- 學位論文
桑黃萃取物Hispolon抑制腦部微膠細胞發炎反應之機制探討
Inhibitory mechanism of Hispolon on the inflammatory responses of microglia cells
摘要
微膠細胞在調控神經發炎反應上扮演重要的角色，它們為各種神經退化性疾病病理發展的中心關鍵要角。因此，可以調節微膠細胞活化狀態的化合物可能具有治療神經退化性疾病的潛力。先前有研究指出，從桑黃中所分離出的Hispolon具有抗腫瘤的活性，然而， Hispolon (HIS)的抗發炎活性主要透過的作用分子標的以及作用機制仍然不清楚。本實驗探討了HIS對脂多醣(LPS)及脂磷壁酸(LTA)誘導微膠細胞株BV-2之iNOS/NO產生的影響，本研究成果發現了HIS能有效抑制LPS及LTA誘導的NO產生且此效果具劑量依存性;同時，在HIS處理下，LPS及LTA誘導iNOS蛋白表現亦顯著被抑制。此外，HIS的給予會造成BV-2細胞株血紅素氧化酵素-1 (HO-1)蛋白表現量的增加，且經由各種Kinase抑制劑證實了HIS誘導HO-1蛋白表現會被ERK抑制劑PD98059所抑制，這結果指出在BV-2細胞中，ERK活化參與了HIS所誘導之HO-1蛋白表現。 此外，LPS及LTA處理之BV-2中,JNK抑制劑SP600125能有效抑制iNOS/NO之產生，結果同樣也發現HIS可抑制LPS及LTA誘導JNK磷酸化並抑制下游轉錄因子NF-kB ,c-Jun的活化最後造成NO生成及iNOS蛋白表現減少;進一步我們發現LPS及LTA能誘導BV-2細胞走向細胞凋亡，此細胞凋亡的結果能被NOS抑制劑L-NAME及HIS所抑制。此外，以HO-1抑制劑SnPP處理下， HIS抑制LPS及LTA誘導NO生成之活性則顯著降低。 因此本研究結果證實了HIS能經由誘導HO-1蛋白表現來抑制LPS及LTA誘導NO產生而達到保護BV-2細胞免於其受LPS及LTA誘導之細胞凋亡。因此, HIS或許可以藉由減輕在各種神經退化性疾病的病程進展過程中的微膠細胞過度發炎狀態而作為一個有潛能的治療藥物。
並列摘要
Elevation of Nitric Oxide (NO) production via stimulating inducible nitric oxide synthase (iNOS) has been shown in activated microglia cells during brain inflammation.Therefore, agents with ability to reduce iNOS/NO production in activated microglia cells possess beneficial effects in the treatment of several neurodegenerative diseases.Hispolon (HIS), a compound derived from Phellinu linteus, has been shown to possess several beneficial effects such as antioxidant, inhibition of tumor growth and metastasis. The anti-inflammatory effects of HIS in microglia,however, remains unclear. In the present study, HIS exhibited concentration-dependent inhibition of LPS/LTA-induced iNOS protein expression/NO production accompanied by Heme Oxygenase-1 (HO-1) induction in microglia cells BV-2. Also, time and concentration-dependent induction of HO-1 protein by HIS was identified in BV-2 cells. Application of ERK inhibitor U0126 reduced HO-1 protein expression elicited by HIS, indicating ERK activation participated in HIS-induced HO-1 protein expression. Additionally, LPS/LTA-induced JNK phosphorylation was suppressed by adding HIS in BV-2 cells. Also, inhibition of LPS/LTA-induced NF-kB P65 nuclear translocation, I?羠 protein degradation and c-Jun protein expression in BV-2 cells under HIS treatment were observed. Furthermore, HIS and NOS inhibitor significantly protected against LPS /LTA-induced apoptosis, characterized by DNA fragmentation assay and Caspase 9 activation. Moreover, inhibitory effect of HIS on LPS/LTA-induced NO production was attenuated by addition of HO-1 inhibitor SnPP, suggesting that HO-1 induction in part participated in anti-inflammatory actions of HIS. In conclusion, data of the present study provided evidence supporting the inhibitory effects of HIS on LPS/LTA-induced inflammatory responses in microglia cells BV-2.Furthermore, beneficial effects of HIS in protection of BV-2 cells from apoptosis via reducing LPS/ LTA-induced iNOS protein expression/NO production were also demonstrated.