| Abstract: | 微神經膠細胞為中樞神經系統主要的常駐型免疫細胞，當有刺激活化壓力時可因應變化大量表現一氧化氮、腫瘤壞死因子-alpha、介白素1beta和介白素6等促發炎相關物質，啟動先天性免疫進而擴展發炎反應。文獻指出，過度活化的微神經膠細胞及所產生的促發炎相關物質會造成神經細胞死亡，導致阿茲海默症等神經退化性疾病。木犀草素為類黃酮成份的一種，具有抗氧化、抗腫瘤、抗過敏及抗發炎等生物活性。研究顯示木犀草素具有神經保護功效而潛在的作用機制可能與抗發炎生物活性有關。木犀草素週邊抗發炎作用及機制已被廣泛討論，然而在中樞神經系統中的抗發炎功效及作用機制仍不明瞭。因此本研究探討木犀草素潛在的中樞抗發炎活性及作用機轉。脂多醣/干擾素-gamma刺激可活化BV-2微神經膠細胞株表現促發炎相關物質，例如腫瘤壞死因子-alpha、誘導型一氧化氮合成?、介白素1beta、環氧?等基因表現及產物釋出。不具細胞毒性的木犀草素可抑制這些基因mRNA及蛋白質表現，意謂著木犀草素的抑制作用是發生在促發炎相關基因的轉錄層次。通常細胞內訊息傳遞分子例如MAPKs、Akt、酪胺酸激?(如Jak家族、Src)等可因應變化而改變生物活性，這些訊息傳遞路徑的下游作用對象之一為轉錄因子。常見NF-kappaB、IRF、STAT等轉錄因子負責調控細胞基因的表現，包括促發炎相關基因。藥理活性分析發現，ERK、JNK、p38、Akt、Jak等訊息傳遞分子活性抑制劑都可降低脂多醣/干擾素-gamma刺激所造成的促發炎物質生合成釋出。藉由蛋白質磷酸化西方轉漬法分析發現，脂多醣/干擾素-gamma刺激會促進ERK、JNK、p38、Akt、Jak1、Jak2、Tyk2、Src等訊息傳遞分子活性，木犀草素可抑制ERK、JNK、p38、Akt、Src等訊息傳遞分子的活化，但是對於Jak1、Jak2、Tyk2等訊息傳遞分子活性則沒有顯著變化。透過IkappaB-alpha磷酸化及NF-kappaB次單元p50、p65、RelB核轉移幅度證實脂多醣/干擾素-gamma刺激會活化NF-kappaB轉錄因子，而木犀草素可藉由抑制IkappaB-alpha磷酸化及NF-kappaB次單元p65及RelB核轉移幅度來降低NF-kappaB轉錄因子活性。脂多醣/干擾素-gamma刺激增加轉錄因子IRF-1蛋白質表現，木犀草素顯著抑制IRF-1的誘導表現。脂多醣/干擾素-gamma刺激可增加STAT-1及STAT-3蛋白質磷酸化導致轉錄活性上升。木犀草素抑制STAT-1及STAT-3蛋白質磷酸化及轉錄活性。進一步分析發現，雖然木犀草素不影響STAT上游活化激?Jak1、Jak2、Tyk2活性，卻會抑制另一個STAT上游活化激?Src活性，此外，木犀草素可誘發STAT活性抑制蛋白質SOCS3的表現。本研究結果發現，木犀草素可透過干擾ERK、JNK、p38、Akt、Src等訊息傳遞分子及NF-kappaB、IRF-1、STAT-1、STAT-3等轉錄因子活性進而顯現中樞抗發炎功效。
Microglia, resident immune cells in the central nervous system (CNS), respond to extracellular insults by releasing diversity of pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF-alpha), interleukin 1beta(IL-1beta), and IL-6, leading to the initiation and promotion of inflammation. Evidence indicates that over-activated microglia and the associated pro-inflammatory mediators cause neural cell damage and contribute to several neurological disorders such as Alzheimer’s disease. Luteolin, a flavonoid compound, exhibits anti-oxidative, anti-neoplastic, anti-allerigc, and anti-inflammatory effects. Study suggests the neuroprotective effect of luteolin and proposes an anti-inflammatory mechanism. While the anti-inflammatory action is well demonstrated in the peripheral system, the role and potential action mechanisms of luteolin against inflammation in the CNS are largely unclear. The study was aimed to elicit the potential anti-inflammatory effect and mechanisms of luteolin against inflammatory responses in BV2 microglia cell lines after lipopolysaccharide (LPS)/interferon (IFN-gamma) stimulation. LPS/IFN-gamma stimulation caused BV2 cells elevating several pro-inflammatory mediators biosynthesis and releasing such as TNF-?, iNOS, IL-6, and cyclooxygenase-2. Non-toxic level of luteolin reduced LPS/IFN-gamma-induced pro-inflammatory mediator production at the level of mRNA and protein. That is, the anti-inflammatory effect of luteolin is mediated by transcriptional regulation. Generally, intracellular signaling molecules such as ERK, JNK, p38, Akt, Jak family members, and Src could transduce extracellular signals to induce gene expression particularly pro-inflammatory genes through the modulation of transcription factors. Transcription factors such as NF-kappaB, IRF, and STAT are commonly downstream effectors of these signaling pathways. The inhibition of ERK, JNK, p38, Akt, and Jak activity by pharmacological inhibitors attenuated LPS/IFN-gamma-induced pro-inflammatory mediator production. LPS/IFN-gamma stimulation increased ERK, JNK, p38, Akt, Jak1, Jak2, Tyk2, and Src activity in BV2 cells, as evidenced by the elevated protein phosphorylation. Luteolin decreased ERK, JNK, p38, Akt, and Src but not Jak1, Jak2, and Tyk2 activity in LPS/IFN-gamma-treated cells. LPS/IFN-gamma stimulation caused IkappaB-alpha? phosphorylation and triggered p50, p65, and RelB NF-kappaB subunit nuclear accumulation, indicating an activation of NF-kappaB. Luteolin attenuated LPS/IFN-gamma-induced IkappaB-alpha phosphorylation and p65 and RelB nuclear accumulation leading to a resolution of NF-kappaB activation. LPS/IFN-gamma increased IRF-1 protein expression resulting in activation and the increased IRF-1 expression was attenuated by luteolin. LPS/IFN-gamma stimulated STAT-1 and STAT-3 activity through the increased protein phosphorylation. Luteolin reduced LPS/IFN-gamma-induced STAT-1 and STAT-3 phosphorylation. Although luteolin had little effect on Jak family protein activity, another STAT upstream activator Src was inactivated in LPS/IFN-gamma stimulated cells by luteolin. Besides, luteolin increased SOCS3 protein expression, a negative regulator of STAT activation. Taken together, our experimental findings indicate that the anti-inflammatory effects of luteolin in microglia cells could be mediated by the down-regulation of ERK, JNK, p38, Akt, Src signaling molecules and NF-kappaB, IRF-1, STAT-1, and STAT-3 transcription factors. |
