以Caco-2細胞模式初探Hepcidin對小腸細胞鐵調節蛋白活性之影響

Abstract

動物體內鐵的恆定受到小腸細胞的嚴密調控，小腸對鐵的吸收能力與體內鐵貯存量與造血組織需求量有關。Hepcidin（Hepc）為一循環胜肽，在肝臟中合成，受鐵貯存、發炎、貧血等因素調節其表現，動物研究中發現，Hepc的表現，與小腸鐵吸收分子的表現有協調關係，推測Hepc可經血液循環，作用於小腸，調控小腸鐵的吸收。Iron regulatory proteins（IRPs）與iron responsive element（IRE）系統是調節細胞鐵恆定的樞紐，隨著細胞鐵量的增加，IRPs的結合活性減少，DMT1、TfR表現減少，減少鐵的吸收與獲取。本實驗以人類大腸腺細胞瘤Caco-2細胞模式，探討Hepc對小腸細胞鐵調節蛋白活性之影響。實驗中以化學合成胺基酸個數為20與25之Hepcidin（Hepc20、Hepc25），經氧化處理形成分子內雙硫鍵，作為細胞處理之Hepc來源；另以市售之氧化態Hepc25處理細胞，觀察Hepc25對小腸細胞IRPs活性之影響，並與自行化學修飾之Hepc對照其效應。化學合成一級結構之Hepc，純化後經質譜分析鑑定分子量為2,199.9與2,797.2。於適當緩衝溶液中進行空氣氧化反應，使完成蛋白質折繞，經質譜鑑定可知氧化態的Hepc分子量為2,191.1（Hepc20）與2,787.9（Hepc25），相差8 Da，表形成4個分子內雙硫鍵。以此三級結構Hepc處理Caco-2細胞：以10 μM濃度Hepc20與Hepc25處理12與24小時，細胞IRP1活性沒有顯著變化；增加處理濃度至20 μM，細胞IRP1自發活性仍無顯著變化。市售氧化態Hepc25以10 μM濃度處理48小時，IRP1自發活性亦不受影響。本研究發現單獨以化學合成，經氧化處理之Hepc20與Hepc25不影響Caco-2細胞之IRP1自發活性。

In mammals, iron balance is maintained by meticulous regulation of iron absorption from the duodenum, which responds to body iron stores and erythropoietic iron demand. Hepcidin (Hepc) is postulated to be a circulating peptide specifically produced in the liver and its expression is regulated by iron storage, inflammation, and anemia. It has been suggested that Hepc could act on the small intestine and regulate the iron absorption. Iron regulatory proteins (IRPs) are crucial components to regulated cellular iron homeostasis. We adopted an in vitro Caco-2 cell model to assess the effect of hepcidin on iron regulatory proteins activity. Hepc20 and Hepc25, composed of 20 and 25 amino acid residues, respectively were synthesized by solid phase peptide synthesis. The primary-structured products were purified and air-oxidized in the presence of the cysteine/cystine redox phosphate buffer at pH 7.4 to form the biologically active molecules containing four disulfide bonds. The molecule weight of primary-structured Hepc20 and Hepc25 were 2,199.9 and 2,797.2; the molecule weight of tertiary -structured Hepc20 and Hepc25 were 2,191.1 and 2,787.9. These tertiary-structured hepcidin were used for cell treatment. Caco-2 cells were exposed to Hepc20 and Hepc25 in the basolateral site prior to the measurement of IRPs activity. Incubation with Hepc20 and Hepc25 at 10 μM for 12 and 24 hours has no effect on IRPs activity. As concentration of treatment increased, the activity of IRPs was still unchanged. IRPs activity was also unaffected by Hepc25 purchased from the market. These results showed that Hepc20 and Hepc25 had no effect on IRPs activity in Caco-2 cells. These phenomena need to be further studied.