| Summary: | 博士 === 國立臺灣海洋大學 === 生物科技研究所 === 97 === Inflammation in the brain leading to many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis and AIDS dementia involve neuronal etiology. Cyclin-dependent kinase (CDK) inhibitors have been shown to inhibit microglia activation and neuronal death. However, the mechanisms of CDK inhibitor on preventing nerve from cell death remain uncovered.
In a pervious study, we demonstrated that olomoucine, a CDK inhibitor, would inhibit ell-cycle progression and lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7macrophages. LPS-induced iNOS expression, NO production, iNOS promoter activity, and pro-inflammatory cytokine expression are reduced by olomoucine. Inhibitory effect is resulted from down-regulation of NF-κB transcriptional activity. We also demonstrated that inhibition of NF-κB expression is due to inhibition of E2F1 transcriptional activity.
In this study, we examined whether CDK inhibitor, olomoucine, may down-regulate LPS-stimulated pro-inflammatory responses and cell death. We demonstrated that the inhibitory effect of olomoucine was shown on cell cycle progression and LPS-induced inflammatory response in microglia cell line BV2 cells. LPS-induced NO production and iNOS expression are reduced by olomoucine. Using reporter gene assay, olomoucine is capable of reducing LPS-stimulated iNOS promoter activity and NF-κB transcriptional activity. These results show that olomoucine may down-regulate NF-κB to reduce pro-inflammatory response in BV2 cells. BV2 cell death is observed after treatment of LPS. To investigate the role of NO, NO scavenger c-PTIO to eliminate NO is able to reduce LPS-stimulated cell death, whereas NO donor, SNAP, results in BV2 cell death. These results indicate that LPS-stimulated cell death may result from NO and the treatment of olomoucine leads to decrease cell death stimulated by LPS
To identify the change of gene expression profile, microarray was used to analyse the BV2 cell after treatment of olomoucine. BNIP3, a Bcl-2 family protein, was found decreased RNA expression after treatment of olomoucine. BNIP3, a pro-apoptotic Bcl2 gene, may involve NO-induced cell death in BV2 cell. To examine the role of BNIP3, knockdown BNIP3 was performed. Our results show that knockdown BNIP3 reduces NO-induced cell death.
Active microglia cells secrete pro-inflammatory molecule to kill neuron cells. Conditional medium (CM) from LPS-stimulated BV2 cells could induce Neuro2a cell death. In addition, olomoucine could also reduce Neuro2a cell death induced by the conditional medium. Moreover, olomoucine also exhibits inhibitory effect on NO-induced cell death in Neuro2a cells.
In the study, these results show that the neuorprotection effects of CDK inhibitor exhibited by: (1) inhibition of microglia activation to reduced pro-inflammatory molecules (2) decrease of pro-inflammatory molecules–induced neuron cell death. (3) down-regulation of BNIP3 expression to inhibit NO-induced cell death. Our results imply that BNIP3 is a potential therapeutic target for neurodegenerative diseases.
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