Mechanism of scutellarin inhibition of astrocyte activation to type A1 after ischemic stroke.

OBJECTIVE: The aim of this study was to investigate the effects of scutellarin on the activation of astrocytes into the A1 type following cerebral ischemia and to explore the underlying mechanism. METHODS: In vivo, a mouse middle cerebral artery wire embolism model was established to observe the regulation of astrocyte activation to A1 type by scutellarin, and the effects on neurological function and brain infarct volume. In vitro, primary astrocytes were cultured to establish an oxygen-glucose deprivation model, and the mRNA and protein expression of C3, a specific marker of A1-type astrocytes pretreated with scutellarin, were examined. The neurons were cultured in vitro to detect the toxic effects of ischemia-hypoxia-activated A1 astrocyte secretion products on neurons, and to observe whether scutellarin could reduce the neurotoxicity of A1 astrocytes. To validate the signaling pathway-related proteins regulated by scutellarin on C3 expression in astrocytes. RESULTS: The results showed that scutellarin treatment reduced the volume of cerebral infarcts and attenuated neurological deficits in mice caused by middle cerebral artery embolism. Immunofluorescence and Western blot showed that treatment with scutellarin down-regulated middle cerebral artery embolism and OGD/R up-regulated A1-type astrocyte marker C3. The secretory products of ischemia-hypoxia-activated A1-type astrocytes were toxic to neurons and induced an increase in neuronal apoptosis, and astrocytes treated with scutellarin reduced the toxic effects on neurons. Further study revealed that scutellarin inhibited the activation of NF-κB signaling pathway and thus inhibited the activation of astrocytes to A1 type.

CX3CR1 mediates motor dysfunction in mice through 5-HTR2a.

CX3CR1 knockout could induce motor dysfunction in several neurological disease models mainly through regulating microglia's function. While CX3CR1 was expressed on neurons in a few reports, whether neuronal CX3CR1 could affect the function of neurons and mediate motor dysfunction under physiological conditions is unknown. To elucidate the roles of neuronal CX3CR1 on motor dysfunction, CX3CR1 knockout mice were created. Rotarod test and Open field test found that the CX3CR1-/- mice's motor capacity was reduced. Immunofluorescence staining detected the expression of CX3CR1 in neurons both in vivo and in vitro. Immunohistochemistry and West blot found that knockout of CX3CR1 did not affect the neurons' number in both spinal cord and brain of mice. While inhibiting the function of CX3CR1 by AZD8797 could decrease the expression of 5-Hydroxytryptamine receptor(5-HTR2a), which involved in the regulation of motor function. Further investigation revealed that CX3CR1 regulated the expression of HTR2a through the NF-κB pathway. For the first time, we reported that neuronal CXCR1 mediates motor dysfunction. Our results suggest that modulating CXCR1 activity offers a novel therapeutic strategy for motor dysfunction.

Identification of m6A methylation-related genes in cerebral ischaemia‒reperfusion of Breviscapus therapy based on bioinformatics methods.

BACKGROUND: Cerebral ischaemia‒reperfusion (I/R) frequently causes late-onset neuronal damage. Breviscapine promotes autophagy in microvascular endothelial cells in I/R and can inhibit oxidative damage and apoptosis. However, the mediation mechanism of breviscapine on neuronal cell death is unclear. METHODS: First, transcriptome sequencing was performed on three groups of mice: the neuronal normal group (Control group), the oxygen-glucose deprivation/ reoxygenation group (OGD/R group) and the breviscapine administration group (Therapy group). Differentially expressed genes (DEGs) between the OGD/R and control groups and between the Therapy and OGD/R groups were obtained by the limma package. N6-methyladenosine (m6A) methylation-related DEGs were selected by Pearson correlation analysis. Then, prediction and confirmation of drug targets were performed by Swiss Target Prediction and UniProt Knowledgebase (UniProtKB) database, and key genes were obtained by Pearson correlation analysis between m6A-related DEGs and drug target genes. Next, gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA) were used to obtain the pathways of key genes. Finally, a circRNA-miRNA‒mRNA network was constructed based on the mRNAs, circRNAs and miRNAs. RESULTS: A total of 2250 DEGs between the OGD/R and control groups and 757 DEGs between the Therapy and OGD/R groups were selected by differential analysis. A total of 7 m6A-related DEGs, including Arl4d, Gm10653, Gm1113, Kcns3, Olfml2a, Stk26 and Tfcp2l1, were obtained by Pearson correlation analysis. Four key genes (Tfcp2l1, Kcns3, Olfml2a and Arl4d) were acquired, and GSEA showed that these key genes significantly participated in DNA repair, e2f targets and the g2m checkpoint. IPA revealed that Tfcp2l1 played a significant role in human embryonic stem cell pluripotency. The circRNA-miRNA‒mRNA network showed that mmu_circ_0001258 regulated Tfcp2l1 by mmu-miR-301b-3p. CONCLUSIONS: In conclusion, four key genes, Tfcp2l1, Kcns3, Olfml2a and Arl4d, significantly associated with the treatment of OGD/R by breviscapine were identified, which provides a theoretical basis for clinical trials.

IL-1ß promotes A7r5 and HASMC migration and invasion via the p38-MAPK/Angpt-2 pathway.

The migration, proliferation, and inflammatory factor secretion of vascular smooth muscle cells (VSMCs) are involved in the important pathological processes of several vascular occlusive diseases, including coronary atherosclerosis (CAS). Interleukin 1ß(IL-1ß), as a bioactive mediator of VSMC synthesis and secretion, can promote the pathological progress of CAS. In this study, we further explored the underlying molecular mechanisms by which IL-1ß regulates VSMC migration, invasion. We pretreated A7r5 and HASMC with IL-1ß for 24 h, and measured the expression of IL-1ß, proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 2 (MMP9) in the cells by Western blotting. Cell migration and invasion ability were measured by Transwell and wound healing assays. Cell viability was measured by an MTT assay. We found that IL-1ß upregulated the expression of proliferation-related proteins (PCNA and Cyclin D1) in A7r5 and HASMC, and induces the secretion of MMP2 and MMP9, promotes cell invasion and migration. In addition, in A7r5 and HASMCs treated with IL-1ß, the expression of Angiopoietin-2 (Angpt-2) increased in a time-dependent manner, transfection with si-Angpt-2 suppressed cell migration and invasion, with downregulated MMP2 and MMP9 expression. Parallelly, we further found that the p38-MAPK pathway is activated in cells induced by IL-1ß, p38-MAPK inhibitors can down-regulate the expression of Angpt-2. Collectively, these data demonstrated that IL-1ß promotes A7r5 and HASMC migration and invasion via the p38-MAPK/Angpt-2 pathway.

RNA disruption indicates CHOP therapy efficacy in canine lymphoma.

BACKGROUND: Assessment of the efficacy of a multi-agent chemotherapy protocol in which cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are administered in canine lymphoma is generally performed by physical measurement of lymph node diameter. However, no consistent correlation has been made with prognostic indicators and the length or absence of clinical remission based on lymph node size. RNA disruption measured mid-therapy has been correlated with increased disease-free survival in recent studies of human cancer and was assessed in this study of canine lymphoma patients. Fine needle aspirate samples were taken before treatment and at weeks 3, 6, and 11 of CHOP therapy. RNA was isolated from these samples and assessed using an Agilent Bioanalyzer. RNA disruption assay (RDA) analysis was performed on the data from the resulting electropherograms. RESULTS: An increased RNA disruption index (RDI) score was significantly associated with improved progression-free survival. CONCLUSIONS: Predicting the risk of early relapse during chemotherapy could benefit veterinary patients by reducing ineffective treatment and could allow veterinary oncologists to switch earlier to a more effective drug regimen.