Hepatic Apoptosis after Hemorrhagic Shock in Rats Can Be Reduced Through Modifications of Conventional Ringer’s Solution

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Abstract
Resuscitation with racemic lactated Ringer's solution induces cellular apoptosis. This study was conducted to determine if the elimination of D-lactate isomer would attenuate apoptosis in the liver, and to investigate the possible mechanisms. Sprague Dawley rats (n=30, 5 per group) were subjected to modified volume-controlled hemorrhage and randomized to the following groups: no hemorrhage (sham); no resuscitation (NR); resuscitation with racemic lactated Ringer's (DL-LR); L-isomer LR (L-LR); ketone (beta-hydroxybuturate) Ringer's (KR); or pyruvate Ringer's (PR). Animals were sacrificed 2 hours later and expressions of proapoptotic proteins (BAD), antiapoptotic (bcl-2) proteins, and poly-ADP ribose polymerase (PARP) cleavage in liver were analyzed by Western blotting. Contribution of the phosphatidylinositol 3-kinase/serine/threonine kinase (PI3k/Akt) pathway was assessed by measuring total and phosphorylated PI3K, Akt, BAD, and endothelial nitric oxide synthase (eNOS) proteins. The terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptotic cells. Liver ATP levels were measured using a luciferase reaction assay. Hemorrhage significantly decreased the hepatic ATP level and resuscitation improved it, but it returned to normal only in the L-isomer lactated Ringer's and ketone Ringer's groups. Expression of proapoptotic proteins was significantly lower in the pyruvate Ringer's and ketone Ringer's groups; L-isomer lactated Ringer's and pyruvate Ringer's resuscitation significantly increased bcl-2 expression. Poly-ADP ribose polymerase fragmentation and total number of apoptotic cells were significantly increased in the racemic lactated Ringer's group. There was no significant induction of Akt activity or changes in phosphorylated BAD, Akt, or eNOS levels. Resuscitation with racemic lactated Ringer's induces hepatic apoptosis, which is decreased if the D-isomer of lactate is eliminated. Apoptosis is reduced even more when lactate is substituted with beta-hydroxybutyrate or pyruvate. The beneficial effects are not through improvements in the energy status or activation of the PI3K/Akt pathway.