脂肪酸-白蛋白複合物於老鼠巨噬細胞發炎及凋亡的角色

The Role of Fatty Acid-Albumin Complex in Murine Macrophage Inflammation and Apoptosis

張綺芬(Chi-Fen Chang)

指導教授 ： 盧國賢

國立臺灣大學/醫學院/解剖學暨生物細胞學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01050

摘要

目的: 血漿中的脂肪酸-白蛋白複合物在生理及病理過程中是非常重要的。脂肪酸-牛血清白蛋白複合物是否可以誘導巨噬細胞的發炎及凋亡反應仍不清楚。在本文中我們檢視脂肪酸-牛血清蛋白的複合物對巨噬細胞的影響並探討其機制。
方法:一氧化氮的測量藉由格里斯方法;前列腺素E2,介白質-6及腫瘤壞死因子-α藉由酶連免疫吸附檢定;誘生型一氧化氮合成酶及環氧合成酶藉由西方墨點法測得;NF-κB及CD14/TLR4藉由西方墨點法及流式細胞儀測得。細胞型態的改變藉由光學顯微鏡觀察。氧化壓力及粒線體膜電位產生透過流式細胞儀做檢視。caspase-9,-3,PARP 的活化及cytochrome c 由粒線體細胞膜間質的釋出量藉由西方墨點法測得。
結果:單獨對無脂多醣體處理的RAW264.7細胞給予牛血清白蛋白,飽和或不飽和脂肪酸-牛血清白蛋白的複合物處理並不會造成發炎反應(一氧化氮,前列腺素E2,細胞激素的釋放或誘生型一氧化氮合成酶及環氧合成酶,CD14的產生, IκB 降解, NF-κB核轉移) 。不飽和脂肪酸-牛血清白蛋白的複合物前處理明顯降低脂多醣體引起的發炎反應。相對的,飽和脂肪酸-牛血清白蛋白的複合物前處理增加了脂多醣體引起的發炎因子及隨後的反應。飽和及不飽和脂肪酸-牛血清蛋白的複合物經由氧化壓力產生,啟動粒線體膜電位的去極化, cytochrome c的釋放及caspase-9, -3,及PARP的活化,最後造成細胞的凋亡。

關鍵字

脂肪酸-牛血清白蛋白複合物 ； 發炎反應 ； 細胞激素 ； 巨噬細胞

並列摘要

Objective Fatty acid (FA)-albumin complexes in plasma are very important for physiological and pathological situations. Whether FA-bovine serum albumin (BSA) complexes affect inflammation and induce cell apoptosis in macrophage is still unknown. In the present study, we examined the effects of FA–BSA complex on macrophages and investigated the underlying mechanisms.
Methods For the inflammation of RAW264.7 cells, the release of nitric oxide (NO) was estimated by the Griess assay, prostaglandin (PG) E2, interleukin (IL)-6 and tumor necrosis factor (TNF)-α were estimated by Enzyme-Linked ImmunoSorbent Assay (ELISA), the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 by Western blotting, nuclear factor (NF)-κB and cluster of differentiation 14 (CD14) / toll-like receptor 4 (TLR4) by Western blotting or flow cytometry, and cell morphology by optical microscopy. For the apoptosis of RAW264.7 cells, the production of Reactive oxygen species (ROS) and mitochondrial membrane potential were estimated by flow cytometry, and the activation of caspase-9, -3, Poly (ADP-ribose) polymerase (PARP) and the amount of cytochrome c (cyt c) released from mitochondrial intermembrane space were examined by Western blot.
Results BSA, saturated FA-BSA (SFA-BSA) or unsaturated FA-BSA (UFA-BSA) pre-treatments could not cause inflammatory responses (release of NO, PGE2, and cytokines; production of iNOS, COX-2, and CD14; inhibitor (I)-κB degradation and NF-κB translocation) in RAW264.7 macrophages. Pre-treatment with UFA-BSA complexes significantly decreased the lipopolysaccharide (LPS)-induced inflammatory responses. On the contrary, pre-treatment with SFA-BSA complexes enhanced LPS-induced inflammatory factors and the subsequent responses. SFA- and UFA-BSA complex can cause cell apoptosis via stimulating the generation of ROS, triggering depolarization of mitochondrial membrane, the release of cyt c, and activation of caspase-9, -3, and PARP.

並列關鍵字

FA-BSA complex ； Inflammatory response ； Cytokine ； Macrophage

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