共軛亞麻油酸對脂多醣體及干擾素-γ誘發小鼠MES13腎絲球細胞發炎反應之效應及相關機轉

腎絲球腎炎 (Glomerulonephritis) 是造成腎衰竭 (Renal failure) 的原因之一。腎絲球 (Glomerulus) 中的腎絲球細胞 (mesangial cells) 可被細菌內毒素LPS (Lipopolysaccharide) 和/或細胞激素 (cytokines) 刺激活化，誘發inducible nitric oxide synthase (iNOS) 基因表現，所以iNOS的誘發及nitric oxide (NO) 的大量合成與腎絲球發炎有著極密切關係。本實驗室證實在活化態的RAW264.7巨噬細胞中，共軛亞麻油酸 (Conjugated linoleic acid, CLA) 可經由抑制NF-κB的活化而減少LPS誘發RAW264.7巨噬細胞iNOS及cyclooxygenase-2 mRNA及蛋白質的表現及NO和PGE2生成而有抗發炎之功效，故本實驗主要目的是探討CLA對LPS及Interferon-γ (IFN-γ) 活化小鼠MES13腎絲球細胞發炎反應之效應和其相關調控機轉。MTT分析顯示，高達100 μM CLA mixture、9Z11E CLA或10E12Z CLA分別在與1μg/ml LPS及5ng/ml IFN-γ共處理18小時下，小鼠MES13腎絲球細胞存活率並不受影響；10-100 μM CLA以劑量依賴關係顯著抑制了在1 μg/ml LPS及5 ng/ml IFN-γ活化下，小鼠MES13腎絲球細胞iNOS mRNA、蛋白質表現及NO生成 (p<0.05)；進一步實驗分析，也顯示在1 μg/ml LPS及5 ng/ml IFN-γ活化下，100 μΜ CLA抑制Inhibitor κB-a (IκB-a) 磷酸化，減少IκB-a降解，且增加核內 NF-κB p65含量 (p<0.05)，並抑制NF-κB與NF-κB response element結合。比較不同CLA同質異構物，結果亦發現：在1 μg/ml LPS及5 ng/ml IFN-γ活化下，100 μM CLA mixture、9Z11E CLA 或10E12Z CLA也可抑制小鼠MES13腎絲球細胞AP-1與AP-1 response element及CREB與cAMP response element之結合。綜合以上結果可知：CLA可抑制LPS及IFN-γ活化小鼠MES13腎絲球細胞iNOS mRNA、蛋白質表現及NO生成，此一作用可能是經由抑制轉錄因子NF-κB、AP-1及CREB的活化，而調節iNOS基因表現。

Glomerulonephritis plays an important role in the process of renal failure. Glomerulus mesangial cells stimulated by lipopolysaccharide (LPS) and/or cytokines could induce inducible nitric oxide synthase (iNOS) gene expression and large amount of nitric oxide (NO) production which are resulted in glomerulonephritis. Data from our laboratory have been proven that in Raw 264.7 macrophage, conjugated linoleic acid (CLA) through inhibiting LPS-induced NF-κB activation and diminishing LPS-induced iNOS and cyclooxygenase-2 mRNA and protein expression as well as subsequent NO and PGE2 synthesis, has anti-inflammatory effect. The purpose of this study is to explore the influence of CLA on modulating LPS and interferon-γ (IFN-γ)-induced inflammatory reactions in mouse MES 13 mesangial cells. MTT assay showed that addition of 100 μM CLA mixture、9Z11E CLA or 10E12Z CLA in the absence or presence of 1μg/ml LPS and 5ng/ml IFN-γ treatment after 18 hours did not influence mouse MES 13 cell viability. CLA dose-dependently inhibited 1μg/ml LPS and 5ng/ml IFN-γ-induced iNOS mRNA and protein expression as well as NO production in mouse MES 13 mesangial cells (p<0.05) compared with cultures treated with LPS and IFN-γ only. Further experiment has been demostrated that addition of 100 μM CLA inhibited LPS and IFN-γ-induced inhibitor κB-a (IκB-a) phosphorylation, IκB-a degradation and nucleus NF-κB p65 protein expression compared with LPS and IFN-γ treatment, additionally, 100 μM CLA decreased LPS and IFN-γ induced NF-κB protein-DNA binding activity. LPS and IFN-γ induced AP-1 protein-DNA and CREB protein-DNA binding activity was also decreased by addition of 100 μM CLA mixture、9Z11E CLA or 10E12Z CLA. Taken together, our results suggest that CLA inhibits LPS and IFN-γ-induced mouse MES 13 mesangial cells iNOS mRNA and protein expression and NO production, and this inhibitory ability of CLA may be resulted from CLA modulating transcription factor NF-κB、AP-1 and CREB trans transcriptional activity.