Japanese encephalitis virus induces matrix metalloproteinase-9 in rat brain astrocytes via NF-ΚB signalling dependent on MAPKs and reactive oxygen species

BACKGROUND AND PURPOSE Japanese encephalitis virus (JEV) is a member of the family Flaviviridae and JEV infection is a major cause of acute encephalopathy in children, which destroys cells in the CNS, including astrocytes and neurons. However, the detailed mechanisms underlying the inflammatory action of JEV are largely unclear. EXPERIMENTAL APPROACH The effect of JEV on the expression of matrix metalloproteinase (MMP)-9 was determined by gelatin zymography, Western blot analysis, real-time PCR and promoter assay. The involvement of the NADPH oxidase and reactive oxygen species (ROS), MAPKs, and the transcription factor NF-ΚB in these responses was investigated by using selective pharmacological inhibitors and transfection with appropriate siRNAs. KEY RESULTS JEV induced the expression of the pro-form of MMP-9 in rat brain astrocytes (RBA-1 cells). In RBA-1 cells, JEV induced MMP-9 expression and promoter activity, which was inhibited by pretreatment with inhibitors of NADPH oxidase (diphenylene iodonium chloride or apocynin), MAPKs (U0126, SB203580 or SP600125) and a ROS scavenger (N-acetylcysteine), or transfection with siRNAs of p47 ^phox, ERK1, JNK2 and p38. In addition, JEV-induced MMP-9 expression was reduced by pretreatment with an inhibitor of NF-ΚB (helenalin) or transfection with p65 siRNA. Moreover, JEV-stimulated NF-ΚB activation was inhibited by pretreatment with the inhibitors of NADPH oxidase and MAPKs. CONCLUSIONS AND IMPLICATIONS MMP-9 expression induced by JEV infection of RBA-1 cells was mediated through the generation of ROS and activation of p42/p44 MAPK, p38 MAPK and JNK1/2, leading to NF-ΚB activation.