. This study aims to further investigate the effects of ISL-mediated neuroprotection and elucidate the underlying molecular mechanism. The findings indicate that ISL treatment significantly alleviated middle cerebral artery occlusion (MCAO)-induced cerebral infarction, neurological deficits, histopathological damage, and neuronal apoptosis in mice. In vitro, ISL effectively mitigated the reduction of cell viability, Na+-K+-ATPase, and MnSOD activities, as well as the degree of DNA damage induced by oxygen-glucose deprivation (OGD) injury in PC12 cells. Mechanistic studies revealed that administration of ISL evidently improved redox homeostasis and restored mitochondrial function via inhibiting oxidative stress injury and ameliorating mitochondrial biogenesis, mitochondrial fusion-fission balance, and mitophagy. Moreover, ISL facilitated the dissociation of Keap1/Nrf2, enhanced the nuclear transfer of Nrf2, and promoted the binding activity of Nrf2 with ARE. Finally, ISL obviously inhibited neuronal apoptosis by activating the Nrf2 pathway and ameliorating mitochondrial dysfunction in mice. Nevertheless, Nrf2 inhibitor brusatol reversed the mitochondrial protective properties and anti-apoptotic effects of ISL both in vivo and in vitro. Overall, our findings revealed that ISL exhibited a profound neuroprotective effect on mice following CIRI insult by reducing oxidative stress and ameliorating mitochondrial dysfunction, which was closely related to the activation of the Nrf2 pathway.

Abstract Background Cerebral ischemic disease is a common cerebrovascular disease, especially ischemic stroke. Exercise has protective functions on brain tissues following cerebral ischemia‐reperfusion injury (CIRI), but its preventive effects and mechanisms in CIRI remain unclear. We aimed to investigate the effects and mechanisms of exercise preconditioning on CIRI. Methods The middle cerebral artery occlusion (MCAO) operation was prepared to establish CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE‐TIMP1, exercise preconditioning, MCAO operation plus intraventricular injection of OE‐TIMP1) groups. Results The results indicated that exercise preconditioning suppressed approximately 66.67% of neurological deficit scores and 73.79% of TIMP1 mRNA expression in MCAO rats, which were partially offset by OE‐TIMP1. The protective effects of exercise against neuron death status and cerebral infarction size in MCAO rats were reversed by OE‐TIMP1. It also confirmed that exercise weakened apoptosis and oxidative stress damage, with notable increases of B‐cell lymphoma‐2, superoxide dismutase, and glutathione peroxidase production, and evident decreases of BCL2‐associated X, caspase 3, and malondialdehyde in MCAO rats, while these effects were partially reversed by OE‐TIMP1. Additionally, the inhibitory effects of exercise on the protein levels of TIMP1, hypoxia‐inducible factor‐alpha, vascular endothelial growth factor receptor 2, vascular endothelial growth factor, and neurogenic locus notch homolog protein 1 in MCAO rats were partially reversed by OE‐TIMP1. Conclusion Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1, which provided new therapeutic strategies for preventing CIRI.

• Inhibition of MyD88 attenuates cerebral ischemia-reperfusion injury in mice. • MyD88 activates microglia via IRF5 following cerebral ischemia-reperfusion (CIR) • MyD88 participates in blood–brain barrier damage following CIR. Myeloid differentiation primary response gene 88 (MyD88), a downstream molecule directly linked to Toll-like receptor (TLRs) and IL1 receptor, has been implicated in ischemia-reperfusion injury across various organs. However, its role in cerebral ischemia-reperfusion injury (CIRI) remains unclear. Five transient middle cerebral artery occlusion (tMCAO) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. We screened these datasets for differentially expressed genes (DEGs) using the GSE35338 and GSE58720 datasets and performed weighted gene co-expression network analysis (WGCNA) using the GSE30655, GSE28731, and GSE32529 datasets to identify the core module related to tMCAO. A protein–protein interaction (PPI) network was constructed using the intersecting DEGs and genes in the core module. Finally, we identified Myd88 was the core gene. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) validated that TNFα, IL17, and MyD88 signaling pathways were significantly enriched in tMCAO. Subsequently, we investigated the mechanistic role of MyD88 in the tMCAO model using male C57BL/6 mice. MyD88 expression increased significantly 24 h after reperfusion. After intraperitoneal administration of TJ-M2010-5, a MyD88-specific inhibitor, during reperfusion, the infarction volumes in the mice were ameliorated. TJ-M2010-5 inhibits the activation of microglia and astrocytes. Moreover, it attenuates the upregulation of inflammatory cytokines TNFα, IL17, and MMP9 while preserving the expression level of ZO1 after tMCAO, thereby safeguarding against blood-brain barrier (BBB) disruption. Finally, our findings suggest that MyD88 regulates the IRAK4/IRF5 signaling pathway associated with microglial activation. MyD88 participates in CIRI by regulating the inflammatory response and BBB damage following tMCAO. [ABSTRACT FROM AUTHOR]

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia–reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection. In a 4-VO rat model, 119 urinary proteins demonstrated significant changes associated with MH. MH is enriched in endopeptidase activity, inflammatory response, oxidative stress, etc, and significantly reversed changes in 12 DEPs. FZD1 and B2M are thought to be involved in the most fundamental molecular biological mechanisms of MH neuroprotection. [ABSTRACT FROM AUTHOR]

BACKGROUND: Inflammation is one of the important factors that induce cerebral ischemia-reperfusion injury. Studies have shown that electroacupuncture can effectively reduce inflammation after ischemic stroke and improve the symptoms of neurological deficits, but the mechanism is not clear. OBJECTIVE: To observe the effect of electroacupuncture on Toll-like receptor 4/nuclear factor-κB in rats with cerebral ischemia-reperfusion injury. METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into sham operation group, model group and electroacupuncture group, with 16 rats in each group. The rat model of cerebral ischemia-reperfusion injury was prepared by middle cerebral artery occlusion. At 24 hours after modeling, the rats in the electroacupuncture group were treated with electroacupuncture, once a day, 20 minutes each time, for a total of 5 days. The sham operation group and the model group did not do any intervention. After 5 days of intervention, Longa method was used to evaluate the degree of neurological injury in rats. Triphenyl tetrazolium chloride staining and hematoxylin-eosin staining were used to measure the volume of cerebral infarction and the pathological changes of brain tissue in rats. Serum interleukin-6, interleukin-18 and tumor necrosis factor-α were detected by ELISA. Expressions of Toll-like receptor 4 and nuclear factor-κB in the cerebral cortex at mRNA and protein levels were detected by fluorescence quantitative PCR and western blot, respectively. RESULTS AND CONCLUSION: Compared with the sham operation group, the neurological function scores, serum interleukin-6, interleukin-18, and tumor necrosis factor-α levels, Toll-like receptor 4 and nuclear factor-κB mRNA and protein expression levels were significantly higher in the model group (P < 0.01). Compared with the model group, electroacupuncture significantly reduced the neurological function scores, serum interleukin-6, interleukin-18, and tumor necrosis factor-α levels, Toll-like receptor 4 and nuclear factor-κB mRNA and protein expression levels (P < 0.05, P < 0.01). Compared with the sham operation group, the volume of cerebral infarction in the model group increased significantly (P < 0.01). Compared with the model group, the volume of cerebral infarction in the electroacupuncture group decreased (P < 0.05). In the model group, the arrangement of neurons was disordered, some nerve cells disappeared, nuclei presented with pyknosis and incomplete structure. After electroacupuncture intervention, the degree of neuronal degeneration and neuronal loss in the cerebral cortex of rats were reduced compared with those in the model group. To conclude, electroacupuncture can significantly improve the neurobehavior of rats with cerebral ischemia-reperfusion injury, reduce brain tissue injury, and effectively reduce the level of serum inflammatory factors. The mechanism may be related to the inhibition of Toll-like receptor 4/nuclear factor-κB signaling pathway. [ABSTRACT FROM AUTHOR]

AIM: To comprehend the mechanism by which ginsenoside Rc protects against cerebral ischemiareperfusion injury (CIRI), with a particular emphasis on pyroptosis. METHODS: The C57BL/6 mice were randomly divided into 6 groups: sham group, middle cerebral artery occlusion/reperfusion (MCAO/R) group, ginsenoside Rc+MCAO/ R group (Rc group), AMP-activated protein kinase (AMPK) agonist acadesine/5-aminoimidazole-4-carboxamide-1-β-Dribofuranoside (AICAR)+MCAO/R group (agonist group), and ginsenoside Rc+MCAO/R+AMPK inhibitor Compound C group (Rc+inhibitor group). The mice in agonist group were given 500 mg/kg of AICAR intraperitoneally, while those in Rc+inhibitor group were given 20 mg/kg of Compound C intraperitoneally. Ginsenoside Rc was gavaged into the mice in Rc and Rc+inhibitor groups at a dose of 40 mg/kg once per day for 7 d after modeling, while the mice in sham and MCAO/ R groups got the same volume of purified water. With the use of the Zea-Longa score, we determined which mice had neurological abnormalities. TTC staining was employed for assessing the cerebral infarct amount in mice, and the dry wet weight technique was utilized for determining the degree of cerebral edema. Moreover, HE staining was used to observe pathological alterations in the brain, and Western blot and RT-qPCR were applied for detecting the expression of AMPK, nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3), caspase-1 and gasdermin-D (GSDMD) in the brains of mice. Lastly, ELISA was employed for measuring the levels of inflammatory factors, interleukin-1β(IL-1β) and IL-18. RESULTS: Brain edema, infarct volume and neurological impairments were all diminished in agonist and Rc groups. Additionally, they demonstrated neuronal damage inhibition. The ratio of p-AMPK/AMPK and AMPK mRNA expression in mouse brain tissues was elevated in both Rc and agonist groups. They showed decreased mRNA and protein levels of NLRP3, caspase-1 and GSDMD, as well as the levels of IL-1β and IL-18. Compared with Rc group, there were remarkable decreases in p-AMPK/AMPK ratio and AMPK mRNA expression in the brain tissue of mice in Rc+inhibitor group (P<0. 05). The mRNA and protein levels of NLRP3, caspase-1 and GSDMD, and the IL-1β and IL-18 expression levels were significantly increased (P<0. 05). Moreover, the neurological deficiency scores and infarct volume were increased, and the degrees of cerebral edema and neuronal pathological damage were enhanced (P<0. 05). CONCLUSION: Ginsenoside Rc may inhibit NLRP3/caspase-1/GSDMD-mediated pyroptosis by activating AMPK pathway, thereby reducing CIRI in mice. [ABSTRACT FROM AUTHOR]

Objective To explore the effect of probiotics interventionon of the expression of Aβ in rats with cerebral ischemia-reperfusion and the protective effect of neurons. Methods Male SD rats were selected as the experimental animals and randomly divided into Sham Group, Model Group, Probiotics Group and Edaravone Group. The common carotid artery was separated only to the Sham Group While a cerebral ischemia-reperfusion model was eatablished to the other groups. The degree of brain injury was evaluated using neurobehavioral scoring; The infarct area of rat brain tissue was observed using TTC staining; HE staining was used to observe the pathological and morphological changes of neurons in the CA1 and cortical regions of the hippocampus in rats; The expression of Aβ proteins was detected using immunohistochemistry experiments. Results Compared with the Sham Group, the Model Group showed an increase in neurobehavioral scores (P < 0.05), an increase in cerebral infarction focus (P < 0.05), neuronal damage in the hippocampal CA1 and cortical regions, and a significant upregulation of Aβ protein expression (P < 0.001, P < 0.05); Compared with the Model Group, the Probiotics Group and Edaravone Group showed a decrease in neurobehavioral scores (P < 0.05) and a reduction in cerebral infarction focus (P < 0.05). The Probiotic Group and the Edaravone Group showed the less neuronal damage in the hippocampal CA1 and cortical regions, and the Aβ protein expression levels were significantly down regulated in both groups (P < 0.001, P < 0.05). The improvement was more significant in Edaravone Group. Conclusion Probiotics intervention can downregulate the expression of Aβ protein, alleviate the brain tissue damage and exert neuroprotective effects in rats with cerebral ischemia-reperfusion. [ABSTRACT FROM AUTHOR]

Autophagy has been involved in protection of ischemia/reperfusion (I/R)-induced injury in many tissues including the brain. The upstream stimulatory factor 2 (Usf2) was proposed as a regulator in aging and degenerative brain diseases; however, the its role in autophagy during cerebral I/R injury remains unclear. Here, the middle cerebral artery occlusion (MCAO) operation was applied to establish an I/R mouse model. We showed that Usf2 was significantly upregulated in I/R-injured brain, accompanied by decreased levels of autophagy. Then, oxygen-glucose deprivation/recovery (OGD/R) treatment was used to establish a cellular I/R model in HT22 neurons, and lentiviral interference vector against Usf2 (LV-sh-Usf2) was used to infect the neurons. Our results showed that Usf2 was significantly upregulated in OGD/R-treated HT22 neurons that displayed an increased level in cell apoptosis and decreased levels in cell viability and autophagy, and interference of Usf2 largely rescued the effects of OGD/R on cell viability, apoptosis, and autophagy, suggesting an important role of Usf2 in neuron autophagy. In the mechanism exploration, we found that, as a transcription factor, Usf2 bound to the promoter of YTHDF1, a famous reader of N6-Methyladenosine (m6A), also induced by OGD/R, and promoted its transcription. Overexpression of YTHDF1 was able to reverse the improvement of Usf2 interference on viability and autophagy of HT22 neurons. Moreover, YTHDF1 suppressed autophagy to induce HT22 cell apoptosis through increasing m6A-mediated stability of Cdc25A, a newly identified autophagy inhibitor. Finally, we demonstrated that interference of Usf2 markedly improved autophagy and alleviated I/R-induced injury in MCAO mice. [ABSTRACT FROM AUTHOR]

Objectives: Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in treatment of cerebral ischemia ‐reperfusion injury (CIRI); however, the mechanisms underlying its benificial effects remain unclear. SUMO‐1ylation and deSUMO‐2/3ylation of dynamin‐related protein 1 (Drp1) results in mitochondrial homeostasis imbalance following CIRI, which subsequently aggravates cell damage. This study investigates the mechanisms by which AST IV combined with Lig protects against CIRI, focusing on the involvement of SUMOylation in mitochondrial dynamics. Methods: Rats were administrated AST IV and Lig for 7 days, and middle cerebral artery occlusion was established to mimic CIRI. Neural function, cerebral infarction volume, cerebral blood flow, cognitive function, cortical pathological lesions, and mitochondrial morphology were measured. SH‐SY5Y cells were subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury. Mitochondrial membrane potential and lactic dehydrogenase (LDH), reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed with commercial kits. Moreover, co‐immunoprecipitation (Co‐IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. The protein expressions of Drp1, Fis1, MFF, OPA1, Mfn1, Mfn2, SUMO1, SUMO2/3, SENP1, SENP2, SENP3, SENP5, and SENP6 were measured using western blot. Results: In rats with CIRI, AST IV and Lig improved neurological and cognitive functions, restored CBF, reduced brain infarct volume, and alleviated cortical neuron and mitochondrial damage. Moreover, in SH‐SY5Y cells, the combination of AST IV and Lig enhanced cellular viability, decreased release of LDH and ROS, increased ATP content, and improved mitochondrial membrane potential. Furthermore, AST IV combined with Lig reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3, suppressed the expressions of Drp1, Fis1, MFF, and SENP3, and increased the expressions of OPA1, Mfn1, Mfn2, SENP1, SENP2, and SENP5. SUMO1 overexpression promoted mitochondrial fission and inhibited mitochondrial fusion, whereas SUMO2/3 overexpression suppressed mitochondrial fission. AST IV combined with Lig could reverse the effects of SUMO1 overexpression while enhancing those of SUMO2/3 overexpression. Conclusions: This study posits that the combination of AST IV and Lig has the potential to reduce the SUMO‐1ylation of Drp1, augment the SUMO‐2/3ylation of Drp1, and thereby exert a protective effect against CIRI. [ABSTRACT FROM AUTHOR]

NLRP3/ASC/Caspase-1 mediated pyroptosis is one of the important causes of cerebral ischemia-reperfusion (I/R) injury. Electroacupuncture (EA) is widely used in clinical treatment of ischemic stroke. However, mechanism of EA on ischemic stroke remains unclear. Therefore, on basis of a previous work, this study used middle cerebral artery occlusion (MCAO) 2 h and then reperfusion 7 days in rats to simulate brain I/R process. EA with Bahui (GV20) and Zusanli (ST36) and VX-765 (a specific inhibitor of Caspase-1) was performed. In this study, we found that EA improved cerebral infarct size and neuronal damage, including ultrastructural injury, and ameliorated nitro/oxidative stress in cerebral I/R. Additionally, EA treatment significantly decreased ASC, Caspase-1, GSDMD, and IL-1β expression and VX-765 treatment significantly decreased NLRP3, Caspase-1, and IL-1β expression. This proved that EA can regulate NLRP3/ASC/Caspase-1 mediated pyroptosis, improve neuronal injury during cerebral I/R, and provide basic experimental data for clinical treatment. [ABSTRACT FROM AUTHOR]

Abnormal accumulation of senescent cells in tissues has been shown to facilitate the onset and progression of various diseases. As an important protein involving in the regulation of cellular senescence process, researches suggested GRSF1 as a potential senolytic target to improve multiple physiological and pathological processes. However, the underlying mechanism of cellular senescence on cerebral ischemia-reperfusion injury (CIRI) has not been revealed. Here, we investigated the effect of GRSF1 on CIRI and delved into its specific mechanisms. In the present study, we established a mouse model of cerebral ischemia-reperfusion (CIR) and observed low expression of anti-aging factor GRSF1, along with greatly increased levels of senescence-related markers p16 and p21 and senescence-associated secretory phenotype TNF-α. Furthermore, we found that the expression of GPX4 was elevated parallel to GRSF1 in CIR mice with overexpression of GRSF1, oxidative stress, and iron metabolism-related proteins were inhibited. Functionally, overexpressing GRSF1 significantly ameliorated infarct volume and neurological function scores and suppressed apoptosis in CIR mice, while administration of GPX4 inhibitors reversed these beneficial phenotypes. Taken together, our results indicate cellular senescence as an important pathological mechanism to exacerbate cerebral injury during CIRI, while GRSF1 could inhibit oxidative stress-mediated ferroptosis through upregulating GPX4 to attenuate reperfusion injury, which makes senolytic treatment, especially GRSF1, a promising therapeutic target for CIRI. [ABSTRACT FROM AUTHOR]

Objective: Research has shown that cerebral ischemia-reperfusion injury (CIRI) involves a series of physiological and pathological mechanisms, including inflammation, oxidative stress, and cell apoptosis. The cannabinoid receptor 2 agonist AM1241 has been found to have anti-inflammatory and anti-oxidative stress effects. However, it is unclear whether AM1241 has a protective effect against brain ischemia-reperfusion injury, and its underlying mechanisms are not yet known. Methods: In this study, we investigated the anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects of AM1241 and its mechanisms in BV2 cells stimulated with H2O2 and in a C57BL/6 mouse model of CIRI in vitro and in vivo, respectively. Results: In vitro, AM1241 significantly inhibited the release of pro-inflammatory cytokines TNF-α and IL-6, reactive oxygen species (ROS), and the increase in Toll-like receptor 4/myeloid differentiation protein 2 (MD2/TLR4) complex induced by H2O2. Under H2O2 stimulation, MD2 overexpression resulted in increased levels of MD2/TLR4 complex, TNF-α, IL-6, NOX2, BAX, and Cleaved-Caspase3 (C-Caspase3), as well as the activation of the MAPK pathway and NF-κB, which were reversed by AM1241. In addition, molecular docking experiments showed that AM1241 directly interacted with MD2. Surface Plasmon Resonance (SPR) experiments further confirmed the binding of AM1241 to MD2. In vivo, AM1241 significantly attenuated neurofunctional impairment, brain edema, increased infarct volume, oxidative stress levels, and neuronal apoptosis in CIRI mice overexpressing MD2. Conclusion: Our study demonstrates for the first time that AM1241 alleviates mouse CIRI by inhibiting the MD2/TLR4 complex, exerting anti-inflammatory, anti-oxidative stress and anti-apoptotic effects.

Aims: Cerebral ischemic preconditioning is a neuroprotective therapy against cerebral ischemia and ischemia-reperfusion injury. This study aims to demonstrate the alternation of gene expression in exosomes from brain tissue of mice after ischemic preconditioning and their potential functions. Methods: Ten mice were divided into the sham and the cerebral ischemic preconditioning groups. Their brain tissues were harvested, from which the exosomes were extracted. The characteristics and protective effects of exosomes were evaluated. Whole transcriptome sequencing was used to demonstrate the gene expression discrepancy between the exosomes from the two groups of mice brains. Volcano graphs and heatmaps were used to picture the difference in expression quantity of mRNA, lncRNA, and circRNA. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to demonstrate the functions of differentially expressed RNAs. Results: Exosomes were successfully extracted, and those from the cerebral ischemic preconditioning group had better protective effects on cells that received oxygen-glucose deprivation and restoration injury. A total of 306 mRNAs and 374 lncRNAs were significantly upregulated, and 320 mRNAs and 405 lncRNAs were significantly downregulated in the preconditioning group. No circRNAs were differentially expressed between the two groups. GO and KEGG pathway analysis indicated that the functions of differentially expressed RNAs were related to both neural protective and injurious effects. Conclusion: The brain-derived exosomes may participate in the neuroprotective effect of cerebral ischemic preconditioning. Thorough research is necessary to investigate exosome functions derived from the ischemic preconditioned brain.

BackgroundThe Chinese ethnic medicine Jie-Du-Huo-Xue Decoction (JDHXD) is used to alleviate neuroinflammation in cerebral ischemia (CI). Our previous studies have confirmed that JDHXD can inhibit microglial pyroptosis in CI. However, the pharmacological mechanism of JDHXD in alleviating neuroinflammation and pyroptosis needs to be further elucidated. New research points out that there is an interaction between autophagy and inflammasome NLRP3, and autophagy can help clear NLRP3. The NLRP3 is a key initiator of pyroptosis and autophagy. The effect of JDHXD promoting autophagy to clear NLRP3 to inhibit pyroptosis on cerebral ischemia-reperfusion inflammatory injury is currently unknown. We speculate that JDHXD can inhibit pyroptosis in CI by promoting autophagy to clear NLRP3.MethodsChemical characterization of JDHXD was performed using LC-MS. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in SD rats. Neurological deficits, neuron damage, and cerebral infarct volume were evaluated. Western Blot and immunofluorescence were used to detect neuronal pyroptosis and autophagy.Results30 possible substance metabolites in JDHXD medicated serum were analyzed by LC-MS (Composite Score > 0.98). Furthermore, JDHXD protects rat neurological function and cerebral infarct size after CI. JDHXD inhibited the expression of pyroptosis and autophagy after CI. Our western blot and immunofluorescence results showed that JDHXD treatment can reduce the expression of autophagy-related factors ULK1, beclin1, and LC3-Ⅱ. The expression of NLRP3 protein was lower in the JDHXD group than in the I/R group. Compared with the I/R group, the expressions of pyroptosis-related factors caspase-1 P 10, GSDMD-NT, IL-18, and IL-1β decreased in the JDHXD group. Furthermore, we observed an unexpected result: immunofluorescence demonstrated that Gasdermin D (GSDMD) was significantly absent in the infarct core, and highly expressed in the peri-infarct and contralateral cerebral hemispheres. This finding challenges the prevailing view that GSDMD is elevated in the ischemic cerebral hemisphere.ConclusionJDHXD inhibited pyroptosis and autophagy after MCAO/R. JDHXD suppressed pyroptosis and autophagy by inhibiting NLRP3, thereby alleviating CI. In addition, we present a different observation from previous studies that the expression of GSDMD in the infarct core was lower than that in the peri-infarct and contralateral non-ischemic hemispheres on day 3 of CI.

Background: Electroacupuncture (EA) has been shown to promote functional recovery after cerebral ischemia–reperfusion (I/R) injury. However, the contribution of mitochondrial dynamics to recovery remains unclear. The aim of this study was to investigate whether mitochondrial dynamics are involved in the effects of EA on cerebral I/R injury. Methods: The rats with cerebral I/R injury were established by the middle cerebral artery occlusion/reperfusion. Subsequently, EA was applied to Baihui (GV20) and Dazhui (GV14) acupoints, with 2 Hz/5 Hz in frequency, 1.0 mA in intensity, 20 min each time, once a day for seven consecutive days. The therapeutic outcomes were assessed by modified neurological severity score (mNSS), 2,3,5-Triphenyte-trazolium chloride (TTC) staining, and hematoxylin-eosin (HE) staining. Mitochondrial morphology was observed under transmission electron microscopy. Adenosine triphosphate (ATP) content and ATP synthases (ATPases) activity were evaluated to measure mitochondrial function using ELISA. Finally, mitochondrial dynamics-related molecules, including dynamin-related protein 1 (Drp1), fission 1 (Fis1), mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy 1 (OPA1), were detected by Western blot and immunofluorescence staining. Results: Cerebral I/R injury induced neurological dysfunction, cerebral infarction and neuronal injury, all of which were ameliorated by EA. And EA improved mitochondrial morphology and function. Moreover, EA altered the balance of mitochondrial dynamics. Specifically, the data showed a significant decrease in the expression of Drp1 and Fis1, leading to the inhibition of mitochondrial fission. Additionally, Mfn1, Mfn2 and Opa1, which are related to mitochondrial fusion, were effectively promoted after EA treatment. However, sham EA did not show any neuroprotective effects in rats with cerebral I/R injury. Conclusions: In summary, our study indicates that the balance of mitochondrial dynamics is crucial for EA therapy to treat cerebral I/R injury.

Cerebral ischemia–reperfusion injury significantly contributes to global morbidity and mortality. Loganin is a natural product with various neuroprotective effects; however, it lacks targeted specificity for particular cells or receptors, which may result in reduced therapeutic efficacy and an increased risk of side effects. To address the limitations of loganin, we developed LA-1, a novel compound incorporating an Arg-Gly-Asp (RGD) peptide to target integrin receptor αvβ3, enhancing brain-targeting efficacy. LA-1 exhibited optimal nanoscale properties, significantly improved cell viability, reduced ROS production, and enhanced survival rates in vitro. In vivo, LA-1 decreased infarct sizes, improved neurological function, and reduced oxidative stress and neuroinflammation. Proteomic analysis showed LA-1 modulates PI3K/Akt and Nrf2/HO-1 pathways, providing targeted neuroprotection. These findings suggest LA-1’s potential for clinical applications in treating cerebral ischemia–reperfusion injury.