Rat Middle Cerebral Artery Occlusion Model Research Articles

Mechanism of Electroacupuncture at Baihui and Shenting Alleviating Cerebral Ischemia-reperfusion Injury via Regulating BNIP3L-Mediated Mitophagy

<sec><title>Objective</title> The purpose of this study is to explore the mechanism of electroacupuncture at Baihui and Shenting acupoints to alleviate cerebral ischemia-reperfusion injury by regulating BNIP3L-mediated mitophagy. </sec><sec><title>Methods</title> A total of 60 SD rats were randomly divided into sham operation group (<italic>n</italic>=15) and operation group (<italic>n</italic>=45) by using the random number table method. Only the blood vessels were separated and exposed in the sham operation group, without ligation and no nylon thread inserted. The rats in the operation group were treated with the middle cerebral artery occlusion (MCAO) method to prepare the focal cerebral ischemia-reperfusion (I/R) model, and the Zea Longa method was used to score the neurological function 2h after the operation. Finally, 30 rats were included in the operation group. The included operation group rats were further randomly divided into the model group, electroacupuncture group, and electroacupuncture+3-MA group, with 10 rats in each group. After grouping, each group was treated with different methods such as electroacupuncture for 7 days, and the Zea Longa method was used to evaluate the neurological function again on the 1, 3, 5, and 7 days. After 7 days of intervention, the left cerebral cortex tissue of each group was obtained, hematoxylin-eosin (HE) staining was used to observe the pathological changes of the brain tissue, the expression level of LC3-Ⅱ/LC3-Ⅰ protein was detected by Western blot, and the tissue immunofluorescence technology was applied to detect the expression and colocalization of mitophagy-related protein BNIP3L and SQSTM1 labeled. The TUNEL method detected the apoptosis rate of rat nerve cells in each group. </sec><sec><title>Results</title> 1) The neurological scores of the rats in the operation group were significantly increased 2 hours after the MCAO, and the rats in the sham-operation group did not show symptoms of neurological deficit. On the 7th day after the intervention, the neurological scores of the rats in the model group and the electroacupuncture+3-MA group still increased significantly, which were significantly different from those in the sham operation group (<italic>P</italic><0.05). However, the neurological scores of rats in the electroacupuncture group reduced after electroacupuncture intervention, which was significantly different from the model group and electroacupuncture+3-MA group (<italic>P</italic><0.05). 2) The results of HE staining showed that the cell body of neurons in the sham operation group was large, polygonal (multiple protrusions), the nucleus was lightly colored, and the characteristic structure of Nissl body could be seen in the cytoplasm. In the model group, neurons shrank, the cytoplasmic structure was unclear, cytoplasmic eosinophilic staining was enhanced, Nissl bodies gathered or disappeared, nuclear pyknosis, and some cell bodies deformed and shrunk to triangles and the spaces around the cells widened. The electroacupuncture group could reduce the pathological damage caused by ischemia-reperfusion to a certain extent. 3) Western blot results showed that the autophagy level (LC3-Ⅱ/LC3-Ⅰ) of the model group decreased on the 7th day after MCAO, there wass a significant difference compared with the sham operation group (<italic>P</italic><0.05), while the electroacupuncture group could activate the level of autophagy, which was significantly different from the model group (<italic>P</italic><0.05). 4) In the model group, the level of mitophagy in the cortical infarction area was absent, as evidenced by the weakened red fluorescence and the intensity of green fluorescence. However, the intervention of electroacupuncture could activate the mitophagy of nerve cells in the infarct area, which was manifested by increased expression of red fluorescence and green fluorescence and enhanced colocalization (orange). 5) TUNEL test results indicated the apoptotic rate of rats in the model group rising, and there was a significant difference compared with the sham operation group (<italic>P</italic><0.05). However, the apoptotic rate of the electroacupuncture group decreased, which was significantly different from that of the model group (<italic>P</italic><0.05). The results of the electroacupuncture+3-MA group showed that the autophagy inhibitor 3-MA could reverse the neuroprotective effect of electroacupuncture in MCAO model rats. </sec><sec><title>Conclusion</title> Electroacupuncture at Baihui and Shenting acupoints can alleviate neurological damage in rats with cerebral ischemia-reperfusion. The specific mechanism is related to regulating BNIP3L-mediated mitophagy to reduce cerebral ischemia-reperfusion injury and cell apoptosis. </sec>

Effect of transcutaneous auricular vagus nerve stimulation on the expressions of GFAP and MAP2 in ischemic penumbra of rats with middle cerebral artery ischemia

To observe the effects of transcutaneous auricular vagus nerve stimulation (taVNS) on the motor function and the expression of glial fibrillary acidic protein (GFAP) and microtubule associated protein 2 (MAP2) in cerebral ischemic penumbra of rats with middle cerebral artery occlusion (MCAO) and explore the mechanism of taVNS in the improvement of motor function in MCAO rats. A total of 48 male SD rats were randomized into a sham-operation group, a model group, a transcutaneous auricular non-vagus nerve stimulation (tnVNS) group and a taVNS group, with 12 rats in each group. The suture-occluded method was adopted to prepare MCAO rat model. The auricular rim was stimulated in the tnVNS group and the concha stimulated in the taVNS group, 2 mA in intensity, 10 Hz in frequency, 30 min each time, once a day, for 14 days consecutively. The nerve functional assessment was recorded in each group. The expressions of nicotinic acetylcholine receptor (α7nAchR) in the cerebral ischemic penumbra and the spleen were detected by using Western blot. With the immunofluorescence, the expressions of GFAP and MAP2 were detected. After modeling, compared with the sham-operation group, the nerve functional score was increased in the model group, the tnVNS group and the taVNS group (P<0.01), suggesting the success of modeling. After treatment, the score was increased in the model group (P<0.01) as compared with the sham-operation group. Compared with the model group, the neurological deficit score was reduced in the taVNS group (P<0.01). Compared with the sham-operation group, GFAP expression was increased and MAP2 expression was reduced remarkably in the cerebral ischemic penumbra in the model group (P<0.05). In comparison with the model group, GFAP expression was reduced, while MAP2 expression was increased remarkably in the cerebral ischemic penumbra in the taVNS group (P<0.05). There were no significant differences in the abovementioned indexes between the model group and tnVNS group (P>0.05). The differences in the expression of α7nAchR in the cerebral ischemic penumbra and the spleen had no statistical significance among groups (P>0.05). TaVNS is effective on neuroprotection in MCAO rats, which may be related to its function of inhibition of GFAP expression and promotion of MAP2 expression in the ischemic penumbra.

Mechanism of Sanhua Decoction in the Treatment of Ischemic Stroke Based on Network Pharmacology Methods and Experimental Verification.

Objective The mechanism of action of Sanhua Decoction (SHD) in the treatment of ischemic stroke (IS) was analyzed based on the network pharmacology technology, and the pharmacodynamics and key targets were verified using the rat middle cerebral artery occlusion (MCAO) model. Methods The GEO database was used to collect IS-related gene set SD, and DrugBank and TTD databases were used to obtain the therapeutic drug target set ST. IS disease gene set SI was collected from DisGeNET, GeneCards, and OMIM databases. These three different gene sets obtained from various sources were merged, duplicates were removed, and the resulting IS disease gene set SIS was imported into the STRING database to establish the protein-protein interaction (PPI) network. Two methods were used to screen the key targets of IS disease based on the PPI network analysis. The TCMSP database and PubChem were applied to retrieve the main chemical components of SHD, and the ACD/Labs software and the SwissADME online system were utilized for ADMET screening. HitPick, SEA, and SwissTarget Prediction online systems were used to predict the set of potential targets for SHD to treat IS. The predicted set of potential targets and the IS disease gene set were intersected. Subsequently, the set of potential targets for SHD treatment of IS was identified, the target information was confirmed through the UniProt database, and finally, the component-target data set for SHD treatment of IS was obtained. clusterProfiler was used for GO function annotation and KEGG pathway enrichment analysis on the target set of SHD active ingredients. A rat MCAO model was established to evaluate the pharmacodynamics of SHD in the treatment of IS, and Western blot analysis assessed the level of proteins in the related pathways. Results This study obtained 1,009 IS disease gene sets. PPI network analysis identified 12 key targets: AGT, SAA1, KNG1, APP, GNB3, C3, CXCR4, CXCL12, CXCL8, CXCL1, F2, and EDN1. Database analyses retrieved 40 active ingredients and 47 target genes in SHD. The network proximity algorithm was used to optimize the six key components in SHD. KEGG enrichment showed that the signaling pathways related to IS were endocrine resistance, estrogen, TNF signal pathway, and AGEs/RAGE. Compound-disease-target regulatory network analysis showed that AKT1, IL-6, TNF-α, TP53, VEGFA, and APP were related to the treatment of IS with SHD. Animal experiments demonstrated that SHD significantly reduces the neurological function of rat defect symptoms (P < 0.05), the area of cerebral avascular necrosis, and neuronal necrosis while increasing the levels of IL-6 and APP proteins (P < 0.05) and reducing the levels of AKT1 and VEGFA proteins (P < 0.05). Conclusion The effective components of SHD may regulate multiple signaling pathways through IL-6, APP, AKT1, and VEGFA to reduce brain damage and inflammatory damage and exert a neuroprotective role in the treatment of IS diseases. Thus, this study provides a feasible method to study the pharmacological mechanism of traditional Chinese medicine compound prescriptions and a theoretical basis for the development of SHD into a new drug for IS treatment.

Ozone treatment alleviates brain injury in cerebral ischemic rats by inhibiting the NF-κB signaling pathway and autophagy

ABSTRACT Stroke is the most frequent cause of disability in developed countries. A common phenomenon of stroke, cerebral ischemia, is threatening many lives worldwide. In addition, ozone treatment was previously reported to exert functions in relieving brain injury. In the current study, the therapeutic effects of ozone on cerebral ischemia are investigated. A rat model of middle cerebral artery occlusion (MCAO) was established. The brain water content was calculated by weighing brain tissues, and the 2, 3, 5-triphenyltetrazolium chloride staining was performed to measure brain infarction volume in rats. A colorimetric assay was conducted to examine expression levels of malondialdehyde, superoxide dismutase, catalase, and glutathione in the rat hippocampus. Reverse transcription quantitative polymerase-chain reaction and Western blot analyses were employed to evaluate expression levels of Beclin1, LC3B, p62, and critical factors implicated in the NF-κB signaling pathway. We found that ozone significantly improved the survival rate of MCAO model rats, reduced the cerebral water content, and decreased the neurological scores of ischemic rats. Ozone markedly reduced cerebral ischemia-induced infarction in ischemic rats. Ozone decreased MDA levels and increased SOD, catalase, and GSH levels in the hippocampus of rats. Ozone significantly inhibited autophagy by decreasing Beclin1 and LC3B expression and increasing p62 expression. The ozone inactivated the NF-κB signaling pathway by decreasing the protein levels of TLR4, p-IKKβ, p-IKBα, and p-p65. We conclude that ozone treatment alleviates the brain injury in ischemic rats by suppressing autophagy and inactivating the NF-κB signaling pathway.

FNDC5 overexpression promotes the survival rate of bone marrow mesenchymal stem cells after transplantation in a rat cerebral infarction model.

BackgroundMost bone marrow mesenchymal stem cell (BMSC) death is caused by the harsh ischemia and hypoxic microenvironment, which impacts the therapeutic effects of transplanted BMSCs. Fibronectin type III domain-containing protein 5 (FNDC5) and its cleaved product, irisin, are reportedly involved in cerebral protective effect. Research into whether FNDC5 plays a key role in the survival rate of BMSCs and cerebral infarction (CI) remains inadequate. The present study aimed to clarify the protective role of FNDC5 on the low viability of transplanted BMSCs and improve CI treatment outcomes.MethodsA lentivirus vector, which drives the expression of FNDC5, was constructed and used to transfect BMSCs. Cell Counting Kit-8 (CCK8), flow cytometry, immunofluorescence, and western blot were performed to evaluate the function of FNDC5-overexpressing BMSCs (BMSCs-OE-FNDC5) exposed to hypoxic and serum deprivation (H/SD) stress. Transmission electron microscopy (TEM) was used to monitor autophagy. In addition, BMSCs were engrafted into a middle cerebral artery occlusion (MCAO) rat model with or without FNDC5-overexpression (OE-FNDC5). The survival rate of transplanted BMSCs was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) labeling. The CI volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining.ResultsH/SD stress caused increased cell autophagy, apoptosis, and decreased cell viability of BMSCs, while OE-FNDC5 alleviated these injuries. The in vivo results showed that transplantation of BMSCs-OE-FNDC5 reduced the infarct volume in the rat MCAO model. Furthermore, OE-FNDC5 decreased neuronal apoptosis. The improved therapeutic efficacy of BMSCs-OE-FNDC5 may be attributable to the obviously increased cell survival number after transplantation.ConclusionsThese results indicated that FNDC5 overexpression promotes BMSC survival in a CI model, which might provide a potential therapeutic target.

Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

Guhong injection (GHI) is a drug for ischemic stroke created by combining safflower, a traditional Chinese medicine, and aceglutamide, a Western medicine. In this study, we investigated the curative effect of GHI on cerebral ischemia–reperfusion (I/R) injury via the PKC/HIF-1α pathway in rats. Adult male Sprague Dawley rats were randomly divided into seven groups: sham-operated, middle cerebral artery occlusion (MCAO), GHI, nimodipine injection (NMDP), MCAO + LY317615 (PKC inhibitor), GHI + LY317615, and NMDP + LY317615. After establishing an MCAO rat model, we performed neurological deficit testing, 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin (HE) staining, enzyme-linked immunosorbent assay, Western blotting, and q-PCR to detect the brain damage in rats. Compared with the MCAO group, the GHI and GHI + LY317615 group showed neurological damage amelioration as well as decreases in serum hypoxia-inducible factor-1α (HIF-1α), protein kinase C (PKC), and erythropoietin levels; brain HIF-1α and inducible nitric oxide synthase protein expression; and brain HIF-1α and NOX-4 mRNA expression. These effects were similar to those in the positive control groups NMDP and NMDP + LY317615. Thus, our results confirmed GHI can ameliorate cerebral I/R injury in MCAO rats possibly via the PKC/HIF-1α pathway.

Propofol Mediated Protection of the Brain From Ischemia/Reperfusion Injury Through the Regulation of Microglial Connexin 43.

Cerebral ischemia/reperfusion (I/R) injury is a serious condition that leads to increased apoptosis of microglial and neurons in the brain. In this study, we identified that Cx43 expression level is significantly increased in the microglial cells during I/R injury. Using an in vitro model (hypoxia/reoxygenation-H/R injury), we observed that H/R injury leads to an increase in activation of microglial cells and increase in levels of pro-inflammatory markers such as IL-1β, IL-6, and TNF-α. Additionally, we could also observe significant increase in phosphorylation of Cx43 and Cav3.2 levels. To assess the role of H/R injured microglial cells on neuronal population, we cultured the neurons with conditioned media (MCS) from H/R injured microglial cells. Interestingly, we observed that microglial H/R injury significantly decreased Map2 expression and affected neuronal morphology. Further, we aimed to assess the effects of propofol on cerebral H/R injury, and observed that 40 μM propofol significantly decreased Cx43, Cx43 phosphorylation, and CaV3.2 levels. Additionally, propofol decreased apoptosis and increased Map2 expression levels in H/R injured neurons. Using silencing experiments, we confirmed that siCx43 could significantly improve the propofol’s rescue after H/R injury in both microglia and neurons. We further developed an in vivo MCAO (middle cerebral artery occlusion) rat model to understand the effect of propofol in I/R injury. Interestingly, propofol treatment and downregulation of Cx43 significantly decreased the infract volume and apoptosis in these MCAO rats. Thus, this study clearly establishes that propofol protects the brain against I/R injury through the downregulation of Cx43 in microglial cells.

Ameliorative Effect of Parishin C Against Cerebral Ischemia-Induced Brain Tissue Injury by Reducing Oxidative Stress and Inflammatory Responses in Rat Model.

BackgroundGastrodia elata Blume (Orchidaceae) is a widely used traditional Chinese herbal medicine in the clinical practice of China, to treat nervous headache, convulsions, dizziness, neurasthenia, and so on. Parishin C (Par C), one of the major bioactive components of Gastrodia elata Blume, is known to exert many different biological activities, including antipsychotic and neuroprotective effects. However, there is little research about its neuroprotective effect in an ischemic stroke model. The objective of the present study is thus to investigate the neuroprotective effects of Par C against cerebral ischemia damage.MethodsRats were pretreated with Par C (25, 50, or 100 mg/kg/day, i.p.) for 21 days, then subjected to 2 h of middle cerebral artery occlusion (MCAO) and 22 h of reperfusion. Neurological deficient scores, brain water content, histopathology, TCC staining were performed to assess the neuroprotective effects of Par C. Meanwhile, the oxidative stress, inflammation and apoptosis-related markers of brain tissue were evaluated by corresponding assay kits. Besides, the antioxidant and pro-inflammatory expression was measured by real-time quantification PCR (RT-qPCR).ResultsOur findings indicated that the pre-treatment with Par C improved nerve function, suppressed oxidative stress, and pro-inflammatory factors release in rats with cerebral ischemia damage. Besides, Par C significantly increased antioxidant expression and declined pro-inflammatory cytokines expression.ConclusionPar C is shown to exert neuroprotective effects partly via inhibiting oxidative stress and inflammation in a rat model of MCAO.

MicroRNA-532-5p protects against cerebral ischemia-reperfusion injury by directly targeting CXCL1.

We investigated the function of microRNA (miR)-532-5p in cerebral ischemia-reperfusion injury (CI/RI) and the underlying mechanisms using oxygen-glucose deprivation and reperfusion (OGD/R)-treated SH-SY5Y cells and middle cerebral artery occlusion (MCAO) model rats. MiR-532-5p levels were significantly downregulated in OGD/R-treated SH-SY5Y cells and the brain tissues of MCAO model rats. MiR-532-5p overexpression significantly reduced apoptosis, reactive oxygen species (ROS), and inflammation in the OGD/R-induced SH-SY5Y cells. Bioinformatics analysis using the targetscan and miRDB databases as well as dual luciferase reporter assays confirmed that miR-532-5p directly binds to the 3’UTR of C-X-C Motif Ligand 1 (CXCL1). Methylation-specific PCR (MSP) analysis showed that miR-532-5p expression was reduced in OGD/R-treated SH-SY5Y cells because of miR-532-5p promoter hypermethylation. Moreover, 5-azacytidine, a methylation inhibitor, restored miR-532-5p expression in OGD/R-treated SH-SY5Y cells. Brain tissues of MCAO model rats showed significantly increased cerebral infarction areas, cerebral water, neuronal apoptosis, and activated CXCL1/CXCR2/NF-κB signaling, but these effects were alleviated by intraventricular injection of miR-532-5p agomir. These findings demonstrate that miR-532-5p overexpression significantly reduces in vitro and in vivo CI/RI by targeting CXCL1. Thus, miR-532-5p is a potential therapeutic target for patients with CI/RI.

Oxytocin improves ischemic stroke by reducing expression of excitatory amino acid transporter 3 in rat MCAO model

ABSTRACT Various molecular mechanisms are activated in neurons during ischemic stroke. Extracellular glutamate secretion into brain tissue causes neurotoxicity and brain damage. Excitatory amino acid transporter 3 (EAAT3) could remove the extracellular glutamate. Neuroprotective activity of oxytocin (OT) in ischemia of various tissues has been reported. This study investigates the neuroprotective effect of OT in an animal model of middle cerebral artery occlusion (MCAO) and the possible role of EAAT3. Transient MCAO was performed as a model of ischemic stroke in male rats and then OT was administrated intra-nasally. Infarct volume was measured by 2, 3, 5-triphenyl tetrazolium chloride staining. Nissl staining method was performed for the evaluation of neuronal cell morphology. Immunohistochemistry assay was performed to analyze the EAAT3 expression in the ischemic region. OT significantly reduced the infarct volume in the cerebral cortex and striatum after ischemia (P< .05). In addition, OT reduces the number of neurons with pyknotic nuclei that are significantly increased in the ischemic region (P< .01) Immunohistochemistry results showed that although EAAT3 expression increased after ischemia, OT therapy increased EAAT3 expression further (P< .05). Therefore, increased EAAT3 expression could be one of the neuroprotective mechanisms of OT after MCAO.

MiR-29a Knockout Aggravates Neurological Damage by Pre-polarizing M1 Microglia in Experimental Rat Models of Acute Stroke.

ObjectiveBy exploring the effects of miR-29a-5p knockout on neurological damage after acute ischemic stroke, we aim to deepen understanding of the molecular mechanisms of post-ischemic injury and thus provide new ideas for the treatment of ischemic brain injury.MethodsmiR-29a-5p knockout rats and wild-type SD rats were subjected to transient middle cerebral artery occlusion (MCAO). miR-29a levels in plasma, cortex, and basal ganglia of ischemic rats, and in plasma and neutrophils of ischemic stroke patients, as well as hypoxic glial cells were detected by real-time PCR. The infarct volume was detected by TTC staining and the activation of astrocytes and microglia was detected by western blotting.ResultsThe expression of miR-29a-5p was decreased in parallel in blood and brain tissue of rat MCAO models. Besides, miR-29a-5p levels were reduced in the peripheral blood of acute stroke patients. Knockout of miR-29a enhanced infarct volume of the MCAO rat model, and miR-29a knockout showed M1 polarization of microglia in the MCAO rat brain. miR-29a knockout in rats after MCAO promoted astrocyte proliferation and increased glutamate release.ConclusionKnockout of miR-29a in rats promoted M1 microglial polarization and increased glutamate release, thereby aggravating neurological damage in experimental stroke rat models.

Carthamin yellow improves cerebral ischemia‑reperfusion injury by attenuating inflammation and ferroptosis in rats.

Carthamin yellow (CY), a flavonoid compound extracted from safflower, has been reported to attenuate cardiac ischemia and reperfusion injury. However, whether CY could ameliorate ischemic stroke is not completely understood. In the present study, the preventive effects of CY on experimental ischemic stroke were investigated using middle cerebral artery occlusion (MCAO) model rats. Neurological scores, brain edema, infarct area and microtubule-associated protein 2 (MAP-2) immunoreactivity were assessed to evaluate the effects of CY on ischemic brain injury. The involvement of inflammation and ferroptosis were examined to investigate the mechanism underlying the effects of CY. The results demonstrated that 2-week CY treatment attenuated the neurological deficit score, brain water content and infarct area, and increased MAP-2 immunoreactivity in the cortex in MCAO model rats. CY administration also deactivated the cortex NF-κB/NLR family pyrin domain containing 3 inflammasome signaling pathway, and decreased serum TNF-α, IL-1β and IL-6 concentrations. Moreover, CY treatment inhibited Fe2+ and reactive oxygen species accumulation, and reversed acyl-CoA synthetase long-chain family member 4, transferrin receptor 1, glutathione peroxidase 4 and ferritin heavy chain 1 protein expression levels in the brain. The levels of glutathione, superoxide dismutase and malondialdehyde in the serum were also reversed by CY treatment. Collectively, the results of the present study demonstrated that CY protected rats against ischemic stroke, which was associated with mitigation of inflammation and ferroptosis.

Effect of Panax notoginseng Saponins on Focal Cerebral Ischemia-Reperfusion in Rat Models: A Meta-Analysis.

With the increase of the aging population, the high mortality and disability rates caused by ischemic stroke are some of the major problems facing the world, and they dramatically burden the society. Panax notoginseng (Burk) F. H. Chen, a traditional Chinese medicine, is commonly used for promoting blood circulation and removing blood stasis, and its main bioactive components are Panax notoginseng saponins (PNS). Therefore, we performed a meta-analysis on focal cerebral ischemia-reperfusion animal models established with middle cerebral artery occlusion (MCAO) surgery to evaluate the therapeutic effect of PNS. We systematically searched the reports of PNS in MCAO animal experiments in seven databases. We assessed the study quality using two literature quality evaluation criteria; evaluated the efficacy of PNS treatment based on the outcomes of the neurological deficit score (NDS), cerebral infarct volume (CIV), and biochemical indicators via a random/fixed-effects model; and performed a subgroup analysis utilizing ischemia duration, drug dosage, intervention time, and administration duration. We also compared the efficacy of PNS with positive control drugs or combination treatment. As a result, we selected 14 eligible studies from the 3,581 searched publications based on the predefined exclusion-inclusion criteria. PNS were significantly associated with reduced NDS, reduced CIV, and inhibited release of the inflammatory factors IL-1β and TNF-α in the focal MCAO rat models. The PNS combination therapy outperformed the PNS alone. In addition, ischemia time, drug dosage, intervention time, and administration duration in the rat models all had significant effects on the efficacy of PNS. Although more high-quality studies are needed to further determine the clinical efficacy and guiding parameters of PNS, our results also confirmed that PNS significantly relieves the focal cerebral ischemia-reperfusion in rat models. In the animal trials, it was suggested that an early intervention had significant efficacy with PNS alone or PNS combination treatment at a dosage lower than 25 mg/kg or 100–150 mg/kg for 4 days or longer. These findings further guide the therapeutic strategy for clinical cerebral ischemic stroke.

Anti-platelet aggregation of Panax notoginseng triol saponins by regulating GP1BA for ischemic stroke therapy

BackgroundPanax notoginseng triol saponins (PTS) has been used clinically for ischemic stroke therapy (IST) in China for more than 17 years due to its anti-platelet aggregation and neuro-protective effects, but its mechanism of action is not fully understand. In this study, anti-platelet aggregation-related protein analysis and computer simulations of drug-protein binding interactions were performed to explore the mechanism of the effects of PTS against ischemic stroke in an ischemia reperfusion model.MethodsThree oral doses of PTS were administered in a model of middle cerebral artery occlusion (MCAO) in rats. Panax notoginseng total saponins (PNS) and a combination of PTS and aspirin were chosen for comparison. To evaluate therapeutic effects and explore possible mechanisms of anti-platelet aggregation, we measured cerebral infarct size and water content in brain tissue, histomorphological changes, expression of related factors (such as arachidonic acid metabolites) and platelet receptors in serum, as well as the binding affinity of PTS for platelet adhesion receptors.ResultsCompared with PNS, PTS showed a stronger and more potent anti-platelet aggregation effect in MCAO model rats. The combination of PTS and aspirin could reduce adverse gastrointestinal effects by regulating the TXA2/PGI2 ratio. We demonstrated for the first time that PTS was able to regulate Glycoprotein Ib-α (GP1BA) in a model animal. The binding of ginsenoside Rg1 and GP1BA could form a stable structure. Moreover, PTS could reduce von Willebrand factor (VWF)-mediated platelet adhesion to damaged vascular endothelium, and thus enhance the probability of anti-platelet aggregation and anti-thrombosis under pathological conditions.ConclusionsOur results showed that GP1BA was closely related to the anti-platelet aggregation action of PTS, which provided new scientific and molecular evidence for its clinical application.

Neuroprotective effects of Ginkgolide B in focal cerebral ischemia through selective activation of prostaglandin E2 receptor EP4 and the downstream transactivation of epidermal growth factor receptor.

The present study was undertaken to identify whether prostaglandin E2 receptor is the potential receptor/binding site for Ginkgolide A, Ginkgolide B, Ginkgolide K, and Bilobalide, the four main ingredients of the Ginkgo biloba L., leaves. Using functional assays, we identified EP4, coupled with Gs protein, as a target of Ginkgolide B. In human neuroblastoma SH-SY5Y cells suffered from oxygen-glucose deprivation/reperfusion, Ginkgolide B-activated PKA, Akt, and ERK1/2 as well as Src-mediated transactivation of epidermal growth factor receptor. These resulted in downstream signaling pathways, which enhanced cell survival and inhibited apoptosis. Knockdown of EP4 prevented Ginkgolide B-mediated Src, epidermal growth factor receptor (EGFR), Akt, and ERK1/2 phosphorylation and neuroprotective effects. Moreover, Src inhibitor prevented Ginkgolide B-mediated EGFR transactivation and the downstream Akt and ERK1/2 activation, while the phosphorylation of PKA induced by Ginkgolide B was not affected, indicating Ginkgolide B might transactivate EGFR in a ligand-independent manner. EP4 knockdown in a rat middle cerebral artery occlusion (MCAO) model prevented Ginkgolide B-mediated infarct size reduction and neurological assessment improvement. At the same time, the increased expressions of p-Akt, p-ERK1/2, p-PKA, p-Src, and p-EGFR and the deceased expression of cleaved capases-3 induced by Ginkgolide B in cerebral cortex were blocked due to EP4 knockdown. In conclusion, Ginkgolide B exerts neuroprotective effects in rat MCAO model through the activation of EP4 and the downstream transactivation of EGFR.

Analysis of regulatory effect of miR-149-5p on Sphingosine-1-phosphate receptor 2 of pericytes and its neuroprotective molecular mechanism after acute cerebral ischemia reperfusion in rats

ABSTRACT To investigate the effect of miR-149-5p on sphingosine-1-phosphate receptor 2 (S1PR2) expression level and contents of matrix metalloproteinase (MMP-9) and superoxide dismutase (SOD) in the pericytes after acute cerebral ischemia reperfusion in rats, so as to clarify the neuroprotective molecular mechanism induced by miR-149-5p and provide references for the treatment of neurological diseases, 60 male SD rats aged 7–8 weeks were selected and divided randomly into test group (establishing middle cerebral artery occlusion (MCAO) model) and control group (no modeling). Rat pericytes and peripheral cerebral infarction tissues were collected 12 h, 1 d, 3 d, 5 d, and 7 d after MCAO modeling, respectively. The pericytes were identified by immunofluorescence assay (IFA) and transfected with miR-149-5p. Fluorescence quantitative PCR (FQPCR) and Western blot were adopted to detect S1PR2 expression level. The expression of S1PR2 in MCAO model rats was detected by IFA. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were used to detect the changes of MMP9 protein and mRNA levels of SOD1, SOD2, and SOD3 in brain tissue. The results showed that mRNA level and protein expression level of S1PR2 in the test group were higher than those in the control group three days after MCAO modeling (P < 0.05); the expression of S1PR2 increased 12 h after MCAO modeling and returned to the normal level on the 5th day, and the content of MMP9 protein in brain tissue of the test group was significantly lower than that of the control group (P < 0.05); the mRNA levels and SODs activity of SOD1, SOD2, and SOD3 in the test group were higher than those in the control group (P < 0.05). Therefore, miR-149-5p played a neuroprotective role by regulating S1PR2 to change the expression levels of SODS and MMP9.

Chrysoeriol promotes functional neurological recovery in a rat model of cerebral ischemia

Background: Stroke is among the leading causes of death worldwide, but current treatment options for cerebral ischemia are limited. Chrysoeriol is a natural isolated agent with bioactive pharmacological properties. Materials and Methods: Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) surgery and chrysoeriol was administrated for the subsequent 3 days. Neurological deficit scores, apoptosis, oxidant stress, and inflammatory cytokines were then measured and the effects on the Wnt/β-catenin pathway were evaluated. Results: Behavioral tests revealed that chrysoeriol promoted recovery from neurological deficits in rats. Hematoxylin and eosin staining demonstrated that chrysoeriol alleviated neurological damage. Triphenyl tetrazolium chloride staining demonstrated that chrysoeriol reduced the area of ischemia. An enzyme-linked immune sorbent assay (ELISA) demonstrated that chrysoeriol inhibited excessive pro-inflammatory cytokine production (tumor necrosis factor-α, interleukin [IL]-1β, and IL-6) and regulated oxidative stress (malondialdehyde, superoxide dismutase, and glutathione). Caspase-3 immunofluorescence and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay showed that chrysoeriol reduced neuronal apoptosis. The ELISA assay and Nissl staining showed that chrysoeriol promoted neuronal survival and growth. Western blotting and an immunofluorescence assay demonstrated that the Wnt/β-catenin signaling pathway plays a crucial role in the neuronal effects of chrysoeriol. The Wnt inhibitor, Dickkopf-related protein-1, validated the role of the Wnt/β-catenin signaling pathway in the neuroprotective effects of chrysoeriol. Conclusion: Chrysoeriol exerts neuroprotective effects in a rat model of MCAO. These effects are mediated by the Wnt/β-catenin signaling pathway.

MiR-10b-3p Protects Cerebral I/R Injury through Targeting Programmed Cell Death 5 (PDCD5).

Previous studies have demonstrated that miR-10b-3p is significantly downregulated in rats after cerebral ischemia injury, and this study aimed to investigate the effect of miR-10b-3p in cerebral ischemia/reperfusion (I/R) injury. The oxygen-glucose deprivation (OGD) induced SH-SY5Y cell model and middle cerebral artery occlusion model (MCAO) rats were constructed to investigate the role of miR-10b-3p and underline the regulatory mechanism of miR-10b-3p/PDCD5 axis in cerebral I/R injury. The expression of miR-10b-3p and PDCD5 was evaluated by qRT-PCR and Western blot. The binding relationship between miR-10b-3p and PDCD5 was determined by bioinformatic analysis and luciferase reporter assay. Cell proliferation was evaluated by MTT assay and Edu staining assay. The apoptosis was assessed by TUNEL staining assay and flow cytometry. MiR-10b-3p was significantly downregulated and PDCD5 was upregulated both in OGD/R induced SH-SY5Y cells and the brain tissues of MCAO/R rats. Luciferase reporter assay determined that miR-10b-3p could directly bind to the 3' UTR of PDCD5 and negatively regulate its expression. MiR-10b-3p overexpression could efficiently inhibit cell viability and proliferation, induce apoptosis of OGD/R-induced SH-SY5Y cells in vitro, and attenuate cerebral I/R injury of MCAO rats in vivo. Silencing of PDCD5 showed similar effect to miR-10b-3p mimics, while PDCD5 overexpression significantly reversed the protective effects of miR-10b-3p mimics on cerebral I/R injury. In summary, our results revealed that miR-10b-3p alleviated cerebral I/R injury partly through targeting PDCD5 and indicated that miR-10b-3p might be a potential target for ischemic stroke.

Melatonin Ameliorates Hemorrhagic Transformation via Suppression of ROS-Induced NLRP3 Activation after Cerebral Ischemia in Hyperglycemic Rats.

Melatonin is a strong antioxidant which beneficially protects against middle cerebral artery occlusion (MCAO) followed by hemorrhagic transformation in rats; protection includes the reduction of neurological deficits, infarction, and hematoma volume. The molecular mechanisms underlying these neuroprotective effects in the MCAO model have not been clearly identified. This study examined the influence and involved mechanism of melatonin on inflammation in hemorrhagic transformation following hyperglycemia MCAO rat model. Compared with the MCAO group, MCAO+dextrose (DX) group showed worse neurological function and higher infarction and hematoma volume. Interestingly, the protein expression of Nod-like receptor protein 3 (NLRP3) inflammasome increased in the MCAO+DX group compared with the MCAO group, which indicated that NLRP3 inflammasome may be involved in the DX-induced hemorrhagic transformation following MCAO. Then, three dosages of melatonin were intraperitoneally injected 2 h after MCAO induction. Melatonin treatment attenuated inflammatory response by inhibiting the reactive oxygen species (ROS) and NLRP3 inflammasome, alleviating neuronal injury, and reducing infarction and hematoma volume, finally improving neurological score. Melatonin also repressed cortical levels of proinflammatory cytokine IL-1β, which were increased 24 h after hyperglycemia MCAO. In order to identify the potential mechanisms, we further revealed that nigericin administration reversed the neuroprotective effect of melatonin by promoting NLRP3 inflammasome activation. In general, this present study reveals that melatonin prevents the occurrence of hyperglycemia-enhanced hemorrhagic transformation, and this effect might be beneficial to attenuate neurological dysfunction via suppressing the inflammatory response after MCAO which possibly associated with the inhibition of the ROS/NLRP3 inflammasome pathway.

Kellerin from Ferula sinkiangensis exerts neuroprotective effects after focal cerebral ischemia in rats by inhibiting microglia-mediated inflammatory responses

Ethnopharmacological relevanceFerula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. Aim of the studyTo explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. Materials and methodsNeurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. ResultsWe found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. ConclusionsOur discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.