Background/Aims: Alcohol consumption has been shown to cause neuroinflammation and increase a variety of immune-related signaling processes. Microglia are a crucial part of alcohol-induced neuroinflammation and undergo apoptosis. Even though the importance of these inflammatory processes in the effects of alcohol-related neurodegeneration have been established, the mechanism of alcohol-induced microglia apoptosis is unknown. In prior research, we discovered that alcohol increases expression of salt-inducible kinase 1 (SIK1) in rodent brain tissue. In this study, we sought to determine what role SIK1 expression plays in alcohol-induced neuroinflammation as well as whether and by what mechanism it regulates microglia apoptosis. Methods: Adult C57BL/6 mice were divided into four groups and for 3 weeks treated with either 0%, 5%, 10%, or 15% alcohol during 3 hour periods. The mice were sacrificed and their brains excised for analysis. Additionally, primary microglia were isolated from neonatal mice. SIK1 expression in alcohol-treated brain tissue and microglia was analyzed via RT-PCR and western blotting. TUNEL staining, caspase-3, and caspase-9 activity assays were performed to evaluate microglial apoptosis. Cell fluorescence staining and NF-κB luciferase activity assays were used to evaluate the effects of SIK1 expression on the NF-κB signaling pathway. Results: SIK1 expression was increased in the brains of mice that consumed alcohol, and this effect was seen in mouse primary microglia. SIK1 knockdown in microglia increased alcohol-induced apoptosis in these cells. Furthermore, SIK1 reduced NF-κB signaling pathway factors, and SIK1 knockdown in microglia promoted alcohol-induced NF-κB activity. TUNEL staining, caspase-3, and caspase-9 activity assays consistently revealed that alcohol-induced microglial apoptosis was inhibited by depletion of p65. Finally, we determined that NF-κB signaling is required for alcohol-induced, SIK1-mediated apoptosis in microglia. Conclusion: This study establishes for the first time not only that SIK1 is crucial to regulating alcohol-induced microglial apoptosis, but also that the NF-κB signaling pathway is required for its activity. Overall, our results help elucidate mechanisms of alcohol-induced neuroinflammation.
It was reported that g-irradiation had a controversial therapeutic effect on glioma cells. We aimed to investigate the cytotoxic effect on the glioma cells induced by g-irradiation and explore the treatment to rescue the phenotype alteration of remaining cells. We used transwell assay to detect the glioma cell invasion and migration capacity. Cell proliferation and apoptosis were tested by the CCK-8 assay and flow cytometry respectively. Western Blot was used to detect the activity of Hedgehog signaling pathway and Epithelial-to-Mesenchymal Transition (EMT) status. g-irradiation showed cytotoxic effect on LN229 cells in vitro, whereas this contribution was limited in U251 cells. However, it could significantly stimulated EMT process in both LN229 and U251. Curcumin (CCM) could rescue EMT process induced by g-irradiation via the suppression of Gli1 and the upregulation of Sufu. The location and expression of EMT markers were also verified by Immunofluorescence. Immunohistochemistry assay was used on intracranial glioma tissues of nude mice. The capacities of cell migration and invasion were suppressed with combined therapy. This research showed Curcumin could rescue the EMT process induced by g-irradiation via inhibiting the Hedgehog signaling pathway and potentiate the cell cytotoxic effect in vivo and in vitro.
Background: In recent years, miRNAs have been suggested to play key roles in the formation and development of human glioma. The aim of this study is to investigate the effect and mechanism of miR-184 expression on the malignant behavior of human glioma cells. Methods: The relative quantity of miR-184 was determined in human glioma cell lines, and the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was explored using western blotting. The effects of miR-184 inhibition on cell viability and apoptosis were explored, and the miR-184 target gene was determined using a luciferase assay and western blotting. Flow cytometry and Hoechst staining were used to evaluate cell growth and apoptosis. Matrigel invasion and scratch assays were performed to measure the ability of cell invasion and migration. Results: miR-184 and HIF-1α protein levels were significantly upregulated in human glioma cells. Downregulation of miR-184 inhibited cell viability and increased the HEB cell apoptotic rate. Luciferase and western blot assays verified that FIH-1 was the target gene of miR-184 and negatively controlled the protein level of HIF-1α. Inhibition of HIF-1α by siRNA facilitated the apoptosis of HEB cells and suppressed A172 cell invasion and migration. Conclusion: miR-184 upregulation enhanced the malignant phenotype of human glioma cancer cells by reducing FIH-1 protein expression.
The role of NADPH oxidases (NOXs) in pathogenesis and development in the Curvularia leaf spot (CuLS) agent Curvularia lunata remains poorly understood. In this study, we identified C. lunata ClNOX2, which localized to the plasma membrane and was responsible for reactive oxygen species (ROS) generation. Scavenging the ROS production inhibited the conidial germination and appressorial formation. The ClNOX2 and ClBRN1 deletion mutants were defective in 1,8-dihydroxynaphthalene (DHN) melanin accumulation, appressorial formation, and cellulase synthesis and exhibited lower virulence. However, disruption of the ClNOX2 and ClBRN1 genes facilitated hyphal growth, enhanced stress adaptation to cell wall-disrupting agents, and promoted developmental processes such as conidiation, conidial germination, and pseudothecium and ascus formation. Interestingly, loss of ClM1, the cell wall integrity (CWI) MAPK gene in C. lunata led to similar morphology and pathogenicity phenotypes with ClNOX2 and ClBRN1 deletion mutants, like abnormal conidia, fewer appressoria, less melanin, increased hyphal growth and enhanced tolerance to congo red (CR). These results indicated that the ClNOX2 gene plays an important role in C. lunata development and virulence via regulating intracellular DHN melanin biosynthesis. RT-qPCR revealed that the ClNOX2-related ROS signaling pathway and ClM1-mediated CWI signaling pathway are cross-linked in regulating DHN melanin biosynthesis. Our findings provide new insights into how ClNOX2 participates in the pathogenesis and development in hemibiotrophic plant fungal pathogens.
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