HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke.

Human antigen R (HuR) is a universally expressed RNA-binding protein that plays an essential role in governing the fate of mRNA transcripts. Accumulating evidence indicated that HuR is involved in the development and functions of several cell types. However, its role in cerebral ischemia/reperfusion injury (CIRI) remains unclear. In this study, we found that HuR was significantly upregulated after CIRI. Moreover, we found that silencing HuR could inhibit the inflammatory response of microglia and reduce the damage to neurons caused by oxygen-glucose deprivation/reperfusion treatment. In vivo, we found that microglial HuR deficiency significantly ameliorated CIRI and reduced NLRP3-mediated inflammasome activation. Mechanistically, we found that HuR could regulate NLRP3 mRNA stability by binding to the AU-rich element (ARE) region within the 3' untranslated region (UTR) of NLRP3 mRNA. In addition, we found that the upregulation of HuR was dependent on the upregulation of NADPH oxidase-mediated ROS accumulation. Collectively, our studies revealed that HuR could regulate NLRP3 expression and that HuR deficiency abrogated the enhanced NLRP3 signaling in experimental ischemic stroke. Targeting HuR may be a novel therapeutic strategy for cerebral ischemic stroke treatment.

Astrocyte-TREM2 alleviates brain injury by regulating reactive astrocyte states following ischemic stroke.

Triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to confer strong neuroprotective effects in acute ischemic stroke (AIS). However, as the vast majority of research findings to date are based on its functions in microglia, the precise role of TREM2 in astrocytes after AIS is unknown. Here, both loss- and gain-of-function experiments were employed to investigate how astrocytic TREM2 influences the pathogenesis of AIS in vivo and in vitro. Our results demonstrated that cerebral ischemia triggered induction of TREM2 expression on reactive astrocytes following AIS. In addition, astrocyte-specific TREM2 knockout mice exhibited much greater brain injury than TREM2 flox/flox controls following AIS, as evidenced by increased cerebral infarct volume, neuronal apoptosis and neurological deficit, which was associated with an increased expression of pro-inflammatory molecule complement component 3 (C3) on reactive astrocytes and activation of microglia/macrophages but decreased expression of S100 calcium binding protein A10 (S100A10) and arginase1 (Arg1) on reactive astrocytes. Mechanistic analyses revealed that astrocytic TREM2 alleviated brain injury by inhibiting detrimental actions of reactive astrocytes but promoting their neuro- and glioprotective actions via the kruppel-like transcription factor-4-nuclear factor-κB axis. Together, this study provides novel evidence for a critical protective role of astrocyte-derived TREM2 in AIS and highlights a potential therapeutic target for the treatment of AIS.

Transplantation of Neural Stem Cells-Overexpressed Ku70 Improves Neurological Deficits in a Mice Model of Cerebral Ischemia Stroke.

Cerebral ischemic stroke is a cerebrovascular disease, which is related to DNA damage. Many researches have shown that Ku70 is a key regulator for DNA damage. Here, we aimed to explore Ku70 roles in cerebral ischemic stroke and its potential molecular mechanism. In our study, neural stem cells (NSCs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) for constructing cerebral ischemic stroke cell model. CCK8 assay, Brdu/GFP staining, flow cytometry and TUNEL staining were performed to examine cell proliferation, cell cycle and apoptosis, respectively. Relative mRNA and protein levels were detected by quantitative real-time PCR and western blot analysis, respectively. Ku70 positive cells were examined by immunofluorescence staining. Comet assay was employed to determine DNA damage. Animal experiments were performed to assess the effect of transplanting NSCs and Ku70-overexpressed NSCs on neurological deficits, infarct volume, brain edema and blood‒brain barrier (BBB) integrity in middle cerebral artery occlusion (MCAO) model. Our data found that Ku70 expression was decreased in NSCs after OGD/R. Overexpression of Ku70 reduced DNA damage and apoptosis of OGD/R-induced NSCs. Knockdown of Ku70 promoted the activity of ATM/p53. Moreover, KU60019 (ATM-specific inhibitor) reversed the promoting effects of Ku70 silencing on DNA damage and apoptosis in OGD/R-induced NSCs. In animal experiments, transplantation of NSCs-overexpressed Ku70 enhanced cell survival, improved motor function, reduced infarct volume, relieved brain edema and alleviated BBB dysfunction in MCAO mice models. In conclusion, Ku70 overexpression repressed the DNA damage and apoptosis in OGD/R-induced NSCs by regulating ATM/p53 pathway, and transplantation of NSCs-overexpressed Ku70 played neuroprotective effects in MCAO mice models.

Protective effects of low-intensity pulsed ultrasound (LIPUS) against cerebral ischemic stroke in mice by promoting brain vascular remodeling via the inhibition of ROCK1/p-MLC2 signaling pathway.

Vascular remodeling is essential for patients with cerebral ischemic stroke (CIS). Our previous study proved that low-intensity pulsed ultrasound (LIPUS) could increase cortical hemodynamics. However, the effects and mechanisms of LIPUS on cerebral vascular remodeling after CIS are still unknown. In this study, we applied LIPUS to the mouse brain at 0.5 h after distal middle cerebral artery occlusion (dMCAO) and subsequently daily for a stimulation time of 30 min. Results showed that compared with the dMCAO group, LIPUS markedly increased cerebral blood flow (CBF), reduced brain swelling, and improved functional recovery at day 3 after CIS. LIPUS promoted leptomeningeal vasculature remodeling, enlarged vascular diameter, and increased the average vessel length and density at day 3 after CIS. Proteomic analysis highlighted that LIPUS mainly participated in the regulation of actin cytoskeleton pathway. Rho kinase 1 (ROCK1) was downregulated by LIPUS and participated in regulation of actin cytoskeleton. Subsequently, we verified that ROCK1 was mainly expressed in pericytes. Furthermore, we demonstrated that LIPUS inhibited ROCK1/p-MLC2 signaling pathway after CIS, which had positive effects on vascular remodeling and cerebral blood circulation. In conclusion, our preliminary study revealed the vascular remodeling effects and mechanism of LIPUS in CIS, provided evidence for potential clinical application of LIPUS.

Expression, Functional Polymorphism, and Diagnostic Values of MIAT rs2331291 and H19 rs217727 Long Non-Coding RNAs in Cerebral Ischemic Stroke Egyptian Patients.

Cerebral ischemic stroke (CIS) is a severe cerebral vascular event. This research aimed to evaluate the role of single-nucleotide polymorphisms (SNPs) of the lncRNAs MIAT rs2331291 and H19 rs217727 and epigenetic methylation in the expression patterns of serum lncRNA H19 in CIS Egyptian patients. It included 80 CIS cases and 40 healthy subjects. Serum MIAT expression levels decreased, whereas serum H19 expression levels increased among CIS compared to controls. For MIAT rs2331291, there were significant differences in the genotypic and allelic frequencies between the CIS and healthy subjects at p = 0.02 and p = 0.0001, respectively. Our findings illustrated a significantly increased MIAT T/T genotype frequency in hypertensive CIS compared to non-hypertensive CIS at p = 0.004. However, H19 rs217727 gene frequency C/C was not significantly higher in non-hypertensive CIS than in hypertensive CIS. The methylation of the H19 gene promoter was significantly higher in CIS patients compared to healthy subjects. The level of MIAT was positively correlated with serum H19 in CIS. Receiver operating characteristics (ROC) analysis revealed that serum MIAT and H19 have a high diagnostic potential for distinguishing CIS subjects from healthy ones. In conclusion, the MIAT-rs2331291 polymorphism might serve as a novel potential indicator of CIS.

Transcranial direct-current stimulation confers neuroprotection by regulating isoleucine-dependent signalling after rat cerebral ischemia-reperfusion injury.

Isoleucine is a branched chain amino acid. The role of isoleucine in cerebral ischemia-reperfusion injury remains unclear. Here, we show that the concentration of isoleucine is decreased in cerebrospinal fluid in a rat model of cerebral ischemia-reperfusion injury, the rat middle cerebral artery occlusion (MCAO). To our surprise, the level of intraneuronal isoleucine is increased in an in vitro model of cerebral ischemia injury, the oxygen-glucose deprivation (OGD). We found that the increased activity of LAT1, an L-type amino acid transporter 1, leads to the elevation of intraneuronal isoleucine after OGD insult. Reducing the level of intraneuronal isoleucine promotes cell survival after cerebral ischemia-reperfusion injury, but supplementing isoleucine aggravates the neuronal damage. To understand how isoleucine promotes ischemia-induced neuronal death, we reveal that isoleucine acts upstream to reduce the expression of CBFB (core binding factor ß, a transcript factor involved in cell development and growth) and that the phosphatase PTEN acts downstream of CBFB to mediate isoleucine-induced neuronal damage after OGD insult. Interestingly, we demonstrate that direct-current stimulation reduces the level of intraneuronal isoleucine in cortical cultures subjected to OGD and that transcranial direct-current stimulation (tDCS) decreases the cerebral infarct volume of MCAO rat through reducing LAT1-depencent increase of intraneuronal isoleucine. Together, these results lead us to conclude that LAT1 over activation-dependent isoleucine-CBFB-PTEN signal transduction pathway may mediate ischemic neuronal injury and that tDCS exerts its neuroprotective effect by suppressing LAT1 over activation-dependent signalling after cerebral ischemia-reperfusion injury.

The critical role of KLF4 in regulating the activation of A1/A2 reactive astrocytes following ischemic stroke.

BACKGROUND: We have previously demonstrated that the expression of kruppel-like transcription factor-4 (KLF-4) is upregulated in astrocytes following acute ischemic stroke (AIS) and found that KLF4 confers vascular protection against cerebral ischemic injury. However, the functional role of KLF4 in astrocyte after AIS is far from clear. METHODS: The intrinsic relationship between KLF4 and A1/A2 reactive astrocytes and the impact of astrocytic KLF4 on the activation of A1/A2 subtype astrocytes were evaluated in middle cerebral artery occlusion (MCAO) mice and oxygen-glucose deprivation and restoration (OGD/R) astrocytes. RESULTS: Our results demonstrated that astrocytic KLF4 expression and complement C3-positive A1 and S100 calcium binding protein A10 (S100A10)-positive A2 astrocytes were induced in the ischemic penumbra following focal cerebral ischemia, and the time course of upregulation of astrocytic KLF4 correlated closely with the activation of A2 astrocytes. The dual immunofluorescent studies displayed that in the ischemic hemisphere, where the high levels of KLF4 were expressed, there were relatively low levels of C3 expressed in the reactive astrocytes and vice versa, but KLF4 was always co-stained well with S100A10. Mechanistic analyses revealed that astrocytic KLF4 inhibited the activation of A1 astrocyte but promoted A2 astrocyte polarization after OGD/R by modulating expressions of nuclear factor-kB. CONCLUSIONS: Astrocyte-derived KLF4 has a critical role in regulating the activation of A1/A2 reactive astrocytes following AIS.

The Mechanism Underlying the Regulation of Long Non-coding RNA MEG3 in Cerebral Ischemic Stroke.

Cerebral ischemic stroke is one of the leading causes of morbidity and mortality worldwide, and rapidly increasing annually with no more effective therapeutic measures. Thus, the novel diagnostic and prognostic biomarkers are urgent to be identified for prevention and therapy of ischemic stroke. Recently, long noncoding RNAs (lncRNAs), a major family of noncoding RNAs with more than 200 nucleotides, have been considered as new targets for modulating pathological process of ischemic stroke. In this review, we summarized that the lncRNA-maternally expressed gene 3 (MEG3) played a critical role in promotion of neuronal cell death and inhibition of angiogenesis in response to hypoxia or ischemia condition, and further described the challenge of overcrossing blood-brain barrier (BBB) and determination of optimal carrier for delivering lncRNA' drugs into the specific brain regions. In brief, MEG3 will be a potential diagnostic biomarker and drug target in treatment and therapy of ischemic stroke in the future.

Sodium Tanshinone IIA Sulfonate Ameliorates Oxygen-glucose Deprivation/Reoxygenation-induced Neuronal Injury via Protection of Mitochondria and Promotion of Autophagy.

Sodium tanshinone IIA sulfonate (STS) has shown significant clinical therapeutic effects in cerebral ischemic stroke (CIS), but the molecular mechanisms of neuroprotection remain partially known. The purpose of this study was to explore whether STS plays a protective role in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by regulating microglia autophagy and inflammatory activity. Co-cultured microglia and neurons were subjected to OGD/R injury, an in vitro model of ischemia/reperfusion (I/R) injury with or without STS treatment. Expression of protein phosphatase 2 A (PP2A) and autophagy-associated proteins Beclin 1, autophagy related 5 (ATG5), and p62 in microglia was determined by Western blotting. Autophagic flux in microglia was observed with confocal laser scanning microscopy. Neuronal apoptosis was measured by flow cytometric and TUNEL assays. Neuronal mitochondrial function was determined via assessments of reactive oxygen species generation and mitochondrial membrane potential integrity. STS treatment markedly induced PP2A expression in microglia. Forced overexpression of PP2A increased levels of Beclin 1 and ATG5, decreased the p62 protein level, and induced autophagic flux. Silencing of PP2A or administration of 3-methyladenine inhibited autophagy and decreased the production of anti-inflammatory factors (IL-10, TGF-ß and BDNF) and induced the release of proinflammatory cytokines (IL-1ß, IL-2 and TNF-α) by STS-treated microglia, thereby inducing mitochondrial dysfunction and apoptosis of STS-treated neurons. STS exerts protection against neuron injury, and the PP2A gene plays a crucial role in improving mitochondrial function and inhibiting neuronal apoptosis by regulating autophagy and inflammation in microglia.

Cross-talk between bioactive lipid mediators and the unfolded protein response in ischemic stroke.

Ischemic cerebral stroke is a severe medical condition that affects about 15 million people every year and is the second leading cause of death and disability globally. Ischemic stroke results in neuronal cell death and neurological impairment. Current therapies may not adequately address the deleterious metabolic changes and may increase neurological damage. Oxygen and nutrient depletion along with the tissue damage result in endoplasmic reticulum (ER) stress, including the Unfolded Protein Response (UPR), and neuroinflammation in the affected area and cause cell death in the lesion core. The spatio-temporal production of lipid mediators, either pro-inflammatory or pro-resolving, decides the course and outcome of stroke. The modulation of the UPR as well as the resolution of inflammation promotes post-stroke cellular viability and neuroprotection. However, studies about the interplay between the UPR and bioactive lipid mediators remain elusive and this review gives insights about the crosstalk between lipid mediators and the UPR in ischemic stroke. Overall, the treatment of ischemic stroke is often inadequate due to lack of effective drugs, thus, this review will provide novel therapeutical strategies that could promote the functional recovery from ischemic stroke.

Up-regulation of lncRNA NEAT1 in cerebral ischemic stroke promotes activation of astrocytes by modulation of miR-488-3p/RAC1.

We aim to research the molecular mechanism of lncRNA NEAT1 in the activation of astrocytes in a cerebral ischemia-reperfusion injury model. Mouse model of cerebral ischemia-reperfusion injury was constructed, and shNEAT1 was transfected. The infarct area, brain water content, and neurological deficiency were detected. Immunofluorescence detection and fluorescence in situ hybridization (FISH) assay were processed to detect glial fibrillary acidic protein (GFAP) expression. Astrocyte cells were cultured for oxygen-glucose deprivation/re-oxygenation (OGD)/re-oxygenation model construction. After treatment by shNEAT1, miR-488-3p mimic, miR-488-3p inhibitor, Q-PCR assay, western blot and ELISA were undertaken to detect the expressions of NEAT1, miR-488-3p, RAC1, inflammatory cytokines, RAC1 and GFAP. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were used to verify the binding of NEAT1, miR-488-3p and RAC1. The expression of NEAT1 in brain tissue was significantly higher than that in Sham operation group. Knockdown of NEAT1 inhibited the brain damage caused by middle cerebral artery occlusion (MCAO) treatment, reduced the inflammatory response, and suppressed the activation of astrocytes. By constructing an in vitro OGD/R cell model, it was found that NEAT1 knockdown also inhibited the activation of astrocytes caused by OGD/R. Knockdown of NEAT1 caused the up-regulation of miR-488-3p and the down-regulation of RAC1. Knockdown of miR-488-3p or over-expression of RAC1 reversed the inhibitory effect of shNEAT1 on OGD/R-induced astrocyte activation. Over-expression of NEAT1 in cerebral ischemic stroke promotes activation of astrocytes by modulation miR-488-3p/RAC1, which is proved in vitro. Our study may provide a new idea for the diagnosis and treatment of MCAO.

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.

MiRNA-190 exerts neuroprotective effects against ischemic stroke through Rho/Rho-kinase pathway.

Ischemic stroke is an urgent public health concern and one of the major causes of deaths and disabilities over the world. MicroRNA (miRNA) has become a key mediator of cerebral ischemia-reperfusion (I/R) injuries. However, whether miR-190 is involved in cerebral I/R-induced neuronal damage remains unknown. This study was to investigate the role of miR-190 in the brain I/R injury. We divided the rats into sham, I/R, control, and miR-190-mim (miR-190 mimics) groups. Quantitative real-time polymerase chain reaction (qRT-PCR), Nissl staining, flow cytometry, and western blot were conducted to examine the expression of miR-190 and cell apoptosis in different groups. The results showed that the expression of miR-190 was greatly decreased in rats suffering with I/R. Overexpression of miR-190 significantly reduced the increased neurological scores, brain water contents, infarct volumes, and neuronal apoptosis in rats suffering with I/R. In addition, we found that the expression of RhoA and Rho kinase was greatly elevated in rats suffering with I/R. Bioinformatics analysis indicated that Rho was a target of miR-190. Moreover, overexpression of miR-190 significantly downregulated the increased mRNA and protein expression of Rho/Rho kinase and cell apoptosis, while inhibition of miR-190 further upregulated the increased mRNA and protein expression of Rho/Rho kinase and cell apoptosis in rats suffering with I/R. Furthermore, knockdown of Rho significantly downregulated the increased mRNA and protein expression of Rho/Rho kinase and cell apoptosis, while these effects were inhibited by miR-190 inhibitors in rats suffering with I/R. These results indicate that miR-190 confers protection against brain I/R damage by modulating Rho/Rho-kinase signaling.

Expression of Slit and Robo during remodeling of corticospinal tract in cervical spinal cord in middle cerebral artery occlusion rats.

BACKGROUND: Slits and Robos were associated with the generation of axons of corticospinal tract during the corticospinal tract (CST) remodeling after the cerebral ischemic stroke (CIS). However, little is known about the mechanism of CST remodeling. In this study, we detected the expression of Slits and Robos in middle cerebral artery occlusion (MCAO) rats to investigate the roles of Slits and Robos in the CIS. METHODS: MCAO model was established using modified Zea Longa method. Beam walking test (BWT) was conducted to evaluate the motor function. The images of the track of cortical spinal cord beam on day 7, 14 and 21 were observed by anterograde CST tracing. Biopinylated dextan amine (BDA) was used to mark CST anterogradely. Expression of GAP-43 mRNA and GAP-43 protein in cervical spinal cord was detected by Real-Time PCR and Western blot analysis, respectively. The expression of Slit1, Slit2 and Robo1 in cervical spinal cord was detected by immunofluorescence staining. RESULTS: The scores in the model group were significantly reduced compared to sham-operation group on day 7 (P < 0.001), 14 (P < 0.001) and 21 (P < 0.001), respectively. There was no significant difference in the score on day 7, 14 and 21 of the sham-operation groups (P > 0.05). In contrast, significant increase was noticed in the scores in model group, presenting a time-dependent manner. More CST staining fibers could be observed at the degenerative side in the model group compared with that of the sham-operation group on day 21. GAP-43 mRNA expression in the model group showed significant increase compared to that of sham-operation group on day 14 (P = 0.015) and 21 days (P = 0.002). The expression of GAP-43 protein in model group showed significant increase compared to that of sham-operation group on day 14 (P = 0.022) and day 21 (P = 0.008), respectively. The expression of Slit1 and Slit2 showed increase on day 14 and day 21, while the expression of Robo1 showed significant decrease in MCAO rats. CONCLUSION: Up-regulation of Slit1 and Slit2 and the downregulation of Robo1 may be related to the axons of CST midline crossing in spinal cord of MCAO rat during the spontaneous recovery of impaired motor function.

A stepwise-targeting strategy for the treatment of cerebral ischemic stroke.

BACKGROUND: Effective amelioration of neuronal damages in the case of cerebral ischemic stroke (CIS) is essential for the protection of brain tissues and their functional recovery. However, most drugs can not penetrate the blood-brain barrier (BBB), resulting in the poor therapeutic outcomes. RESULTS: In this study, the derivatization and dual targeted delivery technologies were used to actively transport antioxidant melatonin (MLT) into the mitochondria of oxidative stress-damaged cells in brain tissues. A mitochondrial targeting molecule triphenylphosphine (TPP) was conjugated to melatonin (TPP-MLT) to increase the distribution of melatonin in intracellular mitochondria with the push of mitochondrial transmembrane potential. Then, TPP-MLT was encapsulated in dual targeted micelles mediated by TGN peptide (TGNYKALHPHNG) with high affinity for BBB and SHp peptide (CLEVSRKNG) for the glutamate receptor of oxidative stress-damaged neural cells.TGN/SHp/TPP-MLT micelles could effectively scavenge the overproduced ROS to protect neuronal cells from oxidative stress injury during CIS occurrence, as reflected by the improved infarct volume and neurological deficit in CIS model animals. CONCLUSIONS: These promising results showed this stepwise-targeting drug-loaded micelles potentially represent a significant advancement in the precise treatment of CIS.

The potential predictive value of salivary cortisol on the occurrence of secondary cognitive impairment after ischemic stroke.

In this study, we aimed to investigate the relationship between salivary cortisol content and secondary mild cognitive impairment (MCI), thereby supporting the prediction of MCI in clinical practice. In this study, the salivary cortisol levels were examined in 120 patients with MCI after cerebral ischemic stroke (CIS) (CIS-MIC) and 80 CIS patients without MIC (CIS). The clinical data were compared among these patients with different cortisol levels. The salivary level of cortisol was significantly higher in patients with CIS-MIC (0.85-3.65 nmol/L) than that in those with CIS (0.52-1.21 nmol/L). The categorized analysis by CIS-MIC quartile showed that patient age, hyperlipidemia, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), etc. were significantly increased with increasing salivary cortisol levels. Moreover, univariate and multivariate logistic regression analyses revealed that the MCI risk of patients in the first quartile was 0.35 and 0.41 times, respectively, of the fourth quartile. Multiple linear regression showed that patient age, the time of rescue, and the salivary cortisol level were independent factors in the Mini-Mental State Exam (MMSE) score of MCI patients. Meanwhile, the receiver operating characteristic (ROC) curve showed that the area under the curve of salivary cortisol as a diagnostic marker for MCI after CIS was 0.982, with sensitivity of 0.973 and specificity of 0.980. In this study, we found that salivary cortisol level was an independent risk factor of MCI after CIS. A higher salivary cortisol level indicated a higher probability of MCI occurrence, and salivary cortisol level can be used as a predictive marker for MCI occurrence.

[The Clinical Efficacy and Possible Mechanism of Combination Treatment of Cerebral Ischemic Stroke with Ginkgo Biloba Extract and Low-Frequency Repetitive Transcranial Magnetic Stimulation].

OBJECTIVE: To study the effect of the combination treatment of ginkgo biloba extract and low-frequency repetitive transcranial magnetic stimulation (LF-rTMS) on the oxidative stress and brain neurotransmitters of patients who had cerebral ischemic stroke (CIS). METHODS: A retrospective analysis was conducted, and 93 CIS patients admitted to the Sichuan Academy of Medical Sciences/Sichuan Provincial People's Hospital from January 2018 to January 2020 were included in the study. They were divided into three groups, the regular treatment group (31 cases), the LF-rTMS group (31 cases), and the combination treatment group (31 cases). Patients in the regular treatment group were given the conventional drug therapy and exercise regimen. The LF-rTMS group received LF-rTMS therapy (for 20-30 min each time, 1 time/d and 5 times/week) in addition to the treatment given to the regular treatment group. The combination treatment group was given ginkgo biloba extract (intravenous drips, once per day) in addition to the treatment given to the LF-rTMS group. The treatment was given continuously for 4 weeks and comparison was made at the end of the 4-week treatment regarding the clinical efficacy, oxidative stress response, cerebral oxygen metabolism, and brain neurotransmitter as shown by the three groups. RESULTS: The treatment efficacy in the combination treatment group (96.77%) was higher than those of the LF-rTMS group (80.65%) and the regular treatment group (54.84%). The LF-rTMS group showed higher treatment efficacy than that of the regular group. The serum superoxide dismutase (SOD) of the combination treatment group was higher than that of the LF-rTMS group and that of the routine group, while the malondialdehyde (MDA) and endothelin-1 (ET-1) of the combination treatment group were lower than those of the LF-rTMS group and the regular treatment group ( P<0.05). The serum SOD of the LF-rTMS group was higher than that of the regular treatment group, while the MDA and ET-1 of the group was lower than those of the regular treatment group ( P<0.05). The arterial oxygen content (CaO 2), arterio-venous oxygen content difference (Ca-vO 2) and cerebral extraction rate of oxygen (CERO 2) in the combination treatment group were lower than those of the LF-rTMS group and the regular treatment group ( P<0.05). The levels of these three indicators of the LF-rTMS group were lower than those of the regular treatment group ( P<0.05). EEG frequencies of gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and dopamine (DA) of the combination treatment group were higher than those of the LF-rTMS group and the regular treatment group, while the acetylcholine (Ach) EEG frequency of the combination treatment group was lower than that of the LF-rTMS group and regular treatment group ( P<0.05). The LF-rTMS group showed higher GABA, 5-HT and DA EEG frequencies than those of the regular treatment group, while the Ach EEG frequency of the group was lower than that of the regular treatment group ( P<0.05). All the patients were followed up for 6 months, and recurrence rate was lower in the combination treatment group (3.23%) than that of the LF-rTMS group (19.35%) and the regular treatment group (25.81%) ( P<0.05). CONCLUSION: The combination treatment of ginkgo biloba extract and LF-rTMS helped to improve the clinical outcome of CIS patients, which may be related to the inhibition of oxidative stress, improvement in cerebral oxygen metabolism, and regulation of brain neurotransmitter.

KLF4 alleviates cerebral vascular injury by ameliorating vascular endothelial inflammation and regulating tight junction protein expression following ischemic stroke.

BACKGROUND: Although inflammatory cell adhesion molecules (CAMs) and anti-inflammation factor Kruppel-like transcription factor (KLF) 4 have all been reported to be induced after cerebral ischemic stroke (CIS), the close temporal and spatial relationship between expressions of CAMs and KLF4 following CIS and whether and how CAMs and KLF-4 contribute to the development of CIS-induced vascular injury are still unclear. METHODS: Here, we first examined the correlation between serum levels of CAMs/KLF4 and infarct volume in acute CIS patients. Then, we determined the relationship between CAMs and KLF4 in mice after focal cerebral ischemia. Finally, we investigated the mechanism of KLF4 in protecting against oxygen-glucose deprivation-induced brain endothelial cell injury. RESULTS: Our results demonstrated that patients with moderate to severe CIS had higher serum levels of three CAMs including E-selectin, inter-cellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) but lower levels of KLF4 at 48 h after an acute event as compared to patients with minor CIS. The expression levels of three CAMs as well as KLF4 all correlated well with the infarct volume in all the CIS subjects at that time. Although the expressions of three CAMs and KLF4 were all induced in the ischemic hemisphere following focal cerebral ischemia, the peak timing and distribution patterns of their expression were different: the induction of KLF4 lagged behind that of the CAMs in the ischemic penumbra; furthermore, the dual immunofluorescent studies displayed that high expression of KLF4 was always associated with relatively less cerebral vascular endothelial inflammation response in the ischemic hemisphere and vice versa. Mechanistic analyses revealed that KLF4 alleviated CIS-induced cerebral vascular injury by regulating endothelial expressions of CAMs, nuclear factor-kB, and tight junction proteins. CONCLUSIONS: These data indicate that KLF4 confers vascular protection against cerebral ischemic injury, suggesting that circulating CAMs and KLF4 might be used as potential biomarkers for predicting the prognosis of acute ischemic stroke and also providing a new proof of concept and potential targets for future prevention and treatment of CIS.

CTRP3 Activates the AMPK/SIRT1-PGC-1α Pathway to Protect Mitochondrial Biogenesis and Functions in Cerebral Ischemic Stroke.

C1q/tumor necrosis factor-related protein-3 (CTRP3) had shown its angiogenesis and enhancement of mitochondrial biogenesis properties in the treatment of myocardial infarction, but its potential roles in cerebral ischemic stroke had not been fully understood. This study aimed to clarify the underlying mechanism of how CTRP3 regulated mitochondrial functions in hippocampal neuronal cells (HPPNCs) after oxygen-glucose deprivation (OGD)/reoxygenation (R) treatment. Results showed that impeded CTRP3 expression and weakened viability were detected in OGD/R treated HPPNCs. CTRP3 showed its ability to enhance the viability and inhibited apoptosis of HPPNCs after OGD/R treatment and it could also promote the mitochondrial biogenesis and physiological functions. Silencing of PGC-1α partially abolished the protective function of CTRP3 on mitochondria and CTRP3 mediated the expression of PGC-1α via the AMPK/SIRT1-PGC-1α pathway. These findings provided information that CTRP3 prevented mitochondria from OGD/R injury through activating the AMPK/SIRT1-PGC-1α pathway. Our study suggested that CTRP3 might have the potential to become an emerging protective agent applied in the reperfusion treatment of ischemic stroke.

MicroRNA-384-5p Promotes Endothelial Progenitor Cell Proliferation and Angiogenesis in Cerebral Ischemic Stroke through the Delta-Likeligand 4-Mediated Notch Signaling Pathway.

BACKGROUND: MicroRNAs (miRs) have a crucial regulatory role in endothelial cell function and tumor angiogenesis by inhibiting the expressions of their target genes. The participation of microRNA-384-5p (miR-384-5p) has been prominently reported in various ischemia-induced diseases such as myocardial ischemia and atherosclerosis. Hence, the present study aimed at exploring the effect of miR-384-5p on proliferation, apoptosis, and angiogenesis of endothelial progenitor cells (EPCs) in cerebral ischemic stroke and investigating the associated underlying mechanism. METHODS: A middle cerebral artery occlusion (MCAO) mouse model was established, with determination of the expression of cluster of differentiation 31 (CD31) and vascular endothelial growth factor (VEGF) proteins. Next, the MCAO mice and EPCs separated from MCAO mice were injected or transfected with mimics or inhibitors of miR-384-5p, or small interference RNA Delta-likeligand 4 (si-DLL4) in order to evaluate their effect on brain infarct size, cell proliferation, apoptosis, and angiogenesis. The relationship among miR-384-5p, DLL4, and the Notch signaling pathway was then verified by a series of experiments. RESULTS: In MCAO mice, an increased brain infarct size and cell apoptosis in brain tissues were evident, with decreased expression of miR-384-5p, VEGF, and CD31, as well as increased DLL4 expression. After miR-384-5p mimic or si-DLL4 treatment, the brain infarct size and cell apoptosis in the brain tissues were reduced in compliance with an increased expression of VEGF and CD31. Our findings demonstrated that miR-384-5p negatively regulated the expression of DLL4, which further downregulated the Notch signaling pathway. When miR-384-5p was overexpressed or DLL4 silenced, the cell proliferation and angiogenesis of EPCs were promoted and cell apoptosis was inhibited. CONCLUSIONS: Our study demonstrated that overexpressed miR-384-5p targeting DLL4 could stimulate proliferation and angiogenesis, while inhibiting apoptosis of EPCs in mice with cerebral ischemic stroke through the Notch signaling pathway.