A Study On Hyperammonemia-induced Hepatocyte Injury And Its Metabonomics

Liver failure is a common critical illness, with a mortality rate as high as 70~80%. The pathogenesis of liver failure is yet unclear and its treatment remains a worldwide challenge. Liver failure involves considerable hepatocyte necrosis, liver function damage, and liver regeneration and differentiation disorders, and is one of the primary causes of death among patients. The remaining hepatocytes cannot meet the body’s needs, resulting in disorders in hepatocyte conversion and metabolism, and leading to the accumulation of metabolites including blood ammonia, lactic acid and indirect bilirubin. At present, the “secondary damage” theory provides one of the important mechanisms for the development of liver failure. The most common metabolic disorder of liver failure is hyperammonemia, the neurotoxicity of which has been studied by many researchers. However, whether or not it could cause secondary damage of the liver has not been reported. In previous studies, we used hyperammonemia as the entry point to investigate liver failure, and established a first hyperammonemia-attacking rat model. We demonstrated hyperammonemia could induce “secondary damage” in rat liver. However, we did not study the damaging effect and the mechanism of hyperammonemia on human hepatocytes at a cellular level. The liver is a vital metabolic organ in the human body. Pathological changes in the liver are always accompanied with related changes in the metabolic network. Thus, research on the mechanism of ammonia in hepatocyte injury cannot focus on hepatocyte damage alone, but should also utilize holistic thinking, or systems biology. Research on the metabonomics of ammonia-induced liver damage has been little reported around the world. Therefore, a study on ammonia-induced hepatocyte injury and its metabonomics will produce a theoretical basis for the molecular biological mechanism of hyperammonemia in liver failure, and provide novel interventional strategy and target for the prevention and treatment of liver failure.Method1 The effect of ammonia on hepatocyte-specific damagesA hyperammonemia cell model was first established. Cell Counting Kit-8(CCK-8) and flow cytometry were used to examine the effects of ammonia on the proliferation and apoptosis of various cell lines. We also investigated whether ammonia’s effects on hepatocyte damage and apoptosis were specific. Real-time PCR and Western Blotting were adopted to examine the expression of proteins related to ammonia transport, RHCG and AQP8, and to investigate their specific mechanisms. Electron microscopy and mitochondrial permeability transition pore(m PTP) were used to observe morphology and membrane permeability of mitochondria, in order to investigate their damage mechanism.2 The effects of ammonia on hepatocyte metabolic profileA metabolomics method based on gas chromatography-mass spectrometry(GC-MS) was established for the hyperammonemia cell model, in order to understand the effects of ammonia on hepatocyte metabolic profile. By combing multivariate data analysis and database comparison, differential metabolites were selected and identified and differential metabolic pathways were found. Based on relevant clues provided by the GC-MS results, in combination with Real-time PCR and western blots, differential metabolic pathways were verified and studied in depth.Result1 Ammonia had specific damage effects on hepatocytes1.1 The effects of ammonia on the proliferation and morphology of hepatocytes, and the choice of modeling concentrationWith increasing ammonia concentration and/or treatment time, the growth and proliferation of primary hepatocytes were significantly suppressed. The hepatocyte growth density observed under a microscope also significantly decreased and eventually resulting in cavitation. The ammonia concentration of 10 m M was determined to be the ideal modeling concentration.1.2 The effects of ammonia on the proliferation and apoptosis of hepatic cells and non-hepatic cells(1) Ammonia inhibited the growth and proliferation of hepatic cells, especially primary hepatocytes. It did not have obvious effect on the growth of non-hepatic cells(2) Ammonia could substantially induce apoptosis in primary hepatocytes, while its apoptotic effect on other cells was not obvious.1.3 The effects of ammonia on ammonia transport proteins in different cell linesAmmonia significantly affected the mRNA and protein expression of ammonia transport protein RHCG and AQP8 in primary hepatocytes, while the expression of ammonia transporters in other cell lines was not affected.1.4 The effect of ammonia on liver function enzymology indicatorsCompared with the control group, glutathione(GSH) in the high-ammonia cell model presented a trend of decrease followed by recovery to normal level; alanine aminotransferase(ALT) presented a trend of significant increase followed by significant decrease; aspartate aminotransferase(AST) showed no significant difference in the first 48 hours and significantly increased afterwards; cyclooxygenase(COX) significantly increased after 24 hours.1.5 The effect of ammonia on the damage and apoptosis of hepatocyte mitochondriaAs observed under an electron microscope, hepatocyte mitochondria in a high ammonia environment presented with morphological changes including swelling, turning round, cristae disorder and cavitation, together with ammonia-induced m PTP, and increased mitochondria permeability. Cell apoptosis was observed at the same time.2 The effect of ammonia on hepatocyte metabolic profile1)A GC-MS method to examine metabolic profile of hyper-ammonia hepatocyte model was established. Multivariate statistical analysis showed significant metabolic differences between ammonia treatment group and no-treatment group.2)GC-MS detected 648 metabolites, 120 of which were differential metabolites. Relevant databases were used to identify these metabolites, and to determine the major differential metabolic pathways. The result showed disorders in Krebs cycle, urea cycle and purine-pyrimidine metabolism in the ammonia treatment group.(1)The effect of ammonia on the Krebs cycleKrebs cycle metabolites, such as citric acid, α-ketoglutarate and malate were all significantly reduced in the ammonia treatment group. The formation of adenosine triphosphate(ATP) also decreased.(2)The effect of ammonia on the urea cycleUrea level in a hyper-ammonia environment did not increase, and had no significant difference compared with the no-treatment group. Arginine level significantly decreased. Ornithine level slightly increased. It was also found that, in a hyper-ammonia environment, the expression of two important enzymes in ammonia metabolism, CPS-I and its isozyme CPS-II, was different from that in a normal physiological environment, and high ammonia induced reduction of CPS-I expression but elevation of CPS-II expression.(3)The effect of ammonia on purine-pyrimidine metabolismThe levels of purine pathway metabolites, inosine and xanthime, were significantly lower in the ammonia treatment group than those in the control group.The levels of orotate, uracil, β-alanine, cytosine and succinate in the pyrimidine pathway were also significantly lower than those in the control group, while the level of thymidylate increased. Orotate is an important precursor of nucleic acid synthesis. Real-time PCR and western blot were combined to detect and verify that the expression of a key enzyme dihydroorotate dehydrogenase(DHODH), which was catalytically synthesized from orotate, was suppressed in a hyper-ammonia environment. RNA synthesis disorder was detected using 14C-UTP-incorporation.3 The effects of differential metabolites on the hyper-ammonia cell modelCompared with the other metabolites, orotate could significantly reverse the damaging effect of ammonia on hepatocytes.Conclusion1.Ammonia could specifically damage hepatocytes. Its specific mechanism is related to the expression of proteins RHCG and AQP8, which are related to ammonia transport at the cell membrane. The damage mechanism is also related to ammonia-initiated mitochondrial apoptosis pathway.2.Ammonia could induce disorders in hepatocyte Krebs cycle, urea cycle and RNA synthesis. We propose for the first time that urea cycle disorder exists in a hyper-ammonia environment, and the theory that insufficient ammonia metabolism leads to excessive ammonia entering ammonia metabolic pathway that involves CPS-II. We also propose that combination of orotate with medicines that can effectively promote endogenous ATP production could be a prospective new direction for the research and development of new pharmaceutical agents for liver failure.