Middle Cerebral Artery Occlusion/reperfusion Research Articles

Arbutin protects brain against middle cerebral artery occlusion-reperfusion (MCAo/R) injury

Focal ischemia causes irreversible brain damage if cerebral blood flow is not restored promptly. Acute phase excitotoxicity and pro-oxidant and inflammatory events in the sub-chronic phase elicit coagulative necrosis, vascular injury, cerebral oedema, and neurobehavioral deficits. Earlier, in pre-clinical studies arbutin protected behavioral functions and improved therapeutic outcomes in different models of brain and metabolic disorders. Arbutin is natural hydroquinone that might protect against ischemia-reperfusion (I/R) injury. In this study, cerebro-protective effects of arbutin were evaluated in the middle cerebral artery occlusion-reperfusion (MCAo/R) mouse model. Mice were administered arbutin (50, 100 mg/kg, i.p.) for 21 days, and subjected to MCAo/R or sham surgery on day 14. Results showed brain infarction, blood-brain barrier dysfunction, oedema, and neurological deficits 24 h post-MCAo/R injury that were prevented by arbutin. Behavioral evaluations over the sub-chronic phase revealed MCAo/R triggered spatial and working memory deficits. Arbutin protected the memory against MCAo/R injury and decreased hydroxy-2′-deoxyguanosine, protein carbonyls, inflammatory cytokines (tumor necrosis factor-α, myeloperoxidase, matrix metalloproteinase-9, inducible nitric oxide synthase), and enhanced glutathione levels in the ischemia ipsilateral hemisphere. Arbutin decreased brain acetylcholinesterase activity, glutamate, and enhanced GABA levels against MCAo/R. Arbutin can alleviate I/R pathogenesis and protects neurobehavioral functions in the MCAo/R mouse model.

PLXNA2 knockdown promotes M2 microglia polarization through mTOR/STAT3 signaling to improve functional recovery in rats after cerebral ischemia/reperfusion injury

Ischemic stroke is an acute cerebrovascular disease characterized by high mortality, morbidity and disability rates. Ischemia/reperfusion is a critical pathophysiological basis of motor and cognitive dysfunction caused by ischemic stroke. Microglia, innate immune cells of the central nervous system, mediate the neuroinflammatory response to ischemia/reperfusion. PlexinA2 (PLXNA2) plays an important role in the regulation of neuronal axon guidance, the immune response and angiogenesis. However, it is not clear whether PLXNA2 regulates microglia polarization in ischemic stroke or the underlying mechanism. In the present study, we investigated the role of PLXNA2 in rats with middle cerebral artery occlusion/reperfusion (MCAO/R) and BV2 microglia cells with oxygen and glucose deprivation/reoxygenation (OGD/R). A battery of behavioral tests, including the beam balance test, forelimb placement test, foot fault test, cylinder test, CatWalk gait analysis and Morris water maze test were performed to evaluate sensorimotor function, locomotor activity and cognitive ability. The expression of M1/M2-specific markers in the ischemic penumbra and BV2 microglia cells was detected using immunofluorescence staining, quantitative real-time PCR analysis and Western blot analysis. Our study showed that PLXNA2 knockdown accelerated the recovery of motor function and cognitive ability after MCAO/R. In addition, PLXNA2 knockdown restrained proinflammatory cytokine release and promoted anti-inflammatory cytokine release, and the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) pathway was involved in PLXNA2 regulated microglia polarization. Taken together, our results indicate that PLXNA2 knockdown reduces neuroinflammation by switching the microglia phenotype from M1 to M2 in the ischemic penumbra of MCAO/R-injured rats, which may be due to the inhibition of mTOR/STAT3 signaling. Treatments targeting PLXNA2 may be a promising therapeutic strategy for ischemic stroke.

Effect of Naoluoxintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats

Naoluoxintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear. To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/RhoA/Rock pathway in a rat model of IS by regulating DNA methylation. Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT+SGI-1027) and blocker group II (NLXT+Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow (rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho-associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue. After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P<0.01 or P<0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P<0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P<0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P<0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P<0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P<0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P<0.01). NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect.

Long Non-coding RNA PVT1 Inhibits miR-30c-5p to Upregulate Rock2 to Modulate Cerebral Ischemia/Reperfusion Injury Through MAPK Signaling Pathway Activation

Long non-coding RNAs (lncRNAs) play a key role in a variety of disease processes. Plasmacytoma variant translocation 1 (PVT1), a lncRNA, is known to regulate cell functions and play a key role in the pathogenesis of many malignant tumors. The function and molecular mechanisms of lncRNA-PVT1 in cerebral ischemia remain unknown. Real-time PCR (qRT-PCR) was used to detect lncRNA-PVT1 and microRNA-30c-5p (miR-30c-5p) expression in the brain tissues of mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reperfusion (OGD/R)-treated mouse primary brain neurons. Gain- or loss-of-function approaches were used to manipulate PVT1, miR-30c-5p, and Rho-associated protein kinase 2 (Rock2). The mechanism of PVT1 in ischemic stroke was evaluated both in vivo and in vitro via bioinformatics analysis, CCK-8, flow cytometry, TUNEL staining, luciferase activity assay, RNA FISH, and Western blot. PVT1 was upregulated in the brain tissues of mice treated with MCAO/R and primary cerebral cortex neurons of mice treated with OGD/R. Mechanistically, PVT1 knockdown resulted in a lower infarct volume and ameliorated neurobehavior in MCAO mice. Consistent with in vivo results, PVT1 upregulation significantly decreased the viability and induced apoptosis of neurons cultured in OGD/R. Moreover, we demonstrated that PVT1 acts as a competitive endogenous RNA (ceRNA) that competes with miR-30c-5p, thereby negatively regulating its endogenous target Rock2. Overexpression of miR-30c-5p significantly promoted cell proliferation and inhibited apoptosis. Meanwhile, PVT1 was confirmed to target miR-30c-5p, thus activating Rock2 expression, which finally led to the activation of MAPK signaling. We demonstrated that PVT1, as a ceRNA of miR-30c-5p, could target and regulate the level of Rock2, which aggravates cerebral I/R injury via activation of the MAPK pathway. These findings reveal a new function of PVT1, which helps to broadly understand cerebral ischemic stroke and provide a new treatment strategy for this disease.

LncRNA NKILA relieves astrocyte inflammation and neuronal oxidative stress after cerebral ischemia/reperfusion by inhibiting the NF-κB pathway

BackgroundIschemic stroke is one of the major diseases of the cerebral vasculature. Currently, Ischemic stroke is the leading cause of neurological disability worldwide and has a high morbidity and mortality rate. The NF-κB interacting lncRNA (NKILA), the recently identified, is a key booster of NF-κB pathway. Accumulating studies have shown that NKILA plays a cancer suppressor in a variety of malignancies by regulating the NF-κB pathway. Nevertheless, the role of NKILA in ischemic stroke remains to be elucidated. MethodsWe constructed a mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R). TTC staining and dry and wet weight method were used to evaluate infarction and water content of brain tissue. RT-qPCR was performed to detect NKILA expression in cerebral infarction tissues. After labeling astrocytes and neurons with GFAP and NeuN, respectively, EDU and TUNEL staining were performed. Inflammatory factor levels were detected by ELISA. Commercial kits were used to detect the levels of oxidative stress-related factors. In in vitro, the HT22/U251 cell co-culture model was used for oxygen-glucose deprivation and re-introduction (OGD/R) to verify the effect of NKILA on neuronal cell inflammation and oxidative stress through astrocytes. ResultsIn in vivo experiments, NKILA significantly reduced cerebral infarction volume, brain water content and neurological score caused by MCAO/R. Moreover, NKILA blocked the activation of the NF-κB pathway, and inhibited astrocyte proliferation and neuron apoptosis as well as inflammation and oxidative stress. In in vitro experiments, NKILA significantly inhibited NF-κB pathway in HT22 cells. In addition, NKILA alleviated the inflammatory response and oxidative stress of U251 cells mediated by HT22 cells after OGD/R, and promoted U251 cell proliferation and inhibit their apoptosis. ConclusionsIn summary, we found for the first time that NKILA alleviates inflammatory response and oxidative stress after cerebral ischemia/reperfusion by blocking the activation of NF-κB pathway.

Destructive Effects of Pyroptosis on Homeostasis of Neuron Survival Associated with the Dysfunctional BBB-Glymphatic System and Amyloid-Beta Accumulation after Cerebral Ischemia/Reperfusion in Rats.

Neuroinflammation-related amyloid-beta peptide (Aβ) accumulation after cerebral ischemia/reperfusion (I/R) accounts for cerebral I/R injuries and poststroke dementia. Recently, pyroptosis, a proinflammatory cell death, has been identified as a crucial pathological link of cerebral I/R injuries. However, whether pyroptosis acts as a trigger of Aβ accumulation after cerebral I/R has not yet been demonstrated. Blood-brain barrier (BBB) and glymphatic system mediated by aquaporin-4 (AQP-4) on astrocytic endfeet are important pathways for the clearance of Aβ in the brain, and pyroptosis especially occurring in astrocytes after cerebral I/R potentially damages BBB integrity and glymphatic function and thus influences Aβ clearance and brain homeostasis. In present study, the method of middle cerebral artery occlusion/reperfusion (MCAO/R) was used for building models of focal cerebral I/R injuries in rats. Then, we used lipopolysaccharide and glycine as the agonist and inhibitor of pyroptosis, respectively, Western blotting for detections of pyroptosis, AQP-4, and Aβ1-42 oligomers, laser confocal microscopy for observations of pyroptosis and Aβ locations, and immunohistochemical stainings of SMI 71 (a specific marker for BBB integrity)/AQP-4 and Nissl staining for evaluating, respectively, BBB-glymphatic system and neuronal damage. The results showed that pyroptosis obviously promoted the loss of BBB integrity and AQP-4 polarization, brain edema, Aβ accumulation, and the formation of Aβ1-42 oligomers and thus increased neuronal damage after cerebral I/R. However, glycine could inhibit cerebral I/R-induced pyroptosis by alleviating cytomembrane damage and downregulating expression levels of cleaved caspase-11/1, N-terminal gasdermin D, NLRP3 (nucleotide-binding domain, leucine-rich repeat containing protein 3), interleukin-6 (IL-6) and IL-1β and markedly abate above pathological changes. Our study revealed that pyroptosis is a considerable factor causing toxic Aβ accumulation, dysfunctional BBB-glymphatic system, and neurological deficits after cerebral I/R, suggesting that targeting pyroptosis is a potential strategy for the prevention of ischemic stroke sequelae including dementia.

Propofol Protects against Cerebral Ischemia/Reperfusion Injury by Down-Regulating Long Noncoding RNA SNHG14.

Cerebral ischemia-reperfusion (CI/R) injury is a serious central nervous system disease. Propofol (PPF) exerts a neuroprotective effect in CI/R injury; the underlying cause is still unclear. Here, we cultured mouse hippocampal neuron (HT22 cells) in oxygen-glucose deprivation/reoxygenation (OGD/R) conditions to mimic CI/R injury in vitro. PPF treatment promoted cell viability and reduced apoptotic cells in the OGD/R-treated HT22 cells, which was effectively abrogated by SNHG14 overexpression. Moreover, we constructed a CI/R injury mouse model on C57BL/6J mice by middle cerebral artery occlusion/reperfusion (MCAO/R), followed by administration of PPF. PPF reduced neuronal damage and loss, enhanced glial cell hyperplasia, and ameliorated cerebral cortex tissue damage and brain infarct in MCAO/R-induced mice. SNHG14 overexpression aggravated MCAO/R-induced CI/R injury in mice. Furthermore, SNHG14 promoted the expression of Atg5 and Beclin 1 via competitively binding miR-30b-5p, which contributed to activate autophagy and apoptosis in HT22 cells. In addition, the levels of p-p38 and p-SP1 were reduced in the OGD/R-treated HT22 cells in the presence of PPF. SP1 interacted with the promoter of SNHG14 and elevated the expression of SNHG14. PPF treatment inhibited the SP1-mediated up-regulation of SNHG14. In conclusion, this work demonstrates that PPF inhibits SNHG14 expression though the p38 MAPK signaling pathway. SNHG14 promotes Atg5 and Beclin 1 expression by sponging miR-30b-5p and thus activates autophagy and aggravates CI/R injury.

CTRP1 Attenuates Cerebral Ischemia/Reperfusion Injury via the PERK Signaling Pathway.

Cerebral ischemic stroke is one of the leading causes of death worldwide. Previous studies have shown that circulating levels of CTRP1 are upregulated in patients with acute ischemic stroke. However, the function of CTRP1 in neurons remains unclear. The purpose of this study was to explore the role of CTRP1 in cerebral ischemia reperfusion injury (CIRI) and to elucidate the underlying mechanism. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen–glucose deprivation/reoxygenation (OGD/R) models were used to simulate cerebral ischemic stroke in vivo and in vitro, respectively. CTRP1 overexpression lentivirus and CTRP1 siRNA were used to observe the effect of CTRP1 expression, and the PERK selective activator CCT020312 was used to activate the PERK signaling pathway. We found the decreased expression of CTRP1 in the cortex of MCAO/R-treated rats and OGD/R-treated primary cortical neurons. CTRP1 overexpression attenuated CIRI, accompanied by the reduction of apoptosis and suppression of the PERK signaling pathway. Interference with CTRP1 expression in vitro aggravated apoptotic activity and increased the expression of proteins involved in the PERK signaling pathway. Moreover, activating the PERK signaling pathway abolished the protective effects of CTRP1 on neuron injury induced by CIRI in vivo and in vitro. In conclusion, CTRP1 protects against CIRI by reducing apoptosis and endoplasmic reticulum stress (ERS) through inhibiting the PERK-dependent signaling pathway, suggesting that CTRP1 plays a crucial role in the pathogenesis of CIRI.

Acupuncture ameliorates neurological function in rats with cerebral ischemia-reperfusion by regulating the opening of large-conductance Ca2+ -activated potassium channels.

Acupuncture has a good effect on improving neurological function after cerebral ischemia‐reperfusion, but there are few studies on the neuroprotective effect of acupuncture from the perspective of ion channel cellular electrophysiology. Studies have shown that the over activation of large‐conductance Ca2+‐activated potassium channel （BKCa） after cerebral ischemia‐reperfusion can reduce the excitability of neurons and induce apoptosis. This study intends to establish middle cerebral artery occlusion/reperfusion (MCAO/R) model, with acupuncture at GV26 as the intervention measure, using patch‐clamp technique to record the electrophysiological changes of BKCa channel. The results showed that the neurological function score of MCAO/R rats was significantly decreased, and the conductance, open dwell time and open probability of BKCa channel in hippocampal CA1 neurons of MCAO/R rats were significantly increased. Acupuncture at GV26 could significantly improve the neurological function scores of MCAO/R rats, and reduce the conductance, open dwell time, and open probability of BKCa channel. The effect of acupuncture at GV26 was significantly better than acupuncture at non‐acupuncture point. The neuroprotective effect of acupuncture at GV26 after cerebral ischemia‐reperfusion may be related to regulating the electrophysiological characteristics of BKCa channel opening.

LINC00665 knockdown protects against cerebral ischemia-reperfusion injury.

LINC00665 has been reported to participate in several human diseases. However, the role of LINC00665 in cerebral ischemia-reperfusion (CI/R) is still unknown. This study is designed to investigate the role of LINC00665 in rats with CI/R injury. We established middle cerebral artery occlusion/ reperfusion (MCAO/R) rats model in vivo. PC12 cells treated with oxygen-glucose deprivation/reperfusion (OGD/R) were used to establish in vitro I/R model. RT-qPCR assay was adopted to assess the mRNA expression of LINC00665 and miR-744-5p. MTT assay was used to determine cell viability. The protein expression of Bax and Bcl-2 were detected by Western blot assay. The relationship between LINC00665 and miR-744-5p was confirmed by dual luciferase reporter assay and RNA immunoprecipitation (RIP). In this study, we found that LINC00665 was sharply up regulated in MCAO/R rats and PC12 cells treated with I/R. Functionally, LINC00665 knockdown attenuated oxidative damage in PC12 cells treated with I/R. Moreover, LINC00665 knockdown promoted cell viability, while inhibited cell apoptosis in PC12 cells treated with I/R. In addition, miR-744-5p was confirmed to be a target of LINC00665. LINC00665 knockdown was validated to project CI/R injury by sponging miR-744-5p expression.

LncRNA-Malat1 down-regulates miR-211-5p expression to promote neuronal damage from cerebral ischemia reperfusion injury

Based on the previous studies, this study was carried out to explore the interaction of LncRNA-Malat1/miR-211-5p in cerebral ischemia reperfusion injury (CIRI). Firstly, the expression changes of LncRNA-MALAT1 and miR-211-5p in ischemia patients’ blood were determined, and the binding sites of them were predicted by bioinformatics. Furthermore, middle cerebral artery occlusion/reperfusion (MCAO/R) injury model was established in adult male SD rats, and primary neuronal oxygen-glucose deprivation/reoxygen-glucose reoxygenation (OGD/R) was established in vitro. The results showed that LncRNA-MALAT1 was significantly up-regulated and miR-211-5p down-regulated in the peripheral blood of patients with ischemic stroke, and the expression changes were negatively correlated. Bioinformatics prediction results showed that LncRNA-MALAT1 had a binding site with miR-211-5p. We also found that LncRNA-Malat1 was significantly up-regulated while miR-211-5p down-regulated in rat cortex tissue and primary neurons treated with OGD/R. In addition, lentivirus interfered with LV-Malat1-RNAi decreased the expression of LncRNA-Malat1 and promoted the up-regulation of miR-211-5p. Combination of LV-Malat1-RNAi and miR-211-5p inhibitor significantly reversed the protective effect of down-regulation of LncRNA-Malat1. Inhibition of LncRNA-Malat1 expression alleviated the neurological deficit score after MCAO/R, improved histopathological damage, and reduced the size of cerebral infarction. Combined administration of LV-Malat1-RNAi + Antagomir-211-5p reversed these effects above. In short, our data suggest that LncRNA-Malat is involved in the occurrence and development of cerebral ischemia reperfusion injury by acting on miR-211-5p and is then regulating the expression of COX-2.

Angiogenesis effects of 4-methoxy benzyl alcohol on cerebral ischemia-reperfusion injury via regulation of VEGF-Ang/Tie2 balance.

Angiogenesis facilitates the formation of microvascular networks and promotes neurological deficit recovery after cerebral ischemia-reperfusion injury (CIRI). This study investigated the angiogenesis effects of 4-methoxy benzyl alcohol (4-MA) on CIRI. The angiogenesis effects of 4-MA and the potential underlying mechanisms were assessed based on a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model and a hind limb ischemic (HLI) mouse model. Immunofluorescence was conducted to detect microvessel density, and Western blotting and polymerase chain reaction were performed to determine the expression of angiogenesis-promoting factors. In addition, we investigated whether the angiogenesis effects of 4-MA caused damage to the blood-brain barrier (BBB). After treatment with 4-MA (20 mg/kg) for 7 days, the neurological deficits recovered and microvessel density in the cerebral cortex increased in the MCAO/R rats. Additionally, 4-MA also regulated the expression of angiogenesis factors, with an increase in vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression and a decrease in angiopoietin 1 (Ang-1), Ang-2, and Tie-2 expression in both MCAO/R rats and HLI mice. Moreover, 4-MA increased the expression of angiogenesis-promoting factors without exacerbating BBB cascade damage in MCAO/R rats. Our results indicated that 4-MA may contribute to the formation of microvascular networks, thus promoting neurological deficit recovery after CIRI.

10-O-(N N-Dimethylaminoethyl)-Ginkgolide B Methane-Sulfonate (XQ-1H) Ameliorates Cerebral Ischemia Via Suppressing Neuronal Apoptosis

Objectives: The 10-O-(N N-dimethylaminoethyl)-ginkgolide B methane-sulfonate (XQ-1H) is an effective novel drug for the treatment of ischemic cerebrovascular disease derived from Ginkgolide B, a traditional Chinese medicine, has been widely used in the treatment of cardiovascular and cerebrovascular diseases. However, whether XQ-1H exerts neuroprotective effect via regulating neuronal apoptosis and the underlying mechanism remain to be elucidated. Materials and methods: This study was aimed to investigate the neuroprotective effect of XQ-1H in rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and the oxygen glucose deprivation/reoxygenation (OGD/R) induced neuronal apoptosis on pheochromocytoma (PC-12) cells. Results: The results showed that administration of XQ-1H at different dosage (7.8, 15.6, 31.2 mg/kg) reduced the brain infarct and edema, attenuated the neuro-behavioral dysfunction, and improved cell morphology in brain tissue after MCAO/R in rats. Moreover, incubation with XQ-1H (1 µM, 3 µM, 10 µM, 50 µM, 100 µM) could increase the cell viability, and showed no toxic effect to PC-12 cells. XQ-1H at following 1 µM, 10 µM, 100 µM decreased the lactate dehydrogenase (LDH) activity and suppressed the cell apoptosis in PC-12 cells exposed to OGD/R. In addition, XQ-1H treatment could significantly inhibit caspase-3 activation both in vivo and in vitro, reciprocally modulate the expression of apoptosis related proteins, bcl-2, and bax via activating PI3K/Akt signaling pathway. For mechanism verification, LY294002, the inhibitor of PI3K/Akt pathway was introduced the expressions of bcl-2 and phosphorylated Akt were down-regulated, the expression of bax was up-regulated, indicating that XQ-1H could alleviate the cell apoptosis through activating the PI3K/Akt pathway. Conclusions: Our findings demonstrated that XQ-1H treatment could provide a neuroprotective effect against ischemic stroke induced by cerebral ischemia/reperfusion injury in vivo and in vitro through regulating neuronal survival and inhibiting apoptosis. The findings of the study confirmed that XQ-1H could be develop as a potential drug for treatment of cerebral ischemic stroke.

DDAH-1, via regulation of ADMA levels, protects against ischemia-induced blood-brain barrier leakage

Dimethylarginine dimethylamino hydrolase-1 (DDAH-1) is an important regulator of nitric oxide (NO) metabolism that has been implicated in the pathogenesis of cardiovascular diseases. Nevertheless, its role in cerebral ischemia still needs to be elucidated. Herein, we examined the expression of DDAH-1 in the brain of rat by double-label immunofluorescence staining. DDAH-1 knock-out (DDAH-1−/−) and wild-type rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). After 24 h, neurological scores, TTC staining and TUNEL assay were used to evaluate neurological damages. 3 and 7-days infarct outcomes were also shown. Blood-brain-barrier (BBB) permeability was examined via Evans blue extravasation and tight junction (TJ) proteins expression and mRNA levels by western blot and RT-qPCR. The levels of plasma asymmetric dimethylarginine (ADMA), NO and ADMA in brain tissue were also assessed. In addition, supplementation of L-arginine to DDAH-1−/− rats was used to explore its role in regulating NO. DDAH-1 was abundantly distributed in cerebral cortex and basal nuclei, and mainly expressed in neurons and endothelial cells. DDAH-1−/− rats showed aggravated neurological damage and BBB disruption, including decrease of TJ proteins expression but indistinguishable mRNA levels after MCAO/R. DDAH-1 depletion and neurological damages were accompanied with increased ADMA levels and decreased NO concentrations. The supplementation with L-arginine partly restored the neurological damages and BBB disruption. To sum up, DDAH-1 revealed to have a protective role in ischemia stroke (IS) and IS-induced leakage of BBB via decreasing ADMA level and possibly via preventing TJ proteins degradation.Dimethylarginine dimethylamino hydrolase-1 (DDAH-1), as the critical enzyme responsible for asymmetric dimethylarginine (ADMA) degradation, serves as a protective factor for ischemic stroke. DDAH-1 protects ischemia-induced disruption of blood-brain-barrier via regulating ADMA level and preventing tight junction proteins degradation. The supplementation of L-arginine helps restore the function of DDAH-1.

The Cerebroprotein Hydrolysate-I Plays a Neuroprotective Effect on Cerebral Ischemic Stroke by Inhibiting MEK/ERK1/2 Signaling Pathway in Rats.

ObjectiveTo investigate the neuroprotective effect and mechanism of cerebroprotein hydrolysate-I (CH-I) on cerebral ischemia/reperfusion injury in rats.MethodsA total of 100 adult healthy male SD rats were randomly divided into a sham group, model group, CH-I treated group, and cerebrolysin (CBL) positive group, consisting of 20 rats in each group. The middle cerebral artery occlusion/reperfusion (MCAO/R) model of rats was built by inserting a suture into the left external carotid artery (ECA) through the internal carotid artery (ICA). Treatment was performed by intraperitoneal injection of CH-I (20 mg/kg). The neurobehavioral function of rats was evaluated by modified neurological severity scores (mNSS). TTC staining was used to detect the cerebral infarction volume (CIV) of rats. The morphological and structural changes of nerve cells were observed by HE staining and the neuronal apoptosis was counted by TUNEL assay. Immunohistochemical (IHC) analysis was used to detect BDNF and pMEK1/2 expressions. The expressions of BDNF, pMEK1/2, pERK1/2, and pCREB were determined with Western blotting.ResultsAfter treatment with CH-I, the mNSS and CIV of rats were improved (P<0.05). And the CH-I can reduce the degeneration and apoptosis of nerve cells in rats (P<0.01). Western blotting showed that the expressions of pMEK1/2, pERK1/2, and pCREB in rats were increased, while the expression of BDNF was decreased after modeling (P<0.05). After treatment, the expressions of pMEK1/2, pERK1/2, and pCREB in the CH-I group were decreased (P<0.05), while the expression of BDNF was significantly increased (P<0.05) compared with the model group. IHC showed that the expression of BDNF and pMEK1/2 was consistent with Western blotting.ConclusionIt is suggested that the CH-I might play a neuroprotective role by inhibiting the expression of MEK-ERK-CREB and enhancing the expression of BDNF after cerebral ischemia/reperfusion injury, thus improving the neurobehavioral function of MCAO/R rats.

Resveratrol-mediated neurorestoration after cerebral ischemic injury - Sonic Hedgehog signaling pathway

AimsResveratrol pretreatment can decrease ischemic cerebral injury and enhance proliferation of neural stem cells via mediation of Sonic Hedgehog signaling. However, it is relatively little known about whether neurorestorative effects of resveratrol are mediated by Shh signaling in ischemic cerebral injury. The present study tests whether the Shh signaling pathway mediates resveratrol to promote neurorestoration of ischemic cerebral injury. Materials and methodsRats or neurons before middle cerebral artery occlusion/reperfusion (MCAO/R) or oxygen-glucose deprivation/reoxygenation (OGD/R) injury were pretreated with resveratrol. Immunohistochemistry is used to be determined BrdU+/DCX+, BrdU+/Nestin+ and BrdU+/NG2+ cell (markers of new proliferated neural stem/progenitor and oligodendrocyte precursor cell, respectively), BrdU+/MAP2+ and BrdU+/CNPase+ cell (markers of new mature neuron and oligodendrocyte, respectively), BrdU+/TUNEL+ cell (marker of apoptosis for new proliferated cell), SY, NF200, Iba-1 and GFAP (markers of synaptogenesis, axon, microglia and astrocyte, respectively). Shh and Gli-1 mRNAs were detected by RT-PCR assay. Iba-1, GFAP, Shh and Gli-1 proteins were detected by Western blot. Key findingsResveratrol pretreatment significantly reduced neurological deficit scores, promoted proliferation, differentiation, migration and survival of neural stem/progenitor and oligodendrocyte precursor cells, inhibited astrocyte and microglia activation, strengthened synaptophysin and NF200 expression, at the same time, promoted neurite outgrowth of neurons. Meanwhile, expression levels of Shh and Gli-1 proteins were significantly increased and Gli-1 translocated into the nucleus. However, cyclopamine, a Smo inhibitor, canceled the above effects of resveratrol. ConclusionsIt may be mediated, at least partly, by the Shh signaling pathway that resveratrol pretreament promote neurorestoration of ischemic cerebral injury.

Long noncoding RNA ANRIL knockdown attenuates neuroinflammation following ischemic stroke via suppressing the expression of NF-κB in vitro and in vivo

ABSTRACT Objective Increasing evidence suggests that long-noncoding RNAs can exert neuroprotective effects in cerebral ischemia-reperfusion injury. Levels of the long noncoding RNA ANRIL (ANRIL) are reportedly altered in ischemic stroke (IS) patients, but its role in IS requires further clarification. This study was designed to explore the mechanistic function of ANRIL in IS. Methods In vitro, HT22 cells was treated with an oxygen-glucose deprivation/reperfusion (OGD/R). In vivo, brain ischemia/reperfusion was induced by 60-minute transient middle cerebral artery occlusion/reperfusion (MCAO/R) IS model in C57/BL6 mice. Additionally, cells were transfected with si-ANRIL, pcDNA3.1-ANRIL, pcDNA3.1-NF-κB, or appropriate negative controls, and si-ANRIL and pcDNA3.1-NF-κB were administered into the lateral ventricles in MCAO/R model mice. Cell viability and apoptosis were detected via MTT and flow cytometry assays. mRNA and protein expression of NF-κB were detected via qRT-PCR and Western blotting. IL-1β, IL-6, TNF-a, and iNOS levels were detected via ELISA. In addition, infarcted area and neuronal injury were evaluated via TTC, Nissl, and immunofluorescent staining. Results We found that ANRIL knockdown increased cell viability and reduced apoptosis in vitro. Additionally, we found that ANRIL knockdown decreased p-P65, P65, IL-1β, IL-6, TNF-a, and iNOS levels, whereas these effects were reversed by NF-κB overexpression both in vitro and in vivo. Conclusion our results suggest that ANRIL knockdown attenuates neuroinflammation by suppressing the expression of NF-κB both in vitro and vivo model of IS, sugguesting that ANRIL might be a potentially viable therapeutictarget to diminish neuroinflammation in IS patients.

Knockdown of XIST Attenuates Cerebral Ischemia/Reperfusion Injury Through Regulation of miR-362/ROCK2 Axis.

Long non-coding RNAs (lncRNAs) are considered as critical regulators in the pathogenesis of cerebral ischemia. In this present study, we aimed to investigate the impact and underlying mechanism of lncRNA X-inactive specific transcript (XIST) in cerebral ischemia/reperfusion (I/R) injury. An oxygen-glucose deprivation/reperfusion (OGD/R) model in PC12 cells was applied to mimic cerebral I/R injury in vitro and middle cerebral artery occlusion/reperfusion (MCAO/R) model was performed in mice to mimic cerebral I/R injury in vivo. Real-time PCR, fluorescence in situ hybridization (FISH) assay, and western blotting assay were carried out to detect the expression levels of XIST, miR-362, and Rho-related coiled-coil containing protein kinase 2 (ROCK2). The functional experiments were measured by CCK-8 assay, immumofluorescence assay, ELISA assay, TUNEL, and TTC staining. Results displayed that XIST was elevated in PC12 cells with OGD/R, as well as in the ischemic penumbra of mice with MCAO/R. In vitro, knockdown of XIST facilitated cell survival, inhibited apoptosis, and alleviated inflammation injury in OGDR PC12 cells. In vivo, inhibition of XIST remarkably reduced the neurological impairments, promoted neuron proliferation, and suppressed apoptosis in MCAO mice. Mechanistically, XIST acted as a competing endogenous RNA of miR-362 to regulate the downstream gene ROCK2. In conclusion, depletion of XIST attenuated I/R-induced neurological impairment and inflammatory response via the miR-362/ROCK2 axis. These findings offer a potential novel strategy for ischemic stroke therapy.

Notoginsenoside R1 activates the NAMPT-NAD+-SIRT1 cascade to promote postischemic angiogenesis by modulating Notch signaling

Nicotinamide phosphoribosyltransferase (NAMPT) maintains mitochondrial function and protects against cerebral ischemic injury by improving energy metabolism. Notoginsenoside R1 (R1), a unique constituent of Panax notoginseng, has been shown to promote the proliferation and tube formation of human umbilical vein endothelial cells. Whether R1 has proangiogenesis on the activation of NAMPT in ischemic stroke remains unclear. The purpose of this study was to investigate the pharmacodynamic effect and mechanism of R1 on angiogenesis after ischemic stroke. We used male Sprague-Dawley (SD) rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered via intraperitoneal (i.p.) injection immediately after ischemia induction. The promotion of R1 on angiogenesis were detected by immunofluorescence staining, 3D stereoscopic imaging and transmission electron microscopy detection. HBMEC cells were pretreated with different concentrations of R1 for 12 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, scratch assay, EdU staining and tube formation were determined. Western blot analyses of proteins, including those involved in angiogenesis, NAMPT-SIRT1 cascade, VEGFR-2, and Notch signaling, were conducted. We showed that R1 significantly restored cerebral blood flow, improved mitochondrial energy metabolism and promoted angiogenesis. More importantly, incubation with 12.5–50 μM R1 significantly increased the migration, proliferation and tube formation of HBMECs in vitro. The promotion of R1 on angiogenesis were associated with the NAMPT-NAD+-SIRT1 cascade and Notch/VEGFR-2 signaling pathway, which was partially eliminated by inhibitors of NAMPT and SIRT1. We demonstrated that R1 promotes post-stroke angiogenesis via activating NAMPT-NAD+-SIRT1 cascade. The modulation of Notch signaling and VEGFR-2 contribute to the post-stroke angiogenesis. These findings offer insight for exploring new therapeutic strategies for neurorestoration via R1 treatment after ischemic stroke.

Long noncoding RNA SOX2OT silencing alleviates cerebral ischemia-reperfusion injury via miR-135a-5p-mediated NR3C2 inhibition

ObjectiveThis study is aimed to investigate the role of the long noncoding RNA SOX2 overlapping transcript (SOX2OT) in cerebral ischemia-reperfusion injury (CIRI) and the underlying regulatory mechanisms. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R)-treated PC12 cells and middle cerebral artery occlusion/reperfusion (MCAO/R)-treated rats were established to simulate CIRI condition in vitro and in vivo. Quantitative real-time polymerase chain reaction was performed to detect the expression of SOX2OT, microRNA-135a-5p (miR-135a-5p), and nuclear receptor subfamily 3 group C member 2 (NR3C2). The cell viability and apoptosis were analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays. The levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) or interleukin (IL)-1β and IL-6 were used to evaluate the oxidative stress or inflammation. Dual-luciferase reporter assay was conducted to validate the interactions among SOX2OT, miR-135a-5p, and NR3C2. Additionally, neurological deficit scores (NDS), infarct volume, and brain edema were used to assess brain impairments in vivo. ResultsThe expression of SOX2OT and NR3C2 was increased, while miR-135a-5p was decreased in OGD/R-treated PC12 cells. SOX2OT silencing repressed the levels of LDH, MDA, ROS, IL-1β, IL-6, reduced the numbers of TUNEL positive cells, and elevated viability and SOD level in OGD/R-treated PC12 cells. Besides, SOX2OT targeted miR-135a-5p, and miR-135a-5p targeted NR3C2. Both miR-135a-5p downregulation and NR3C2 upregulation reversed the suppressive effects of SOX2OT knockdown on oxidative stress, apoptosis, and inflammation of OGD/R-treated PC12 cells. Furthermore, injection of sh-SOX2OT reduced the NDS, cerebral infarct, and cerebral edema in MCAO/R-treated rats. ConclusionsSilencing of SOX2OT attenuated CIRI via regulation of the miR-135a-5p/NR3C2 axis, which may provide a novel therapeutic target for CIRI.