| BackgroundAstrocytes are the most abundant non-neuronal cells in central nervous system. In addition to their trophic and structural role, astrocytes play an important role in the maintenance of homeostatic balance of pH, neurotransmitters and ions, as well as regulation of cell communication and inflammation. Morphological and functional alterations of astrocytes may lead to disruption of brain homeostasis and induce various brain diseases. Gap junctions (GJs) are important intercellular channels at the plasma membrane, which are expressed in almost all kinds of species, provided direct channels for ions and small molecules between adjacent cells. Multiple lines of evidence suggest that GJs play a pivotal role in the regulation of cell communication, proliferation and differentiation, along with responsing to inflammation reaction and hypoxic-ischemic damage. Pannexins (Panxs) have been described as a new family member of GJs in 2000. As one of its subtypes, Panx-1 is abundantly expressed in the brain and forms hemi-channels at the plasma membrane of astrocytes. Panx-1 channels are permeable to ions, metabolic molecules, second messengers (Ca2+、ATP and so on) and neurotransmitters which are smaller than 1kDa. Connexins (Conxs), the classical GJs in vertebrates, had been studied extensively over the last decade. Animal and clinical studies have shown that there existed some overlap in functions between Panx-1 and Conxs, and most inhibitors of Conxs have an inhibition effect on Panx-1. Therefore it was reasonable to consider that Panx-1 might be responsible for the release of cytokines and glutamate. Inflammation and disruption of the balance between excitatory and inhibitory are involved in the onset and development of many neurological disorders, such as cerebrolvascular diseases, epilepsy, neurodegenerative diseases and so on. Elucidating the effect of Panx-1 in the release of cytokines and glutamate from astrocytes will provide futher insights into the mechanism underlying the pathogenesis of various neurological diseases. In addition, in this study, we found that cells in mitosis of U87-MG cells had a higher expression of Panx-1 protein. Since astrogliosis is a process that involves electrophysiological and biochemical changes associated with astrocyte activation,next we do the transfection work to evaluate the role of Panx-1 in cell proliferation.Materials and MethodsThis research is divided into two sections.In the first section, immunocytochemistry was used to detect the expression of Panx-1 in U87-MG cells. Then cells were transfected with small interfering RNA (siRNA) or stimulated with LPS or ATP seperately. The expression of Panx-1 was examined by Real-time PCR and Western Blot. At the same time, levels of cytokines (IL-1β, IL-6, IL-8, IL-10 and TNF-α) and glutamate in culture supernatants were determined using ELISA, Bio-Plex(?) suspension array system and HPLC.In the second section, CCK-8 assay was carried out to evaluate the proliferation afer U87-MG cells transfected with Panx-1-siRNA.ResultsThe results of the first section:1. Panx-1 positive staining was located in the cytoplasm, predominantly in mitosis cells.2. Compared with the control siRNA transfection group, mRNA and protein expressions of Panx-1 were decerased (P< 0.05, resp.). Moreover, lower levels of IL-6, IL-8 and glutamate were found in the supernatants of U87-MG cells transfected with Panx-1-siRNA (P< 0.05, resp.). Level of IL-1β was also decreased, but there was no statistical difference (P>0.05).3. Compared with the control group, no change was found in the expression of Panx-1 protein in neither LPS-stimulated nor ATP-stimulated groups. Additionally, levels of IL-1β, IL-6, IL-8 and glutamate were similar to protein levels, which had no statistically significant compared with the control group (P> 0.05, resp.)The results of the second section:1. Compared with the control siRNA transfection group, the expressions of Panx-1 and PCNA proteins in cells transfected with Panx-1-siRNA were decreased (P< 0.05, resp.). PCNA is one of the well-known markers of cell proliferation.2. Compared with the control siRNA transfection group, Panx-1-siRNA significantly inhibited cell proliferation (P< 0.05).Conclusions1. Panx-1 knockdown leaded to a decreased release of IL-6, IL-8 and glutamate in U87-MG cells, suggested that Panx-1 takes part in the inflamation reaction and glutamate release.2. Panx-1 knockdown reduced the proliferation of U87-MG cells, indicating that Panx-1 was involved in cell proliferation. |
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