Nitric oxide and Cytokine Production by Rat Glial cell Stimulated with Coxsackievirus B3 in vitro

• Main Authors: Ching-Ping,Yang, 楊境評
• Other Authors: Chun-Jung Chen,Ph.D.
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/38127326786597245779

碩士 === 國立中興大學 === 獸醫微生物學研究所 === 93 === Abstract Coxsackievirus group B3 (CVB3) within the genus enterovirus, which is a member of the Picornavirus family. Neonates are particularly susceptible to coxsackievirus infections of the central nervous system (CNS), which can cause meningitis, encephalitis, and long-term neurological deficits. The mechanisms by which CVB3 causes neurological diseases are not fully understood. The activation of inducible nitric oxide synthase (iNOS) and consequent production of nitric oxide (NO) in neural cells play important roles in the regulation of brain inflammation. NO exerts two opposite biological functions in the CNS including neuroprotective and neurotoxic effects. In animal study, the infection with HSV-1 or Junin virus in mice is characterized at the pathologic level by significant infiltration of inflammatory cells and the production of pro-inflammatory factors such as NO, TNF-alpha, IL-1beta and IFN-gamma. In the beginning of our study, we found that CVB3-infected neuron/glia cells, consisting of neuron, astrocyte, and microglia, produced NO. The generation of NO in in vitro neural cells after CVB3 infection is mimicked the in vivo animal study showing the activation of nitric oxide synthase activity in brain tissues after virus inoculation. Generally, large amounts of NO are produced by activated glial cells. Therefore, we assessed whether glial cells played roles in the induction of NO synthesis after CVB3 infection and tried to elucidate the underlying mechanisms. In addition to neuron/glia, mixed glia and microglia were able to produce NO after CVB3 infection. However, astrocytes were unable to release NO. Next, we examined whether CVB3 infection stimulated glial activation by the following assays including morphological change and cytokine expression. Obvious change in morphology was only observed in microglia after CVB3 infection. CVB3-infected mixed glia, astrocyte, or microglia all released various cytokines with variable efficiency. In short conclusion, CVB3 infection activated glial cells, especially microglia, and the activated microglia could produce NO. The mechanisms of CVB3-induced NO production were further characterized. Both UV and heat-inactivated CVB3 lost their ability to stimulate glial cells release NO. These findings imply that the event of viral amplification is essential to NO production in CVB3-infected glial cells. Regarging the intracellular signaling pathways, CVB3 infection markedly increased the phosphorylation level of ERK and JNK in glial cells. We found that treatment of glial cells with ERK/MEK inhibitor U0126 and JNK inhibitor SP600125 reduced the production of NO and cytokines. Based on the western blot analysis, the importance of ERK and JNK signaling pathway in the induction of iNOS and cytokine production after CVB3 infection in glial cells was appreciated. CVB3 infection increased the DNA binding ability of NF-κB, and the reduction of NO and cytokine production by the treatment of NF-κB inhibitor, PDTC, indicated the requirement of NF-κB activate in CVB3-metiated gene induction. Our current study provides evidence showing the underlying signaling pathways for NO and cytokine expression in CVB3-infected glial cells, which will shed light on the regulation of brain inflammation.