硫酸吲哚酚和硫酸對甲酚影響血管內皮功能之比較

Comparison of indoxyl sulfate and p-cresyl sulfate on endothelial function

林裕祥(Yu-Hsiang Lin)

指導教授 ： 謝政穎

共同指導教授 ： 許準榕

臺北醫學大學/醫學院/醫學科學研究所/碩士(2014年)

摘要

硫酸吲哚酚 (indoxyl sulfate, IS) 以及硫酸對甲酚 (p-cresyl sulfate, PCS) 分別為色胺酸及酪胺酸之代謝產物，當病患之腎功能下降時，IS及PCS會滯留於人體內，可作為腎功能下降之指標。臨床統計研究發現腎臟透析病患的心血管疾病死亡率為正常人的五倍，而近來的研究亦發現IS會促進動脈粥狀硬化病程之進行。IS及PCS對血管內皮細胞直接性的作用，被認為可能與此現象相關，然而其詳細作用機轉尚未清楚。本篇論文將探討並比較IS及PCS對血管內皮細胞的作用及其可能的機轉。
首先利用細胞存活率實驗(MTT assay)探討IS及PCS對人類臍帶血管靜脈內皮細胞(HUVEC)細胞存活率的影響，實驗結果發現IS(50-1000 ?嵱)呈濃度相關性地降低HUVEC的細胞存活率，而PCS僅在最高濃度(1000 ?嵱)時有些微的影響。同樣地，在血管新生(tube formation assay)及細胞老化(??-galactosidase assay)實驗中亦發現，IS有意義並濃度相關性地抑制血管內皮細胞的活性，而PCS並沒有明顯的抑制作用。進一步地去探討可能的機轉發現，無論是IS或PCS均會刺激活性氧自由基(reactive oxygen species, ROS)於血管內皮細胞中的產生，並顯著地刺激p47phox蛋白的磷酸化。此外，過去的研究中亦指出IS會與芳香烴受體(aryl hydrocarbon receptor, AHR) 結合並刺激AHR進入細胞核中作用，並經由此一現象影響血管內皮細胞的活性；本研究發現PCS也有相同的作用。IS已被證實會經由抑制endothelial nitric oxide synthase(eNOS)的磷酸化而影響內皮細胞的功能，本次實驗結果亦印證了此一結果；然而相較於IS，PCS並不會抑制eNOS的磷酸化，反而有促進的現象，而給予NOS抑制劑(L-NAME)會有意義地減少給予PCS組別的血管內皮細胞存活率。
綜合以上實驗結果，本研究發現相較於IS明顯地抑制血管內皮細胞功能，PCS對血管內皮細胞並無顯著的抑制作用，這可能是因為PCS會經由刺激eNOS的磷酸化而有保護血管內皮細胞的效果。根據本研究成果發現，相較於PCS，IS可能是監控腎臟病患其心血管疾病發生率較主要的指標尿毒素蛋白。

關鍵字

硫酸吲哚酚 ； 硫酸對甲酚

並列摘要

Indoxyl sulfate and p-cresyl sulfate were tryptophan and tyrosine metabolites when patients with renal function decline, p-cresyl sulfate and indoxyl sulfate will remain in the human body. It can be used as indicators of decline of renal function. Clinical statistical study found that kidney dialysis patients from cardiovascular disease mortality was five times normal,and recent studies have also found that indoxyl sulfate will be promoting the course of atherosclerosis. Indoxyl sulfate and p-cresyl sulfate direct effect of vascular endothelial cells that were considered to be associated with this phenomenon, but its detailed mechanisms are unclear. This paper will explore and compare the effect of indoxyl sulfate and p-cresyl sulfate on vascular endothelial cells. To investigate the difference between IS and PCS on physiological functions of vascular endothelial cells, the human umbilical vein endothelial cells (HUVEC) were used.
The cell viability was detected by MTT assay. IS (50 – 1000 ?嵱) had concentration dependent manner inhibition on cell viability, but PCS (1000 ?嵱) just had minor inhibition in HUVEC. In addition, IS (100 – 1000 ?嵱) showed concentration dependent manner inhibition angiogenesis and cell senescence, but PCS did not. According to signaling pathway, not only IS but also PCS enhanced reactive oxygen species (ROS) and phosphorylation of p47 phox in HUVEC. In previous studies, the results showed IS binding on aryl hydrocarbon receptor (AHR) and AHR translocating from cytoplasm to nucleus that unregulated activity of HUVEC, and our study showed same effect of PCS. It had been proved that IS inhibited phosphorylation of endothelial nitric oxide synthanse (eNOS) that regulated cell functions of vascular endothelial cells, and we had same results. Furthermore, L-NAME is the inhibitor of eNOS that significantly reduced viability of HUVEC that pretreated PCS.
In conclusion, we found that IS significantly inhibits physiological functions of vascular endothelial cells, but PCS did not. We demonstrate that PCS protect vascular endothelial cells through phosphorylation of eNOS. Those results indicated that IS are major indicator of risk factor on cardiovascular disease of patients with renal disease compared with PCS.

並列關鍵字

indoxyl sulfate ； p-cresyl sulfate

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