精胺酸的可利用性對內皮細胞之一氧化氮生成和細胞存活的影響

Abstract

代謝體學係分析體內液體或組織的代謝物，以了解疾病或治療對各種代謝物的影響。本研究第一部分以分析胺基酸為例，尤其是與一氧化氮（nitric oxide，NO）生成有關的胺基酸，即精胺酸（L-arginine，L-arg）和瓜胺酸（L-citrulline，L-cit），以期了解末期腎臟病（end-stage renal disease，ESRD）對血漿中胺基酸濃度及NO生成的變化。 首先建立超高速液相層析法分析胺基酸之條件，分析正常腎功能（控制組）和ESRD族群的血漿中胺基酸濃度。全部76個志願者，其中26個人具正常腎功能，50個人有ESRD且接受腎替代性治療。另外，以Griess法測量血漿中一氧化氮代謝物（NOx）之濃度。 在17個分析的胺基酸中，經獨立t檢定發現有7個胺基酸在控制組和ESRD病人其血漿中濃度有顯著不同（p<0.05）。控制組血漿中L-cit的濃度比ESRD組低，34.3 ± 2.4 μM v.s. 80.4 ± 4.7 μM（p<0.001）。然而，血漿中L-arg濃度在兩組沒有顯著差異。至於NOx濃度，結果發現個體間分布差異大，控制組及ESRD組並無統計上明顯差異，排除界外值後，雖然ESRD組高於控制組（p<0.05），但此差異並沒有在ESRD進行腹膜透析的病人看到。 本研究建立了利用UPLC分析胺基酸之方法，較過去以HPLC分析之方法提高了解析度，並節省許多分析時間和溶劑使用量。腎臟細胞可利用精胺琥珀酸合成酶（argininosuccinate synthase，AS）和精胺琥珀酸裂解酶（argininosuccinate lyase，AL）將L-cit生合成L-arg。而AS是L-arg的合成速率限制酵素，AS的表現與胞內L-arg的再生應有重要關係，此是否影響NO的產生及細胞之存活，是第二部分研究的重點。 NO與許多生理功能及病理生理過程有關，內皮細胞一般存在endothelial nitric oxide synthase（eNOS）及inducible NOS（iNOS）蛋白，通常eNOS-NO可以維持內皮細胞的功能及完整性，而iNOS-NO通常都是受到發炎刺激引起過量生成造成病理現象。第二部分的研究探討在內皮細胞中，細胞外及細胞內生合成的L-arg之可利用性對不同NOS生成之NO和細胞存活的影響。本實驗以TR-BBB（transgenic rat blood-brain barrier）內皮細胞株當作體外細胞實驗的模型，TR-BBB內皮細胞可表現AS、eNOS和iNOS蛋白質，藉由培養於含L-arg或L-arg-free/L-cit-containing之培養液，以及利用小片段干擾核糖核酸（small interfering RNA，siRNA）短暫抑制AS基因，來調控細胞外、內L-arg之來源。 結果發現不論在regular medium或L-arg-free/L-cit-containing medium，抑制AS時，測得之eNOS-NO產量皆減少，尤其在L-arg-free/L-cit-containg medium中更少； iNOS-NO則不受AS影響，但L-arg-free/L-cit-containing medium中iNOS-NO產量較少，剩約三分之一。至於西方墨點法之結果顯示抑制AS後，兩種培養液中細胞的AS蛋白表現量皆減少；而eNOS蛋白表現僅在regular medium顯著增加。同時，也發現抑制AS，使細胞內L-cit濃度增加的趨勢。此外，AS基因沉默的TR-BBB內皮細胞，無外來刺激活化eNOS和iNOS時，在regular medium和L-arg-free/L-cit-containing medium中，細胞存活率分別減少約一成五和二成；A23187刺激eNOS後，細胞存活率分別增加約四成五和五成；而cytokines刺激iNOS後，細胞存活不受AS影響。 總結，本研究第二部分成功建立了針對AS基因進行沉默之內皮細胞株，有效地抑制AS蛋白質表現約七成。AS的表現顯著影響eNOS-NO生成，證實eNOS不僅將細胞外L-arg作為受質，也將細胞內從L-cit重新合成之L-arg做為受質；而AS的表現對於iNOS-NO生成沒有顯著影響，iNOS主要依賴細胞外L-arg作為受質。此外，在無外來刺激情況下，抑制AS表現會使內皮細胞之存活率降低。 AS蛋白表現對於eNOS-NO生成及內皮細胞存活具有重要影響，腎臟衰竭病人是否能藉由AS蛋白表現的調控，進而擴張血管或延緩內皮細胞的傷害，值得未來進一步的研究探討。

Metabolomics is to analyze the metabolites in body fluids or tissues in order to understand the effect of diseases or treatments on various metabolites. The first part of this study is a targeted metabolomics amino acid study in end-stage renal disease (ESRD) patients. We focus on nitric oxide (NO) production related amino acids, i.e. L-arginine (L-arg) and L-citrulline (L-cit) to understand the influence of end-stage renal disease (ESRD) on plasma concentrations of amino acids and NO. We established a UPLC (ultra-performance liquid chromatography) method to analyze plasma concentrations of amino acids in normal renal function (control) and ESRD populations. Total 76 volunteers were recruited, 26 people with normal renal functions, 50 people under renal replacement therapy (ESRD). In addition, we measured plasma concentration of NO metabolites (NOx) by Griess assay. Among the 17 analyzed amino acids, plasma concentration of 7 amino acids were significantly different between control and ESRD patients by independent student-t test (p<0.05). The plasma L-cit concentration was lower in control than in ESRD group, 34.3 ± 2.4 μM v.s. 80.4 ± 4.7 μM (p<0.001). However, there was no difference in plasma L-arg concentration in both groups. From our results, the NOx concentration was distributed widely among individuals, and there was no difference between control and ESRD group. After excluded the outliers, the NOx concentration was higher in ESRD than in control (p<0.01), but there was no difference between control and ESRD patients under continuous ambulatory peritoneal dialysis (CAPD). Here we established the method for analyzing amino acids by UPLC, which increased the resolution and reduced the expenditure of time and solvents. L-cit can be converted to L-arg by argininosuccinate synthase (AS) and argininosuccinate lyase (AL) in renal. AS is a rate-limiting enzyme in the pathway of the regeneration of L-arg. Therefore, there is an important correlation between AS expression and intracellular L-arg regeneration. The effect of AS expression on NO production and cell viability would be investigated in the second part. NO is related to various physiological functions and pathophysiological processes. There are endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) protein in endothelial cells. It is known that eNOS-NO can maintain function and integrity of endothelial cells, whereas iNOS-NO is overproduced under the inflammatory status. Therefore, the second part of this study is to study the impact of the availability of extracellular L-arg and intracellular regenerated L-arg on NO production via different NOSs and cell viability in cultured endothelial cells. We used TR-BBB (transgenic rat blood-brain barrier) endothelial cell line, expressing AS, eNOS, and iNOS proteins, as an in vitro model. The cells were cultured in the presence and absence of L-arg medium, respectively, and silenced AS gene by siRNA (small interference RNA) to control the availability of L-arg. From our results, they showed that no matter in regular or L-arg-free/L-cit-containing medium, when AS were down-regulated, eNOS-NO production was decreased, especially in the latter medium. On the contrary, AS did not affect iNOS-NO production. However, in L-arg-free/L-cit-containing medium, iNOS-NO production was decreased, remaining about 1/3 of that in regular medium. From results of western blotting, they indicated that AS protein expression decreased in both mediums when AS were down-regulated by AS-siRNA, whereas eNOS protein expression only increased in regular medium. Besides, the intracellular L-cit concentration had an increased trend when AS were down-regulated. In addition, AS silencing reduced 15% and 20% cell viability of cells grown in regular medium and L-arg-free/L-cit-containing medium, respectively, without any stimulation. Under A23187 stimulation, the cell viability was increased about 45% and 50% in respective medium. Under cytokines stimulation, the cell viability was not affected by AS silencing. To conclude, in the second part study, we established an AS silencing endothelial cell line whose AS protein was efficiently down-regulated to 30%. AS knockdown affects the amount of eNOS-NO production. This demonstrated that eNOS not only used extracellular L-arg as substrate, but also used regenerated L-arg from L-cit as substrate. However, AS silencing had no effect on iNOS-NO production indicating that iNOS mainly relied on extracellular L-arg as substrate. In addition, the cell viability was decreased in AS silencing endothelial cells without stimulation. AS protein expression is important for eNOS-NO production and endothelial cell viability. It is necessary to evaluate possible beneficial effect in maintaining vasodilatation and viable endothelial via regulation of AS protein expression in renal failure patients in the future.