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.

Soluble Epoxide Hydrolase Inhibitor Ameliorates Olfactory Dysfunction, Modulates Microglia Polarization, and Attenuates Neuroinflammation after Ischemic Brain Injury.

Olfactory bulb (OB) microglia activation and inflammation can lead to olfactory dysfunction, which often occurs after an ischemic stroke. Inhibition of soluble epoxide hydrolase (sEH) attenuates neuroinflammation in brain injuries by reducing the degradation of anti-inflammatory epoxyeicosatrienoic acids. However, whether sEH inhibitors can ameliorate olfactory dysfunction after an ischemic stroke remains unknown. Ischemic brain injury and olfactory dysfunction were induced by middle cerebral artery occlusion (MCAO) in Wistar Kyoto rats. The rats were administered 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a selective sEH inhibitor. Olfactory function, cerebral infarct volume, and the degree of degeneration, microglial polarization and neuroinflammation in OB were evaluated. Following treatment with AUDA, rats subjected to MCAO displayed mild cerebral infarction and OB degeneration, as well as better olfactory performance. In OB, AUDA triggered a modulation of microglial polarization toward the M2 anti-inflammatory type, reduction in proinflammatory mediators, and enhancement of the antioxidant process. The effectiveness of AUDA in terms of anti-inflammatory, neuroprotection and anti-oxidative properties suggests that it may have clinical therapeutic implication for ischemic stroke related olfactory dysfunction.

Immune Cells Promote BDNF Expression by Infiltrated Macrophages via Interleukin 4 in the Cerebral Ischemia of Male Rats.

We reported that infiltrated Ly6C+ macrophages express brain-derived neurotrophic factor (BDNF) only at the cerebral cortex infarct in a rat dMCAO model. However, the changein neuron-expressed BDNF, the niche components that induce the Ly6C+ cells to express BDNF, and the cellular sources of these components, remain unclear. In this study, immunofluorescence double staining was performed to label BDNF and Ly6C on brain sections at 3, 24, and 48 h following distal middle cerebral artery occlusion (dMCAO) of male rats, and to stain BDNF with Ly6C, IL-4R, and IL-10R. A neutralizing anti-IL-4 antibody was injected into the infarct, and the IL-4 and BDNF concentrations in the subareas of the infarct were determined using enzyme-linked immunosorbent assay. To find out the cellular sources of IL-4, the markers for microglia, T cells, and neurons were co-stained with IL-4 separately. In certain infarct subareas, the main BDNF-expressing cells shifted quickly from NeuN+ neurons to Ly6C+ cells during 24-48 h post-stroke, and the Ly6C+/BDNF+ cells mostly expressed IL-4 receptor. Following IL-4 neutralizing antibody injection, the BDNF, IL-4 protein levels, and BDNF+/Ly6C+ cells decreased significantly. The main IL-4-expressing cell type in this infarct subarea is not neuron either, but immune cells, including microglia, monocyte, macrophages, and T cells. The neurons, maintained BDNF and IL-4 expression in the peri-infarct area. In conclusion, in a specific cerebral subarea of the rat dMCAO model, IL-4 secreted by immune cells is one of the main inducers for Ly6C+ cells to express BDNF.

Acid sphingomyelinase inhibition induces cerebral angiogenesis post-ischemia/reperfusion in an oxidative stress-dependent way and promotes endothelial survival by regulating mitochondrial metabolism.

Acid sphingomyelinase (ASM) inhibitors are widely used for the treatment of post-stroke depression. They promote neurological recovery in animal stroke models via neurorestorative effects. In a previous study, we found that antidepressants including amitriptyline, fluoxetine, and desipramine increase cerebral angiogenesis post-ischemia/reperfusion (I/R) in an ASM-dependent way. To elucidate the underlying mechanisms, we investigated the effects of the functional ASM inhibitor amitriptyline in two models of I/R injury, that is, in human cerebral microvascular endothelial hCMEC/D3 cells exposed to oxygen-glucose deprivation and in mice exposed to middle cerebral artery occlusion (MCAO). In addition to our earlier studies, we now show that amitriptyline increased mitochondrial reactive oxygen species (ROS) formation in hCMEC/D3 cells and increased ROS formation in the vascular compartment of MCAO mice. ROS formation was instrumental for amitriptyline's angiogenic effects. ROS formation did not result in excessive endothelial injury. Instead, amitriptyline induced a profound metabolic reprogramming of endothelial cells that comprised reduced endothelial proliferation, reduced mitochondrial energy metabolism, reduced endoplasmic reticulum stress, increased autophagy/mitophagy, stimulation of antioxidant responses and inhibition of apoptotic cell death. Specifically, the antioxidant heme oxygenase-1, which was upregulated by amitriptyline, mediated amitriptyline's angiogenic effects. Thus, heme oxygenase-1 knockdown severely compromised angiogenesis and abolished amitriptyline's angiogenic responses. Our data demonstrate that ASM inhibition reregulates a complex network of metabolic and mitochondrial responses post-I/R that contribute to cerebral angiogenesis without compromising endothelial survival.

MicroRNA-199a-5p attenuates blood-brain barrier disruption following ischemic stroke by regulating PI3K/Akt signaling pathway.

OBJECTIVE: To explore whether miR-199a-5p regulated BBB integrity through PI3K/Akt pathway after ischemia stroke. METHODS: Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion(MCAO) were used in experiment. The Ludmila Belayev 12-point scoring was used to measure the neurological function of MCAO rats. The Evans Blue Stain, immunofluorescence staining, western-blotting and RT-PCR were performed to evaluate the effects of miR-199a-5p mimic on BBB integrity in rats following MCAO. RESULTS: The result suggested that miR-199a-5p mimic treatment possessed the potential to boost proprioception and motor activity of MCAO rats. MiR-199a-5p decreased the expression of PIK3R2 after MCAO, activated Akt signaling pathway, and increased the expression of Claudin-5 and VEGF in the ischemic penumbra. Furthermore, miR-199a-5p alleviated inflammation after cerebral ischemia. BBB leakage and neurocyte apoptosis were cut down in MCAO rats treated with miR-199a-5p mimic. CONCLUSIONS: MiR-199a-5p mimic decreased the expression of PIK3R2 and activated Akt signaling pathway after ischemia stroke, reduced the expression of inflammatory cytokines, and attenuated BBB disruption after ischemic stroke.

Synergism of salvianolic acid B and ginsenoside Rg1 magnifies the therapeutic potency against ischemic stroke.

Even though considerable progress has been made to reduce insult, ischemic stroke is still a significant cause of mortality and morbidity in the world, and new therapeutic strategies are urgently needed. In the present study, the magnesium salt of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) combination as a multicomponent strategy against stroke was evaluated. The synergistic effect of Sa1B and Rg1 was evaluated by Bliss independence analysis on the middle cerebral artery occlusion model. The infarct volume, neuroethology, cerebral structure, and neurocyte number were evaluated by 3,5-triphenyltetrazolium chloride staining, Longa score, Garcia score, hematoxylin-eosin staining, and Nissl staining, respectively. Metabolomics was used to search for potential biomarkers and explore the mechanism of Sa1B/Rg1. First, the superior effects of SalB/Rg1 than SalB or Rg1 at the same dose were evaluated. Compared with SalB ( P  < 0.001) or Rg1 ( P  < 0.01), SalB/Rg1 significantly decreased infarct volume through 3,5-triphenyltetrazolium chloride staining and protected the structural integrity of cortex and striatum. The superior effect of SalB/Rg1 on neurological behavior was also detected compared with SalB or Rg1 significantly. Accompanying behavioral improvement, a considerable increase of SalB/Rg1 on neurons detected by Nissl staining was found on the cortex compared with SalB ( P  < 0.05) or Rg1 ( P  < 0.01). Second, the synergistic effect between SalB and Rg1 was strictly verified by Bliss independence analysis ( P  < 0.01) based on infarct volume. Finally, alleviation of cerebral metabolic disorders may be the possible mechanism of SalB/Rg1. Our study provided a multicomponent strategy against ischemic stroke, with not only dose reduction but also improved efficacy relative to single agents.

Ischemic Microenvironment-Targeted Bioinspired Lipoprotein Sequentially Penetrates Cerebral Ischemic Lesions to Rescue Ischemic Stroke.

Reperfusion injury represents a significant impediment to recovery after recanalization in an ischemic stroke and can be alleviated by neuroprotectants. However, inadequate drug delivery to ischemic lesions impairs the therapeutic effects of neuroprotectants. To address this issue, an ischemic microenvironment-targeted bioinspired lipoprotein system encapsulating lipoic acid (LA@PHDL) is herein designed to sequentially penetrate ischemic lesions and be readily taken up by neurons and microglia. In transient middle cerebral artery occlusion (tMCAO) mouse models, LA@PHDL accumulates rapidly and preferentially in the ischemic brain, with a 2.29-fold higher than the nontargeted nanoplatform in the early stage. Furthermore, LA@PHDL effectively restores neurological function, reduces infarct volume to 17.70%, prevents brain cell necrosis and apoptosis, and attenuates inflammation in tMCAO mouse models. This design presents new opportunities for delivering neuroprotectants to cerebral ischemic lesions to improve the outcome of an ischemic stroke.

Mitochondrial ATP Synthesis and Proton Transport Synergistically Mitigate Oligodendrocyte Progenitor Cell Dysfunction Following Transient Middle Cerebral Artery Occlusion via the Pbx3/Dguok/Kif21b Signaling Pathway.

In the realm of this study, obtaining a comprehensive understanding of ischemic brain injury and its molecular foundations is of paramount importance. Our study delved into single-cell data analysis, with a specific focus on sub-celltypes and differentially expressed genes in the aftermath of ischemic injury. Notably, we observed a significant enrichment of the "ATP METABOLIC PROCESS" and "ATP HYDROLYSIS ACTIVITY" pathways, featuring pivotal genes such as Pbx3, Dguok, and Kif21b. A remarkable finding was the consistent upregulation of genes like Fabp7 and Bcl11a within the MCAO group, highlighting their crucial roles in regulating the pathway of mitochondrial ATP synthesis coupled proton transport. Furthermore, our network analysis unveiled pathways like "Neuron differentiation" and "T cell differentiation" as central in the regulatory processes of sub-celltypes. These findings provide valuable insights into the intricate molecular responses and regulatory mechanisms that govern brain injury. The shared differentially expressed genes among sub-celltypes emphasize their significance in orchestrating responses post-ischemic injury. Our research, viewed from the perspective of a medical researcher, contributes to the evolving understanding of the molecular landscape underlying ischemic brain injury, potentially paving the way for targeted therapeutic strategies and improved patient outcomes.

Therapeutic and Mechanistic Effects of Granulocyte Colony Stimulating Factor and Neurotrophin Receptor on Cerebral Ischemia-Reperfusion Injury: An Experimental Pilot Study.

OBJECTIVE: To study the therapeutic effect and protective mechanism of granulocyte colony stimulating factor (G-CSF) and neurotrophin receptor (NTR) on cerebral ischemia-reperfusion injury. METHODS: Rat models of permanent focal middle cerebral artery occlusion (MCAO) were constructed by using a modified suture method, and the rats were assigned into three groups such as treatment group (the rats were injected with mixed G-CSF and NTR once), sham operation group and PBS control group. The volume of the cerebral infarction was detected using Triphenyltetrazolium Chloride (TTC) staining method; the motor function in rats was evaluated; and qRT-PCR detection, double immunofluorescence histochemistry and immunohistochemistry were performed to observe various effects. RESULTS: After G-CSF and NTR treatment, the infarct volume induced by MCAO in the treatment group was significantly lower than that in the PBS control group (P<0.05). The motor function in the treatment group was significantly improved on day 7 and day 14 compared to the PBS control group (P<0.05). The levels of MCP-1, TNF-α, TGF-ß and IL-10 mRNA in the treatment group decreased by 22% compared with PBS control group, and the difference was statistically significant (P<0.05). The Bcl-2 protein level in the treatment group was greater than that in the PBS control group, while the Bax level in the treatment group was lower than in the control group; and both the differences were statistically significant (P<0.05). The number of BrdU + cells in the treatment group was significantly greater than that in the PBS control group (P<0.05). CONCLUSION: G-CSF can promote the regeneration of neurons, promote the formation of new blood vessels, promote the reconstruction of neural network in rat MCAO models through anti apoptosis, anti-inflammation and mobilization of bone marrow hematopoietic cells to exert its powerful protective effect on neurons, and contribute to the repair of neural function and improvement of prognosis.

Phillygenin attenuates cell apoptosis and microglia activation in cerebral ischaemia-reperfusion rats through activation of peroxisome proliferator-activated receptor γ.

Ischaemic stroke is a common condition that can lead to cerebral ischaemia-reperfusion injury. Phillygenin (PHI), a natural bioactive compound derived from Forsythia suspensa, has been shown to play a crucial role in regulating inflammation across various diseases. However, its specific regulatory effects in ischaemic stroke progression remain unclear. In this study, we established a middle cerebral artery occlusion (MCAO) rat model. Treatment with PHI (50 or 100 mg/kg) significantly reduced cerebral infarction in MCAO rats. PHI treatment also mitigated the increased inflammatory response observed in these rats. Additionally, PHI suppressed microglial activation by reducing iNOS expression, a marker of M1-type polarization of microglia, and attenuated increased brain tissue apoptosis in MCAO rats. Furthermore, PHI's anti-inflammatory effects in MCAO rats were abrogated upon co-administration with GW9662, a peroxisome proliferator-activated receptor γ (PPARγ) inhibitor. In summary, PHI attenuated microglial activation and apoptosis in cerebral ischaemia-reperfusion injury through PPARγ activation, suggesting its potential as a therapeutic agent for mitigating cerebral ischaemia-reperfusion injury.

Comparative Evaluation of Neuroprotective Activity of Tryptanthrin and Its Oxime in Middle Cerebral Artery Occlusion in Rats.

The neuroprotective activity of tryptanthrin and its oxime was compared in male Wistar rats with a model of intraluminal occlusion of the middle cerebral artery. Neurobehavioral tests were performed 4, 24, and 48 h after focal cerebral infarction (FCI) using a modified neurological severity score (mNSS); additionally, the horizontal stability test, the plantar sensitivity test of the fore and hind limbs, holding on the tilted cage top test, and negative geotaxis test were performed. The size of FCI and the severity of brain tissue swelling were examined on day 2 after occlusion. Tryptanthrin and its oxime were administered at a dose of 10 mg/kg intraperitoneally during FCI, then daily for 2 days. In the control group, the mean score of neurological deficit remained at a high level for 2 days. FCI size was 43.8±3.4% of hemisphere area, and the hemisphere volume increased by 18.5±2.0% due to brain tissue swelling and edema. Administration of tryptanthrin and its oxime significantly decreased neurological deficits at all control points and reduced FCI size (by 24.2 and 30.4%, respectively) and brain tissue swelling of the affected hemisphere (by 64.9 and 62.7%, respectively). Therefore, the neuroprotective effect of tryptanthrine and its oxime in the acute period of FCI is largely determined by their anti-inflammatory activity.

Oltipraz attenuated cerebral ischemia-reperfusion injury through inhibiting the oxidative stress and ferroptosis in mice.

Oltipraz (OPZ) is a synthetic dithiolethione and is considered a novel activator of nuclear factor E2-related factor 2 (Nrf2). Increasing evidence indicates that Nrf2 protects against cerebral ischemia/reperfusion (I/R) injury by antagonizing ferroptosis and lipid peroxidation. However, the protective effects of OPZ on cerebral I/R injury remain to be elucidated. We investigated the in vitro and in vivo neuroprotective effects of OPZ. Mice were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) to construct an in vivo model and PC12 cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) to establish an in vitro model. OPZ administration reduced the infarct volume and brain water content, and alleviated the neurological deficit of MCAO/R mice. Moreover, OPZ ameliorated MCAO/R-induced oxidative stress by decreasing the levels of 4-HNE and MDA and increasing the activities of SOD and GSH. We also found that OPZ ameliorated MCAO/R-induced ferroptosis by increasing SLC7A11 and GPX4 protein expression and downregulating ACSL4 protein expression. Similarly, the in vitro results revealed that OGD/R-induced oxidative stress and ferroptosis. Finally, mechanistic analysis revealed that OPZ significantly upregulated the Nrf2 expression and Nrf2 knockout (Nrf2 KO) abolished the OPZ-mediated protective effects. Taken together, these findings demonstrate that OPZ ameliorates cerebral I/R injury by suppressing the oxidative stress and ferroptosis.

ALKBH5 deficiency attenuates oxygen-glucose deprivation-induced injury in mouse brain microvascular endothelial cells in an m6A dependent manner.

Structural and functional alterations in brain microvascular endothelial cells (BMECs) caused by oxygen-glucose deprivation (OGD) are involved in the pathogenesis of various brain disorders. AlkB homolog 5 (ALKBH5) is a primary m6A demethylase that regulates various cell processes, but its distinct roles in BMEC function remain to be clarified. In the present study, in mouse middle cerebral artery occlusion (MCAO) model, knockout of ALKBH5 reduced neurological deficits, infarct volumes and tissue apoptosis caused by ischemia/reperfusion injury. Evans blue leakage and decreased expression of the tight junction protein ZO-1 and Occludin were also attenuated by ALKBH5 knockout. During the exploration of the underlying mechanisms of the role of ALKBH5 in BMECs, we found that the expression of ALKBH5 was induced at both the mRNA and protein levels by hypoxia; however, its protein stability was impaired by OGD treatment. Knockdown of ALKBH5 expression increased total m6A levels and alleviated OGD-induced BMEC injury. At the same time, the selective ALKBH5 inhibitor Cpd 20m also exhibited a protective effect on cell injury. In contrast, overexpression of ALKBH5 increased the sensitivity of BMECs to OGD. Interestingly, the m6A sequencing data revealed that knockdown of ALKBH5altered the expression of many genes via m6A upregulation. The gene expression alterations were verified by real-time PCR. Taken together, our results suggest that ALKBH5, as well as its target genes, plays important roles in the regulation of brain microvascular endothelial cell function through its RNA demethylase activity.

Nrf1 Reduces COX-2 Expression and Maintains Cellular Homeostasis After Cerebral Ischemia/Reperfusion By Targeting IL-6/TNF-α Protein Production.

Neuroinflammation has been considered involved in the process of cerebral ischemia-reperfusion injury (CIRI). Transcription factors play a crucial role in regulating gene transcription and the expressions of specific proteins during the progression of various neurological diseases. Evidence showed that transcription factor nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as Nrf1) possessed strong biological activities including antioxidant, anti-inflammatory and neuroprotective properties. However, its role and potential molecular mechanisms in CIRI remain unclear. In our study, we observed a significant elevation of Nrf1 in the cerebral cortex following cerebral ischemia-reperfusion in rats. The Nrf1 downregulation markedly raised COX-2, TNF-α, IL-1ß, and IL-6 protein levels during middle cerebral artery occlusion/reperfusion in rats, which led to worsened neurological deficits, higher cerebral infarct volume, and intensified cortical histopathological damage. In subsequent in vitro studies, the expression of Nrf1 protein increased following oxygen-glucose deprivation/reperfusion treatment on neurons. Subsequently, Nrf1 knockdown resulted in a significant upregulation of inflammatory factors, leading to a substantial increase in the cell death rate. Through analyzing the alterations in the expression of inflammatory factors under diverse interventions, it is indicated that Nrf1 possesses the capacity to discern variations in inflammatory factors via specific structural domains. Our findings demonstrate the translocation of the Nrf1 protein from the cytoplasm to the nucleus, thereby modulating the protein expression of IL-6/TNF-α and subsequently reducing the expression of multiple inflammatory factors. This study signifies, for the first time, that during cerebral ischemia-reperfusion, Nrf1 translocases to the nucleus to regulate the protein expression of IL-6/TNF-α, consequently suppressing COX-2 expression and governing cellular inflammation, ultimately upholding cellular homeostasis.

ß-Caryophyllene mitigates ischemic stroke-induced white matter lesions by inhibiting pyroptosis.

ß-Caryophyllene (BCP), a selective agonist for cannabinoid receptor 2 (CB2R), has demonstrated promising protective effects in various pathological conditions. However, the neuroprotective effects of BCP on white matter damage induced by ischemic stroke have not been elucidated previously. In this study, we find that BCP not only improves sensorimotor and cognitive function via CB2R but also mitigates white matter lesions in mice following ischemic stroke. Furthermore, BCP enhances the viability of MO3.13 oligodendrocytes after oxygen-glucose deprivation and reoxygenation (OGD/R), attenuating OGD/R-induced cellular damage and pyroptosis. Notably, these protective effects of BCP are partially enhanced by the NLRP3 inhibitor MCC950 and counteracted by the NLRP3 activator nigericin. In addition, nigericin significantly exacerbates neurological outcomes and increases white matter lesions following BCP treatment in middle cerebral artery occlusion (MCAO) mice. These results suggest that BCP may ameliorate neurological deficits and white matter damage induced by cerebral ischemia through inhibiting NLRP3-mediated pyroptosis.

AD16 attenuates neuroinflammation induced by cerebral ischemia through down-regulating astrocytes A1 polarization.

A1 polarization of astrocytes mediated prolonged inflammation contributing to brain injury in ischemic stroke. We have previously shown that AD16 protects against neonatal hypoxic-ischemic brain damage in vivo and oxygen-glucose deprivation in vitro. More recently, AD16 has demonstrated safety, tolerability, and favorable pharmacokinetics in a randomized controlled phase I trial. In this study, we utilized a rat model of transient middle cerebral artery occlusion (tMCAO) to explore whether the anti-inflammatory compound AD16 protects against ischemic brain injury by regulating A1 polarization and its underlying mechanisms. Our results showed that AD16 treatment significantly reduced the brain infarcted volume and improved neurological function in tMCAO rats. GO analysis results show that differential genes among the Sham, tMCAO and AD16 treatment groups are involved in the regulation of cytokine and inflammatory response. KEGG enrichment pathways analysis mainly enriched in cytokine-cytokine receptor interaction, viral protein interaction with cytokine-cytokine receptor, TNF, chemokine, NF-κB and IL-17 signaling pathway. Furthermore, AD16 treatment decreased the permeability of the blood-brain barrier and suppressed neuroinflammation. AD16 treatment also significantly reduced the polarization of A1 and inhibited NF-κB and JAK2/STAT3 signaling pathways. This study demonstrates that AD16 protects against brain injury in ischemic stroke by reducing A1 polarization to suppress neuroinflammation through downregulating NF-κB and JAK2/STAT3 signaling. Our findings uncover a potential molecular mechanism for AD16 and suggest that AD16 holds promising therapeutic potential against cerebral ischemia.

Minocycline alleviates microglia ferroptosis by inhibiting HO-1 during cerebral ischemia-reperfusion injury.

OBJECTIVE: Ischemic stroke is a leading cause of death and disability in individuals worldwide. Cerebral ischemia-reperfusion injury (CIRI) typically results in severe secondary injury and complications following reperfusion therapy. Microglia play critical roles in the inflammatory reaction of CIRI. However, less attention has been given to microglial death in this process. Our study aims to explore microglial death in CIRI and the effects and mechanism of minocycline treatment on microglia. METHODS: A middle cerebral artery occlusion (MCAO) model was applied to induce CIRI in rats. At 0 h, 24 h and 48 h post-operation, rats were intraperitoneally injected with 45 mg/kg minocycline. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, assessment of activated microglia and examination of mitochondrial structure were conducted and checked at 72 h after reperfusion. Additionally, an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) model was established. BV-2 cells were treated with various pharmacological inhibitors of cell death or minocycline. Cell viability, lipid peroxidation, mitochondrial structure and function, and labile Fe2+ and ferroptosis-associated gene/protein levels were measured. Hemin was used for further validation after transcriptome analysis. RESULTS: In the MCAO and OGD/R models, ferroptosis was identified as a major form of microglial death. Minocycline inhibited microglia ferroptosis by reducing HO-1 expression. In addition, minocycline improved mitochondrial membrane potential, mitochondrial structures and microglial survival in vivo. Minocycline also decreased labile Fe2+ levels, lipid peroxidation, and expression of ferritin heavy chain (FTH) and it improved mitochondrial structure and function in vitro. Upregulation of HO-1 counteracted the protective effect of minocycline. CONCLUSION: Ferroptosis is a major form of microglial death in CIRI. The protective mechanism of minocycline in CIRI partially hinges on its ability to effectively ameliorate microglia ferroptosis by downregulating HO-1 expression. Consequently, targeting microglia ferroptosis is a promising treatment for CIRI.

Microglial heterogeneity in the ischemic stroke mouse brain of both sexes.

BACKGROUND: Ischemic stroke elicits a complex and sustained immune response in the brain. Immunomodulatory treatments have long held promise for improving stroke outcomes, yet none have succeeded in the clinical setting. This lack of success is largely due to our incomplete understanding of how immune cells respond to stroke. The objective of the current study was to dissect the effect of permanent stroke on microglia, the resident immune cells within the brain parenchyma. METHODS: A permanent middle cerebral artery occlusion (pMCAO) model was used to induce ischemic stroke in young male and female mice. Microglia were sorted from fluorescence reporter mice after pMCAO or sham surgery and then subjected to single-cell RNA sequencing analysis. Various methods, including flow cytometry, RNA in situ hybridization, immunohistochemistry, whole-brain imaging, and bone marrow transplantation, were also employed to dissect the microglial response to stroke. Stroke outcomes were evaluated by infarct size and behavioral tests. RESULTS: First, we showed the morphologic and spatial changes in microglia after stroke. We then performed single-cell RNA sequencing analysis on microglia isolated from sham and stroke mice of both sexes. The data indicate no major sexual dimorphism in the microglial response to permanent stroke. Notably, we identified seven potential stroke-associated microglial clusters, including four major clusters characterized by a disease-associated microglia-like signature, a highly proliferative state, a macrophage-like profile, and an interferon (IFN) response signature, respectively. Importantly, we provided evidence that the macrophage-like cluster may represent the long-sought stroke-induced microglia subpopulation with increased CD45 expression. Lastly, given that the IFN-responsive subset constitutes the most prominent microglial population in the stroke brain, we used fludarabine to pharmacologically target STAT1 signaling and found that fludarabine treatment improved long-term stroke outcome. CONCLUSIONS: Our findings shed new light on microglia heterogeneity in stroke pathology and underscore the potential of targeting specific microglial populations for effective stroke therapies.

Cooperation Between Platelet ß1 and ß3 Integrins in the Arrest of Bleeding Under Inflammatory Conditions in Mice-Brief Report.

BACKGROUND: Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how ß1 and ß3 integrins are involved. METHODS: The impact of isolated or combined platelet deficiency in ß1 and ß3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery. RESULTS: Mice with platelet-directed inactivation of Itgb1 (PF4Cre-ß1-/-) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-ß3-/-) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-ß3-/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of ß1 integrins, PF4Cre-ß1-/-/ß3-/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-ß1-/-/ß3-/- platelets to inflamed microvessels. Unlike PF4Cre-ß1-/- and PF4Cre-ß3-/- mice, PF4Cre-ß1-/-/ß3-/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-ß1-/-/ß3-/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-ß3-/- animals. CONCLUSIONS: Altogether, these results show that the requirement for and degree of functional redundancy between platelet ß1 and ß3 integrins in inflammation-associated hemostasis vary with the inflammatory situation.

Long noncoding RNA H19 knockdown promotes angiogenesis via IMP2 after ischemic stroke.

AIMS: This study aimed to explore the effects of long noncoding RNA (lncRNA) H19 knockdown on angiogenesis and blood-brain barrier (BBB) integrity following cerebral ischemia/reperfusion (I/R) and elucidate their underlying regulatory mechanisms. METHODS: A middle cerebral artery occlusion/reperfusion model was used to induce cerebral I/R injury. The cerebral infarct volume and neurological impairment were assessed using 2,3,5-triphenyl-tetrazolium chloride staining and neurobehavioral tests, respectively. Relevant proteins were evaluated using western blotting and immunofluorescence staining. Additionally, a bioinformatics website was used to predict the potential target genes of lncRNA H19. Finally, a rescue experiment was conducted to confirm the potential mechanism. RESULTS: Silencing of H19 significantly decreased the cerebral infarct volume, enhanced the recovery of neurological function, mitigated BBB damage, and stimulated endothelial cell proliferation following ischemic stroke. Insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) is predicted to be a potential target gene for lncRNA H19. H19 knockdown increased IMP2 protein expression and IMP2 inhibition reversed the protective effects of H19 inhibition. CONCLUSION: Downregulation of H19 enhances angiogenesis and mitigates BBB damage by regulating IMP2, thereby alleviating cerebral I/R injury.