- 學位論文
探討中國橄欖萃取物對TNBS誘導腸炎小鼠的改善功效
Effects of Chinese Olive (Canarium album L.) Fruit Extract on Attenuating TNBS-Induced Colitis in Mice
摘要
發炎性腸道疾病是指消化道的慢性發炎疾病。主要分為潰瘍性結腸炎（UC）和克隆氏症（CD）。發炎性腸道疾病的發病率在亞洲持續增加，但發病的根本原因仍不清楚。目前最常使用的治療藥物有抗生素，例如：5-ASA，更嚴重的患者會使用生物製劑，例如：TNFα 的抑製劑英利昔單抗（Infliximab）。然而，長期使用這些藥物可能造成嚴重的副作用，因此仍迫切需要探究可以有效緩解發炎性腸道疾病且降低副作用的新療法。在本篇研究中，我們評估中國橄欖（Canarium album L.）是否具有可以緩解發炎性腸道疾病的潛力。由於在過去的研究中，我們已經發現中國橄欖的乙酸乙酯層萃取物（在本文中稱為COE）具有抗發炎的特性，在本研究中，我們首先使用 RAW264.7 小鼠巨噬細胞的平台來再次確認 COE 的抗發炎能力。我們觀察了幾種促炎介質的表現量，包含一氧化氮 (NO)、誘導型一氧化氮合成酶 (iNOS)、環氧合酶-2 (COX-2)、TNFα、IL-1β和 IL-6。冷光素酶檢測法的結果表明，與脂多醣（LPS）誘導組相比，以400 mg/L濃度的COE處理可顯著抑制iNOS啟動子的活性。此外，亞硝酸鹽檢測試驗的結果表明，濃度為 100、200、400 和 800 mg/L的COE 均能顯著抑制 LPS 誘導產生的 NO含量。 COE對LPS誘導的iNOS和COX-2蛋白質的表現量也有抑制作用。除此之外，我們也發現COE可以抑制RAW264.7細胞中促炎細胞因子的mRNA表現量，包含IL-1β和 IL-6。這些細胞實驗的發現促使我們更進一步研究 COE 對 2,4,6-三硝基苯磺酸（TNBS）誘導的急性克隆氏症模式小鼠的改善作用。在TNBS誘導的結腸炎組中，可觀察到疾病活動指數（DAI）相較於對照組有顯著的上升，然而COE治療組與結腸炎組之間無顯著差異。另一方面，與結腸炎組相比，治療組的組織學活性指數（HAI）則有顯著的降低，表明 COE 降低了結腸組織的發炎反應和上皮損傷的嚴重程度。我們還分析了 TNFα、IL-1β和 IL-6 在結腸組織中和血清中的表現量。與結腸炎組相比，治療組在近端結腸組織中的TNF-α 和IL-1β 的mRNA表現量均降低。儘管COE沒有降低IL-6在結腸組織中的mRNA表現量，但IL-6在血清中的濃度則在治療組中有所降低，甚至低於對照組。此外，COE 也降低了結腸組織中的 COX-2 蛋白質表現量。總結來說，我們的研究證實了中國橄欖萃取物表現出抗發炎的能力，並具有改善 TNBS 誘導結腸炎小鼠發炎症狀的潛力，表明 COE 可能作為預防和治療發炎性腸道疾病的天然療法。未來我們將進一步評估 COE 和現有治療發炎性腸道疾病的生物製劑（如Infliximab）之協同治療是否有更好的療效。
並列摘要
Inflammatory bowel disease (IBD) is featured by chronic immune activation and inflammation in the gastrointestinal tract. The two major forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). Incidences of IBD have been increasing in Asia but the underlying cause of IBD is still unclear. Currently, the most commonly-used treatments for IBD are antibiotics such as 5-ASA, and biologics such as TNF-α inhibitor infliximab for more severe patients. However, long-term usage of these drugs still causes inevitable side effects. Therefore, exploring new treatments to effectively relieve colitis is urgently needed. In this study, we evaluate whether Chinese olive (Canarium album L.) is a potential functional food ingredient in ameliorating IBD since we have revealed that the Chinese olive fruit extract (referred to as “COE” in this thesis) exhibited anti-inflammatory activities in the previous studies. In this study, we first used RAW264.7 mouse macrophage-based platform to reconfirm the anti-inflammatory ability of COE. We investigated the expression of several pro-inflammatory mediators, including nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF, IL-1, and IL-6. The results of luciferase assay showed that the treatment of COE with the concentration of 400 mg/L could significantly inhibit the iNOS promoter activity compared to the lipopolysaccharide (LPS)-stimulated group. Also, the results of Griess assay showed that the treatment of COE with the concentrations of 100, 200, 400, and 800 mg/L all significantly suppressed the LPS-induced NO production. COE also exerted an inhibitory effect on the expression of iNOS and COX-2 protein in LPS-induced RAW264.7. Moreover, we have found that COE can inhibit the mRNA levels of pro-inflammatory cytokines, including IL-1 and IL-6, in RAW264.7 cells. These findings motivated us to further examine the ameliorative effects of COE on acute CD murine models induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS). The disease activity index (DAI) in the TNBS-induced colitis group was significantly increased compared to the control group, but there is no significant difference between the colitis group and the COE treatment group. However, the histological activity index (HAI) of colon tissue in the treatment group was significantly decreased compared to the colitis group, suggesting that COE reduced the severity of inflammatory responses and epithelial damages. We also analyzed the expression of TNF, IL-1, and IL-6 in colon tissue and in serum. In the proximal colon, the mRNA expression of TNF-α and IL-1β was reduced in the treatment group compared to that in the colitis group. Even though the mRNA expression of IL-6 was not downregulated by the treatment of COE, its concentration in serum was restored in the treatment group and even lower than in the control group. Also, COE treatment downregulated the protein expression of COX-2 induced in the colitis group in colon tissue. In conclusion, our data demonstrated that Chinese olive fruit extract exhibited the ability to ameliorate the inflammatory condition in the proximal colon of TNBS-induced colitis mice, suggesting that COE could be targeted as a potential natural therapeutic for the prevention and treatment of inflammatory colitis diseases. In our future work, we will further evaluate the effect of COE on enhancing the therapeutic effect of the biologics, such as Infliximab (IFX).