Middle Cerebral Artery Occlusion And Reperfusion Research Articles

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.

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.

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.

Comparison of the Effects of Constraint-Induced Movement Therapy and Unconstraint Exercise on Oxidative Stress and Limb Function-A Study on Human Patients and Rats with Cerebral Infarction.

Most conventional post-stroke rehabilitation treatments do not involve imposed constraints of the unaffected limb. In contrast, Constraint-Induced Movement Therapy (CIMT) is comprised of massed task practice with the affected limb and constraint of the unaffected limb. CIMT is a promising rehabilitation technique used for motor recovery of affected limbs after stroke, but its effectiveness and mechanism are not fully understood. We compared the effects of the two exercise modes on limb function post-stroke in animal models and human subjects, and investigated whether oxidative stress response was involved in regulating the effects. We first conducted a randomized controlled trial (RCT), in which 84 subjects with cerebral infarction were assigned to dose-matched constraint-induced movement therapy (CIMT), or unconstraint exercise (UE), or conventional rehabilitation treatment. Motor functions of the limb are primary outcomes of the RCT measured using Brief Fugl-Meyer upper extremity score (FMA-UE), Ashworth score, and Barthel scale. Psychological influence of CIMT and UE was also examined using Self-Rating Depression Scale (SDS). Next, we investigated the effects of CIMT and UE in rats undergoing middle cerebral artery occlusion and reperfusion (MCAO/R). Motor function, infarct volume, and pathohistological changes were investigated by mNSS, MRI, and histological studies. The role of Keap1-Nrf2-ARE was investigated using qRT-PCR, Western blot, immunochemistry, immunofluorescence, and ELISA experiments. In RCT, patients taking CIMT had a higher score in FMA-UE, Barthel index, and SDS, and a lower score in modified Ashworth, compared to those taking UE. In rats receiving CIMT, motor function was increased, and infarct volume was decreased compared to those receiving UE. The expression of Keap1 protein and mRNA in the peri-infarct tissue was decreased, and Nrf2 and ARE protein and mRNA were increased in rats receiving CIMT compared with UE. Nrf2 agonist t-BHQ increased the benefits of CIMT. In conclusion, CIMT is more effective than UE in improving upper limb motor function, reducing muscle spasm in patients with cerebral infarction compared to UE, but patients receiving CIMT may feel depressed. Moreover, both CIMT and UE are beneficial to limb function recovery and limit the infarct expansion in MCAO/R rats, but CIMT was more effective than UE. Oxidative stress reaction has an essential role in regulating the CIMT induced benefits.

Leptin alleviates endoplasmic reticulum stress induced by cerebral ischemia/reperfusion injury via the PI3K/Akt signaling pathway.

Cerebral ischemic/reperfusion injury (CIRI) is a key factor for the prognosis of ischemic stroke (IS), the leading disease in terms of global disability and fatality rates. Recent studies have shown that endoplasmic reticulum stress (ERS) may be a target against CIRI and that leptin, a peptide hormone, has neuroprotective activity to mitigate CIRI. An in vitro CIRI model was induced in primary cortical neurons by oxygen-glucose deprivation and reoxygenation (OGD/R) after pretreatment with LY294002 (10 µmol/L) and/or leptin (0.4 mg/L), and cell viability, neuronal morphology and endoplasmic reticulum (ER) dysfunction were evaluated. An in vivo CIRI model was established in rats by middle cerebral artery occlusion and reperfusion (MCAO/R) after the injection of LY294002 (10 μmol/L) and/or leptin (1 mg/kg), and neurological function, infarct volume, cerebral pathological changes, the expression of ERS-related proteins and cell apoptosis were examined. In vitro, leptin treatment improved the cell survival rate, ameliorated neuronal pathological morphology and alleviated OGD/R-induced ERS. In vivo, administration of leptin significantly reduced the infarct volume, neurological deficit scores and neuronal apoptosis as well as pathological alterations. In addition, leptin suppressed MCAO/R-induced ERS and may decrease apoptosis by inhibiting ERS-related death and caspase 3 activation. It also regulated expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax in the cortex. Furthermore, the inhibitory effect of leptin on ERS was significantly decreased by the effective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results confirm that ERS plays an important role in CIRI and that leptin can inhibit the activation of ERS through the PI3K/Akt pathway, thereby alleviating CIRI. These findings provide novel therapeutic targets for IS.

Ocimum gratissimum Leaves Aqueous Extract Can Minimize Middle Cerebral Artery Occlusion and Reperfusion-Induced Injury in Rat’s Brain

Background and Aim: Ocimum gratissimum is widely used in traditional medicine for its good antioxidant and anti-inflammatory activity. Stroke is an important factor of disability and death worldwide causing brain injury through oxidative stress and inflammation. The present study aimed to evaluate the neuroprotective effect of Ocimum gratissimum on middle cerebral artery occlusion and reperfusion-induced brain injury in rat.
 Experimental Procedure: The rats (48) were divided into 8 groups of 6 animals each and treated as followed: 3 groups receiving distilled water (10 mL/kg); one piracetam (250 mg/kg) and 4 groups receiving the plant extract (30, 60, 120 and 240 mg/kg) for 3 days. On the fourth day, ischemia by middle cerebral artery occlusion and reperfusion (MCAO) surgery was performed on the groups receiving piracetam and extract. As for the 3 groups receiving distilled water, one was not operated; one was operated without MCAO (sham operated) while the last one was operated with MCAO. The temperature, neurological function through behavioral tests, heat sensibility, and open arena tests were assessed. Inflammatory and oxidative stress markers were analyzed in the region of the brain affected by MCAO.
 Results and Conclusion: Three days pretreatment with the plant extract prevented the drop of temperature and heat sensibility, reduced the neurological score and associated anxiety-like behavior. The extract also prevented inflammation and oxidative stress induced by MCAO in the brain. All these observations suggest that Ocimum gratissimum by its antioxidant and anti-inflammatory potencies can protect the brain against ischemic stroke-induced brain injury.

The effect of head-down tilt in experimental acute ischemic stroke.

Collateral therapeutics exert a promising protective effect on the outcome of acute ischemic stroke. Cerebral blood flow (CBF) may be modulated by different head positioning. The current study aimed to determine the effect of head-down tilt (HDT) on stroke in a rodent model. The model of middle cerebral artery occlusion and reperfusion (MCAO/R) was used in this study. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride staining, brain water content, perivascular aquaporin protein-4 (AQP4) localization, pericyte marker platelet-derived growth factor receptor β (PDGFRβ), and CBF velocity were evaluated at 24 h after MCAO/R and HDT treatment. In the rat model of MCAO/R, brain infarct volume and neurological deficit score were significantly alleviated in the -30° and -60° groups compared to those in the lying flat (0°) group. Compared with the 0° group, an increase in CBF velocity was detected in the -30° group through two-photon microscopy imaging at 24 h after MCAO/R. Compared with the SHAM group, a decrease in PDGFRβ was observed in both the MCAO/R and HDT treatment (-30°) groups. The integrated optical density of PDGFRβ was found to be higher in the HDT treatment (-30°) group than in the MCAO/R group. An impairment in perivascular AQP4 polarity and an increase in brain water content were observed after MCAO/R, which were not exacerbated by HDT treatment (-30°). Our findings suggest that HDT treatment at certain degrees may exert a neuroprotective effect after MCAO/R through improving CBF velocity and the protection of pericytes.

Edaravone Ameliorates Cerebral Ischemia-Reperfusion Injury by Downregulating Ferroptosis via the Nrf2/FPN Pathway in Rats.

Edaravone, an antioxidant protective agent, has anti-cerebral ischemic reperfusion injury (CIRI) effects, but its anti-CIRI mechanism is unclear. The aim of this study is to investigate the anti-CIRI mechanism of edaravone based on the nuclear factor-E2-related factor 2 (Nrf2)/ferroportin (FPN) pathway that regulates ferroptosis-mediated cerebral ischemia-reperfusion injury. We evaluated the brain injury by constructing a middle cerebral artery occlusion and reperfusion (MCAO/R) model in rats. The results showed that cerebral infarct volume and neurological impairment scores were increased in cerebral ischemia-reperfusion rats, with impaired sensorimotor ability; furthermore, brain tissue glutathione (GSH) content was decreased, Fe2+, malondialdehyde (MDA) and lipide peroxide (LPO) content were increased, and the expression level of glutathione peroxidase 4 (GPX4), a key protein of ferroptosis, was also decreased. Meanwhile, the Nrf2 expression level was increased and the FPN expression level was decreased after cerebral ischemia-reperfusion, while the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and myeloperoxidase (MPO) were increased. However, edaravone exhibited a protective effect on cerebral infarct and neurological and sensorimotor function in relevant tests. In addition, we also found that edaravone decreased the contents of Fe2+, MDA, and LPO in the brain tissue of MCAO/R rats and increased GSH content to inhibit ferroptosis. Furthermore, Western blot showed that after treatment with edaravone, the expression of Nrf2, GPX4, and FPN was up-regulated, the nuclear location of Nrf2 was increased, and the levels of inflammation-related indicators IL-6, IL-1β, TNF-α, and MPO were lower than in the MCAO/R group. Our results demonstrated that edaravone inhibits ferroptosis to attenuate CIRI, probably through the activation of the Nrf2/FPN pathway.

Astrocyte-Derived TNF-α-Activated Platelets Promote Cerebral Ischemia/Reperfusion Injury by Regulating the RIP1/RIP3/AKT Signaling Pathway

Ischemic stroke is a clinical syndrome caused by the disruption of blood flow into cerebral tissues and is associated with high disability and mortality rates. Studies have established the pathological role of platelets in cerebral ischemia/reperfusion (I/R) injury, although the underlying mechanism of action remains largely unclear. In this study, we created an I/R mouse model via middle cerebral artery occlusion and reperfusion (MCAO/R) and analyzed the transcriptomic profiles of the ipsilateral and contralateral cortices using RNA-seq. We found that cerebral I/R injury induced platelet invasion and accumulation in the cerebral cortex by stimulating TNF-α secretion from activated astrocytes in the ischemic region, while TNF-α expression enhanced platelet reactivity through the RIP1/RIP3/AKT pathway. Furthermore, the inoculation of TNF-α-stimulated platelets aggravated I/R injury in mice, whereas the administration of anti-TNF-α antibodies at the onset of reperfusion alleviated ischemic damage. The RNA-seq results further showed that AP-1 transcriptionally activated TNF-α in the I/R-injured cortex by directly binding to the promoter region. These findings provide novel insights into the pathological role of platelets activated by reactive astrocyte-derived TNF-α in cerebral I/R injury.

Luteolin alleviates inflammation and autophagy of hippocampus induced by cerebral ischemia/reperfusion by activating PPAR gamma in rats

BackgroundLuteolin, a flavonoid compound with anti-inflammatory activity, has been reported to alleviate cerebral ischemia/reperfusion (I/R) injury. However, its potential mechanism remains unclear.MethodsThe binding activity of luteolin to peroxisome proliferator-activated receptor gamma (PPARγ) was calculated via molecular docking analysis. Rats were subjected to middle cerebral artery occlusion and reperfusion (MCAO/R). After reperfusion, vehicle, 25 mg/kg/d luteolin, 50 mg/kg/d luteolin, 10 mg/kg/d pioglitazone, 50 mg/kg/d luteolin combined with 10 mg/kg/d T0070907 (PPARγ inhibitor) were immediately orally treatment for 7 days. ELISA, TTC staining, H&E staining, immunohistochemistry, immunofluorescence and transmission electron microscope methods were performed to evaluate the inflammation and autophagy in damaged hippocampal region. The PPARγ, light chain 3 (LC3) B-II/LC3B-I and p-nuclear factor-κB (NF-κB) p65 proteins expression levels in damaged hippocampal region were analyzed.ResultsLuteolin showed good PPARγ activity according to docking score (score = − 8.2). Luteolin treatment downregulated the infarct area and the pro-inflammatory cytokines levels caused by MCAO/R injury. Moreover, luteolin administration ameliorated neuroinflammation and autophagy in damaged hippocampal region. Pioglitazone plays protective roles similar to luteolin. T0070907 concealed the neuroprotective roles of 50 mg/kg/d luteolin.ConclusionsLuteolin exerts neuroprotective roles against inflammation and autophagy of hippocampus induced by cerebral I/R by activating PPARγ in rats.

Chuanzhi Tongluo Capsules antagonizes cerebral ischemia-reperfusion injury in mice by inhibiting neuroinflammation and oxidative stress

Chuanzhi Tongluo Capsules(CZTL) is effective in activating blood, resolving stasis, replenishing Qi, and dredging collaterals, which has been widely used in clinical treatment of stroke(cerebral infarction) differentiated into the syndrome of wind striking meridian and collateral in the recovery stage characterized by blood stasis and Qi deficiency. However, its modern pharmacological mechanisms of action remain unclear. This study duplicated the middle cerebral artery occlusion and reperfusion(MCAO/R) model in mice using the suture-occluded method to explore the protective effect and mechanism of CZTL on ischemic stroke. The mice were divided into the sham-operation group, model group, and CZTL group. The ones in the CZTL group were gavaged with 0.3 g·kg~(-1)·d~(-1) CZTL for three successive days. One hour after the last intragastric administration, those in the model and CZTL groups were subjected to MCAO/R. After 24 h reperfusion, the effects of CZTL on neurological deficit score, cerebral infarction area, brain edema, and brain histopathology were evaluated. The levels of reactive oxygen species(ROS), malondialdehyde(MDA), interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) and the activity of superoxide dismutase(SOD) in brain tissue homogenate were detected using corresponding assay kits. The expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), Toll like receptor 4(TLR4), and phosphorylated nuclear factor-κB P65 subunit(p-NF-κB P65) were assayed by Western blot. The results indicated that CZTL significantly reduced the neurological deficit score, brain edema, and infarct volume, improved the brain histopathology, inhibited the expression of ROS, MDA, IL-6, IL-1β, and TNF-α in the brain tissue, and up-regulated the activity of SOD, down-regulated the expression of pro-apoptotic protein Bax, promoted the expression of anti-apoptotic protein Bcl-2, and suppressed the expression of TLR4 and p-NF-κB P65 phosphorylation of MCAO/R mice. All these have demonstrated that CZTL has a significant protective effect against MCAO/R injury in mice, which may be related to its resistance to neuroinflammation and oxidative stress.

N-myc Downstream-Regulated Gene 2 (Ndrg2): A Critical Mediator of Estrogen-Induced Neuroprotection Against Cerebral Ischemic Injury.

Growing evidence indicates that estrogen plays a pivotal role in neuroprotection against cerebral ischemia, but the molecular mechanism of this protection is still elusive. N-myc downstream-regulated gene 2 (Ndrg2), an estrogen-targeted gene, has been shown to exert neuroprotective effects against cerebral ischemia in male mice. However, the role of Ndrg2 in the neuroprotective effect of estrogen remains unknown. In this study, we first detected NDRG2 expression levels in the cortex and striatum in both female and male mice with western blot analyses. We then detected cerebral ischemic injury by constructing middle cerebral artery occlusion and reperfusion (MCAO-R) models in Ndrg2 knockout or conditional knockdown female mice. We further implemented estrogen, ERα, or ERβ agonist replacement in the ovariectomized (OVX) Ndrg2 knockout or conditional knockdown female mice, then tested for NDRG2 expression, glial fibrillary acidic protein (GFAP) expression, and extent of cerebral ischemic injury. We found that NDRG2 expression was significantly higher in female than in male mice in both the cortex and striatum. Ndrg2 knockouts and conditional knockdowns showed significantly aggravated cerebral ischemic injury in female mice. Estrogen and ERβ replacement treatment (DPN) led to NDRG2 upregulation in both the cortex and striatum of OVX mice. Estrogen and DPN also led to GFAP upregulation in OVX mice. However, the effect of estrogen and DPN in activating astrocytes was lost in Ndrg2 knockout OVX mice and primary cultured astrocytes, but partially retained in conditional knockdown OVX mice. Most importantly, we found that the neuroprotective effects of E2 and DPN against cerebral ischemic injury were lost in Ndrg2 knockout OVX mice but partially retained in conditional knockdown OVX mice. These findings demonstrate that estrogen alleviated cerebral ischemic injury via ERβ upregulation of Ndrg2, which could activate astrocytes, indicating that Ndrg2 is a critical mediator of E2-induced neuroprotection against cerebral ischemic injury.

The crosstalk signals of Sodium Tanshinone ⅡA Sulfonate in rats with cerebral ischemic stroke: Insights from proteomics

BackgroundStroke could cause long-term disability, even mortality around the world. Recently, Sodium tanshinone IIA sulfonate (STS), identified from Salvia miltiorrhiza Bunge and was found to have unique efficiency in clinical practice as a potential therapeutic agent for ischemic cerebral infarction. However, systematic investigation about the biological mechanism is still lacking. Herein, we utilized high-throughput proteomics approach to identify the underlying targets for the treatment of STS in stroke. MethodsWe investigated the effect of STS on stroke outcomes on rat model of the Middle Cerebral Artery Occlusion and Reperfusion (MCAO/R), assessing by Z-Longa score, infarct volume and HE staining. Pharmacoproteomic profiling of ischemic penumbra in cortical (IPC) was performed using DIA-based label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Bioinformatics analysis was processed for further investigation. The expression of core proteins was semi-quantified by DIA, and the major protein correlating with stroke was examined using parallel reaction monitoring (PRM). ResultsRats in the MCAO/R group showed neurological function deterioration, which was improved by STS. There were 423 differentially expressed proteins (DEPs) in IPC being detected and quantified in both the sham group and the MCAO/R group. Meanwhile, 285 proteins were significantly changed in the STS treated group, compared to the MCAO/R model. Protein-protein interaction (PPI) network, pathway and biological function enrichment were processed for the DEPs across each two groups, the results of which were integrated for analysis. Alb, mTOR, Dync1h1, Stxbp1, Cltc, and Sptan1 were contained as the core proteins. Altered molecules were discovered to be enriched in 18 signal pathways such as phosphatidylinositol signaling system, PI3K/AKT signal pathway and HIF-1 signal pathway. The results also showed the correlation with sleep disturbances and depression post-stroke. ConclusionsWe concluded that STS could prevent penumbra from progressively ongoing damage and improve neurological deficits in MCAO/R model rats. The intersected pathways and protein networks predicted by proteomics might provide much more detailed information for the therapeutic mechanisms of STS in the treatment of CIS.

Beta-Boswellic Acid Protects Against Cerebral Ischemia/Reperfusion Injury via the Protein Kinase C Epsilon/Nuclear Factor Erythroid 2-like 2/Heme Oxygenase-1 Pathway.

Ischemic strokes are associated with a high rate of disability and death globally. Cerebral ischemia/reperfusion (I/R) injury is a type of brain damage associated with oxidative stress after an ischemic stroke. Beta-boswellic acid (β-BA) reportedly exerts antioxidant and neuroprotective effects, but its role in cerebral I/R injury is unclear. The aim of this research was to investigate the neuroprotective effects, as well as the mechanisms of β-BA in cerebral I/R injury. In vivo experiments were conducted using a rat middle cerebral artery occlusion and reperfusion (MCAO/R) model, and in vitro experiments were performed using a rat neuronal oxygen-glucose deprivation and reoxygenation (OGD/R) model. Triphenyltetrazolium chloride staining, neurological function scores, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, hematoxylin and eosin staining, and antioxidant levels in the brain were used to assess the effects of β-BA. Flow cytometry was used to detect reactive oxygen species and apoptotic cells. Western blotting and immunofluorescence staining were used to measure protein levels. The results showed that β-BA markedly improved neurological deficits and decreased infarct volume and necrotic neurons in rats. The in vitro results showed that β-BA protected neurons against OGD/R-induced injury. Additionally, β-BA significantly increased the phosphorylation of protein kinase C epsilon (PRKCE) at S729, the translocation of nuclear factor erythroid 2-like 2 (NFE2L2), and expression of heme oxygenase-1 (HMOX1). This study demonstrates that β-BA exerts neuroprotective effects against cerebral I/R via the activation of the PRKCE/NFE2L2/HMOX1 pathway and is a potential therapeutic candidate for ischemic stroke.

Ligustilide ameliorates hippocampal neuronal injury after cerebral ischemia reperfusion through activating PINK1/Parkin-dependent mitophagy

BackgroundMitophagy plays a critical role in cerebral ischemia/reperfusion by timely removal of dysfunctional mitochondria. In mammals, PINK1/Parkin is the most classic pathway mediating mitophagy. And the activation of PINK1/Parkin mediated mitophagy exerts neuroprotective effects during cerebral ischemia reperfusion injury (CIRI). Ligustilide (LIG) is a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis (Oliv.) diels that exerts neuroprotective activity after cerebral ischemia reperfusion injury (CIRI). However, it still remains unclear whether LIG could attenuates cerebral ischemia reperfusion injury (CIRI) through regulating mitophagy mediated by PINK1/Parkin. PurposeTo explore the underlying mechanism of LIG on PINK1/Parkin mediated mitophagy in the hippocampus induced by ischemia reperfusion. MethodsThis research used the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model and oxygen-glucose deprivation and reperfusion (OGD/R) as in vitro model. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of LIG in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP) and activities of Na+-K+-ATPase were detected to reflect mitochondrial function. Moreover, transmission electron microscope (TEM) and fluorescence microscope were used to observe mitophagy and the western blot was performed to explore the changes in protein expression in PINK1/Parkin mediated mitophagy. Finally, exact mechanism between neuroprotection of LIG and mitophagy mediated by PINK1/Parkin was explored by cell transfection. ResultsThe results show that LIG improved mitochondrial functions by mitophagy enhancement in vivo and vitro to alleviate CIRI. Whereas, mitophagy enhanced by LIG under CIRI is abolished by PINK1 deficiency and midivi-1, a mitochondrial division inhibitor which has been reported to have the function of mitophagy, which could further aggravate the ischemia-induced brain damage, mitochondrial dysfunction and neuronal injury. ConclusionLIG could ameliorate the neuronal injury against ischemia stroke by promoting mitophagy via PINK1/Parkin. Targeting PINK1/Parkin mediated mitophagy with LIG treatment might be a promising therapeutic strategy for ischemia stroke.

Inhibition of Autophagy Facilitates XY03-EA-Mediated Neuroprotection against the Cerebral Ischemia/Reperfusion Injury in Rats.

Objective L-3-n-Butylphthalide (NBP) is used to treat moderate and severe acute ischemia stroke. A previous screening study indicates that XY03-EA, a novel derivative of NBP, is more potent than NBP in the oxyradical scavenging capacity. In this study, in vivo and in vitro ischemia/reperfusion (I/R) models were used to test whether the XY03-EA offered therapeutic benefits in the ischemic stroke and explore the underlying mechanism of action. Methods For this purpose, behavioral scores, cerebral infarct volume, cerebral blood flow, oxidative stress levels, inflammatory factor expression, energy metabolism levels, and autophagy activation were estimated in the rat middle cerebral artery occlusion and reperfusion (MCAO/R) model. The nonhuman primate MCAO/R model was conducted to validate the therapeutic effect of XY03-EA applied for 3 weeks. The neurological deficit score (NDS) progression rate and the infarct volume were continuously recorded on days 3, 7, 14, and 21. The PC-12 cell OGD/R model was used to assess the cell survival rate, reactive oxygen species (ROS) levels, the expression of autophagy execution molecules, and the activation of autophagy-related signaling pathways. Results XY03-EA decreased the cerebral injuries and NDS by increasing cerebral blood flow, improving brain energy metabolism, accelerating ROS clearance, suppressing inflammatory responses, and inhibiting autophagy in the MCAO/R model rats. In the nonhuman primate MCAO/R model, the treatment of XY03-EA for 3 weeks could significantly inhibit the NDS progression rate and indicate a positive trend to reduce the infarct volume in a dose-dependent way. Mechanistically, XY03-EA inhibited ROS-dependent autophagy activation and thereby protected the PC-12 cells from the autophagic cell death induced by OGD/R. Conclusions In this study, we found that XY03-EA alleviated the cerebral I/R injuries in rats and nonhuman primates. Our results demonstrated that XY03-EA exerted neuroprotective effects against the ROS-mediated autophagic neurocyte death and had great potential for the treatment of ischemic stroke.

Curcumin nanoemulsion ameliorates brain injury in diabetic rats.

Diabetes mellitus has been implicated in the exacerbation of cerebral ischemic injuries. Among the most promising therapeutic approaches is the combination of nutraceuticals and nanotechnology. Curcumin has been termed "the magic molecule", and it was proven to exert several therapeutic actions. Therefore, the aim of the presented work was to investigate the therapeutic effects of curcumin nanoemulsion (NC) administered orally on the middle cerebral artery occlusion and reperfusion (MCAO/Re)-induced cerebral damage in rats with streptozotocin-induced diabetes. The cerebral injury was induced in rats by MCAO/Re 6 weeks after single intraperitoneal STZ injection (50mg/kg; i.p.). MCAO/Re diabetic rats were then treated with NC (50 and 100mg/kg; bw; p.o.) for two consecutive weeks. The results of the present study showed that oral treatment of MCAO/Re diabetic rats with NC was associated with a marked attenuation of the neurological deficit score as well as the brain imbalance of the redox homeostasis. NC treatment was also associated with decline in the brain expression of tumor necrosis factor, interleukin-1β, COX-2, cleaved caspase-3, and nuclear factor kappa B. In addition, the expression of glucose transporter 1 proteins upon treatment was restored. PRACTICAL APPLICATIONS: From all these results, it can be concluded that oral supplementation of curcumin nanoemulsion (NC) in diabetic rats reduced the brain injury via augmentation of the expression of glucose transporter 1, as well as its antioxidant and anti-inflammatory properties. Therefore, NC could be delineated as a promising treatment option for cerebral ischemia in diabetic patients.

Rehmannioside A improves cognitive impairment and alleviates ferroptosis via activating PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathway after ischemia

Ethnopharmacological relevanceRehmannioside A is derived from Rehmannia glutinosa Libosch, which is widely used as an important ingredient in diverse traditional Chinese medicines to treat diseases caused by “kidney deficiency” such as cerebral arteriosclerosis, aging-related stroke and dementia in China. Recent studies have proved that Rehmannia glutinosa Libosch and Rehmannioside A can improve memory capability and recover nerve damage. Aim of the studyTo investigate the effect of Rehmannioside A on cognitive impairment after ischemia in rats and SH-SY5Y cells, and further evaluate the anti-oxidative and anti-ferroptosis mechanisms. Materials and methodsDifferentially expressed proteins (DEPs) in patients after cerebral ischemic stroke were revealed by a RayBio protein array. Cognitive impairment model was established by middle cerebral artery occlusion and reperfusion (MCAO) 14 days in rats. Rehmannioside A was administered intraperitoneally injection at dose of 80 mg/kg. The SH-SY5Y cells were exposed to H2O2 for 24 h and treated with Rehmannioside A (80 μM) for 24 h. The neuroprotecion of Rehmannioside A were evaluated by infarct volume (TTC), neurological defects (Garcia score) and learning memory (Morris water maze test) in vivo, and cell viability (CCK-8 or LDH) in vitro. Superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO) activity of rats, glutathione (GSH), oxidized glutathione (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH) of cells were detected by biochemical assay. Intracellular reactive oxygen species (ROS) were measured by DCFH-DA assay. Myeloperoxidase (MPO), PI3 kinase (PI3K), p-PI3K, Akt, p-Akt, heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2), SLC7A11, glutathione peroxidase 4 (GPX4) of the cerebral cortex in rats or SH-SY5Y cells were examined by western blotting. ResultsCompared with model group, the cognitive impairment and neurological deficits of Rehmannioside A group were significantly improved, and the cerebral infarction was reduced in MCAO rats. Moreover, the cell viability obviously increased and the H2O2-induced toxicity was reduced in Rehmannioside A group. Further research indicated that the expression of p-PI3K, p-Akt, nuclear Nrf2, HO-1 and SLC7A11 in Rehmannioside A group was significantly higher than model group. ConclusionRehmannioside A has neuroprotection effect and improves cognitive impairment after cerebral ischemia by inhibiting ferroptosis and activating PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathway. These findings provide valuable insight into the pathogenesis and therapeutic target of ischemic stroke.

Buyang Huanwu Decoction promotes neurogenesis via sirtuin1/autophagy pathway in a cerebral ischemia model.

Stroke is one of the main causes of disease-related mortality worldwide. Buyang Huanwu Decoction (BHD) has been used to protect against stroke and stroke-induced disability for several years in China. Studies have shown that BHD can relieve neuronal damage in rats with cerebral ischemia/reperfusion (I/R) injury. However, the mechanism remains unclear. A middle cerebral artery occlusion and reperfusion (MCAO-R) model was used in the present study. The animals were treated with BHD (5, 10 and 20 g/kg) or rapamycin. Infarct size and modified neurological severity score were calculated on day 5 following MCAO-R surgery. Cellular changes around the ischemic penumbra were revealed by hematoxylin and eosin and Nissl staining. The protein expression levels of nestin, brain-derived neurotrophic factor (BDNF), doublecortin on the X chromosome (DCX) and autophagy-related proteins (beclin 1, LC3-II and p62) in the peri-ischemic area of the brain were detected. The results demonstrated that post-surgical treatment with BHD reduced the brain infarct size and improved neurological deficits in MCAO-R rats. BHD protected against MCAO-R-induced neuronal impairment and promoted neurogenesis, increased the protein expression of nestin, BDNF and DCX and markedly enhanced autophagy by increasing beclin 1 and LC3-II and decreasing p62. Meanwhile, BHD promoted the expression of sirtuin 1 (SIRT1), an important regulator of autophagy. In conclusion, the present study suggested that post-surgical treatment with BHD could protect rat brains from I/R injury, potentially through the SIRT1/autophagy pathway.