探討黃嘌呤衍生物於肝臟之抗發炎作用機轉以及在 3T3-L1 脂肪細胞的脂肪分解作用

Anti-inflammation Signaling in Livers and Lipolysis in 3T3-L1 Adipocytes by Xanthine Derivative

陳諭勳(Yu-Hsun Chen)

指導教授 ： 吳炳男

高雄醫學大學/醫學院/醫學系藥理學科碩士班/碩士(2015年)

摘要

本研究主要探討茶鹼衍生物 KMUP-1調控HSL/p-HSL/PKA/PKG/ATGL和MMP-9/TNF-的路徑，以及此藥物是否可以會影響脂肪肝中肥胖和發炎相關的吞噬細胞穿透性。
此次實驗使用兩個研究脂肪代謝的模型，分別為高脂肪飼料誘導C57BL/6J肥胖小鼠動物模型以及3T3-L1前驅纖維母細胞(3T3-L1 pre-adipocyte) 分化為脂肪細胞模型。在高脂肪飼料誘導C57BL/6J肥胖小鼠動物模型中，自第0週到第14週，KMUP-1預防組給予KMUP-1-HCl(2.5 mg/200 ml)，KMUP-1治療組則是在第8週到14週給予KMUP-1-HCl (2.5 mg/200 ml)。3T3-L1前驅纖維母細胞(3T3-L1 pre-adipocyte) 分化為脂肪細胞模型則是被用來研究油滴脂質累積及促進成熟脂肪細胞的脂解作用，其標準的誘導試劑為加入insulin (10 g/ml)、dexamethasone (0.25 M) 、3-isobutyl-1 methylxanthine (0.5 mM) (總稱為IDM)之細胞生長促進劑48小時後，充分分化細胞，形成油滴脂質累積，且使用紅油染色法(oil red-O stain)觀察細胞內油滴之大小及分布情形。在3T3-L1脂肪細胞的脂解作用期，以免疫細胞化學法(Immunofluorescence)來偵測脂肪細胞中phoso-Hormone sensitive lipase (p-HSL)之表現，以評估藥物促進脂解作用的能力。
在高脂肪飼料誘導C57BL/6J肥胖小鼠動物模型中，KMUP-1可以減輕肝臟和小鼠其他的的重量，且其肝臟中的油滴直徑和數量亦有減少的。KMUP-1減少肝臟中的TNFα 和MMP-9表現，以及染CD11c的M1吞噬細胞。而在肝臟中，KMUP-1 活化p-HSL、ATGL 和染上CD209a的M2吞噬細胞。在高脂肪飼料誘導C57BL/6J肥胖小鼠動物的肝臟中，可以看見馬洛氏小體(Mallory's Hyaline Bodies)，並且和HFD 組別相較之下，在治療組以及預防組都可以看到其減少。在3T3-L1脂肪細胞中可以看到，KMUP-1、 Caffeine和Theophylline對於增加p-HSL都有劑量依賴性。在紅油染色法(oil red-O stain)中，可以減少3T3-L1脂肪細胞的油滴累積程度。
我們的研究結果顯示KMUP-1在高脂肪飼料誘導C57BL/6J肥胖小鼠動物模型中，有減肥和減少脂質的累積及分布的效果。並且，KMUP-1可以減少3T3-L1脂肪細胞的油滴脂質累積作用以及促進其脂解作用。

關鍵字

3T3-L1 脂肪細胞 ； 脂肪分解 ； 抗發炎

並列摘要

Background:
Our study aims to investigate whether the xanthine derivative, KMUP-1 regulates HSL/p-HSL/PKA/PKG/ATGL and MMP-9/TNF- pathways, and also macrophage infiltration that are associated with the modulation of adiposity and inflammation in fatty liver.

Materials and Methods:
High-fat diet (HFD)-induced obese mice (in vivo) and 3T3-L1 pre-adipocyte differentiated to adipocyte (in vitro), both models were used to investigate lipid metabolisms. From week 0 to week 14, the HFD-induced obese C57BL/6J mice were administered with KMUP-1-HCl (2.5 mg/200 ml) for prophylaxis and the other group for treatment were treated with KMUP-1-HCl (2.5 mg/200 ml) from week 8 to week 14. 3T3-L1 pre-adipocytes were used to study adipogenesis and lipolysis. The standard hormonal medium containing insulin (10 μg/ml), dexamethasone (0.25 μM) and isobutyl-methylxanthine (0.5 mM) was used to induce cell adipogenesis. Immunofluorescence technique was used to detect the phospho-hormone sensitive lipase (p-HSL) protein expression in 3T3-L1 adipocytes and to estimate the responses of lipolysis.
Results:
In HFD-induced obese C57BL/6J mice, KMUP-1 attenuated the liver and body weight gain and the diameter and number of hepatic oil globulets. KMUP-1 also decreased the expression of TNFα and MMP-9. M1 macrophages were stained with CD11c. Activated p-HSL, ATGL and M2 macrophages were stained with CD209a. Mallory's Hyaline Bodies were found in HFD-induced hyperadiposity of liver slices, and decreased in the treatment and protection groups. In 3T3-L1 adipocytes, KMUP-1 increased p-HSL in a dose-dependent manner similar to caffeine and theophylline. In the Oil Red O staining, KMUP-1 decreased the oil globulet accumulation in a dose-dependent manner.
Conclusion:
Our result showed that KMUP-1 not only decreased adiposity, lipid accumulation and mobilization in HFD-induced obese mice, but also induced lipolysis of 3T3-L1 adipocytes. Thus, KMUP-1 could have potential benefits in preventing HFD-induced obesity and fatty liver.

並列關鍵字

3T3-L1 Adipocytes ； Lipolysis ； Anti-inflammation

參考文獻

參考文獻

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