硫辛酸影響小鼠腦微膠細胞株BV-2對寡聚態乙型類澱粉樣蛋白吞噬作用之研究

Abstract

本研究探討維生素B群、硫辛酸與牛磺酸等對小鼠腦微膠細胞株(microglia) BV-2吞噬及清除寡聚態類澱粉樣蛋白(oligomeric beta-amyloid, oAβ1-42)之影響，評估這些營養素是否能藉由降低過多的oAβ1-42堆積以延緩阿茲海默氏症之進展。 實驗首先建立體外小鼠微膠細胞株BV-2攝入oAβ1-42之細胞模式。將Aβ1-42粉末溶於F12 medium中24小時進行聚集反應以製備oAβ1-42。穿透式電子顯微鏡(transmission electron microscopy, TEM)型態觀察顯示所製備的oAβ1-42為聚集型球狀構造；以分析型超高速離心機(analytical ultracentrifugation, AUC)進行沉降係數分析，結果顯示製備的oAβ1-42分子量大多介於100-300 kDa之間。在細胞實驗方面，MTT分析結果發現，在0.1 mM的樣品濃度下測試樣品thiamin、riboflavin、nicotinamide、pyridoxine、cobalamin、folic acid、taurine、α-lipoic acid (ALA)對BV-2細胞皆無明顯之毒性產生。接著，以此樣品濃度處理經1 μM螢光分子標定的oAβ1-42 (FAM-oAβ1-42) 共培養24小時之小鼠微膠細胞株BV-2以進行攝入試驗，結果發現，在所有樣品中以ALA對BV-2細胞具有最佳的促進攝入FAM-oAβ1-42之效果。進一步以real-time PCR對microglia細胞表面上與oAβ吞噬作用相關之接受器(receptor)，包括scavenger receptor A1 (SR-A1)、scavenger receptor B1 (SR-B1)、CD36、receptor for advanced glycation end-products (RAGE)之基因表現進行分析。結果發現，與 oAβ1-42共同培養的BV-2細胞其CD36基因表現會隨著ALA處理濃度增加而升高，然而其他接受器則無；再則，以ALA處理未與oAβ1-42共同培養的BV-2細胞時也觀察到其CD36基因的表現有上升之情形。免疫細胞染色法(immunocytochemistry, ICC)結果顯示，無論是否與oAβ1-42共同培養，ALA的處理均會增加BV-2細胞CD36接受器之表現量，但添加CD36接受器阻斷劑(CD36抗體)則會顯著地減少ALA提升BV-2細胞對FAM-oAβ1-42之攝入能力。ICC分析結果亦發現，ALA處理會增加BV-2細胞中CD36的轉錄因子PPAR-γ (peroxisome proliferator-activated receptor-γ)之轉位作用(translocation)。Enzyme-linked immunosorbent assay (ELISA)分析發現，ALA處理會增加BV-2細胞對15-deoxy-△12,14- prostaglandin J2的產生。Real-time PCR以及western blotting分析結果發現，經ALA處理的BV-2細胞其COX-2基因與蛋白質之表現均有明顯增加之趨勢。根據上述結果，我們推測ALA可能是透過COX-2/15-deoxy-△12,14-prostaglandin J2/PPAR-γ/CD36路徑，提升BV-2細胞膜上CD36接受器之表現、增加其對oAβ1-42進行辨識，進而促進對oAβ1-42之吞噬作用。

The aim of this study is to investigate the effect of vitamin B complex (including thiamin, riboflavin, nicotinamide, pyridoxine, cobalamin, folic acid), vitamin-like α-lipoic acid (ALA), and taurine on enhancing phagocytosis of oAβ1-42 in BV-2 mouse microglia cells. An in vitro model was established to investigate phagocytosis of oAβ1-42 in BV-2 mouse microglia cells. The image of transmission electron microscopy (TEM) indicated that the morphology of prepared oAβ1-42 was spherical particles. In addition, the data from analytical ultracentrifugation (AUC) showed that Aβ1-42 assembled by themselves to form oligomers with molecular size of 100-300 kDa principally. The MTT test revealed non-cytotoxicity of all tested compounds at concentration up to 100 μM on BV-2 cells. The BV-2 microglia cells were incubated with 1 μM 5(6)-carboxyfluorescein-labeled oAβ1-42 (FAM-oAβ1-42) for 24 h followed by flow cytometer analysis to assess the in vitro phagocytosis ability of oAβ1-42. The results showed that ALA exhibits the highest enhancing effect on phagocytosis of FAM-oAβ1-42 in BV-2 mouse microglia cells among all tested samples. Furthermore, the mRNA expressions of oAβ1-42 phagocytosis-related receptors of microglia were analyzed by real-time PCR for elucidating the phagocytosis mechanisms, and the results indicated that ALA significantly increases the mRNA expression of CD36 receptor in BV-2 cells. However, no significant difference was observed for other tested receptors, including scavenger receptor A1, scavenger receptor B1, and the receptor for advanced glycation end-products (RAGE). Interestingly, when treated with ALA alone, CD36 mRNA expression also increased in BV-2 cells even though without oAβ1-42 stimulation. The immunocytochemistry (ICC) analysis showed that ALA significantly elevated CD36 protein expression in BV-2 cells whether with or without oAβ1-42 treatment. Moreover, the results from flow cytometry analysis indicated CD36 receptor blocking antibody, the CD36 receptor inhibitor, significantly attenuated ALA-promoted phagocytosis of FAM-oAβ1-42 in BV-2 cells. The ICC analysis revealed that ALA caused translocation of PPAR-γ, which was known to regulate expression of CD36 mRNA, in BV-2 cells obviously. The real-time PCR and western blotting also revealed that ALA elevated both mRNA and protein expression of cyclooxygenase-2 (COX-2), a key enzyme that involved in the synthesis process of 15-deoxy-△12,14- prostaglandin J2, in BV-2 mouse micrgolia cells. Conclusively, we postulated that ALA enhances oAβ1-42 phagocytosis via up-regulation of COX-2/15-deoxy-△12,14-prostaglandin J2/PPAR-γ/CD36 pathway in BV-2 cells.