探討丙酮酸對HEK 293細胞抗氧化能力之影響

組織缺血與再灌流(ischemia/reperfusion)是指血液供應受到阻礙與隨後的血流恢復，這種現象帶給細胞組織許多負面影響，其中粒線體失去正常功能而導致的氧化壓力將帶給細胞嚴重的傷害，在動物模式中發現外加高於生理濃度的丙酮酸可以有效的救援氧化壓力帶來的傷害。以往對於中間代謝的觀點只是提供細胞養分以及生合成的材料，然而，越來越多的證據顯示有些代謝物質可能扮演訊息傳遞的角色，這些代謝物質可能會藉著與目標蛋白的直接作用而影響其功能，為了驗證此觀點，本實驗室發展了能夠鑑定與丙酮酸直接作用的蛋白質體的方法學(AETHIC)，本研究利用AETHIC與代謝體分析，探討外加的丙酮酸對於HEK細胞抗氧化能力的影響，主要針對我們鑑定到的蛋白：檸檬酸合成酶以及硫氧還蛋白還原酶(TrxR)。前者與異檸檬酸脫氫酶(NADP+-dependent isocitrate dehydrogenase, NADP+-IDH)所構成的代謝途徑可以產生細胞還原力的來源NADPH，而後者與抗氧化能力有直接的關係。在代謝體分析中顯示丙酮酸加入HEK細胞培養液中導致檸檬酸循環流量的提高，另外NADPH/NADP+與NADH/NAD+也有明顯的增加，酵素活性分析結果顯示相同的處理下檸檬酸合成酶活性降低，所以檸檬酸合成酶活性的下降可看做對TCA cycle的調控。接著觀察檸檬酸合成酶的下游代謝：異檸檬酸脫氫酶，在in cellulo活性測試發現丙酮酸可以增加異檸檬酸脫氫酶的活性，但在in vivo實驗中，只在較丙酮酸高濃度下才觀察到，故推測細胞內有其他的機制來調控異檸檬酸脫氫酶活性。在硫氧還蛋白還原酶的研究中，觀察到氧化壓力會導致此蛋白的失活，但丙酮酸可以快速的恢復其活性至正常值，所以推測丙酮酸具有保護硫氧還蛋白還原酶的功效。由以上實驗顯示，丙酮酸對於細胞抗氧化壓力是有幫助的，可以快速增加細胞的還原能力，同時也有維持TrxR/Trx還原系統的功能。

關鍵字

組織缺血與再灌注；丙酮酸；檸檬酸合成酶異檸檬酸脫氫酶硫氧還蛋白還原酶

並列摘要

Ischemia/reperfusion is a pathophysiological condition of which blood flow is blocked and then restored. It will cause profound pathological effects such as oxidative stress caused by the mitochondria dysfunction. In some animal models, supplement of pyruvate effectively mitigates ischemia/reperfusion injury. There is growing evidence that some metabolites may play a new role as a signaling messenger and regulate the function of proteins in addition to their traditional roles of energy supplement and biosynthesis. To verify this, our laboratory developed a method for identification of pyruvate-interacting proteome (AETHIC). And the purpose of this study is to investigate the role of pyruvate in counteracting oxidative stress. According to AETHIC and metabolome results, we focused on citrate synthase and thioredoxin reductase (TrxR). The former enzyme and NADP+-dependent isocitrate dehydrogenase (NADP+-IDH) contribute a pathway of NADPH production, and the latter, TrxR, is directly associated with anti-oxidative function. The flux of TCA cycle, NADPH/NADP+ and NADH/NAD+ increased after addition of pyruvate to HEK cell. Also, the activity of citrate synthase decreased under the same treatment, which indicates the regulatory role of pyruvate in TCA cycle. Next, I measured the activity of NADP+-IDH and found that pyruvate increased the activity of IDH in vitro but was less effective in cellulo. The activity of TrxR was significantly decreased under H2O2 treatment, but pyruvate completely recovered the activity to normal levels. Consequently, it is suggested that pyruvate may protect TrxR and restore its activity under oxidative stress. Above all, pyruvate rescue cells from oxidative stress by elevation of the reducing power of cells and sustaining the TrxR/Trx system.

並列關鍵字

Ischemia/reperfusion ； pyruvate ； citrate synthase ； NADP+-dependent isocitrate dehydrogenase ； thioredoxin reductase

參考文獻

1. Eltzschig, H.K. and T. Eckle, Ischemia and reperfusion--from mechanism to translation. Nat Med, 2011. 17(11): p. 1391-401.

2. Dhalla, N.S., et al., Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res, 2000. 47(3): p. 446-56.

3. Rouslin, W., Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis. Am J Physiol, 1983. 244(6): p. H743-8.

4. Lesnefsky, E.J., et al., Mitochondrial dysfunction in cardiac disease: ischemia--reperfusion, aging, and heart failure. J Mol Cell Cardiol, 2001. 33(6): p. 1065-89.

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探討丙酮酸對HEK 293細胞抗氧化能力之影響

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