- 學位論文
TLR2及TLR4在高糖及乙型轉型生長因子所誘發腎間質細胞之生物效應所扮演的角色
Roles of TLR2 and TLR4 in high glucose and TGF-β1 induced effects in mesangial cells
摘要
腎細胞過度增生與肥大所導致的腎組織間隙纖維化及細胞外間質(extracellular matrix, ECM) 堆積是糖尿病腎病變 (diabetic nephropathy, DN) 的主要病理特徵。高糖會促使活性氧群 (Reactive oxygen species, ROS) 生成並誘導乙型轉型生長因子 (transforming growth factor-β1, TGF-β1) 表現，而以 TGF-β1刺激小鼠腎間質細胞 (Mes 13) 則會導致ECM 蛋白，如 fibronectin堆積以及發炎。Toll-like receptors (TLRs) 是當微生物入侵時負責辨識微生物表面物質並引發免疫及發炎反應的受體，近年來研究發現，TLRs 除了與這些外來配體結合外，亦會與細胞產生之內生性配體結合，包括ROS、heat shock protein 70、fibronectin 等，而這些內生性的物質在形成 DN 的過程中皆會大量表現，因此我們想探討 TLRs 在 DN 中所扮演的角色及其關連性。我們以 real-time PCR 及 Western blotting發現，高糖及 TGF-β1 刺激 Mes13 細胞時，TLR 2 及 TLR 4 的 mRNA 和蛋白質表現量皆會增加。另外，在高脂肪飲食誘導小鼠產生第二型糖尿病初期症狀的動物模式中，以免疫組織染色觀察其腎臟中 TLR2 分佈的情形，結果顯示，在第二型糖尿病的老鼠腎臟中腎小管及腎絲球之 TLR2 確實有增加的情形。抗氧化劑 N-acetylcysteine (NAC) 可以逆轉 TGF-β1 在 Mes13 中所誘導之TLR2 及 TLR4表現，顯示 TGF-β1 是透過增加 ROS 而誘導 TLR2 及 TLR4 大量表現。當加LY294002 (PI3K inhibitor)、PD98059 (ERK inhibitor)、SB203580 (p38 MAPK inhibitor) 以及SB431542 (TGFβ1R1 inhibitor) 後，TGF-β1 刺激 Mes13 細胞產生的TLR2 及 TLR4表現被抑制，顯示 TGF-β1是經由PI3K、p38 MAPK、ERK、Smad等訊息分子而活化TLR2 及 TLR4。最後，我們以能專一性抑制TLR2 及TLR4 表現的siRNA進行研究，結果發現在抑制了TLR2 及 TLR4後，TGF-β1 刺激 Mes13 細胞所造成的發炎 (TNFα、IL-1β及IL-6)及纖維化 (fn1 及COL1α2)都有回復的情形。綜合以上結果，在 DN 形成的過程中，TLR 2 及 TLR 4的確在促進發炎反應及纖維化上扮演重要的角色。
並列摘要
Renal interstitial fibrosis and extracellular matrix (ECM) accumulation resulted from cellular hypertrophy / hyperplasia are characteristics of diabetic nephropathy (DN). Previous studies suggested that reactive oxygen species (ROS) production induced by hyperglycemia may lead to the induction of TGF-β1 which promotes fibronectin accumulation and inflammation in Mes13 cells. Toll-like receptors (TLRs) are able to recognize surface substance , such as lipopolysaccharide (LPS) and lipoteic acid (LTA), of invading microbes. Recently, several invastigators showed that TLRs exist some endogenous ligands, i.e. ROS、heat shock protein 70 (HSP 70) and fibronectin, which were induced in the pathogenesis of DN. In this study, we found that hyperglycemia and TGF-β1 up-regulate mRNA and protein expression of TLR2 and TLR4 , and TGF-β1-induced TLR2 and TLR4 mRNA expression is attenuated by LY294002 (PI3K inhibitor)、PD98059 (ERK inhibitor)、SB203580 (p38 MAPK inhibitor) and SB431542 (TGFβ1R1 inhibitor). These data implied that TLRs might involve in the pathogenesis in DN. To elucidate if the induction of TLRs by hyperglycemia and TGF-β1 is through ROS production, Mes13 cells were pretreated with N-acetylcysteine (NAC), an antioxidant, to block ROS production. We found that hyperglycemia and TGF-β1-induced TLR2 and TLR4 expression is blocked by NAC pretreatment, indicating a ROS-mediated up-regulation of TLR2 and TLR4 by TGF-β1. In addition, we set up an animal model with early stage type 2 DM, which was induced by feeding of high-fat diet. By immunohistochemistry assay, it appears that TLR2 expression is elevated in renal tubules and glomeruli of type 2 DM mice. Further more, we found TGF-β1-induced inflammation (TNFα, IL-1β, and IL-6) and fibrosis (fn-1 and COL1α2) in Mes13 cells are both reversed by expressing TLR2- and TLR4-specific siRNA. In conclusion, hyperglycemia and TGF-β1-induced expression of TLR2 and TLR4 is through ROS production, and the role of TLR2 and TLR4 in the pathogenesis of DN is promoting renal inflammation and fibrosis.
參考文獻
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