This study was aimed to investigate nitric oxide-dependent mechanisms of L-ornithine-L-aspartate (LOLA) action in acute toxic liver injury in rats. Acute hepatitis was induced in Wistar rats using 50% oil solution of tetrachloromethane (CCl₄₎ intragastrically (2 g/kg) twice in a 24 hour interval. Intraperitoneal treatment with LOLA (200 mg/kg) was started 6 hours after the second CCl₄ administration and maintained for 3 consecutive days. L-Nω-Nitroarginine Methyl Ester (L-NAME) was used intraperitoneally (10 mg/kg). In CCl₄-induced hepatitis, LOLA restores the structure of hepatocytes and prevents aminotransferases, alkaline phosphatase and gamma-glutamyl transferase elevation. It decreases total bilirubin concentration but does not affect increased cholesterol level. LOLA augments urea concentration, total protein level in blood and liver as well as serum and liver content of nitrite anions. LOLA enhances activity of catalase, glutathione S-transferase, manganese superoxide dismutase, increases reduced glutathione level and total antioxidant capacity and decreases thiobarbituric acid reactive substances level. The concomitant use of L-NAME inhibits the action of LOLA to enhance nitrite anions synthesis both in serum and liver, to delay the recovery of hepatocytes, to counteract LOLA effect against blood total protein reduction, to prevent the decline in aminotransferases, alkaline phosphatase,, gamma-glutamyl transferase and glutathione S-transferase activity and to reduce catalase activity and reduced glutathione level. Therefore, in CCl₄-induced hepatitis, LOLA effectively prevents cytolysis and cholestasis, improves liver metabolism and protects against oxidative stress. Partially, these changes occur in nitric oxide-mediated mechanism since the use of L-NAME declines most of LOLA effects.

L-ornithine-L-aspartate, hepatoprotection, nitric oxide

L-ornithine-L-aspartate (LOLA) is a well tolerated medicine effective in the treatment of patient with liver cirrhosis and chronic hepatic encephalopathy (HE) (Butterworth and Canbay 2018). LOLA induces hepatic urea synthesis, increases glutamine production in muscles and regulates the relationship between branched aromatic amino acids. The components of LOLA participate in the reactions of the ammonia molecule incorporation into urea and glutamine which underlies the application of LOLA as an effective ammonia-lowering strategy widely used for the management and treatment of HE (Kircheis and Lüth 2019).

LOLA exerts hepatoprotective effects by stabilizing peroxidant/antioxidant balance in the liver cells as it provides L-ornithine and L-aspartate as substrates for glutamate production (Jalan et al. 2007). The product of LOLA-derived glutamate, namely, glutathione (GSH), is a potent antioxidant that has the requisite properties for the control of oxidative damage (Najmi et al. 2010).

Finally, since LOLA administration results in the accumulation of L-glutamate and L-arginine, it leads to the increase in nitric oxide synthase (NOS) production with the consequent enhancement in hepatic microperfusion (Ijaz et al. 2005). This mechanism was confirmed in patients with cirrhosis (Staedt et al. 1993) and in an experimental model of chronic liver failure (Rose et al. 1999). However, nitric oxide (NO)-dependent mechanism of LOLA action in acute toxic liver injury is not well understood.

Acute liver injury is associated with higher blood ammonia levels than in cirrhosis which correlates with increased risk of mortality, more severe encephalopathy, intracranial hypertension and cerebral herniation. This differences between ammonia levels are explained by the fact that in cirrhosis, there is some remaining liver cell mass that retains some capacity to detoxify ammonia, while in acute hepatitis large amounts of ammonia escape hepatic clearance (Acharya et al. 2009).

The experimental animal model, in which acute liver toxicity is induced by tetrachloromethane (CCl₄), is characterized by the specific biochemical and histopathological changes which represent the common features of liver injury in humans. CCl₄ causes direct liver injury due to cell necrosis caused by altering hepatocyte membrane permeability and induces up-regulation of pro-inflammatory mediators resulting in secondary hepatic injury (Díaz-Gómez et al. 2011). The toxicity of CCl₄ is also associated with free radical formation by the liver endoplasmic reticulum leading to a persistent oxidative stress (Martinelli et al. 2021).

Our previous research demonstrates that in 7 days after CCl4-induced toxic hepatitis, LOLA prevents cytolysis and cholestasis, improves liver metabolism and protects against oxidative damage (Oleshchuk et al. 2021). Multiple randomized trials in patients with cirrhosis using LOLA have demonstrated its efficacy in lowering systemic ammonia levels (Stauch et al. 1998; Ytrebø et al. 2009) and suggested the effectiveness of LOLA in HE (Vela and Ramírez 2011; Kircheis and Lüth 2019), while its benefits in acute liver toxicity still remain unclear. Therefore, this study is aimed to investigate NO-dependent mechanisms of LOLA action in acute toxic liver injury induced by CCl₄ administration in rats.

LOLA is a stable salt of ornithine and aspartate which activates urea cycle in the liver (Kircheis and Lüth 2019), decreases blood ammonia levels (Ytrebø et al. 2009) and delays the onset of HE (Rose et al. 1999). However, to date, there is no study regarding the mechanism of LOLA action in acute toxic liver injury. In this study we evaluated hepatoprotective effects of LOLA in acute hepatitis induced by CCl₄ administration which has caused the major changes common for acute toxic hepatitis: hepatocellular and cholestatic damage, specific histomorphological features as well as activation of lipid peroxidation process. We have found that LOLA prevents hepatic cytolysis and cholestasis, improves metabolic functions of the liver and exerts significant antioxidant action. Furthermore, we provide the first evidence that LOLA protects liver, which undergoes acute toxic injury, mainly in NO-mediated mechanism.

During liver injury, hepatocellular permeability is increased, and consequently AST and ALT are released from the intracellular space into plasma (Lescot et al. 2012). Thus ALT and AST enzymes are of major importance in assessing and monitoring liver cytolysis (Yakubu et al. 2005; Datsko et al. 2020). Our findings suggest that LOLA’s hepatoprotective activity might be due to its effect against cellular leakage and loss of functional integrity of the cell membrane in hepatocytes as it reduces CCL₄-induced rise in ALT and AST activity and restores the structure of the liver.

Intrahepatic cholestasis develops due to swelling of hepatocytes, which may occur in different liver diseases, causing occlusion of the canaliculi and bile ductules. The hepatocytes become overloaded with substances that cannot be adequately excreted, thus, they enter the blood circulation (Rothuizen 2009). Cholestasis results in increased concentration of all bile constituents, such as cholesterol, bile acids, and bilirubin (Rothuizen 2009), and also of enzymes such as alkaline phosphatase and γ-GT (Malaguarnera et al. 2012). Our data show that in rats with CCL₄-induced hepatitis, the major features of cholestasis developed while LOLA prevented the increase in ALP and γ-GT activity and reduced total bilirubin concentration, although total cholesterol concentration was not affected.

We have found significant antioxidant properties of LOLA as well. The oxidative damage is thought to be a basic mechanism underlying liver injury (Malaguarnera et al. 2012). The free radicals attack on cell membrane-bound polyunsaturated fatty acids resulting in formation of TBARS which are the markers of lipid peroxidation (Tóthová et al. 2013). Our study demonstrates the protective action of LOLA against LP since content of TBARS is decreased under its influence.

The body has a defense mechanism against oxidative stress which includes the antioxidant enzymes which can catalytically remove the reactive species. Mn-SOD is one of the mitochondrial targets of ROS, and thus its activity might become reduced with ROS exposure (Nandi et al. 2019). SOD converts superoxide into hydrogen peroxide which is in turn degraded by catalase, which maintains an optimum level of the H₂O₂ molecule in the cell and regulates cellular signaling processes. Catalase deficiency or malfunctioning is associated with the variety of diseases (Nandi et al. 2019). We have demonstrated that exposure to CCl₄ induces the failure of potent antioxidant enzymes such as catalase and Mn-SOD while LOLA-treatment enhances their activity in rats with hepatitis as compared to untreated rats.

Hepatoprotective effects of LOLA could be explained, in part, by antioxidant properties of its metabolic products glutamine and GSH (Butterworth and Canbay 2018). The mitochondrial GSH pool presents an important defense system against oxidative stress as it reacts covalently with ROS, and serves as a substrate for GST which provides protection against membrane LP by scavenging peroxides and their end products (Malaguarnera et al. 2012). Our data show that in rats with hepatitis, LOLA treatment increases the level of reduced GSH and activity of GST which were inhibited by CCl₄. This confirms previously published findings suggesting that LOLA corrects the loss of GSH in the serum of animals with liver failure (Najmi et al. 2010).

The measure of TAC considers the cumulative action of all the antioxidants present in plasma and body fluids (Ghiselli et al. 2000). TAC is decreased in conditions associated with oxidative stress (Young 2001). Our results show that in rats with hepatitis, TAC is significantly inhibited, whereas LOLA improves this parameter confirming its potent antioxidant effects.

The liver performs numerous biochemical functions. It is the site of metabolism for carbohydrates, fats and proteins where they are all broken down and synthesized. Metabolism in the liver usually leads to detoxification of environmental compounds (Allouche et al. 2011). We established that in CCl₄-induced hepatitis liver metabolism is significantly altered, which results in the reduction of blood and liver total protein content and urea concentration. LOLA administration prevented inhibition of liver metabolism.

Since urea synthesis is stimulated by LOLA, it confirms the other authors’ conclusions (Kircheis et al. 1997; Ytrebø et al. 2009) suggesting this drug might serve as a potent tool in HE prevention. In acute liver injury, liver detoxification function is abruptly reduced due to the massive necrosis of hepatocytes. This leads to the hyperammonemia and development of HE (Ciećko-Michalska et al. 2012). Mode of ammonia‑lowering actions of LOLA is defined by its ability to provide critical substrates for both urea genesis and glutamine synthesis. Activation of the urea cycle leads to the detoxification of ammonia (Kircheis and Lüth 2019).

Urea is a final metabolite of NO. The latter displays the liver synthesizing function and serves as an indicator of protein metabolism and correlates with the level of residual nitrogen in the body (Oleshchuk et al. 2019). NO is formed in the liver under the action of two isoforms – endothelial (eNOS) and inducible (iNOS) and the pattern of their expression and activity differs among physiological and pathological processes. In liver diseases, there is a significant increase in the activity of iNOS isoform (Oleshchuk 2014).

In the biological systems, NO exists as an unstable compound which could be quickly transformed into nitrite anion (NO₂-). Products of nitrites, in turn, could be further involved into the process of free NO synthesis and release and take part in NO-related mechanisms. In liver injury, changes in the content of NO₂⁻ are frequently observed (Prochazkova et al. 2001). We have found that the liver production of NO₂⁻ in acute hepatitis is reduced while serum NO₂- is augmented.

Decline in liver NO₂- production could be explained by a profound change in the cellular distribution of eNOS, which leads to its translocation into hepatocyte nuclei. At the same time the high serum nitrite level is likely to be due to the increased concentration of NO, which synthesis is mediated by iNOS. Increased activity of iNOS is the result of proinflammatory cytokines release which is a prominent feature of in liver injury (Datsko et al. 2020). The increased concentration of NO in the flowing blood by the feedback mechanism dramatically inhibits the expression of eNOS. eNOS-induced deficiency of vasodilation develops, contributing to a decreased sinusoid diameter and increased overall portal vascular resistance. Thus, despite the overproduction of nitric oxide, there is a relative lack of intrahepatic microcirculation mediator, as evidenced by reduction of nitrite anion in the liver.

Notably, LOLA treatment resulted in the increased production of NO₂- in the serum and liver. Previously it was established that in patients with cirrhosis (Staedt et al. 1993) and in an experimental model of chronic liver failure (Rose et al. 1999), LOLA treatment enhanced NOS synthesis. NO, produced due to the function of endothelial NOS (eNOS), exerts protective properties in liver injury through the regulation of sinusoidal diameter, prevention of neutrophil adhesion, inhibition of platelet aggregation and adhesion, and scavenging of reactive oxygen species (Peralta et al. 2001). The relevance of NO in hepatic microcirculation is supported by the fact that administration of eNOS inhibitors reduces microvascular perfusion and aggravates I/R injury, while the supplementation with L-arginine (NO precursor) or NO donors improves microcirculatory status and minimizes liver damage after I/R (Shah and Kamath 2003). However, NO-dependent mechanisms of LOLA action in acute toxic liver injury are not well understood.

To determine if the mechanisms of LOLA action in acute toxic liver injury depends on NO synthesis, we used L-NAME which is a competitive inhibitor of all NOSes with high selectivity to eNOS (Víteček et al. 2012).

The concomitant use of L-NAME in animals with CCl₄-induced hepatitis inhibited the action of LOLA to enhance NO₂- synthesis both in serum and liver. Besides, we have found that the use of L-NAME delayed the recovery of hepatocytes and affected the reduction of AST and ALT, ALP and γ-GT caused by LOLA, counteracted LOLA effect against blood total protein reduction, prevented the decline in сatalase and GST activity and reduced GSH level caused by LOLA.

Altogether these results indicate that in CCl₄-induced acute hepatitis, LOLA effectively prevents major syndromes which accompany acute liver injury, such as cytolysis and cholestasis, improves liver metabolism and protects against oxidative stress. Partially, these changes develop in NO-mediated mechanism since the use of L-NAME declines most of LOLA effects.