薑烯酚對脂多醣體所誘發的小鼠微膠細胞發炎之抗發炎機轉研究

Investigating anti-inflammatory effects of 6-shogaol on Lipopolysaccharide-stimulated murine microglial cell BV-2

林琬羚(Wan-Ling Lin)

指導教授 ： 高紹軒

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2012年)

https://doi.org/10.6834/CSMU.2012.00003

摘要

多種神經退化性疾病幾乎發現伴隨腦部發炎，而神經細胞的死亡已證實和活化的微膠細胞有關。微膠細胞為常駐在中樞神經系統中的巨噬細胞，微膠細胞被活化會釋放許多細胞激素：TNF-α、IL-1β也產生發炎相關的酵素：iNOS、COX-2。本論文
在探討薑烯酚(6-shogaol) 對細菌脂多醣體 (lipopolysaccharide，LPS) 所誘導的小鼠微膠細胞BV-2 (murine microglial cell BV-2) 發炎之抗發炎機轉。在本篇研究中，我們發現以不同濃度之6-shogaol (0.05, 0.1, 0.25, 0.5μM) ，可分別有不同之效率降低脂多醣體誘導之發炎反應；以及6-shogaol處理正常的小鼠微膠細胞也可引發特定之發炎因子及前趨物，包含 iNOS 以及 COX-2之下游訊息傳遞機轉、核酸轉錄以及蛋白質的生成。進一步的，探討6-shogaol對細菌脂多醣體誘導小鼠微膠細胞前發炎的因子。發現6-shogaol有抗發炎的效果而抑制IL-6和TNF-α訊息核酸及蛋白質的生成。由此，我們更進一步探討6-shogaol抑制脂多醣體所造成的發炎反應是否透過MAPKs和PI3K / Akt路徑。確實發現可以降低脂多醣體所誘導的磷酸化Akt和MAPK家族(包含了ERK、JNK、P38) 。此外，也發現6-shogaol抗發炎的效果，是經由影響NF-κB無法轉入到細胞核內，而抑制轉錄因子NF-κB訊息的活化。綜合以上結果，可以得知在6-shogaol刺激下，明顯降低脂多醣體所誘導的前發炎的因子IL-6和TNF-α 的mRNA，是透過抑制MAPK家族和AKT這兩條路徑，使NF-κB無法轉入到細胞核內，而抑制下游的發炎基因無法開啟；最後，針對6-shogaol也會經由NO降低發炎的途徑進行探討，再次證明了6-shogaol 對於iNOS之核酸及其訊息傳遞之生合成能力。

關鍵字

薑烯酚 ； 脂多醣體 ； 微膠細胞 ； 發炎反應

並列摘要

Inflammation in brain has been manifested in many neuro degenerative disorders.Though the molecular mechanism is not clear,evidences link neuron death to the activation of microglia.Microglia are resident macrophages with wide distribution in nervous tissues. The activation of microglial cells in response to pathological stimuli is associated with the production of various cytokines, such as TNF-α and IL-1β, and the induction of inflammation-related enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2. In the present study, we investigated the effects and mechanisms of 6-shogaol on the inflammatory effect induced by bacterial lipopolysaccharide (LPS) on murine microglial cell BV-2 . We found that 6-shogaol (0.05, 0.1, 0.25, 0.5μM) exerted a concentration-dependently inhibited the inflammatory mediator expression including iNOS and COX-2 mRNA and protein expression upon stimulation by LPS (1μg/mL) on BV-2. And the continued focused on the pro-inflammatory factors of 6-shogaol effect on LPS with induced murine microglial cell BV-2 inflammation. We found that 6-shogaol attenuated inflammatory effect by inhibiting IL-6 and TNF-α mRNA and protein expression. Accordingly,we further investigated investigated the effect of 6-shogaol on LPS-induced mitogen-activated protein kinases (MAPKs) and PI3K / Akt cascades. Our results revealed that 6-shogaol significantly inhibited the LPS-induced phosphorylation of MAPKs and Akt,including extracellular signal-regulated kinase1/2 ( ERK1/2) ,p38 MAPK(p38), c-jun NH2-terminal kinase(JNK)and Akt. In addition,we found that andrographolide attenuated inflammatory effect by inhibiting NF-κB tranlocation therefore down-regulated transcription factor NF-κB signal activation. Taken together,our finings indicated that 6-shogaol significantly diminished the LPS- induced mRNA expression of pro-inflammatory factors IL-6 and TNF-α in BV-2 cells,which may result from inhibition of MAPKs and Akt activation and suppressing NO production,and the following reduced translocation of NF-κB into nucleus.

並列關鍵字

6-shogaol ； lipopolysaccharide ； microglial cell ； inflammation

參考文獻

1. 吳才元 and 劉衡生, Acute and Chronic Inflammation: Effect of the Risk Factor (s) on the Progression of the Early Inflammatory Response to the Oxidative and Nitrosative Stress. Journal of Biomedical & Laboratory Sciences, 2007. 19(3): p. 71-75.

2. Streit, W.J., Microglia and macrophages in the developing CNS. Neurotoxicology, 2001. 22(5): p. 619-24.

3. Chan, W.Y., S. Kohsaka, and P. Rezaie, The origin and cell lineage of microglia: new concepts. Brain Res Rev, 2007. 53(2): p. 344-54.

4. Monier, A.,Adle-Biassette, H.,Delezoide, A. L.,Evrard, P.,Gressens, P.,Verney, C.,Entry and distribution of microglial cells in human embryonic and fetal cerebral cortex. J Neuropathol Exp Neurol, 2007. 66(5): p. 372-82.

5. Calvo, M. and D.L. Bennett, The mechanisms of microgliosis and pain following per al nerve injury. Exp Neurol, 2012. 234(2):