Huang-Qi-Long-Dan Granule alleviates ischemic stroke injury by regulating the crosstalk between Nrf2 and NF-κB signaling.

Introduction: Ischemic stroke (IS) is the 5 th leading cause of death in the USA, but only one FDA approved drug exists. IS causes widespread neuron death and inflammatory injury. Microglial efferocytosis, the process to remove dead cells, limits inflammation and enhances brain recovery. Extracellular cold-inducible RNA-binding protein (eCIRP) is a pro-inflammatory alarmin that is deleterious in IS, increases infarct volume, neuroinflammation and microglial activation. eCIRP decreases efferocytosis and induces pro-inflammatory micro-RNA 155 (miR-155) in IS by binding to TLR4 on microglia. Our objective is to develop C23, a small peptide inhibitor of eCIRP/TLR4 binding, in a murine model of IS to reduce neuroinflammation and improve efferocytosis and IS outcomes. Methods: Primary microglia were isolated from mouse pups, seeded, and treated with eCIRP with or without C23, lysed for RNA, and RT-qPCR measurement of micro-RNA. 8-10-week-old male C57BL/6 mice, were subjected to transient middle cerebral artery occlusion (tMCAO) model of IS for 1 h, followed by reperfusion and retroorbitally injected with either normal saline (vehicle) or C23 (10 µg/g b.w.). 24 h post-reperfusion, behavior was scored, mice were sacrificed and tissues were collected for infarct volume measurement, immunofluorescence microscopy, or miRNA quantification. Results: As summarized in Table 1, eCIRP treated microglia upregulated miR-155 by 29-fold compared to controls, and pretreatment with C23 significantly attenuated eCIRP-induced miR-155 by 33%. Brain levels of miR-155 increased 1.8-fold in the vehicle tMCAO group vs. sham brains but were unchanged in the C23 tMCAO group. Similarly, plasma miR-155 levels increased 2.1-fold in vehicle tMCAO mice and returned to sham levels in C23 tMCAO mice. C23 treatment significantly increased efferocytosis in penumbra compared to vehicle in tMCAO brains (Fig. 1). Infarct volume was reduced in the C23 treated tMCAO group by 39% (100.3 mm 3 in vehicle tMCAO vs. 61.2 mm 3 in C23 tMCAO; Table 1&Fig. 2). Finally, C23 tMCAO mice had mild motor impairment with a mean Bederson score of 1.8 vs. severe impairment of vehicle tMCAO mice scoring 3.1 (Table 1). Conclusion: eCIRP induces pro-inflammatory miR-155 in primary microglia in-vitro and in tMCAO mice, and C23 attenuates this detrimental effect. C23 treatment improves microglial efferocytosis and reduces infarct volume and behavioral deficit in IS. Thus, C23 is a promising candidate therapeutic agent in IS.

Background.Ischemic stroke remains a leading cause of mortality and disability worldwide, but efforts to develop efficacious neuroprotective therapy face ongoing challenges. Efferocytosis, the phagocytic clearance of dying cells, by microglia is crucial for limiting neuroinflammation and promoting stroke resolution. Extracellular cold-inducible RNA-binding protein (eCIRP) is an inflammatory mediator which impairs macrophage bacterial phagocytosis in sepsis and radiation injury, but its role in microglial efferocytosis in ischemic stroke has not yet been studied.Results.Using a transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke, we demonstrate that eCIRP is released into the cerebrospinal fluid and microglial expression of crucial efferocytic receptor MerTK decreases in tMCAO mice. CIRP deficiency significantly improved neurological deficit, MerTK expression and microglial efferocytosis in tMCAO mice. Utilizing tMCAO, hippocampal injections, and primary microglia, we show that eCIRP induces pro-inflammatory micro-RNA 155 (miR-155) via TLR4, which suppresses its target pro-efferocytic transcription factor MAF bZIP (MafB), downregulating MerTK and microglial efferocytosis. Pharmacological blockade of eCIRP–TLR4 interaction using small peptide C23 attenuates miR-155 induction, restores MerTK expression, rescues microglial efferocytosis, and improves outcomes in tMCAO mice.Conclusion.We show that eCIRP causes microglial efferocytic dysfunction in ischemic stroke via TLR4/miR-155/MafB axis. These findings uncover a previously unknown pathway through which eCIRP signaling impairs neuroprotective function in microglia and suggest that targeting eCIRP may promote functional recovery after stroke.

Small extracellular vesicles (sEVs) miRNAs are promising diagnosis and prognosis biomarkers for ischemic stroke (IS). This study aimed to determine the impact of IS on the serum sEVs miRNA profile of IS patients and a transient middle cerebral artery occlusion (tMCAO) mouse model. Small RNAseq was used to define the serum sEVs miRNA profile in IS patients and healthy controls (HC), and tMCAO mice and sham controls. Among the 1,444 and 1,373 miRNAs identified in human and mouse serum sEVs, the expression of 424 and 37 miRNAs was significantly altered in the IS patients and tMCAO mice, respectively (| Log2FC| ≥ 1, p < 0.01). Notably, five of the top 25 upregulated miRNAs in IS patients were brain-specific or enriched, including hsa-miR-9-3p, hsa-miR-124-3p, hsa-miR-143-3p, hsa-miR-98-5p, and hsa-miR-93-5p. Upregulation of these four miRNAs was further validated by qPCR. Nine of the 20 upregulated miRNAs in tMCAO mice were also brain-specific or enriched miRNAs. Temporal analysis indicated that the dynamics of mmu-miR-9-5p, mmu-miR-124-3p, mmu-miR-129-5p, and mmu-miR-433-3p were closely correlated with the evolution of ischemic brain injury, as their expression increased at 0.5 days after the onset of ischemia, peaked at day 1 or 3, and returned to normal levels at day 7 and 14. Notably, with the exceptions of mmu-miR-128-3p, the expression of the other eight miRNAs in the mouse serum sEVs was unaffected in the lipopolysaccharide (LPS)-induced neuroinflammation model. Together, in this study, we provided a comprehensive view of the influences of IS on the serum sEVs miRNA profile of IS patients and tMCAO mice and demonstrated the increment of a set of brain-specific miRNAs in serum sEVs after acute cerebral ischemia, which could be promising candidates directly reflecting the ischemic brain injury.

Anti-edema action of thyroid hormone in MCAO model of ischemic brain stroke: Possible association with AQP4 modulation

NSUN2-mediated m⁵C methylation of HSPB1 mRNA suppresses inflammation and ferroptosis in ischemic stroke via activating the NRF2/HO-1/NQO-1 pathway.

Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here ona mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. NL-1 decreased hydrogen peroxide production with an IC50 of 5.95μM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10mg/kg, i.p.) at time of reperfusion and allowed to recover for 24h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

The incidence of ischemic stroke, involving neuronal cell death and damaged blood vessels, is gradually increasing. In this study, we investigated the effects of a miR-495-3p-I on neuronal viability and recovery using both oxygen–glucose deprivation and recovery (OGD/R) and in transient middle cerebral artery occlusion (tMCAo) models of ischemic stroke. Cell viability increased with miR-495-3p-I treatment compared with the OGD/R group, and both early and late apoptosis decreased through by flow cytometry analysis. In the tMCAo model, tissues injected with miR-495-3p-I showed upregulation of canonical Wnt pathway genes (cyclin D1 and c-Myc) and neuronal marker (Tuj1), while non-canonical Wnt/MAPK pathway genes (caspase-3 and -7) were downregulated. Expression of the pro-apoptotic marker Bax was reduced, whereas the Bcl-2 family protein Mcl-1 increased. Behavioral tests revealed improved motor function compared to the sham group and treatment with miR-495-3p-I reduced brain infarct volume. This research explored the function of miR-495-3p-I in Wnt and MAPK pathways by using the miR-495-3p-I in a model of ischemic stroke, a neurological disease. Thus, we demonstrated that miR-495-3p-I reduced apoptosis and enhanced neuronal cell survival, suggesting it could be a potential therapeutic target.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-025-05443-x.

Dynamic changes in Beclin-1, LC3B, and p62 in aldose reductase-knockout mice at different time points after ischemic stroke

NADPH exerts neuroprotection in ischemic stroke by reinforcing blood-brain barrier integrity and stimulating angiogenesis.

ALPK1 signaling pathway activation by HMGB1 drives microglial pyroptosis and ferroptosis and brain injury after acute ischemic stroke.

Salvia miltiorrhiza (SM) has been used in oriental medicine for its neuroprotective effects against cardiovascular diseases and ischemic stroke. In this study, we investigated the therapeutic mechanism underlying the effects of SM on stroke using a transient middle cerebral artery occlusion (tMCAO) mouse model. Our results showed that SM administration significantly attenuated acute brain injury, including brain infarction and neurological deficits, 3 days after tMCAO. This was confirmed by our magnetic resonance imaging (MRI) study, which revealed a reduction in brain infarction with SM administration, as well as our magnetic resonance spectroscopy (MRS) study, which demonstrated the restoration of brain metabolites, including taurine, total creatine, and glutamate. The neuroprotective effects of SM were associated with the reduction in gliosis and upregulation of inflammatory cytokines, such as interleukin-6 (IL-6) and Tumor necrosis factor-α (TNF-α), along with the upregulation of phosphorylated STAT3 in post-ischemic brains. SM also reduced the levels of 4-Hydroxynonenal (4-HNE) and malondialdehyde (MDA), which are markers of lipid peroxidation, induced by oxidative stress upregulation in the penumbra of the tMCAO mouse brain. SM administration attenuated ischemic neuronal injury by inhibiting ferroptosis. Additionally, post-ischemic brain synaptic loss and neuronal loss were alleviated by SM administration, as demonstrated by Western blot and Nissl staining. Moreover, daily administration of SM for 28 days after tMCAO significantly reduced neurological deficits and improved survival rates in tMCAO mice. SM administration also resulted in improvement in post-stroke cognitive impairment, as measured by the novel object recognition and passive avoidance tests in tMCAO mice. Our findings suggest that SM provides neuroprotection against ischemic stroke and has potential as a therapeutic agent.

Wnt-3a alleviates neuroinflammation after ischemic stroke by modulating the responses of microglia/macrophages and astrocytes

Danlou tablet attenuates ischemic stroke injury and blood‒brain barrier damage by inhibiting ferroptosis

Receptor-interacting serine/threonine kinase 2 (RIPK2) is an important modulator of the inflammatory response in many disease states, including ischemic stroke. Here, we utilized, for the first time, a proteolysis targeting chimera (PROTAC) to specifically degrade RIPK2 in vivo in a rodent model of ischemic stroke. First, we established a dose-response curve for RIPK2 degradation following treatment with the RIPK2 PROTAC in naïve mice. Then, we assessed the time course of RIPK2 degradation and resolution to determine our optimal dosing paradigm for the acute ischemic stroke study. Interestingly, we saw significant degradation in peripheral organs but not in the brain. Using the transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke, we assessed the efficacy of RIPK2 PROTAC degradation as a potential therapeutic approach for ischemic stroke. While we did not see reductions in infarct volume, we did find significant improvements in behavioral outcomes using the open field test, weight grip test, vertical grid test, and neurological deficit scoring. This study adds to the literature supporting RIPK2 as an important mediator of the post-stroke inflammatory response and further demonstrates the importance of the peripheral immune response, specifically involving the spleen, in secondary injury after ischemic stroke.

Ischemic stroke (IS) is the most recorded case of stroke that is caused by decreased blood flow to the brain. Nowadays, therapeutical agents for IS are limited and they have not shown maximum clinical results. Therefore, the exploration of new candidates for IS treatment continues to be done. Zebrafish as one of the animal models has its advantages and currently is being developed to be incorporated into the drug discovery pipeline of IS. This review explores the latest applications of the zebrafish model in screening potential therapeutic agents for IS. Key factors related to the experimental design such as developmental stage and strain, routes of drug administration, induction methods, and experimental parameters are also elaborated. Finally, this review offers future recommendations for the use of zebrafish in the pre-clinical study of IS. This review is beneficial as a reference for establishing drug screening protocols using the zebrafish IS model.