High glucose stimulates GRO secretion from rat microglia via ROS, PKC, and NF‐κB pathways

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Abstract
Hyperglycemia causes direct neuronal damage in diabetic encephalopathy. Microglia have been found to be activated in diabetic encephalopathy, presumably mediating and amplifying neuron degeneration. Chemokine IL-8 plays an important role in the pathogenesis of encephalopathy. Therefore, we investigated whether high glucose could activate microglia and stimulate IL-8 secretion and if so, the possible mechanisms that were involved. ELISA results showed that treatment with high glucose (35 mM) compared with treatment with low glucose (10 mM) time-dependently elevated secretion of GRO (the rat ortholog of human IL-8) in primary cultured rat microglia. Real-time PCR results showed GRO mRNA expression also increased in response to high glucose in a time-dependent manner. These effects were specific to high glucose because the osmolality control had no such effect. High-glucose treatment stimulated the formation of ROS, as seen in the DCF fluorescence assay, increased phosphorylation of PKC, as seen in the Western blot analysis, and activated NF-κB, as seen in the luciferase reporter assay. In addition, treatment with the ROS scavenger NAC (2 mM) significantly reduced the high glucose–induced phosphorylation of PKC and GRO secretion. Treatment with the PKC activator PMA (10–50 nM) stimulated GRO secretion, and the PKC inhibitors calphostin C (300 nM) or chelerythrine (1 μM) attenuated the high glucose–induced GRO secretion. Furthermore, the NF-κB inhibitors MG132 (10 μM) or PDTC (5 μM) completely blocked the high glucose–induced GRO secretion. In conclusion, high glucose induces GRO secretion and mRNA expression in activated rat microglia, which is mediated by the ROS, PKC, and NF-κB pathways. High glucose–induced IL-8 production by microglia may contribute to diabetic encephalopathy.