Melastoma dodecandrum lour. Protects against cerebral ischemia-reperfusion injury by ameliorating oxidative stress and endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Melastoma dodecandrum Lour. (MD), a traditional Chinese medicine used by the She ethnic group, has been used to treat cerebral ischemia-reperfusion (CIR) injury due to its efficacy in promoting blood circulation and removing blood stasiss; however, the therapeutic effects and mechanisms of MD in treating CIR injury remain unclear. AIM: To investigate the protective effects of MD on CIR injury, in addition to its impact on oxidative stress, endoplasmic reticulum (ER) stress, and cell apoptosis. MATERIALS AND METHODS: The research was conducted using both cell experiments and animal experiments. The CCK-8 method, immunofluorescence staining, and flow cytometry were used to analyze the effects of MD-containing serum on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cell viability, reactive oxygen species (ROS) clearance, anti-inflammatory, neuroprotection and inhibition of apoptosis. Furthermore, 2,3,5-Triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, Nissl staining, and immunohistochemistry were used to detect infarct size, pathological changes, Nissl corpuscula and neuronal protein expression in middle cerebral artery occlusion (MCAO) rats. Polymerase chain reaction and Western Blotting were conducted in cell and animal experiments to detect the expression levels of ER stress-related genes and proteins. RESULTS: The MD extract enhanced the viability of PC12 cells under OGD/R modeling, reduced ROS and IL-6 levels, increased MBP levels, and inhibited cell apoptosis. Furthermore, MD improved the infarct area in MCAO rats, increased the number of Nissl bodies, and regulated neuronal protein levels including Microtubule-Associated Protein 2 (MAP-2), Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP), and Neurofilament 200 (NF200). Additionally, MD could regulate the expression levels of oxidative stress proteins malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Both cell and animal experiments demonstrated that MD could inhibit ER stress-related proteins (GRP78, ATF4, ATF6, CHOP) and reduce cell apoptosis. CONCLUSION: This study confirmed that the therapeutic mechanism of the MD extract on CIR injury was via the inhibition of oxidative stress and the ER stress pathway, in addition to the inhibition of apoptosis.

Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

Astragaloside IV plays a neuroprotective role by promoting PPARγ in cerebral ischemia-reperfusion rats.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) usually occurs during the treatment phase of ischemic disease, which is closely related to high morbidity and mortality. Promoting neurogenesis and synaptic plasticity are effective neural recovery strategies for CIRI. Astragaloside IV (AS-IV) has been shown to play a neuroprotective role in some neurological diseases. In the current study, we evaluated the effect and possible mechanism of AS-IV in CIRI rats. METHODS: The middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats to simulate the occurrence of human CIRI. First, we determined the cerebral injury on the 1st, 3rd, 5th and 7th day after cerebral ischemia-reperfusion (I/R) surgery by neurological deficit detection, TTC staining, TUNEL staining and Western blot analysis. Furthermore, rats were pre administered with AS-IV and then subjected to cerebral I/R surgery. Brains were collected on the 3rd day to evaluate the neuroprotective effect of AS-IV. RESULTS: Our results showed that on the 3rd day after I/R, the neurological impairment score and infarct volume were highest, the levels of apoptosis and expression of Caspase3 and Bax reached the peak. AS-IV treatment apparently attenuated neurological dysfunction, reduced infarct volume and pathological damage, promoted the neurogenesis, and alleviated the pathological damage caused by cerebral I/R involved in thickening and blurring of synaptic membranes, reduction of microtubules and synaptic vesicles, and loss of synaptic cleft. Our study also showed that AS-IV promoted the transcription and expression of the peroxisome proliferators-activated receptors γ (PPARγ) and brain-derived neurotrophic factor (BDNF), increased the expression of phosphorylation of tyrosine kinase receptor B (TrkB) and downstream PI3K/Akt/mTOR pathway proteins. Notably, when GW9662, an inhibitor of PPARγ was administered with AS-IV, the neuroprotective effect of AS-IV was reduced. CONCLUSIONS: These findings suggested that AS-IV has neuroprotective function in CIRI rats, and its molecular mechanism may depend on the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB)/Akt signalling pathway activated by PPARγ. AS-IV could be an effective therapeutic drug candidate for CIRI treatment.

Galangin Regulates Astrocyte Phenotypes to Ameliorate Cerebral Ischemia-reperfusion Injury by Inhibiting the RhoA/ROCK/LIMK Pathway.

PURPOSE: This study aimed to explore whether Galangin (Gal) could improve cerebral Ischemia- reperfusion (I/R) injury by regulating astrocytes, and clarify its potential molecular mechanism. METHODS: An I/R injury model of rats was established using the Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R) method, followed by the administration of Gal (25, 50, 100 mg/kg) via gavage for 14 consecutive days. Besides, astrocytes were isolated from the rats to construct an Oxygen-Glucose Deprivation/Re-oxygenation (OGD/R) cell model, with treatments of Gal or the Ras homolog gene family member A (RhoA)/Rho-associated Coiled-coil containing protein Kinase (ROCK) inhibitor Y-27632. Subsequently, the severity of nerve injury was assessed using the modified Neurological Severity Score (mNSS) test; behavioral disorders in I/R rats were observed through the open field and ladder-climbing tests. Pathological damages and neuron survival in the peri-infarct zone were examined by hematoxylin and eosin staining and NeuN staining, respectively. Additionally, immunofluorescence staining was employed to determine astrocyte polarization and TUNEL staining was carried out to measure the level of cell apoptosis; also, western blot was performed to detect the expression of proteins related to the RhoA/ROCK/LIM domain Kinase (LIMK) pathway. RESULTS: Gal significantly ameliorated the neurological and motor dysfunctions caused by I/R in rats, reduced pathological damage in the peri-infarct zone, and promoted neuronal survival. Additionally, Gal increased the number of A2 astrocytes, while it decreased the number of A1 astrocytes. In vitro experiments revealed that the effect of Gal was consistent with that of Y-27632. Additionally, Gal significantly enhanced the survival of OGD/R cells, increased the number of A2 astrocytes, and inhibited the expression of proteins associated with the RhoA/ROCK pathway. CONCLUSION: Gal could reduce the level of apoptosis, promote the polarization of A2 astrocytes, and improve cerebral I/R injury, and its mechanism may be related to the inhibition of the RhoA/ROCK pathway.

Inhibiting Mitochondrial Damage for Efficient Treatment of Cerebral Ischemia-Reperfusion Injury Through Sequential Targeting Nanomedicine of Neuronal Mitochondria in Affected Brain Tissue.

Oxidative stress, predominantly from neuronal mitochondrial damage and the resultant cytokine storm, is central to cerebral ischemia-reperfusion injury (CIRI). However, delivering drugs to neuronal mitochondria remains challenging due to the blood-brain barrier (BBB), which impedes drug entry into affected brain tissues. This study introduces an innovative tannic acid (TA) and melanin-modified heteropolyacid nanomedicine (MHT), which highly specifically eliminates the neuronal mitochondrial reactive oxygen radicals burst to efficiently reduce neuronal mitochondrial damage through a strategically designed sequential targeting strategy from affected brain tissue to neuronal mitochondria. TA endows MHT with sequential targeting ability by binding to matrix proteins exposed to the damaged BBB and mitochondrial outer membrane proteins of neurons, while melanin significantly enhances the antioxidant capacity of MHT. Consequently, MHT effectively inhibits neuronal apoptosis by protecting mitochondria and reversing the inflammatory immune environment through the deactivation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. MHT demonstrated a strong therapeutic effect on CIRI, with an ultralow dose (2 mg kg-1) proving effective in reversing the condition. This work not only introduces a new avenue to significantly reduce CIRI through sequential targeting therapy but also offers a new paradigm for treating other ischemia-reperfusion injury diseases.

Dopamine receptor agonist pramipexole exerts neuroprotection on global cerebral ischemia/reperfusion injury by inhibiting ferroptosis.

OBJECTIVE: To explore the mechanism of dopamine receptor agonist pramipexole in exerting neuroprotection on global cerebral ischemia/reperfusion injury (GCI/R). MATERIAL AND METHOD: Male Sprague-Dawley rats were randomly divided into four groups (n = 36 in each group), and the Pulsinelli's four-vessel occlusion method was used to establish the rat model of GCI/R injury. Pramipexole administration group was intraperitoneally injected with pramipexole 0.5 mg/kg once a day for 14 days. Pramipexole combined with levodopa administration group was intraperitoneally injected with pramipexole 0.5 mg/kg and levodopa 50 mg/kg once a day for 14 days. The mNSS scores and Y maze test were used to evaluate neurological behaviors. Nissl staining and transmission electron microscopy were used to respectively observe hippocampal neurons and mitochondrial ultrastructure. Molecular biological tests including tissue iron concentration, GSH, MDA were used to detect the degree of ferroptosis. Western blotting was used to detect the expression levels of Nrf2, GPX4, X-CT and p53 proteins at 3 days, 7 days and 14 days after GCI/R injury. RESULTS: Pramipexole alone or combined with levodopa for 14 days improved neurological behaviors, improved the morphology of neurons, increased the number of surviving neurons in the hippocampal CA1 region of GCI/R rats, which showed similar neuroprotective effects. Pramipexole alone or combined with levodopa for 14 days restored mitochondrial ultrastructure, decreased malondialdehyde concentration and increased glutathione concentration in the brain of GCI/R rats, which also induced the relative expressions of Nrf2, GPX4 and X-CT proteins and reduced p53 protein. CONCLUSION: Pramipexole alone or combined with levodopa exert neuroprotection by inhibiting ferroptosis after GCI/R injury via Nrf2/GPX4/SLC7A11 pathway, and long-term intervention could be applied as an effective therapeutic strategy for neuroprotection against GCI/R injury.

Astrocytic Gap Junctions protein Cx43/Cx30 modulate EAAT1 and glutamate to mediate cerebral ischemia-reperfusion injury.

The gap connexins of astrocytes play a crucial role in facilitating neuronal coordination and maintaining the homeostasis of the central nervous system. Cx30/Cx43 are the main proteins constituting these gap junctions, and the glutamate transporter EAAT1 associates with nerve injury. However, the role and mechanism underlying the changes of astrocytic connexins and EAAT1 during cerebral ischemia-reperfusion injury remain unclear. In this study, we investigated the expressions of Cx30, Cx43, and EAAT1 in OGD/R-treated astrocytes and in a MCAO/R animal model using gap junction inhibitors and siRNAs targeting Cx43 and Cx30. The differences of cell viability, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reactive oxygen species (ROS) and glutamate in cells and tissues were detected. Our results indicate that OGD/R exposure leads to the decline of astrocyte activity, which, in turn, adversely affects neuronal health. Ischemia-reperfusion induced increasing Cx43 and EAAT1 expression and decreasing Cx30 expression in astrocytes and animal brain tissue. Moreover, ischemia-reperfusion resulted in heightened MDA and ROS levels and reduced CAT and SOD activities in both astrocytes and the surrounding brain tissue. The release of glutamate from astrocytes and its concentration in animal brain tissue significantly increased following ischemia-reperfusion. Inhibition Cx43 expression through Gap26 or siRNA effectively mitigated the increase in EAAT1 and glutamate levels, as well as the oxidative stress changes induced by ischemia-reperfusion. Therefore, Brain astrocytes may mediate the effects of cerebral ischemia-reperfusion injury by influencing glutamate transporters and glutamate dynamics in response to oxidative stress through Cx30/Cx43.

Pathophysiology of cerebral ischemia-reperfusion injury: An overview of oxidative stress and plant-based therapeutic approaches.

Stroke is a debilitating neurological disorder that causes substantial morbidity and mortality on a global scale. Ischemic stroke, the most common type, occurs when the brain's blood supply is interrupted. Oxidative stress is a key factor in stroke pathology, contributing to inflammation and neuronal cell death. As a result, there is increasing interest in the potential of plant extracts, which have been used in traditional medicine for centuries and are generally considered safe, to serve as alternative or complementary treatments for stroke. The plant extracts can target multiple pathological processes, including oxidative stress, offering neuroprotective effects. The development of highly efficient, low-toxicity, and cost-effective natural products is crucial for enhancing stroke treatment options. In this review, we examine 60 plant extracts that have been focused on the studies published from year 2000 to 2024 along with the studies' experimental models, dosages, and results. The plant extracts hold promise in modulating cerebral ischemia-reperfusion injury through counteraction of relevant pathophysiologic processes such as oxidative stress.

Polyphyllin VII ameliorates neuroinflammation and brain injury via modulating Treg/Th17 balance in a mouse model of cerebral ischemia-reperfusion injury.

Dysregulation of Th17 and Treg cells contributes to the pathophysiology of cerebral ischemia. Metabolic changes of peripheral CD4+ T cells lead to the imbalance of Treg/Th17 polarization, which represents a promising strategy for post-stroke therapy. Polyphyllin VII (PVII), a steroidal saponin extracted from traditional Chinese herb Rhizoma Paridis, has multiple bioactivities, but the potential function of PVII in cerebral ischemia-reperfusion injury is not elucidated yet. In our study, a mouse transient middle cerebral artery occlusion (MCAO) model was constructed. TTC staining, H&E staining, TUNEL staining, ELISA assay, flow cytometry, western blot, RT-qPCR, Open-field test, Morris water maze test, hanging wire test, rotarod test and foot-fault test were performed to evaluate the potential function of PVII in MCAO mice. We found that PVII showed protective effects on cerebral ischemia-reperfusion injury by reducing infarct volume, ameliorating brain injury and neuroinflammation, and improving long-term functional recovery of MCAO mice. PVII promoted Treg infiltration and suppressed infiltration of Th1/Th17 cells in ischemic brain in vivo. Moreover, PVII impaired peripheral CD4+ T cell activation and modulated Treg/Th17 differentiation in vitro. Mechanistically, PVII suppressed mTORC1 activation to influence glycolytic metabolism and ROS generation of T cells, thus leads to the imbalance of Treg/Th17 polarization towards Treg skewed. Furthermore, reactivation of mTORC1 by MHY1485 abolished the influence of PVII on brain injury and neuroinflammation in MCAO mice. Our data provided a novel role of PVII in cerebral ischemia-reperfusion injury via manipulating Treg/Th17 imbalance.

Orexin A protects against cerebral ischemia-reperfusion injury by enhancing reperfusion in ischemic cortex via HIF-1α-ET-1/eNOS pathway.

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.

The deubiquitinase OTUD3 stabilizes IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to ameliorate cerebral ischemia-reperfusion injury.

BACKGROUND: Ischemic stroke (IS) is known for its high morbidity, disability and mortality rates, and studies designed to explore its pathophysiological mechanisms and identify novel therapeutic strategies are urgently needed. We aimed to probe the effects of the deubiquitinase OTUD3-IRP2-p53/PTGS2 pathway on cerebral ischemia‒reperfusion (I/R) injury and hippocampal neuron ferroptosis. METHODS: A cerebral I/R mouse model was established. Furthermore, lentiviral vectors that overexpressed OTUD3 and knocked down IRP2 were constructed, and a series of assays were performed to probe the OTUD3/IRP2/p53/PTGS2 mechanism. An oxygen‒glucose deprivation and reoxygenation (OGD/R) model of mouse hippocampal neurons was constructed. Then, OTUD3 and IRP2 were knocked down and overexpressed, and p53 was overexpressed to explore the mechanism of the OTUD3/IRP2/p53/PTGS2 pathway. RESULTS: OTUD3 and IRP2 were expressed at low levels in cerebral I/R models. OTUD3 promoted IRP2 expression to protect damaged hippocampal neurons. Moreover, IRP2 affected ferroptosis in hippocampal neurons. In addition, IRP2 inhibited p53. After IRP2 and p53 were overexpressed, IRP2 regulated the p53/PTGS2 pathway and affected ferroptosis in hippocampal neurons. In vivo, after overexpressing OTUD3 and knocking down IRP2, we found that overexpression of OTUD3 promoted IRP2 expression to reduce ferroptosis in hippocampal neurons and improve cerebral I/R injury via the inhibition of the p53/PTGS2 pathway. CONCLUSIONS: The deubiquitinase OTUD3 stabilized IRP2 expression to reduce hippocampal neuron ferroptosis via the p53/PTGS2 pathway to subsequently ameliorate cerebral I/R injury.

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OBJECTIVES: To observe the effect of "Xingnao Kaiqiao" needling on the expression of ferroptosis-related proteins in neurons of rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanism underlying improvement of CIRI. METHODS: Male Wistar rats were randomly divided into sham operation, model, acupuncture and deferoxamine (DFO) groups, with 18 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery. In the acupuncture group, "Xingnao Kaiqiao" needling was applied to "Shuigou" (GV26), "Neiguan" (PC6) and "Sanyinjiao"(SP6) for 20 min with electroacupuncture (2 Hz/15 Hz, 1 mA) at PC6 and SP6, twice daily for continuous 3 days. Rats of the DFO group received intraperitoneal injection of iron chelator DFO (0.1 g/kg, once daily). The severity of neurological impairment (neurological deficit score, 0-5 points, the lower the score, the severer is the neurological impairment) was evaluated by using Zausinger 6-poins scaling method. The cerebral infarct volume was measured after 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and the histopathological changes of the ischemic brain tissue were observed after H.E. staining. The mitochondrial structure of the hippocampal neurons on the ischemic side of the brain was observed by using transmission electron microscope. The levels of iron deposition rate (%) in the ischemic penumbra of the brain tissue and hippocampus were observed after Prussian blue staining, and the reactive oxygen species (ROS) content of the cerebral ischemic penumbra was assayed using flow cytometry, and the content of glutathione (GSH) content in the ischemic penumbra was detected by using microplate method. The real-time quantitative PCR was used to detect the expression of glutathione peroxidase 4 (GPX4), divalent metal transporter 1 (DMT1), transferrin (TF), transferrin receptor 1 (TFR1), and ferroportin 1 (FPN1) mRNAs in the ischemic penumbra, and the Western blot was used to detect the expression of GPX4, DMT1, TF, TFR1, FPN1, and ferritin (FER) proteins in the ischemic penumbra. RESULTS: Compared with the sham operation group, the neurological deficit score, GSH content, expression of GPX4 and FPN1 mRNAs and proteins were significantly decreased (P<0.01), while the percentage of cerebral infarct volume, iron deposit rates of the cerebral ischemic penumbra and hippocampus, ROS content, and the expression levels of DMT1, TF, and TFR1 mRNAs and proteins and FER protein were considerably increased (P<0.01) in the model group. In comparison with the model group, the decrease of neurological deficit score, GSH content, expression of GPX4 and FPN1 mRNAs and proteins, and the increase of the percentage of cerebral infarct volume, iron deposit rates of the cerebral ischemic penumbra and hippocampus, ROS content, and the expression levels of DMT1, TF, and TFR1 mRNAs and proteins and FER protein were all reversed in both DFO and acupuncture groups (P<0.01, P<0.05). The effects of acupuncture were significantly superior to those of DFO in lowering the levels of cerebral cortical and hippocampal iron deposit rates, ROS content and in elevating the expression of GPX4 mRNA and protein (P<0.01, P<0.05). H.E. staining showed large necrotic cells, disordered arrangement of cells in the cerebral cortex and hippocampus, with hyperchromic nuclei, vacuole-like changes, widening of cellular space, and cell swelling in the model group, which was relatively milder in the cell damage in both acupuncture and DFO groups. In addition, the ultrastructure of cells in the hippocampus showed irregular cellular nuclear morphology, atrophy of some mitochondria in the cytoplasm, partial mitochondrial membrane rupture and edema, and loosening of the ridge structure in the model group, which was milder in the mitochondrial impairment (including reduced number of mitochondria, broken mitochondrial membrane and reduced ridge structure in fewer cells) in the acupuncture group. CONCLUSIONS: The "Xingnao Kaiqiao" needling intervention has a neuroprotective effect in CIRI rats, which may be related to its functions in regulating ferroptosis-related targets and iron metabolism in cerebral ischemic penumbra, reducing oxidative stress injury, and suppressing neuronal ferroptosis.

Enforced HCELL Expression Enhances Bone Marrow Stromal Cells Homing and Amelioration of Cerebral Ischemia-Reperfusion Injury via induction of PGE2.

Ischemic stroke (IS) is a significant and potentially life-threatening disease with limited treatment options, often resulting in severe disability. Bone marrow stromal cells (BMSCs) transplantation has exhibited promising neuroprotection following cerebral ischemia-reperfusion injury (CIRI). However, the effectiveness is hindered by their low homing rate when administered through the vein. In this study, we aimed to enhance the homing ability of BMSCs through lentivirus transfection to express fucosyltransferase 7. This glycosylation engineered CD44 on BMSCs to express hematopoietic cell E-selectin/L-selectin ligand (HCELL), which is the most potent E-selectin ligand. Following enforced HCELL expression, the transplantation of BMSCs was then evaluated in a middle cerebral artery occlusion (MCAO) model. Results showed that HCELL+BMSCs significantly ameliorated neurological deficits and reduced the volume of cerebral infarction. Furthermore, the transplantation led to a decrease in apoptosis by up-regulating BCL-2 and down-regulating BAX, also reduced the mRNA levels of inflammatory factors, such as interleukin-1ß (IL-1ß), IL-2, IL-6 and tumor necrosis factor-alpha (TNF-α) in the ischemic brain tissue. Notably, enforced HCELL expression facilitated the migration of BMSCs towards cerebral ischemic lesions and their subsequent transendothelial migration through the up-regulation of PTGS-2, increased production of PGE2 and activation of VLA-4. In summary, our study demonstrates that transplantation of HCELL+BMSCs effectively alleviates CIRI, and that enforced HCELL expression enhances the homing of BMSCs to cerebral ischemic lesions and their transendothelial migration via PTGS-2/PGE2/VLA-4. These findings indicate that enforced expression of HCELL on BMSCs could serve as a promising therapeutic strategy for the treatment of ischemic stroke.

A coloaded liposome in situ gel as a novel therapeutic strategy to treat cerebral ischemia reperfusion injury.

BACKGROUND: Ischemic stroke has become one of the leading causes of death and disability worldwide in individuals aged 60 and above. However, currently available drugs show limited efficacy. Therefore, research to find more effective and safer therapeutic strategies is an urgent requirement for the treatment of cerebral ischemia reperfusion injury (CIRI). METHODS: First, the free radical scavenger Edaravone and a Ginseng active ingredient were coloaded into liposomes (aER@Lip), followed by optimization and characterization. Pluronic F127 and F68 at different concentrations were mixed and stored at 4 °C for more than 24 h to obtain gel solutions. Then, aER@Lip was added to the gel solutions to prepare the drug-loaded in situ gel, termed aER@Lip-TSG. RESULTS: In vitro experiments showed that aER@Lip-TSG was taken up by cells and had a good protective effect on oxygen-glucose deprivation/reoxygenation in pheochromocytoma 12 cells. In a rat CIRI model, aER@Lip-TSG delivered by intranasal administration not only decreased the apoptosis in brain tissue induced by CIRI, but also decreased the resultant inflammatory response. Moreover, the results suggested that aER@Lip-TSG had good biosafety. CONCLUSION: This delivery system provides a promising multi-factor combination, synergistic effects, sustained-release capabilities, and is a non-invasive treatment strategy for CIRI. It thus meets the urgent need for effective treatments of central nervous system diseases.

M6A Modification and Transcription Analysis of LncRNA in Cerebral Ischemia/Reperfusion Injury.

LncRNA is a major factor in the occurrence and development of many diseases. However, its mechanism in cerebral ischemia/reperfusion injury (CIRI) is yet unknown. In this study, the transcriptional level and methylation modification level of LncRNAs before and after mechanical thrombectomy were compared by high-throughput sequencing. Venn diagram, Spearman correlation analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, TargetScan, and miRanda were used to analyze the experimental data. The results showed that four key LncRNAs changed at both transcription and methylation levels. Specifically, LncRNA FAR2, LINC02431, and AL357060.1 were downregulated and hypomethylated, while LncRNA FOXD2-AS1 was upregulated and hypomethylated. Moreover, positive regulation of angiogenesis, protein domain-specific binding, autophagy pathway, PPAR signaling pathway, and MAPK signaling pathway were co-enriched between LncRNAs with different expression levels and different methylation levels. Finally, a LncRNA-miRNA-mRNA network was constructed. Therefore, this study explored the potential key LncRNAs and regulatory mechanisms of CIRI.

Exploring Copper's role in stroke: progress and treatment approaches.

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

The nitration of SIRT6 aggravates neuronal damage during cerebral ischemia-reperfusion in rat.

Ischemic stroke is a major cause of death and disability. The activation of neuronal nitric oxide synthase (nNOS) and the resulting production of nitric oxide (NO) via NMDA receptor-mediated calcium influx play an exacerbating role in cerebral ischemia reperfusion injury. The NO rapidly reacts with superoxide (O2-) to form peroxynitrite (ONOO-), a toxic molecule may modify proteins through tyrosine residue nitration, ultimately worsening neuronal damage. SIRT6 has been proven to be crucial in regulating cell proliferation, death, and aging in various pathological settings. We have previous reported that human SIRT6 tyrosine nitration decreased its intrinsic catalytic activity in vitro. However, the exact role of SIRT6 function in the process of cerebral ischemia reperfusion injury is not yet fully elucidated. Herein, we demonstrated that an increase in the nitration of SIRT6 led to reduce its enzymatic activity and aggravated hippocampal neuronal damage in a rat model of four-artery cerebral ischemia reperfusion. In addition, reducing SIRT6 nitration resulted in increase the activity of SIRT6, alleviating hippocampal neuronal damage. Moreover, SIRT6 nitration affected its downstream molecule activity such as PARP1 and GCN5, promoting the process of neuronal ischemic injury in rat hippocampus. Additionally, treatment with NMDA receptor antagonist MK801, or nNOS inhibitor 7-NI, and resveratrol (an antioxidant) diminished SIRT6 nitration and the catalytic activity of downstream molecules like PARP1 and GCN5, thereby reducing neuronal damage. Finally, in the biochemical regulation of SIRT6 activity, tyrosine 257 was essential for its activity and susceptibility to nitration. Replacing tyrosine 257 with phenylalanine in rat SIRT6 attenuated the death of SH-SY5Y neurocytes under oxygen-glucose deprivation (OGD) conditions. These results may offer further understanding of SIRT6 function in the pathogenesis of cerebral ischemic diseases.

Exercise preconditioning mitigates Ischemia-Reperfusion injury in rats by enhancing mitochondrial respiration.

Cerebral ischemia and subsequent reperfusion damage are prevalent in clinical practice, linked to numerous neurodegenerative diseases. Cerebral ischemia deprives brain tissue of essential oxygen and nutrients, disrupting energy metabolism and causing cellular dysfunction. Although reperfusion theoretically aids recovery, it instead initiates complex injury responses such as oxidative stress, apoptosis, and inflammation, worsening brain damage. Recent research suggests that enhancing neuronal energy status by modulating energy metabolism pathways can effectively counter these effects. For instance, boosting mitochondrial function, improving energy provision, and decreasing harmful metabolites can mitigate oxidative stress and cellular injury. This study investigated the protective effects of exercise preconditioning against ischemia-reperfusion injury in rats. It was observed that exercise enhances energy levels and mitochondrial respiration by upregulating the expression of COX4 and NAMPT proteins and activating AMPK and mitochondrial complex V. This process facilitates metabolic reprogramming characterized by the promotion of oxidative phosphorylation (OXPHOS) and the pentose phosphate pathway (PPP), alongside a reduction in glycolysis. Such reprogramming reduces harmful metabolites, mitigating apoptosis and oxidative stress, and is a key factor in alleviating acute ischemic hypoxia-induced brain damage. These findings introduce a novel therapeutic approach for ischemic brain reperfusion injury, underscoring the crucial role of ATP production and metabolic regulation in neuroprotection.

Isoliquiritigenin alleviates cerebral ischemia-reperfusion injury by reducing oxidative stress and ameliorating mitochondrial dysfunction via activating the Nrf2 pathway.

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.

Drug pairs of Huangqi and Dnggui alleviates pyroptosis by promoting autophagy activity via AMPK/mTOR signaling pathway in middle-cerebral artery occlusion/reperfusion in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemia-reperfusion (I/R) injury is a common complication of ischemic stroke, with autophagy and pyroptosis playing key roles. Huangqi and Danggui (HQDG) are a commonly used drug pair of Chinese traditional medicine for clinical treatment of ischemic stroke. AIM OF THE STUDY: The study aims to investigate the interaction between autophagy and pyroptosis regulated by HQDG through the AMPK/mTOR signaling pathway during cerebral I/R injury. MATERIALS AND METHODS: Model of middle-cerebral artery occlusion/reperfusion (MCAO/R) in SD rats was established using the Longa suture method. The components of traditional Chinese medicine were detected by liquid chromatography coupled to quadrupole orbitrap high resolution mass spectrometry (LC/MS). Neurological deficits were evaluated by neurological function score. Changes of cerebral blood flow were detected by a laser speckle blood flow imaging instrument. The volume of cerebral infarction was observed by 2,3,5-Chlorotriphenyltetrazolium (TTC) staining. The permeability of the blood-brain barrier was measured by Evans blue test. Neurovascular unit and autophagosomes in brain tissue were assessed by transmission electron microscopy. Neuronal pyroptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)/Caspase-1 staining. The expression of autophagy related proteins, pyroptosis related proteins, and AMPK/mTOR pathway related proteins were detected by Western blot. RESULTS: After cerebral I/R injury, autophagy and pyroptosis, were characterized by increased number of autophagosomes and pyroptosis cells, upregulated expression of Beclin 1, LC3-II/LC3-I, NLRP3, cleaved Caspase-1, IL-1beta, IL-18 proteins, and downregulated expression of P62 proteins. HQDG significantly improved neurological function, reduced the volume of cerebral infarction, increased cerebral blood flow, improved blood-brain barrier permeability and the function of neurovascular units. Autophagy was further activated and pyroptosis was significantly inhibited by HQDG, which promoted increased number of autophagosomes, enhanced expression of Beclin 1, LC3-II/LC3-I proteins, reduced expression of P62, NLRP3, cleaved Caspase-1, IL-1beta, and IL-18 proteins, and downregulated the number of pyroptosis cells. On the other hand, after administering 3-Methyladenine (3-MA) to inhibit autophagy, the above effects of HQDG were significantly inhibited. Besides, HQDG promoted AMPK phosphorylation, and weakened mTOR phosphorylation. However, after the administration of AMPK inhibitor Compound C, HQDG caused increase in Beclin 1 and LC3-II/LC3-I, reduced P62 and NLRP3, and cleaved Caspase-1 protein expression, whereas cerebral blood flow decreased. CONCLUSION: HQDG alleviated pyroptosis by promoting autophagy via AMPK/mTOR signaling pathway after middle-cerebral artery occlusion/reperfusion in rats, showing its potential for treatment of cerebral I/R injury in humans.