探討迷迭香酸如何抑制巨噬細胞分泌組織因子並改善糖腎病

Abstract

糖腎病是一種常見的慢性糖尿病併發症，也是造成末期腎臟衰竭的原因之一，伴隨的尿毒症狀更是讓洗腎人口數向上攀升。約有20-30%的糖尿病患者會發展成糖腎病，其中第一型糖尿病在10-15年內會演變成糖腎病，並具有50%的機會進展到末期腎病變；而第二型糖尿病則有20%-40%的糖腎病進程，然而20年後僅有20%的機率形成末期腎病變。根據美國腎臟資料系統的末期腎臟病年報中顯示，台灣糖腎病患者的盛行率位居世界第二，發生率則居全球之冠，在國人十大死因裡更是排名第五，為此研發出延緩或改善糖尿病腎病變的治療方法是極其迫切的議題。 糖尿病患者體內的高血糖環境會促進最終糖化蛋白的合成 (Advanced Glycosylation End Products, AGEs) ，進而引發體內細胞產生有害的活性氧物 (Reactive oxygen species, ROS) 以及釋放促發炎因子，造成內皮細胞受損並吸引巨噬細胞前來浸潤。接著巨噬細胞會釋放出組織因子，造成腎臟基底膜增厚、基質膨脹與腎凝血等病理變化。迷迭香酸 (rosmarinic acid, RA) 已被研究指出具有抗發炎、抗氧化和抗纖維化等生物活性，而且還能促進抗發炎因子IL-10的表現量，進而去抑制巨噬細胞表現和分泌組織因子。因此在本研究中，我們在C57BL/6小鼠身上注射STZ誘發糖腎病以建立體內實驗的模型，另外也使用RAW264.7細胞株作為體外實驗的模型，藉此來探討迷迭香酸是否能透過抑制巨噬細胞釋放組織因子，來改善糖腎病並試圖找出可能參與其中的訊號路徑。 根據實驗結果，迷迭香酸不但可以抑制LPS誘導RAW264.7所分泌組織因子和發炎因子的表現量，也可以使其從發炎的M1表現型轉變回抗發炎的M2表現型。有給予迷迭香酸治療的小鼠，不僅體重有回升，血糖濃度和排尿量也有明顯降低，而有改善腎臟功能的能力。觀察腎臟切片和染色結果，可發現迷迭香酸除了可以減輕基底膜增厚和基質膨脹，也可以減少巨噬細胞的浸潤和轉化生長因子β (Transforming growth factor beta, TGF-β) 的表現，進而緩和發炎反應和纖維化的現象，達到保護腎臟的功效。

Diabetic nephropathy is a common complication of diabetes and is the primary cause of end-stage Renal Disease worldwide. The following uremia accounts for the increasing amount of hemodialysis population. Approximately 20% to 30% of all diabetics will develop diabetic nephropathy. Type 1 diabetes will become overt nephropathy after 10 to 15 years and get 50% chance to result in ESRD. However, there is about 30% of type 2 diabetes progressing to overt nephropathy and then just 20% develop ESRD after 20 years. According to Annual Data Report presents data on end–stage renal disease, in addition to the highest incidence of diabetic nephropathy in Taiwan, the prevalence of it ranks second in the world. This makes diabetic nephropathy occupy the fifth leading causes of deaths in Taiwan. Thus, it is urgent issue to find the efficient therapy to preventive intervention. Hyperglycemia in diabetic patients promoted the production of AGEs, so that AGEs stimulated cells to generate harmful ROS and secrete proinflammatory cytokines, which damaged endothelial cells and recruited macrophages. Then, releasing tissue factors by macrophages resulted in glomerular basement membrane(GBM) thickening, mesangial expansion, renal coagulation and so on. Rosmarinic acid had been reported to possess a range of biological activities such as anti-inflammation, antioxidant and antifibrosis. In addition, IL-10 expression up-regulated by rosmarinic acid could inhibit macrophages secreting tissue factor. Therefore, in order to investigate whether rosmarinic acid ameliorated diabetic nephropathy through inhibiting Monocyte tissue factor expression and found out the possible signaling pathway, we injected streptozotocin (50mg/kg) in C57BL/6 mice to induce diabetic nephropathy as our animal model; meanwhile, we chose LPS-induced RAW264.7 cell line to establish our cell model. Our data showed that rosmarinic acid not only inhibited LPS-induced RAW264.7 expressing tissue factor and inflammatory cytokines, but also transformed M1 phenotype into M2 phenotype. Treatment with RA recovered body weight, decreased blood glucose concentration and reduced urine excretion in STZ-DN mice. Section observation indicated that RA is able to improve GBM thickening, moderate mesangial expansion, regulate macrophage recruitment and decrease renal fibrosis. Consequently, our studies showed that RA ameliorated the inflammatory response and fibrosis to protect kidney from the development of diabetic nephropathy.