菸鹼酸可以回復受到吲哚酚硫酸鹽傷害的內皮前驅細胞功能

Abstract

【研究背景及目的】 內皮功能障礙在慢性腎衰竭早期就開始進展，不僅因為內皮細胞損害增加，具修復系統的內皮前驅細胞 (endothelial progenitor cells, EPCs) 數量也降低，且功能不良。研究發現尿毒素-吲哚酚硫酸鹽 (indoxyl sulfate ,IS)具有腎毒性及血管毒性，會抑制血管內皮細胞增殖和降低修復能力，損害內皮功能。因此降低尿毒素可以減低慢性腎臟病人心臟血管的傷害。菸鹼酸（Niacin) 是最早用來降血脂肪的藥物，並有增強細胞抗氧化能力，可能對於阻止內皮細胞的損害以及恢復修復系統有所助益。本研究針對 IS 對於 EPCs 的不良影響，探討 Niacin 是否可以拮抗 IS 的作用，回復血管修復能力以降低內皮功能障礙的發生或惡化。 【研究方法】 以體外細胞實驗，由健康成人之週邊血分離出單核細胞，種植於纖連蛋白塗佈 (Fibronectin coating )的培養皿，具貼附性單核細胞分化為EPCs，並鑑定其特徵。培養的 EPCs 加入相當於慢性腎臟病人血中 IS 的濃度，觀察細胞增埴能力及可能機制包括自我凋零 ( Apoptosis )、老化(Senescence)、自噬( Autophage ) 的變化；EPCs的功能評估則是移行能力（ migration ）和管形成作用（ tube formation ）。其後探討 Niacin 拮抗IS 對 EPCs增殖和功能毒性作用的機轉。細胞大致分為三組：控制組、IS作用組、IS合併Niacin組。 【結果】 IS 降低 EPCs 細胞活性（viability）是呈現濃度依賴 (p= 0.0012) 及時間依賴(p＜0.0001) 的效果。1mM 的 IS 降低 28% ( p=0.024 ) 的 EPCs 細胞增殖能力，1mM Niacin對回復 IS 降低 EPCs 增殖作用效果最好( p=0.0495 )。IS（1mM）並不影響細胞凋亡標幟 Caspase-3 的表現量，但增加 1.8倍自噬標幟 LC3b 的表現，以及100% 的老化 EPCs 數量( p<0.05 )。1mM 的 Niacin對於自噬標幟 LC3b 的表現可以降低 70% 同時可以減少 IS 增加 EPCs 的老化 (減少54 %; p=0.03)。累計性細胞族群總數倍增程度 ( Cumulative Population Doublings Assay ) 檢測結果顯示於第一天(Day1)時增加的細胞數在 1mM IS 降低約 58% ，於第二天(Day2)時增加的細胞數降低約 61% ( p=0.0097 ) ，加入 1mM濃度 Niacin 後於 Day1 可以較 IS 組增加約 46% 的細胞數; 於 Day2 可以增加約 96% 的細胞數 ( p=0.007 )。 功能上，1mM 的 IS 抑制 EPCs 45% 的移行能力（p<0.001），和49% 管形成作用（p=0.0042），1mM Niacin 會增加 EPCs 的移行能力 40%（p=0.021），但管形成作用的恢復並無顯著差別（p=0.416）。 分析影響的蛋白質變化，IS（1mM） 可以增加原血紅素氧化酵素一型 ( Heme oxygenase-1，HO-1 ) 的表現量達 89 倍，1mM Niacin 可以減少約 50% HO-1的表現量。IS (1mM) 會增加 p-AKT 表現量 (增加3.26倍)，1mM Niacin 加入後對 AKT ,p-AKT表現量無影響。IS (1mM) 減少 77% ( p<0.05 ) 內皮細胞一氧化氮合成脢（ endothelial nitroide synthase，eNOS ）及70 % ( p<0.05 ) 血管內皮生成因子（ vascular endothelial growth factor，VEGF ）的表現量，但1mM Niacin 可以提升 eNOS (增加 130% ; p=0.0016)及 VEGF的表現量(增加140% ; p=0.00030) ,並可抑制 IS (1mM) 誘發活性氧化自由基（reactive oxygen species；ROS）的生成。但 Niacin 專屬接受器 GPR109A 不存在於 EPCs 細胞上。 【結論】 吾人的研究顯示IS對EPCs的傷害包括增生降低及功能受損的情況可以因Niacin的存在而明顯改善，拮抗機制可能來自於(1)降低EPCs細胞的老化及自噬，(2)提升細胞eNOS及VEGF的整體表現量，以及(3)降低IS誘發的ROS生成量。我們設計的體外細胞實驗，無法完全模擬臨床上慢性腎衰竭病人體內尿素素的傷害影響，但對於臨床上改善慢性腎衰竭病人的內皮功能不良，提出治療的思考。以細胞初步研究結果，提供更進一步的動物試驗和臨床試驗計畫。

【Background and Objective】 Substantial evidence has confirmed that endothelial dysfunction starts in the early stage of CKD,not only increasing damage to endothelial cell but affect of endothelial progenitor cells (EPCs) in the repair system.The uremic toxins−indoxyl sulfate (IS) exhibit renal and vascular toxicity, impaired endothelial cell proliferation and repair capacity. Nicotinic acid (Niacin) is the earliest lipid –lowering drug and has antioxidant capacity might helpful to prevent endothelial cell injury and restore the function of repair system. In this study, we focus on the adverse impact of IS on EPCs and to investigate if Niacin can antagonize the deleterious effect of IS to reduce the occurrence or deterioration of endothelial dysfunction. 【Material and Method】 In vitro study, mononuclear cells were isolated from peripheral blood from healthy donators and plated on culture dishes coated with fibronectin,adherent cells were differentiation to EPCs then confirmation was determined.EPCs were incubated with an additional IS to investigate the changes including apoptosis、senescence、autophage. EPCs function was tested by migration and tube formation ability.The restorative effects and mechanism of Niacin on damaged EPCs were survey. Study groups:were divided to three categories:control group, IS along group, and IS combined Niacin group. 【Result】 The effect on proliferation induced by .IS was in dose and time-dependent manner.IS at concentrations of 1mM induced a decreased proliferation in EPCs of 28% (p = 0.024) but was blunted by adding Niacin that the best effective dose is 1mM (p=0.0495).The presence of apoptosis biomarker-activated caspase-3 did not change after 1mM IS infusion but the autophagy marker LC3b expression were increased 80% and the percentage of senescence EPCs increases 100% (P<0.05).However, 1mM Niacin administration attenuated the IS-induced senescence reduced by 54% and decreased LC3b expression by 70%. Cumulative population doublings assay results showed that 1mM IS reduced the cell number about 61% (p=0.0097) but increased approximately 46%−96%. by 1mM Niacin (p =0.007).EPCs transwell migration ability decreased 45% (p <0.001) and tube formation capacity reduced by 49% (p = 0.0042) when 1mM IS was added. 1mM Niacin administration improved the transwell migration ability of 40% (p = 0.021),but tube formation capacity was not significant recovery (p = 0.416).Analysis of protein altered by IS(1mM),the HO-1 increased to 89 times attenuated approximately 50% by 1mM Niacin treatment.IS (1mM) increases the expression of p-AKT to 3.26 times, addition of 1mM Niacin had no effect on AKT, p-AKT expression. eNOS and VEGF expression were decreased by 77% (p <0.05) and 70% (p <0.05) respectively by IS (1mM) stimulation but 1mM Niacin treatment can enhance eNOS and VEGF expression with an increase of 130% ( p = 0.0016 ) and increase of 140%(p = 0.00030 ) respectively. IS (1mM) prompted ROS generation was blunted by 1 mM Niacin treatment. Niacin exclusive receptors GPR109A was not exist on EPCs. 【Conclusion】 Our study showed after Niacin treated,the impairment of EPCs proliferative capacity and function ability induced by the deleterious effect of IS is repaired. A antagonist mechanism of Niacin on damaged EPCs induced by IS may derive from the decrease of cell aging and autophagy,the elevation of the eNOs and VEGF expression, the decrease of the generation of free radicals .In the future,we hope to apply Niacin in clinical practice to improve endothelial function in patients with CKD.