Ringer's lactate improves liver recovery in a murine model of acetaminophen toxicity

Acetaminophen toxicity is the leading cause of drug-induced acute liver failure (ALF) in the US and Europe [1]; however the underlying mechanisms of APAP-induced acute liver injury (ALI) are still not clear. The toxic response to APAP is triggered by a highly reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which reacts with and depletes glutathione (GSH), after which, it forms covalent adducts and initiates mitochondrial oxidative stress [2, 3]. This increases the membrane permeability transition and causes the collapse of the mitochondrial membrane potential, which resultantly diminishes the mitochondrial capacity to synthesize ATP [4], and ATP depletion leads to massive necrosis of the hepatocyte, a characteristic feature of APAP hepatotoxicity [5].

Liver regeneration is a vital process for survival after a toxic insult [6, 7]. Regeneration ensures the replacement of necrotic cells and the full recovery of organ function. Since hepatocytes are mostly in a quiescent state (G₀), the regeneration process requires entry into the highly regulated cell cycle [8]. The first step of this process is the priming of hepatocytes by cytokines such as TNF-α and IL-6 [9, 10], which makes cells more responsive to growth factors [8]. The exposure to growth factors such as hepatocyte growth factor results in the expression of cell cycle proteins [8]. The induction of cyclin D1 is the most reliable marker for cell cycle (G ₁ phase) progression in hepatocytes [8]. Once hepatocytes express cyclin D1, they have passed the G₁ restriction point and are committed to DNA replication [8].

Many factors can influence liver regeneration. TNF-α and IL-6 are important pro-regenerative cytokines, which can prime hepatocytes to facilitate liver regeneration [8]. IL-8 and MIP-2, the CXC (CXC motif, the two N-terminal cysteines of CXC chemokines are separated by one amino acid, represented in this name with an "X") pro-inflammatory cytokines, are able to enhance hepatocyte regeneration in acute liver injury induced by APAP even when the treatment is delayed [11]. Currently, NF- κB is thought to play a major role in the initiation of liver regeneration after cell or tissue loss (such as by hepatotectomy) [8]. In addition, nutrients and metabolic status can also influence regeneration, because APAP induces massive hepatocytes necrosis. After the loss of a large number of parenchymal cells, the metabolic work of surviving hepatocytes is increased and more ATP is needed for maintaining homeostasis and regeneration [8].

Ringer's lactate solution (RLS), a frequently used resuscitative fluid, has been shown to increase serum IL-6, IL-8 [12, 13] and TNF-α [13, 14] in patients and experimental animals; in addition, RLS can provide lactate as an alternative metabolic fuel [15‐21]. "Lactic acid" was thought to be responsible for tissue damage, and as a consequence, lactate is frequently considered to be a "toxic" compound. These concepts are now being reexamined as metabolic evidence has emerged in favor of lactate reassessment [22, 23]. Lactate provides a satisfactory alternative to glucose as the primary energy in brain tissue during recovery from hypoxia [24, 25], and lactate infusion can improve the recovery of neuron damage following brain injury [20]. Moreover, lactate improves cardiac efficiency during shock, and it has recently been shown that lactate deprivation during shock impairs heart metabolism [26]. Such evidence indicates that lactate can be used as an energy substrate and resuscitative fluid to improve liver recovery from APAP-induced hepatotoxicity.

Based on this information, we hypothesize that RLS might improve liver recovery in ALI induced by APAP. To evaluate this idea, ALI was induced in mice by APAP i.p. injection, and the mice were observed over a 72-h period.

The purpose of this study was to test the hypothesis that RLS treatment improves liver recovery in ALI induced by APAP. The major and the novel findings of this investigation are: (a) RLS-treated mice demonstrate decreased serum ALT and AST and improved liver recovery 72 hrs after APAP administration; (b) the beneficial effect is associated with an augmented NF-κB DNA binding; and (c) RLS-treated mice demonstrate significantly increased expression of cell cycle protein cyclin D1 in liver tissue.

In this study, after RLS treatment, hepatic inflammatory cell infiltration was not reduced as seen in histopathology; neutrophils infiltration was not decreased by measuring MPO activity in total liver extracts. These data indicate that improved liver recovery by RLS therapy is not via changing hepatic inflammatory cells infiltration.

To elucidate the molecular basis of liver recovery in RLS-treated mice, we investigated its effect on the NF-κB signaling pathway because activation of NF-κB is linked strongly not only to the inflammatory response [30], but also to liver regeneration [8]. In addition, NF- κB is currently thought to play a major role in the initiation of liver regeneration after cell or tissue loss (such as partial hepatectomy) [8, 10]. NF- κB activation also induces increased expression of survival genes, including BCL_XL and A1 [33]. Our data suggested that RLS treatment is associated with a beneficial response characterized by activation of NF-κB. Although NF-κB activation modulates inflammation [34], it is also known to protect hepatocytes from cell death, and inhibition of NF-κB after partial hepatectomy results in massive hepatocyte apoptosis, worsens liver injury and decreases survival [35]. Enhanced NF-κB activation is also seen in mice that are protected from hepatic I/R following blockade of the receptor for advanced glycation end products [31]. There is evidence suggesting that the impact of APAP toxicity ensues, at least in part, by dramatic modulation of inflammatory and/or regeneration programs [36]. It is possible that in RLS treated mice subjected to APAP overdose, enhanced NF-κB activation diverts intracellular pathways from those associated with inflammation and cell death to mechanisms linked to recruitment and activation of pro-regenerative programs, therefore, activation of NF-κB by RLS treatment might facilitate regeneration in this ALI induced by APAP.

Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury. Tissue repair is an important determinant of final outcome of toxicant-induced injury [7], and cyclin D1 is an important cell cycle protein. In current investigation, our western blot data showed that RLS treatment markedly increased the level of cyclin D1 in the APAP challenged liver tissue. The change in cyclin D1 expression was associated with decreased serum AST and improved liver regeneration in RLS -treated mice receiving APAP, suggesting that RLS therapy likely facilitates cyclin D1-mediated regeneration pathway, and the increased cyclin D1 expression might be modulated by enhanced NF-κB DNA binding [8].

RLS-treated mice demonstrated lower serum ALT/AST and improved liver recovery at the 72 h time point. This could be due to the increased pro-regenerative cytokine TNF-α, at an early time point by RLS therapy and increased TNF-α, might prime hepatocytes for regeneration. RLS therapy also provides lactate as an alternative metabolic fuel [16‐21] to meet the increased energy demand for regeneration after the massive necrosis induced by APAP. Taken together, the increase in pro-regenerative cytokine and in fuel supply might hasten the process of liver recovery from APAP overdose.

In this study, the number of PCNA positive cells was large, and the vast majority of PCNA positive cells were inflammatory cells, this is different from the other studies which have shown hepatocytes at bordering area of necrosis express PCNA during injury progression [37, 38]. There are two possibilities for this phenomenon: (1) The specificity and the sensitivity of the first antibody to PCNA might markedly influence the results, because the other two studies used monoclonal antibody to PCNA from DakoCytomation (Carpinteria, CA, USA) and Abcam, (Cambridge, MA), our PCNA staining kit was purchased from Invitrogen (Camarillo, CA, USA). (2) Animal species might also influence the results, because 24 hrs after APAP administration, PCNA expression was apparent in B6C3F1 mice [37] and B6J129 SVF2 mice [38], however, PCNA staining was negative or occasional in C57/BL6 mice in our study at 24 h time point, and our result was consistent with another study [32] in which BrdU (5-bromo-2-deoxyuridine) immunohistochemistry was performed to detect hepatocyte regeneration in C57/BL6 mice.

Currently NAC, a glutathione precursor, is the antidote for APAP overdose [39]. However, this antidotal therapy is effective for early-presenting patients, and is less effective for late-presenting patients [39, 40], additional therapies are needed. Treatment with RLS would serve as an adjuvant to NAC therapy. Further experiments designed to compare the two therapies are needed in the future.

Since RLS treatment is able to improve liver recovery from APAP overdose in which massive necrosis of the hepatocyte is a characteristic feature, it is possible that RLS therapy may improve hepatic regeneration following liver surgery (such as partial hepatectomy). Further study is needed to confirm this idea in the future.

RLS treatment improves liver recovery after APAP overdose and RLS may present a novel therapy to treat APAP hepatotoxicity.