Jaskille Amín, Alam Hasan B, Rhee Peter, Hanes William, Kirkpatrick John R, Koustova Elena
Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
J Trauma. 2004 Aug;57(2):262-69; discussion 269-70. doi: 10.1097/01.ta.0000133841.95455.73.
Resuscitation with racemic lactated Ringer's solution (containing equal amounts of D and L isomers of lactate) has been shown to induce pulmonary apoptosis. Substitution of DL-isomer lactate with ketone bodies (beta-hydroxybutyrate, BHB), sodium pyruvate, or L-isomer of lactate decrease this injury without changing the energy status of the tissues or the expression of apoptotic genes. These modified solutions however alter the function of apoptotic proteins through an unknown mechanism. We postulated that DL-LR induces apoptosis by restricting the phosphorylation of key apoptotic proteins.
Male Sprague Dawley rats (n = 30, 5/group) were subjected to a three stage, volume-controlled hemorrhage and randomized to the following groups. 1) No hemorrhage (Sham); 2) Hemorrhage and no resuscitation (NR); 3) Resuscitation with 3x shed blood volume of racemic LR (DL-LR); 4) Resuscitation with 3x shed blood volume of LR containing only the L-isomer of lactate (L-LR); 5) Resuscitation with 3s shed blood volume of pyruvate Ringer's (PR); 6) Resuscitation with 3s shed blood volume of ketone Ringer's (KR). The modified Ringer's solutions were identical to racemic LR except for equimolar substitution of DL-lactate for L-lactate, pyruvate and BHB respectively. Lung tissue was obtained 2 hours later and subjected to Western Blotting. The levels of Akt, Bad, and eNOS (total and phosphorylated) proteins were measured. Finally, the expression of gene coding for protein 14-3-3 was measured using RT-PCR.
Resuscitation with DL-LR caused a significant (p < 0.05) increase in the total Bad and a decrease in phosphorylated Bad protein expression in the lung. It also caused an increase in the phosphorylated Akt levels and a decrease in gene coding for protein 14-3-3. These changes were consistent with signaling imbalances that favor apoptosis. Modified LR solutions, on the other hand, did not cause these alterations. Phosphorylation pattern of eNOS supported the involvement of PI3K/Akt pathway in this process.
Racemic lactate plays a role in the induction of pulmonary apoptosis by restricting phosphorylation of Bad and eNOS proteins.
已证明用消旋乳酸林格氏液（含有等量的D型和L型乳酸异构体）进行复苏会诱导肺细胞凋亡。用酮体（β-羟基丁酸酯，BHB）、丙酮酸钠或L型乳酸异构体替代DL型乳酸异构体可减少这种损伤，而不会改变组织的能量状态或凋亡基因的表达。然而，这些改良溶液通过未知机制改变凋亡蛋白的功能。我们推测DL-LR通过限制关键凋亡蛋白的磷酸化来诱导细胞凋亡。
将雄性Sprague Dawley大鼠（n = 30，每组5只）进行三阶段、容量控制的出血，并随机分为以下几组。1）未出血（假手术组）；2）出血且未复苏（NR组）；3）用3倍失血量的消旋LR（DL-LR）进行复苏；4）用3倍失血量的仅含L型乳酸异构体的LR（L-LR）进行复苏；5）用3倍失血量的丙酮酸林格氏液（PR）进行复苏；6）用3倍失血量的酮体林格氏液（KR）进行复苏。改良的林格氏液与消旋LR相同，只是分别用等摩尔的L型乳酸、丙酮酸和BHB替代了DL型乳酸。2小时后获取肺组织并进行蛋白质印迹分析。测量Akt、Bad和eNOS（总蛋白和磷酸化蛋白）的水平。最后，使用逆转录-聚合酶链反应（RT-PCR）测量编码蛋白14-3-3的基因表达。
用DL-LR进行复苏导致肺中总Bad蛋白显著增加（p < 0.05），磷酸化Bad蛋白表达减少。它还导致磷酸化Akt水平增加，以及编码蛋白14-3-3的基因减少。这些变化与有利于细胞凋亡的信号失衡一致。另一方面，改良的LR溶液未引起这些改变。eNOS的磷酸化模式支持PI3K/Akt途径参与这一过程。
消旋乳酸通过限制Bad和eNOS蛋白的磷酸化在诱导肺细胞凋亡中起作用。
