Middle Cerebral Artery Occlusion And Reperfusion Research Articles

Mechanism of astragaloside IV regulating NLRP3 through LOC102555978 to attenuate cerebral ischemia reperfusion induced microglia pyroptosis

Astragaloside IV(ASⅣ), the main component of Radix Astragali, has been used to treat cerebral ischemia reperfusion injury (CIRI). However, the molecular mechanism of ASIV in CIRI needs to be further elucidated. Long non-coding RNA (lncRNA) is considered to be an important kind of regulatory molecule in CIRI. In this work, the biological effect and molecular mechanism of ASIV in CIRI through lncRNA were analyzed by using rat middle cerebral artery occlusion and reperfusion (MCAO/R) model and primary rat microglia (RM) cells oxygen and glucose deprivation/reoxygenation (OGD/R) model. The neurological deficit score was evaluated, the volume of cerebral infarction was calculated, and pyroptosis related molecules were detected by qPCR and western blot. Then, high-throughput sequencing was performed in sham and MCAO/R groups. The competitive endogenous RNA (ceRNA) networks associated with pyroptosis were constructed by functional enrichment analysis. CCK-8 detection of cell survival rate, qPCR and western blot were used to determine the specific molecular mechanism of ASⅣ through ceRNA in vitro. Results showed thatASⅣ could decrease the neurological deficit score, reduce the volume of cerebral infarction, inhibit inflammatory reaction and pyroptosis in MCAO/R model rats. Next, the ceRNA network was established, including the LOC102555978/miR-3584-5p/NLRP3 regulatory network. In vitro experiments showed that LOC102555978 promotes NLRP3 mediated pyroptosis of RM cells through sponge adsorption of miR-3584-5p, which may provide a potential therapeutic target for post-CIRI inflammation regulation. ASⅣ could inhibit pyroptosis of RM cells by down-regulating LOC102555978. LOC102555978/miR-3584-5p/NLRP3 may be the molecular mechanism of ASⅣ's CIRI protective effect.

Unveiling the neuroprotection effects of Volvalerenic acid A: Mitochondrial fusion induction via IDO1-mediated Stat3-Opa1 signaling pathway

BackgroundIschemic stroke is a leading cause of death and long-term disability worldwide. Studies have suggested that cerebral ischemia induces massive mitochondrial damage. Valerianic acid A (VaA) is the main active ingredient of valerianic acid with neuroprotective activity. PurposeThis study aimed to investigate the neuroprotective effects of VaA with ischemic stroke and explore the underlying mechanisms. MethodIn this study, we established the oxygen-glucose deprivation and reperfusion (OGD/R) cell model and the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model in vitro and in vivo. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of VaA in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP), and activities of NAD+ were detected to reflect mitochondrial function. Mechanistically, gene knockout experiments, transfection experiments, immunofluorescence, DARTS, and molecular dynamics simulation experiments showed that VaA bound to IDO1 regulated the kynurenine pathway of tryptophan metabolism and prevented Stat3 dephosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. ResultsWe showed that VaA decreased the infarct volume in a dose-dependent manner and exerted neuroprotective effects against reperfusion injury. Furthermore, VaA promoted Opa1-related mitochondrial fusion and reversed neuronal mitochondrial damage and loss after reperfusion injury. In SH-SY5Y cells, VaA (5, 10, 20 μM) exerted similar protective effects against OGD/R-induced injury. We then examined the expression of significant enzymes regulating the kynurenine (Kyn) pathway of the ipsilateral brain tissue of the ischemic stroke rat model, and these enzymes may play essential roles in ischemic stroke. Furthermore, we found that VaA can bind to the initial rate-limiting enzyme IDO1 in the Kyn pathway and prevent Stat3 phosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. Using in vivo IDO1 knockdown and in vitro IDO1 overexpressing models, we demonstrated that the promoted mitochondrial fusion and neuroprotective effects of VaA were IDO1-dependent. ConclusionVaA administration improved neurological function by promoting mitochondrial fusion through the IDO1-mediated Stat3-Opa1 pathway, indicating its potential as a therapeutic drug for ischemic stroke.

Transcription Factor TFAP2B Exerts Neuroprotective Effects Targeting BNIP3-Mediated Mitophagy in Ischemia/Reperfusion Injury.

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.

Kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside): a novel neuroprotective agent from Diospryros kaki against cerebral ischemia-induced brain injury.

Our previous study demonstrated neuroprotective and therapeutic effects of a standardized flavonoid extract from leaves of Diospyros kaki L.f. (DK) on middle cerebral artery occlusion-and-reperfusion (MCAO/R)-induced brain injury and its underlying mechanisms. This study aimed to clarify flavonoid components responsible for the effects of DK using in vitro and in vivo transient brain ischemic models. Organotypic hippocampal slice cultures (OHSCs) subjected to oxygen- and glucose-deprivation (OGD) were performed to evaluate in vitro neuroprotective activity of DK extract and nine isolated flavonoid components. MCAO/R mice were employed to elucidate in vivo neuroprotective effects of the flavonoid component that exhibited the most potent neuroprotective effect in OHSCs. DK extract and seven flavonoids [quercetin, isoquercetin, hyperoside, quercetin-3-O-(2″-O-galloyl-β-D-galactopyranoside), kaempferol, astragalin, and kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside) compound (9)] attenuated OGD-induced neuronal cell damage and compound (9) possessed the most potent neuroprotective activity in OHSCs. The MCAO/R mice showed cerebral infarction, massive weight loss, characteristic neurological symptoms, and deterioration of neuronal cells in the brain. Compound (9) and a reference drugs, edaravone, significantly attenuated these physical and neurological impairments. Compound (9) mitigated the blood-brain barrier dysfunction and the change of glutathione and malondialdehyde content in the MCAO mouse brain. Edaravone suppressed the oxidative stress but did not significantly affect the blood-brain barrier permeability. The present results indicated that compound (9) is a flavonoid constituent of DK with a potent neuroprotective activity against transient ischemia-induced brain damage and this action, at least in part, via preservation of blood-brain barrier integrity and suppression of oxidative stress caused by ischemic insult.

D-allose Inhibits TLR4/PI3K/AKT Signaling to Attenuate Neuroinflammation and Neuronal Apoptosis by Inhibiting Gal-3 Following Ischemic Stroke

BackgroundIschemic stroke (IS) occurs when a blood vessel supplying the brain becomes obstructed, resulting in cerebral ischemia. This type of stroke accounts for approximately 87% of all strokes. Globally, IS leads to high mortality and poor prognosis and is associated with neuroinflammation and neuronal apoptosis. D-allose is a bio-substrate of glucose that is widely expressed in many plants. Our previous study showed that D-allose exerted neuroprotective effects against acute cerebral ischemic/reperfusion (I/R) injury by reducing neuroinflammation. Here, we aimed to clarify the beneficial effects D-allose in suppressing IS-induced neuroinflammation damage, cytotoxicity, neuronal apoptosis and neurological deficits and the underlying mechanism in vitro and in vivo.MethodsIn vivo, an I/R model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 N mice, and D-allose was given by intraperitoneal injection within 5 min after reperfusion. In vitro, mouse hippocampal neuronal cells (HT-22) with oxygen–glucose deprivation and reperfusion (OGD/R) were established as a cell model of IS. Neurological scores, some cytokines, cytotoxicity and apoptosis in the brain and cell lines were measured. Moreover, Gal-3 short hairpin RNAs, lentiviruses and adeno-associated viruses were used to modulate Gal-3 expression in neurons in vitro and in vivo to reveal the molecular mechanism.ResultsD-allose alleviated cytotoxicity, including cell viability, LDH release and apoptosis, in HT-22 cells after OGD/R, which also alleviated brain injury, as indicated by lesion volume, brain edema, neuronal apoptosis, and neurological functional deficits, in a mouse model of I/R. Moreover, D-allose decreased the release of inflammatory factors, such as IL-1β, IL-6 and TNF-α. Furthermore, the expression of Gal-3 was increased by I/R in wild-type mice and HT-22 cells, and this factor further bound to TLR4, as confirmed by three-dimensional structure prediction and Co-IP. Silencing the Gal-3 gene with shRNAs decreased the activation of TLR4 signaling and alleviated IS-induced neuroinflammation, apoptosis and brain injury. Importantly, the loss of Gal-3 enhanced the D-allose-mediated protection against I/R-induced HT-22 cell injury, inflammatory insults and apoptosis, whereas activation of TLR4 by the selective agonist LPS increased the degree of neuronal injury and abolished the protective effects of D-allose.ConclusionsIn summary, D-allose plays a crucial role in inhibiting inflammation after IS by suppressing Gal-3/TLR4/PI3K/AKT signaling pathway in vitro and in vivo.

Pathogenic role of NAMPT in the perivascular regions after ischemic stroke in mice with type 2 diabetes mellitus

Ischemic stroke in patients with abnormal glucose tolerance results in poor outcomes. Nicotinamide phosphoribosyltransferase (NAMPT), an adipocytokine, exerts neuroprotective effects. However, the pathophysiological role of NAMPT after ischemic stroke with diabetes and the relationship of NAMPT with cerebrovascular lesions are unclear. The purpose of this study was to clarify the pathophysiological role of NAMPT in cerebral ischemia with diabetes, using db/db mice as a type 2 diabetes animal model. The number of degenerating neurons increased after middle cerebral artery occlusion and reperfusion (MCAO/R) in db/db mice compared with the degenerating neurons in db/+ mice. Extracellular NAMPT (eNAMPT) levels, especially monomeric eNAMPT, increased significantly in db/db MCAO/R mice but not db/+ mice in isolated brain microvessels. The increased eNAMPT levels were associated with increased expression of inflammatory cytokine mRNA. Immunohistochemical analysis demonstrated that NAMPT colocalized with GFAP-positive cells after MCAO/R. In addition, both dimeric and monomeric eNAMPT levels increased in the conditioned medium of primary cortical astrocytes under high glucose conditions subsequent oxygen/glucose deprivation. Our findings are the first to demonstrate the ability of increased monomeric eNAMPT to induce inflammatory responses in brain microvessels, which may be located near astrocyte foot processes.

Neuroprotective Effects of Water Extract from Brown Algae Petalonia binghamiae in an Experimental Model of Focal Cerebral Ischemia In Vitro and In Vivo.

Focal cerebral ischemia (fCI) can result in brain injury and sensorimotor deficits. Brown algae are currently garnering scientific attention as potential therapeutic candidates for fCI. This study investigated the therapeutic effects of the hot water extract of Petalonia binghamiae (wPB), a brown alga, in in vitro and in vivo models of fCI. The neuroprotective efficacy of wPB was evaluated in an in vitro excitotoxicity model established using HT-22 cells challenged with glutamate. Afterward, C57/BL6 mice were administered wPB for 7 days (10 or 100 mg/kg, intragastric) and subjected to middle cerebral artery occlusion and reperfusion (MCAO/R) operation, which was used as an in vivo fCI model. wPB co-incubation significantly inhibited cell death, oxidative stress, and apoptosis, as well as stimulated the expression of heme oxygenase-1 (HO-1), an antioxidant enzyme, and the nuclear translocation of its upstream regulator, nuclear factor erythroid 2-related factor 2 (Nrf2) in HT-22 cells challenged with glutamate-induced excitotoxicity. Pretreatment with either dose of wPB significantly attenuated infarction volume, neuronal death, and sensorimotor deficits in an in vivo fCI model. Furthermore, the attenuation of oxidative stress and apoptosis in the ischemic lesion accompanied the wPB-associated protection. This study suggests that wPB can counteract fCI via an antioxidative effect, upregulating the Nrf2/HO-1 pathway.

Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia

BackgroundThe urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. MethodsThe effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model.Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. ResultsWe initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1β, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. ConclusionCrebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

Glycosides of Buyang Huanwu decoction inhibits pyroptosis associated with cerebral ischemia-reperfusion through Nrf2-mediated antioxidant signaling pathway both in vivo and in vitro

BackgroundGlycosides are the pharmacodynamic substances of Buyang Huanwu Decoction (BYHWD) and they exert a protective effect in the brain by inhibiting neuronal pyroptosis of cerebral ischemia-reperfusion (CIR). However, the mechanism by which glycosides regulate neuronal pyroptosis of CIR is still unclear. PurposeA significant part of this study aimed to demonstrate whether glycosides have an anti-pyroptotic effect on CIR by nuclear factor erythroid 2-related factor (Nrf2)-mediated antioxidative mechanism. MethodsRats were used in vivo models of middle cerebral artery occlusion and reperfusion (MCAO/R). Neuroprotective effect of glycosides after Nrf2 inhibiting was observed by nerve function score, Nissl staining, Nrf2 fluorescence staining and pyroptotic proteins detection. SH-SY5Y cells were used in vitro models of oxygen-glucose deprivation/reperfusion (OGD/R). Glycosides was evaluated for their effects by measuring cell morphology, survival rate, lactate dehydrogenase (LDH) rate and expression of pyroptotic proteins. Nrf2 si-RNA 54–1 interference with lentivirus was used to create silenced Nrf2 SH-SY5Y cells (si-Nrf2-SH-SY5Y). Glycosides were evaluated on si-Con-SH-SY5Y and si-Nrf2-SH-SY5Y cells based on the expression of Nrf2 signaling pathway, pyroptotic proteins and cell damage manifestation. ResultsIn vivo, glycosides significantly promoted the fluorescence level of nuclear Nrf2, added more Nissl bodies, reduced neurological function scores and inhibited the pyroptotic proteins level. In vitro, glycosides significantly repaired the morphological damage of cells, promoted the survival rate, reduced the LDH rate, inhibited the pyroptosis. Moreover, antioxidant activity of glycosides was enhanced via Nrf2 activation. Both Nrf2 blocking in vivo and Nrf2 silencing in vitro significantly weakened the pyroptosis inhibitory and neuroprotective effects of glycosides. ConclusionThese results suggested for the first time that glycosides inhibited neuronal pyroptosis by regulating the Nrf2-mediated antioxidant stress pathway, thereby exerting brain protection of CIR. As a result of this study, This study improved understanding of the pharmacodynamics and mechanism of BYHWD, as well as providing a Traditional Chinese Medicine (TCM) treatment strategy for CIR .

Optimizing intra-arterial hypothermia scheme for acute ischemic stroke in an MCAO/R rat model

Hypothermia is a promising neuroprotective treatment. This study aims to explore and optimize the intervention scheme of intra-arterial hypothermia (IAH) in a middle cerebral artery occlusion and reperfusion (MCAO/R) rat model. The MCAO/R model was established with a thread that could be retracted 2 h after occlusion. Cold normal saline was injected into the internal carotid artery (ICA) through a microcatheter in different infusion conditions. Grouping followed an orthogonal design (L9[34]) based on three critical factors closely associated with IAH: perfusate temperature (4, 10, 15 °C), infusion flow rate (1/3, 1/2, 2/3 blood flow rate of ICA), and duration (10, 20, 30 min), resulting in 9 subgroups (H1, H2 to H9). A myriad of indexes were monitored, such as vital signs, blood parameters, changes in local ischemic brain tissue temperature (Tb), ipsilateral jugular venous bulb temperature (Tjvb), and the core temperature of the anus (Tcore). After 24 h and 72 h of cerebral ischemia, cerebral infarction volume, cerebral water content, and neurological function were assessed to explore the optimal IAH conditions. The results revealed that the three critical factors were independent predictors for cerebral infarction volume, cerebral water content, and neurological function. The optimal perfusion conditions were 4 °C, 2/3 RICA (0.50 ml/min) for 20 min, and there was a significant correlation between Tb and Tjvb (R = 0.994, P < 0.001). The vital signs, blood routine tests and biochemical indexes showed no significant abnormal changes. These findings revealed that IAH was safe and feasible with the optimized scheme in an MCAO/R rat model.

Effects of electroacupuncture on NLRP3/Caspase-1/GSDMD axis and neurological function in rats with cerebral ischemic reperfusion

To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi" (LI 11) and "Zusanli" (ST 36) in the rats with cerebral ischemic reperfusion and the potential mechanism of microglia pyroptosis. Sixty SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 20 rats in each group. The Zea Longa method was employed to establish the rat model of the middle cerebral artery occlusion and reperfusion (MACO/R) in the left brain. In the EA group, since the 2nd day of modeling, EA was given at "Quchi" (LI 11) and "Zusanli" (ST 36) of right side with disperse-dense wave, 4 Hz/20 Hz in frequency and 0.2 mA in current intensity, 30 min each time, once a day for lasting 7 consecutive days. The reduction rate of cerebral blood flow was measured with laser Doppler flowmetry during operation. The neurological function of rats was observed using Zea Longa neurobehavioral score. The cerebral infarction volume was detected by TTC staining method. The microglia positive expression in the ischemic side of the cortex was detected with the immunofluorescence method. Under transmission electron microscope, the ultrastructure of cell in the ischemic cortex was observed. The mRNA expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1) and gasdermin D (GSDMD) in the ischemic cortex were detected using real-time PCR. Compared with the sham-operation group, in the model group, the reduction rate of cerebral blood flow was increased during operation (P<0.001); Zea Longa neurobehavional score and the percentage of cerebral infarction volume were increased (P<0.001), the numbers of M1-type microglia marked by CD68+ and M2-type microglia marked by TMEM119+ were elevated in the ischemic cortex (P<0.001), the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was increased (P<0.001, P<0.01); the cytomembrane structure was destroyed, with more cell membrane pores formed in the ischemic cortex. Compared with the model group, after intervention, Zea Longa neurobehavioral score and the percentage of cerebral infarction volume were reduced (P<0.05), the number of M1-type microglia marked by CD68+ was reduced (P<0.05) and the number of M2-type microglia marked by TMEM119+ was increased (P<0.05); and the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was decreased (P<0.01, P<0.05) in the EA group. Even though the cytomembrane structure was incomplete, there were less membrane pores presented in the ischemic cortex in the EA group after intervention. The intervention with EA attenuates the neurological dysfunction and reduces the volume of cerebral infarction in the rats with cerebral ischemic reperfusion. The underlying mechanism is related to the inhibition of microglia pyroptosis through modulating NLRP3/Caspase-1/GSDMD axis.

M6A modification promotes blood-brain barrier breakdown during cerebral ischemia/reperfusion injury through increasing matrix metalloproteinase 3 expression

Blood-brain barrier (BBB) breakdown is a critical event in cerebral ischemia-reperfusion (I/R) injury, and matrix metalloproteinases (MMPs), which are proteolytic enzymes, play essential roles in BBB breakdown through degrading the extracellular matrix. N6-Methyladenosine (m6A), the most common and reversible mRNA modification, has an important role in the progression of cerebral I/R injury. However, whether m6A is related to BBB breakdown and MMPs expression in cerebral I/R injury is still not clear. In this study, we explored the potential effects of m6A modification on BBB breakdown in cerebral I/R injury and its underlying mechanisms using mice subjected to transient middle cerebral artery occlusion and reperfusion (MCAO/R), and mouse brain endothelial cells treated with oxygen-glucose deprivation and reoxygenation (OGD/R). We find that MMP3 expression is highly expressed and positively associated with the m6A writer CBLL1 (Cbl proto-oncogene like 1) in cerebral I/R injury in vivo and in vitro. Furthermore, MMP3 mRNA occurs m6A modification in mouse brain endothelial cells, and the m6A modification level of MMP3 mRNA is significantly increased in cerebral I/R injury. Moreover, inhibition of m6A modification reduces MMP3 expression and ameliorates BBB breakdown in cerebral I/R in vivo and in vitro. In conclusion, m6A modification promotes BBB breakdown in cerebral I/R injury through increasing MMP3 expression, indicating that m6A may be a potential therapeutic target for cerebral I/R injury.

TRIM67 alleviates cerebral ischemia‒reperfusion injury by protecting neurons and inhibiting neuroinflammation via targeting IκBα for K63-linked polyubiquitination

BackgroundExcessive and unresolved neuroinflammation plays an important role in the pathophysiology of many neurological disorders, such as ischemic stroke, yet there are no effective treatments. Tripartite motif-containing 67 (TRIM67) plays a crucial role in the control of inflammatory disease and pathogen infection-induced inflammation; however, the role of TRIM67 in cerebral ischemia‒reperfusion injury remains poorly understood.ResultsIn the present study, we demonstrated that the expression level of TRIM67 was significantly reduced in middle cerebral artery occlusion and reperfusion (MCAO/R) mice and primary cultured microglia subjected to oxygen–glucose deprivation and reperfusion. Furthermore, a significant reduction in infarct size and neurological deficits was observed in mice after TRIM67 upregulation. Interestingly, TRIM67 upregulation alleviated neuroinflammation and cell death after cerebral ischemia‒reperfusion injury in MCAO/R mice. A mechanistic study showed that TRIM67 bound to IκBα, reduced K48-linked ubiquitination and increased K63-linked ubiquitination, thereby inhibiting its degradation and promoting the stability of IκBα, ultimately inhibiting NF-κB activity after cerebral ischemia.ConclusionTaken together, this study demonstrated a previously unidentified mechanism whereby TRIM67 regulates neuroinflammation and neuronal apoptosis and strongly indicates that upregulation of TRIM67 may provide therapeutic benefits for ischemic stroke.Graphical

Puerarin inhibited oxidative stress and alleviated cerebral ischemia-reperfusion injury through PI3K/Akt/Nrf2 signaling pathway.

Introduction: Puerarin (PUE) is a natural compound isolated from Puerariae Lobatae Radix, which has a neuroprotective effect on IS. We explored the therapeutic effect and underlying mechanism of PUE on cerebral I/R injury by inhibiting oxidative stress related to the PI3K/Akt/Nrf2 pathway in vitro and in vivo. Methods: The middle cerebral artery occlusion and reperfusion (MCAO/R) rats and oxygen-glucose deprivation and reperfusion (OGD/R) were selected as the models, respectively. The therapeutic effect of PUE was observed using triphenyl tetrazolium and hematoxylin-eosin staining. Tunel-NeuN staining and Nissl staining to quantify hippocampal apoptosis. The reactive oxygen species (ROS) level was detected by flow cytometry and immunofluorescence. Biochemical method to detect oxidative stress levels. The protein expression related to PI3K/Akt/Nrf2 pathway was detected by using Western blotting. Finally, co-immunoprecipitation was used to study the molecular interaction between Keap1 and Nrf2. Results: In vivo and vitro studies showed that PUE improved neurological deficits in rats, as well as decreased oxidative stress. Immunofluorescence and flow cytometry indicated that the release of ROS can be inhibited by PUE. In addition, the Western blotting results showed that PUE promoted the phosphorylation of PI3K and Akt, and enabled Nrf2 to enter the nucleus, which further activated the expression of downstream antioxidant enzymes such as HO-1. The combination of PUE with PI3K inhibitor LY294002 reversed these results. Finally, co-immunoprecipitation results showed that PUE promoted Nrf2-Keap1 complex dissociation. Discussion: Taken together, PUE can activate Nrf2 via PI3K/Akt and promote downstream antioxidant enzyme expression, which could further ameliorate oxidative stress, against I/R-induced Neuron injury.

Modulation of gut microbiota alleviates cerebral ischemia/reperfusion injury in rats by inhibiting M1 polarization of microglia.

Gut microbiota affects the gut-brain axis; hence, the modulation of the microbiota has been proposed as a potential therapeutic strategy for cerebral ischemia/reperfusion injury (CIRI). However, the role and mechanism of the gut microbiota in regulating microglial polarization during CIRI remain poorly understood. Herein, using a middle cerebral artery occlusion and reperfusion (MCAO/R) rat model, we evaluated changes in the gut microbiota after CIRI and the potential effects of fecal microbiota transplant (FMT) on the brain. Rats underwent either MCAO/R or sham surgery, and then they received FMT (started 3days later; continued for 10days). 2,3,5-Triphenyltetrazolium chloride staining, neurological outcome scale, and Fluoro-Jade C staining showed that MCAO/R induced cerebral infarction, neurological deficits, and neuronal degeneration. In addition, immunohistochemistry or real-time PCR assay showed increased expression levels of M1-macrophage markers-TNF-α, IL-1β, IL-6, and iNOS-in the rats following MCAO/R. Our finding suggests that microglial M1 polarization is involved in CIRI. 16S ribosomal RNA gene sequencing data revealed an imbalance in the gut microbiota of MCAO/R animals. In contrast, FMT reversed this MCAO/R-induced imbalance in the gut microbiota and ameliorated nerve injury. In addition, FMT prevented the upregulation in the ERK and NF-κB pathways, which reversed the M2-to-M1 microglial shift 10days after MCAO/R injury in rats. Our primary data showed that the modulation of the gut microbiota can attenuate CIRI in rats by inhibiting microglial M1 polarization through the ERK and NF-κB pathways. However, an understanding of the underlying mechanism requires further study.

DUSP10 alleviates ischemic stroke-induced neuronal damage by restricting p38/JNK pathway

Neuronal apoptosis is considered one of the hallmarks of ischemic stroke. Dual specificity phosphatase 10 (DUSP10), a member of the dual-specificity phosphatase family, which is involved in the regulation of apoptosis process. This study aimed to investigate the effect of on apoptosis in primary cortical neurons exposed to oxygen-glucose deprivation and reoxygenation (OGD/R) and mice suffered from transient middle cerebral artery occlusion and reperfusion (MCAO/R). The results showed that DUSP10 overexpression improved survival and reduced apoptosis in neurons subjected to OGD/R, which was manifested by decreased apoptotic proteins (cleaved caspase 3 and bax) and TUNEL+ cells, as well as increased the anti-apoptotic protein (bcl-2). DUSP10 overexpression inhibited the p38/JNK signaling pathway after OGD/R treatment, whilst DUSP10 knockdown had opposite effects. In addition, the p38 inhibitor SB203580 or JNK inhibitor SP600125 attenuated the increased apoptosis of OGD/R-stimulated neurons treated with DUSP10 silencing. Consistently, DUSP10 knockdown exacerbated infarct volume in MCAO/R injury. The data of Nissl staining and TUNEL-NeuN double staining revealed that DUSP10 interference aggravated neuronal damage in the ischemic penumbra of mice. Furthermore, DUSP10 inhibition activated the p38/JNK axis accompanied by enhanced phosphorylation of p38 and JNK in vivo. In summary, DUSP10 is a neuroprotective agent against ischemic stroke-induced neuronal damage via suppressing the p38/JNK signaling pathway.

Scutellarin Alleviates Ischemic Brain Injury in the Acute Phase by Affecting the Activity of Neurotransmitters in Neurons.

Cerebral ischemic stroke is a common neuron loss disease that is caused by the interruption of the blood supply to the brain. In order to enhance the CIS outcome, both identifying the treatment target of ischemic brain damage in the acute phase and developing effective therapies are urgently needed. Scutellarin had been found to be beneficial to ischemic injuries and has been shown to have potent effects in clinical application on both stroke and myocardial infarction. However, whether scutellarin improves ischemic brain damage in the acute phase remains unknown. In this study, the protective effects of scutellarin on ischemic brain damage in the acute phase (within 12 h) were illustrated. In middle cerebral artery occlusion and reperfusion (MCAO/R) modeling rats, the Z-Longa score was significantly down-regulated by 25% and 23.1%, and the brain infarct size was reduced by 26.95 ± 0.03% and 25.63 ± 0.02% when responding to high-dose and low-dose scutellarin treatments, respectively. H&E and TUNEL staining results indicated that the neuron loss of the ischemic region was improved under scutellarin treatment. In order to investigate the mechanism of scutellarin's effects on ischemic brain damage in the acute phase, changes in proteins and metabolites were analyzed. The suppression of scutellarin on the glutamate-inducing excitatory amino acid toxicity was strongly indicated in the study of both proteomics and metabolomics. A molecular docking experiment presented strong interactions between scutellarin and glutamate receptors, which score much higher than those of memantine. Further, by performing a parallel reaction monitoring-mass spectrometry (PRM-MS) study on both the cortex and hippocampus tissue of the ischemic region, we screened the scutellarin-regulating molecules that are involved in both the release and transportation of neurotransmitters. It was found that the aberrant levels of glutamate receptors, including EAAT2, GRIN1, GRIN2B, and GRM1, as well as of other glutamatergic pathway-involving proteins, including CAMKK2, PSD95, and nNOS, were significantly regulated in the ischemic cortex. In the hippocampus, EAAT2, GRIN1, nNOS, and CAM were significantly regulated. Taken together, scutellarin exerts potent effects on ischemic brain damage in the acute phase by regulating the activity of neurotransmitters and reducing the toxicity of excitatory amino acids in in neurons.

Fasudil alleviates cerebral ischemia‑reperfusion injury by inhibiting inflammation and improving neurotrophic factor expression in rats.

The Rho kinase inhibitor fasudil exerts neuroprotective effects. We previously showed that fasudil can regulate M1/M2 microglia polarization and inhibit neuroinflammation. Here, the therapeutic effect of fasudil on cerebral ischemia‑reperfusion (I/R) injury was investigated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model in Sprague‑Dawley rats. The effect of fasudil on the phenotype of microglia and neurotrophic factors in the I/R brain and its potential molecular mechanism was also explored. It was found that fasudil ameliorated neurological deficits, neuronal apoptosis, and inflammatory response in rats with cerebral I/R injury. Fasudil also promoted the polarization of microglia into the M2 phenotype, in turn promoting the secretion of neurotrophic factors. Furthermore, fasudil significantly inhibited the expression of TLR4 and NF‑κB. These findings suggest that fasudil could inhibit the neuroinflammatory response and reduce brain injury after I/R injury by regulating the shift of microglia from an inflammatory M1 phenotype to an anti‑inflammatory M2 phenotype, which may be related to the regulation of the TLR4/ NF‑κB signal pathway.

P‑hydroxybenzyl alcohol ameliorates neuronal cerebral ischemia‑reperfusion injury by activating mitochondrial autophagy through SIRT1.

Mitochondrial autophagy serves a key role in clearing damaged mitochondria. P‑hydroxybenzyl alcohol (pHBA) can improve neuronal injury induced by cerebral ischemia‑reperfusion (I/R). However, the mechanism of pHBA improving I/R damage through the mitochondrial pathway remains unclear. A rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) was used in the present study. The rats were treated with sirtuin 1 (SIRT1) inhibitor EX527 and pHBA for 7 days, followed by reperfusion. At 24h after reperfusion, the infarct size was calculated and the severity of nerve damage was evaluated. Hematoxylin and eosin and Nissl staining revealed cellular changes in the ischemic penumbra. Changes in mitochondrial structure were observed using electron microscopy. Mitochondrial function was evaluated by detecting mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP) and ATP levels using commercially available kits. In addition, the ischemic penumbra tissues were used for immunofluorescence staining for p62 and LC3 proteins. The expression of SIRT1 and mitochondrial autophagy‑related proteins, PTEN‑induced kinase 1 (PINK1) and Parkin, were detected by western blotting. Finally, apoptosis was analyzed by TUNEL staining and the expression of apoptosis‑related proteins (Bax, Bcl‑2 and Caspase‑3) by western blotting. The results suggested that postoperative pHBA treatment may reduce the size of cerebral infarction and damage to the nervous system, and may improve cell damage in the ischemic penumbra of MCAO/R rats. Compared with rats in the untreated MCAO/R group, the mitochondrial structure of the pHBA‑treated group was improved, the levels of MMP and ATP were increased, and the degree of opening of mPTP was decreased. Simultaneously, immunofluorescence and western blotting results showed that compared with the MCAO/R group, the number of LC3‑ and TUNEL‑positive cells increased, the number of p62‑positive cells decreased, SIRT1 and autophagy protein (PINK1, Parkin and LC3 II/I) expression levels increased and p62 expression decreased in the pHBA group. However, these improvements were blocked by treatment with EX527. In summary, results from the present study suggested that pHBA may improve neuronal injury in the ischemic penumbra of MCAO/R rats through SIRT1‑activated mitochondrial autophagy and mitochondrial‑mediated neuronal apoptosis.

Triptolide promotes nerve repair after cerebral ischemia reperfusion injury by regulating the NogoA/NgR/ROCK pathway.

Activation of the NogoA/NgR/ROCK pathway limits nerve repair after brain ischemia-reperfusion (I/R) injury. Triptolide displays anti-inflammatory, anti-oxidant, and immunosuppressive effects and is derived from the traditional Chinese medicine Tripterygium wilfordii Hook F. This agent can also penetrate the blood-brain barrier, where it has a neuroprotective effect and ameliorates cerebral I/R injury via an as yet unknown mechanism(s). Here, an animal model of middle cerebral artery occlusion and reperfusion (MCAO/R) was employed to assess triptolide's therapeutic impact on brain I/R injury and the possible mechanism of action. The results indicate that triptolide treatment can decrease cerebral infarction and nerve injury after cerebral I/R injury. Importantly, in vivo and in vitro experiments revealed that treatment with triptolide decreased NogoA, NgR, p75NTR and ROCK2 expression, and upregulated the expression of GAP43 and PSD-95, thus suggesting improved synaptic function. These results indicate that triptolide can promote nerve repair following brain I/R injury by inhibiting NogoA/NgR/ROCK signalling.