Systematic Analysis of RNA Expression Profiles in Different Ischemic Cortices in MCAO Mice.

The prognosis of ischemic stroke patients is highly associated with the collateral circulation. And the competing endogenous RNAs (ceRNAs) generated from different compensatory supply regions may also involve in the regulation of ischemic tissues prognosis. In this study, we found the apoptosis progress of ischemic neurons in posterior circulation-supplied regions (close to PCA, cortex2) was much slower than that in anterior circulation-supplied territory (close to ACA, cortex1) in MCAO-3-h mice. Using the RNA sequencing and functional enrichment analysis, we analyzed the difference between RNA expression profile in cortex1 and cortex2 and the related biological processes. The results indicated that the differential expressed ceRNAs in cortex1 were involved in cell process under acute injury, while the differential expressed ceRNAs in cortex2 was more likely to participate in long-term injury and repair process. Besides, by establishing the miRNA-ceRNA interaction network we further sorted out two specifically distributed miRNAs, namely mmu-miR446i-3p (in cortex1) and mmu-miR3473d (in cortex2). And the specifically increased mmu-miR3473d in cortex2 mainly involved the angiogenesis and cell proliferation after ischemic stroke, which may be the critical reason for the longer therapeutic time window in cortex2. In conclusion, the present study reported the specific changes of ceRNAs in distinct compensatory regions potentially involved in the evolution of cerebral ischemic tissues and the unbalance prognosis after stroke. It provided more evidence for the collateral compensatory effects on patients' prognosis and carried out the new targets for the ischemic stroke therapy.

Beclin­1 exerts protective effects against cerebral ischemia­reperfusion injury by promoting DNA damage repair through a non­autophagy­dependent regulatory mechanism.

Cerebral ischemia­reperfusion (I/R) can result in severe brain injury, for which there are no optimal treatment options. I/R is often accompanied by increased autophagy. Beclin­1, a central player in autophagy, has been extensively studied in I/R; however, to date, at least to the best of our knowledge, there are no definitive descriptions of its specific role. Thus, the aim of the present study was to explore the regulatory role played by Beclin­1 in I/R. In vivo experiments were performed using an animal model of brain I/R with male Sprague­Dawley rats. Brain tissue damage was observed using 2,3,5­triphenyltetrazolium chloride, and hematoxylin and eosin staining. Tissue apoptosis levels were evaluated using a TUNEL assay, as well as western blot analysis. Immunofluorescence together with western blot analysis was used to detect autophagy in the tissues. Immunohistochemistry and western blot analysis were used to analyze DNA double­stranded breaks (DSBs). Moreover, HT22 cells overexpressing Beclin­1 were subjected to oxygen glucose deprivation/reoxygenation injury to simulate I/R pathological damage in vitro. Apoptosis was assessed using TUNEL and flow cytometric assays in this in vitro model, and autophagy was assessed using immunofluorescence and western blot analysis. The DSBs of the cells were analyzed using western blot analysis. I/R activated autophagy and induced DSBs. Autophagy inhibitors decreased brain tissue damage and reduced cell apoptosis; however, the degree of decrease in damage and apoptosis was not highly associated with the change in autophagy, and the frequency of DSBs slightly increased. The overexpression of Beclin­1 in neurons significantly attenuated I/R­induced damage and promoted DSB repair. On the whole, the present study demonstrates that Beclin­1 protects neurons from ischemic damage through the non­autophagy­dependent regulation of DNA repair processes.

Up-regulation of SETD3 may contribute to post-stroke depression in rat through negatively regulating VEGF expression.

Post-stroke depression (PSD) is one of the most familiar complications of stroke, which refers to stroke patients who have varying degrees of depression (lasts for >2 weeks). SET domain-containing 3 (SETD3) is a conserved histone H3 methyltransferase, and the role of SETD3 in some diseases is increasingly being explored. However, the effects of SETD3 in PSD remain unclear. In this study, the PSD rat model was firstly constructed by Endothelin-1 injection combined with chronic unpredictable mild stress, and we discovered that SETD3 expression was up-regulated in PSD rat model. Additionally, SETD3 knockdown relieved the depressive symptom of PSD. Moreover, SETD3 knockdown promoted proliferation and differentiation of neural stem cells (NSCs). Due to the critical role of vascular endothelial growth factor (VEGF) in antidepressant and SETD3 can negatively regulate VEGF, we speculated that SETD3 may regulate PSD progression through VEGF. Our results demonstrated that SETD3 knockdown up-regulated VEGF expression. Furthermore, SETD3 modulated the proliferation and differentiation of NSCs through regulating VEGF expression. In conclusion, our study indicated that up-regulation of SETD3 contributed to PSD progression in rats through negatively regulating VEGF expression. The findings of this work suggest that SETD3 may be a promising target for treating PSD in the future.

Genes Induced by Panax Notoginseng in a Rodent Model of Ischemia-Reperfusion Injury.

Stroke is a cerebrovascular disease that results in decreased blood flow. Although Panax notoginseng (PN), a Chinese herbal medicine, has been proven to promote stroke recovery, its molecular mechanism remains unclear. In this study, middle cerebral artery occlusion (MCAO) was induced in rats with thrombi generated by thread and subsequently treated with PN. After that, staining with 2,3,5-triphenyltetrazolium chloride was employed to evaluate the infarcted area, and electron microscopy was used to assess ultrastructural changes of the neurovascular unit. RNA-Seq was performed to determine the differential expressed genes (DEGs) which were then verified by qPCR. In total, 817 DEGs were identified to be related to the therapeutic effect of PN on stroke recovery. Further analysis by Gene Oncology analysis and Kyoto Encyclopedia of Genes and Genomes revealed that most of these genes were involved in the biological function of nerves and blood vessels through the regulation of neuroactive live receptor interactions of PI3K-Akt, Rap1, cAMP, and cGMP-PKG signaling, which included in the 18 pathways identified in our research, of which, 9 were reported firstly that related to PN's neuroprotective effect. This research sheds light on the potential molecular mechanisms underlying the effects of PN on stroke recovery.

Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

Treatment with Panax notoginseng saponin (PNS) can prevent neurological damage in middle cerebral artery occlusion model rats to promote recovery after a stroke. However, the exact molecular mechanisms are unknown and require further study. In the present study, mRNA sequencing was employed to investigate differential gene expression between model and sham groups, and between model and PNS­treated groups. Enrichment of gene data was performed using Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes database. Hub genes were identified and networks were constructed using Cytoscape that were further verified by reverse transcription­quantitative PCR. A total of 1,104 genes of interest were found, which included 690 upregulated and 414 downregulated genes that were identified when the model was compared with the sham group. Additionally, 817 genes of interest, which included 390 upregulated and 427 downregulated genes, were identified when the PNS­treated group was compared with the model group. There were 303 overlapping genes of interest between the analysis of model to sham groups, and the analysis of model to PNS­treated groups. The top 10 genes from the 303 aberrantly expressed genes of interest included ubiquitin conjugating enzyme E2 variant 2, small ubiquitin­related modifier 1, small RNA binding exonuclease protection factor La, Finkel­Biskis­Reilly murine sarcoma virus (FBR­MuSV) ubiquitously expressed, centrosomal protein 290 kDa, DNA­directed RNA polymerase II subunit K, cullin­4B, matrin­3 and vascular endothelial growth factor receptor 2. In conclusion, these genes may be important in the underlying mechanism of PNS treatment in ischemic stroke. Additionally, the present data provided novel insight into the pathogenesis of ischemic stroke.

Inhibition of PDE1-B by Vinpocetine Regulates Microglial Exosomes and Polarization Through Enhancing Autophagic Flux for Neuroprotection Against Ischemic Stroke.

Exosomes contribute to cell-cell communications. Emerging evidence has shown that microglial exosomes may play crucial role in regulation of neuronal functions under ischemic conditions. However, the underlying mechanisms of microglia-derived exosome biosynthesis are largely unknown. Herein, we reported that the microglial PDE1-B expression was progressively elevated in the peri-infarct region after focal middle cerebral artery occlusion. By an oxygen-glucose-deprivation (OGD) ischemic model in cells, we found that inhibition of PDE1-B by vinpocetine in the microglial cells promoted M2 and inhibited M1 phenotype. In addition, knockdown or inhibition of PDE1-B significantly enhanced the autophagic flux in BV2 cells, and vinpocetine-mediated suppression of M1 phenotype was dependent on autophagy in ischemic conditions. Co-culture of BV2 cells and neurons revealed that vinpocetine-treated BV2 cells alleviated OGD-induced neuronal damage, and treatment of BV2 cells with 3-MA abolished the observed effects of vinpocetine. We further demonstrated that ischemia and vinpocetine treatment significantly altered microglial exosome biogenesis and release, which could be taken up by recipient neurons and regulated neuronal damage. Finally, we showed that the isolated exosome per se from conditioned BV2 cells is sufficient to regulate cortical neuronal survival in vivo. Taken together, these results revealed a novel microglia-neuron interaction mediated by microglia-derived exosomes under ischemic conditions. Our findings further suggest that PDE1-B regulates autophagic flux and exosome biogenesis in microglia which plays a crucial role in neuronal survival under cerebral ischemic conditions.

[Association of single nucleotide polymorphisms of SCN1A gene with therapeutic effect of carbamazepine among ethnic Zhuang Chinese patients with epilepsy].

OBJECTIVE: To assess the association of single nucleotide polymorphisms of SCN1A gene with therapeutic effect of carbamazepine among ethnic Zhuang Chinese patients with epilepsy. METHODS: Peripheral blood samples were taken from 186 epileptic patients for whom 66 cases standard regime of carbamazepine treatment was effective. Genotypes of rs3812718 and rs1813502 loci of the SCN1A gene were determined by Mass ARRAY-IPLEX and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Correlation between genotypes of patients and efficacy of carbamazepine treatment was analyzed. RESULTS: Three genotypes (GG, GA and AA) were detected at both rs3812718 and rs1813502 loci of the SCN1A gene. A significant difference was found in allelic distribution (chi-square=17.810, P=0.000) and genotypic distribution (chi-square=17.873, P=0.000) of the rs3812718 locus between the effective group and ineffective group. No such difference was found with the rs1813502 locus (chi-square=1.606, P=0.206; chi-square=1.546, P=0.462, respectively). Compared with the GG+GA genotype, the AA genotype at rs3812718 locus significantly reduced the antiepileptic efficacy of carbamazepine (OR=3.776, 95%CI: 2.007-7.105). Among the 66 patients who were responsive to carbamazepine treatment, those with the AA genotype for rs3812718 or rs1813502 shown no significant difference in their blood concentration of carbamazepine compared with those with the GG+GA genotype (t=1.562, P=0.125; t=0.843, P=0.562, respectively). rs3812718 and rs1813502 were not in strong linkage disequilibrium. CONCLUSION: Polymorphisms of rs3812718 of the SCN1A gene is associated with carbamazepine resistance among ethnic Zhuang Chinese epilepsy patients from Baise region.

CDA-2 induces cell differentiation through suppressing Twist/SLUG signaling via miR-124 in glioma.

Cell differentiation agent-2 (CDA-2) is an extraction from healthy human urine consisting of primary organic acids and peptides, and it has been demonstrated to inhibit growth and induce differentiation in glioma and other cell lines. But the mechanism of CDA-2 remains unclear. In this study, we demonstrated that CDA-2 inhibited cell growth and induced differentiation of glioma cells, accompanied with decreased expression of SLUG, Twist and Vimentin in both SWO-38 and U251 cell lines. Overexpression of SLUG or Twist greatly eliminated the efficiency of CDA-2 in inducing differentiation. Further study showed that CDA-2 treatment resulted in great changed microRNAs (miRNAs) detected by quantitative PCR, in which miR-124 was one of the most changed miRNAs and its level was increased by fourfold. The result of miRNA target prediction showed that miR-124 could regulate hundreds of genes which were relative to cell differentiation, such as SLUG, Vimentin, actin cytoskeleton, focal adhesion, tight junction. Inhibition of miR-124 up-regulated SLUG, Twist and Vimentin proteins, and partly eliminated the function of CDA-2 on these mesenchymal markers. Our findings demonstrated for the first time that CDA-2 induced cell differentiation through suppressing Twist and SLUG via miR-124 in glioma cells.