The Influence Of Different Drugs On Energy Metabolism Of Ammonia Chloride-induced Liver Cell Model

BackgroundThe liver is a pivotal organ of human body in the metabolism of glucose, lipids, proteins, vitamins and hormones, which has a crucial role in bioconversion, detoxification, metabolism, digestion, secretion, excretion and immunity. When liver function is impaired, "remnants" of the liver cells are unable to meet the body’s metabolic needs, it will produce a series of metabolic disorders, including lactic acidosis, elevated blood ammonia,increased levels of indole and so on. Acute-on-chronic liver failure(ACLF) is defined as a short-term(2-4 weeks) liver function sharp deterioration developing from chronic liver disease and it is manifested as a group of clinical syndromes including blood coagulation disturbance, jaundice, hepatic encephalopathy and ascites [1].ACLF is featured with acute onset, severe illness situation, difficult treatment and high mortality[2]. Currently, it is believed that the increase of blood ammonia level is closely correlated with the occurrence of hepatic encephalopathy, which is the most common complication of ACLF [3]. Blood ammonia in vivo originates from the decomposition and metabolism of various amino acids by all tissues, and from the absorption by the intestinal tubes. It is accepted that blood ammonia is excreted as nontoxic urea mainly through the ornithine cycle metabolism in the liver [4].Upon the onset of ACLF, the ability of liver to synthesize ammonia into urea is weakened, and thus the ammonia in the intestinal tracts, without detoxifcation by the liver, directly enters the body circulation, thus increasing the blood ammonia level.Via inducing cerebral astrocyte swelling, damaging the synthesis and release of neurotransmitters, and enhancing oxidative stress, the high blood ammonia thereby increases the incidence rates of hepatic encephalopathy, hydrocephalus, cerebral hernia, and epilepsy, thus interfering with the prognosis [5]. Because elevated arterial blood ammonia levels, patients with liver failure occurs brain energy metabolism disorder, including glycolysis, the citric acid cycle and oxidative respiratory electron chain process, leading to lack of energy.As reported, artery blood ammonia>124 umol/L is correlated with the occurrence of severe hepatic encephalopathy and hydrocephalus [6]. It is generally believed that the increase of blood ammonia level is only related with hepatic encephalopathy, but no evidence proves that blood ammonia can directly or indirectly affect the production of liver cells.However, our team found that the effects of blood ammonia on ACLF patients were beyond our imagination. As is well-known, the occurrence of ACLF will induce the necrosis of abundant liver cells, and thus the hope to save and treat the patients without liver transplantation is to maintain the functions of residual liver cells. Thus, how to better maintain the functions of residual liver cells is also a hotspot in the field of supportive therapy of liver failure. Our preliminary results suggest that the increase of blood ammonia level induced by liver failure will not only cause hepatic encephalopathy, but also will injure liver cells(mainly apoptosis), inhibit cell regeneration.Our retrospective study showed that early-stage or full-course use of blood-ammonia-lowering drugs significantly relieved liver injuries, reduced mortality rate, and improved prognosis [7].In this study, through the establishment of high ammonia cell model, we reveal the role of ammonia in the liver cell energy metabolism, as well as to comparing the effects of L-carnitine, diisopropylamine dichloroacetate and trimetazidine on liver cell with NH4 Cl treatment。 ObjectiveEstablish high ammonia hepatocyte model and test the toxicity of ammonia on liver cells. Normal hepatocytes chang liver were cultured with concentration gradient of ammonium chloride, and detect the level of compounds, enzyme and ROS in glycolysis and the Krebs cycle process, thus clarifying the role of ammonia on energy metabolism and oxidative damage of liver cell.At the same time, we compare the effects of L-carnitine, diisopropylamine dichloroacetate and trimetazidine on liver cell with NH4 Cl treatment, thus providing a new theoretical basis for clinical treatment of acute on chronic liver failure. MethodsCell model of ammonia poisoning1. Detect toxicity of ammonia or different drugs to liver cells by method of CCK-82. Detect the level of lactic acid, pyruvic acid, pyruvate kinase, lactate dehydrogenase, pyruvate dehydrogenase, acetyl-COA during glycolysis and the TCA cycle by ELISA and automatic biochemistry analyzer. Results:With the increasing of ammonia chloride and time,the inhibition rate of ammonia to chang liver cells increased gradually(P<0.05); With the concentrations of ammonium chloride increasing, levels of intracellular pyruvate, pyruvate kinase and lactate dehydrogenase increased, while the expression of pyruvate dehydrogenase, acetyl COA decreased(P <0.05); Ammonia chloride can cause lactic acid increasing; Chang liver cells were treated with 5m M ammonia chloride and L-carnitine, diisopropylamine dichloroacetate, trimetazidine, the inhibition rate of ammonia to chang liver cells has decreased in L-carnitine, diisopropylamine dichloroacetate group(P <0.05),while has no signficant changes in trimetazidine group. Lactate levels were lower than the ammonia treatment(P <0.05) in L-carnitine, diisopropylamine dichloroacetate and ammonia chloride combination groups, while increased in trimetazidine and ammonia chloride combination group.Conclusion:1. Ammonia can inhibit the growth of liver cells, and with ammonia concentration increasing, the inhibition rate also increase.2. Ammonia can promote liver cell glycolysis, and inhibite the citric acid cycle.3. L-carnitine and diisopropylamine dichloroacetate can both decreased lactate levels and the inhibition rate of ammonia to chang liver cells and the L-carnitine has the best effects, while trimetazidine can’t decreased the inhibition rate but increased lactate levels.