從物理化學特性探討魷魚皮磷脂質微脂體對抗發炎及抗蝕骨細胞新生作用之影響

Abstract

在臺灣及許多開發中和已開發國家，發炎相關慢性病被認為是各國醫療及健康照護系統中最沉重的負擔。有效的解除炎症反應是降低發炎相關疾病發生的基礎。近年來，研究指出受損組織中的凋亡細胞是啟動發炎緩解程序的關鍵，因此，本論文主要從物理化學特性上，探討利用美洲大赤魷(jumbo flying squid, Dosidicus gigas)皮膜萃取的磷脂質(phospholipids)，製備含有磷脂醯絲胺酸(phosphatidylserine, PS)的魷魚皮微脂體(squid-skin liposome, 以下簡稱為SQ liposome)來模擬凋亡細胞的可行性，並分別在一些病原因子(pathogen-associated molecular pattern, PAMP)例如：細菌脂多醣(lipopolysaccharide, LPS)及鹿藻菜膠(carrageenan)，或組織損傷因子(damage-associated molecular pattern, DAMP)例如：核因子-B配體受體致活劑(receptor activator of nuclear factor kappa-B ligand, RANKL)的誘導發炎模式中，研究SQ liposome對緩解發炎的功效。本論文第一部分的研究中，首先SQ liposome的物理化學及免疫學特性被仔細的研究，其主要組成分包含：磷脂醯肌醇(phosphatidylinositol, PI)、磷脂醯絲胺酸(phosphatidylserine, PS)、磷脂醯乙醇胺(phosphatidylethanolamine, PE)、磷脂醯膽鹼(phosphatidylcholine, PC)及溶血磷脂醯膽鹼(Lyso-phosphatidylcholine, Lyso-PC)，其大小粒徑約100 nm。添加濃度7.5 mg/mL以下，對小鼠腹腔巨噬細胞株(RAW 264.7)不具明顯細胞毒性(p>0.05)；在脂多醣誘導RAW 264.7細胞發炎的實驗中，預處理0.09～7.5 mg/mL SQ liposome顯著的調降(p<0.05)第一型介白素-(interleukin-1 beta, IL-1)、第六型介白素(interleukin-6, IL-6)及腫瘤壞死因子-(tumor necrosis factor-α, TNF-α)、前列腺素E2(prostaglandin E2, PGE2)、一氧化氮(nitric oxide, NO)的分泌；並且吞噬0.28～7.5 mg/mL SQ liposome的RAW 264.7細胞，因LPS誘導在細胞質中所產生的活性氧自由基濃度明顯較低。此外，給予每公斤老鼠體重60 mg的SQ liposome加速緩解鼠爪因注射鹿藻菜膠所引起腫脹。本論文的第二部分研究更進一步解析了SQ liposome內的磷脂質上脂肪酸組成及其微脂體表面所帶膜電位的差異，同時探討SQ liposome對抑制蝕骨細胞新生的功能及機制。結果顯示：單層的SQ liposome表面帶有負電荷，組成微脂體的磷脂質中含有28.7 %的二十二碳六烯酸(docosahexaenoic acid, DHA)及11.8%的二十碳五烯酸(eicosapentaenoic acid, EPA)；在抑制蝕骨細胞新生的實驗中，核因子κ-B配體受體致活劑(receptor activator of nuclear factor kappa-Β ligand, RANKL)與巨噬細胞選殖生長因子(macrophage colony stimulating factor, M-CSF)共同誘導的RAW 264.7細胞會分化為成熟的蝕骨細胞，同時會促進PGE2和乙型細胞轉型生長因子(transforming growth factor beta, TGF-)的分泌；然而添加1 mg/mL的SQ liposome共處理顯著的回復RANKL/M-CSF誘導壓力下的細胞活存率(p<0.05)，添加0.33～1 mg/mL SQ liposome則有效調降PGE2 (p<0.05)，但對TGF-的調節則不顯著(p>0.05)。另外，添加0.33～1 mg/mL SQ liposome則顯著抑制蝕骨細胞的成熟，例如：減少多核蝕骨細胞的數目及減少蝕骨斑塊的面積、抑制抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase)活性，並且下調多種蝕骨細胞新生相關訊息核醣核酸(mRNA)的表現(p<0.05)。總結，SQ liposome有如擬態凋亡細胞一樣能啟動發炎的緩解程序，亦可能藉由抑制蝕骨細胞的新生作用來避免骨質流失。在本研究結果的基礎上，未來則需要更多的動物試驗來驗證SQ liposome在活體內的實際保健功效。

Not only in Taiwan but also in other developing or developed countries, chronic inflammatory diseases have been recognized as the major burden of the health care system. An effective resolution on the inflammatory reaction is the fundamental prevention against inflammation-induced diseases. Recently, an apoptotic cell from the injured tissues has been reported to trigger the initiation of inflammatory resolution. Hence, this dissertation focused on the manufacture and physicochemical properties of an apoptotic mimicry of squid-skin liposome (SQ liposome) from the phospholipid extract of the squid (jumbo flying squid, Dosidicus gigas) skin. The inflammatory resolving effects of the SQ liposomes were further investigated on the pathogen-associated molecular pattern (PAMP) induced inflammatory models such as lipopolysaccharide or carrageenan, and on the damage-associated molecular pattern (DAMP) induced inflammatory model like as receptor activator of nuclear factor kappa-B ligand (RANKL). In the first study of this dissertation, the physicochemical and immunological properties of the SQ liposomes were completely investigated. This SQ liposome was majorly composed of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and lyso-phosphatidylcholine (Lyso-PC) while its size was an approximate diameter of 100 nm. There was no (p>0.05) cytotoxicity on RAW 264.7 cells below a concentration of 7.5 mg mL−1 of SQ liposome. The SQ liposome pretreatment on lipopolysaccharide (LPS)-induced RAW 264.7 cells could decrease (p<0.05) secretions of prostaglandin E2 (PGE2), nitric oxide (NO), interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α). The engulfment of SQ liposomes by the RAW 264.7 cells resulted in lower (p<0.05) LPS-induced intracellular levels of reactive oxygen species. Furthermore, the SQ liposome administration demonstrated an amelioration (p<0.05) of carrageenan-induced paw edema in mice. In the second study of this dissertation, the intermolecular fatty-acid composition and zeta potential (ζ) of the SQ liposomes were further analyzed. Simultaneously, the function and mechanism of the SQ liposome on anti-osteoclastogenesis effects were elucidated in an in-vitro cell-based assay. The results indicated that SQ liposomes contain 28.7% docosahexaenoic acid (DHA) and 11.8% eicosapentaenoic acid (EPA) and own a single layer with negative charges on the surface. An anti-osteoclastogenesis cell-based assay indicated that co-treatment with RANKL/macrophage colony-stimulating factor (M-CSF) induces RAW 264.7-cell differentiation into mature osteoclasts, thus enhancing PGE2 and TGF- secretion. However, co-treatment with one mg mL-1 of SQ liposome restored (p<0.05) the cell viabilities under the RANKL stress. Increased PGE2 levels were decreased (p<0.05) by co-treating 0.11 and 0.33 mg mL-1 of SQ liposome, but the TGF- levels were not (p>0.05) influenced in the SQ liposome cotreatments. Co-treatments with 0.331 mg mL-1 of SQ liposome suppressed (p<0.05) the osteoclast maturation (such as decreased multinucleated cells [MNCs] and bone pit formation), inhibited TRAP activities, and down-regulated the osteoclastogenesis related gene expressions. In summary, the SQ liposomes may act as an apoptotic mimicry to elicit the resolution of inflammation and prevent bone loss through the suppression of osteoclastogenesis. Based on the current results, more in vivo studies warrant further investigation.