| Background: Liver is one of the largest digestive gland in the body and the largest substantive organs. The function of the liver is very complicated and varied.It occupies an extremely important position In the body’s metabolism, It is closely related to the function of metabolism,transformation and detoxification of the three organic matter. When all sorts of reasons Such as severe hepatitis, liver cirrhosis and hepatocellular carcinoma cause serious liver disease,Loss of the liver’s synthetic and detoxification function lead to jaundice, hepatic encephalopathy, blood coagulation disorders, and multiple organ failure,all of them are seriously threat to the patient’s life. While it is common that The blood ammonia increases in liver failure, In clinical practice in recent years,as a ndicator of the metabolic capacity about liver,it is drawing more and more attention. It not only have diagnostic value of hepatic encephalopathy,but also can reflect the severity of hepatic lesions. blood ammonia is the norma metabolic product in the human body,the source of Blood ammonia mainly contains intestinal decomposition, Kidney secreted and muscle to produce ammonia. The normal value of blood ammonia in the human body is 18 ~ 72 umol/L. The liver is the main place to metabolism ammonia. In the normal condition the ammonia in human body is mainly compounded to ureain the liver through urea cycle,and thendrain out of the body with the urine.while in the brain tissue, heart and muscle, the blood ammonia is generated to glutamine catalyzed by glutamine synthetase. and then the glutamine is moved into the liverwith blood circulation,and resolved to be ammonia again catalyzed by glutaminase. Finally, the ammonia participate in Ornithine Cycle to generate urea in liver cells. And glutamine in cytoplasm of liver cell can also participate in De novo synthesis of pyrimidine through CPSII pathway,which Is the basic materials of DNA and RNA synthesis in the cell.When the liver is damaged due to pathological changes, the ability to metabolize of the liver itself Will be affected, it can’t effectively to metabolize ammonia out of the human body, Leading to constantly accumulate in the body. Ammonia has large toxic effect to the human body, if it cannot be timely transformed into non or lower toxic product and drained out,it will further aggravate hepatic injury and more serious complications such as hepatic encephalopathy will appear, even induce hepatic failure. When the liver failure happens, A large amount of liver cells encounter necrosis and apoptosis,the normal function of the cell is badly damaged, But the "residual" of liver cells are unable to meet the normal needs of the human body through the compensatory resulting in a series of functional disorders, Including high blood ammonia, Lactic acidosis, disorder of glucose metabolism and i high livel of ndole and so on. And after accumulating in the body,the impact of the ammonia mainly reflects in the following respects.1. Ammonia can cause swelling of the astrocytes in the body.the research findings indicate that the swelling astrocytes is an important pathological indicator of the brain edema happened in acute liver failure.2, Ammonia can cause transformation of mitochondrial permeability.3, Ammonia can also cause disfunction of energy metabolism in cells.4. Oxidative stress effect caused by ammonia.In the past the research on high blood ammonia mainly focus on the toxic effects to nerve cells that is research of hepatic encephalopathy. Now the study about the pathogenesis of hepatic encephalopathy mainly contains ammonia intoxication theory, the metabolism theory of amino acid in plasma, Pseudo neurotransmitter theory and so on. And a variety of toxins in the body work together, mutual influence, each other cause and effect, promoting the occurrence and progress of encephalopathy sensibility together.Among them the most research proceedingaround ammonia intoxication theory. Blood ammonia mainly relies on the glutamine synthetase in the astrocytes of the brain tissue to complete metabolism. However, astrocytes is the main target cells of the neurotoxicity from ammonia. Ammonia can induce brain edema and change the gene expression through a variety of approaches. The end result is neurons lost the nutrition support role of astrocytes, thus lead to the occurance of hepatic encephalopathy.We found that through clinical practice, in based on the theory of "second strike",given patients with HBV related acute on chronic liver failure the same therapy of antivirus, protect liver, diuresis, etc at the same time, using the nmda ornithine treatment through the whole caurse can significantly put off the disease progression, it can obviously alleviate the liver injury and greatly promote the secretion, detoxification, synthesis and transformation and other functions of the liver cells by reducing the patients’ blood ammonia level in the body,it can also reduce mortality and improve prognosis of patients. And with the application of drugs to drop blood ammonia, the serum bilirubin level of patients get down with it, there are correlations between the two. Animal experiment later also confirmed that after building the model of high blood ammonia on mice through lavage with ammonium chloride, we are able to observe that blood ammonia can be directly through the apoptosis of liver cells inducing liver injury, it may be mediated through affecting the expression of genes. Also dealing with human normal liver cells with ammonium chloride,we can find that it can significantly inhibit the growth of normal liver cells. Further experiments showed that ammonia can disturb the bilirubin metabolism process by interfering with he energy metabolism of normal liver cells. Metabonomics studies show that the glutamine level in the liver cells increased after treated with ammonium chloride,while RNA synthesis in cells is suppressed, the raw material increased but the product is reduced. Further results show that as an important intermediate of de novo synthesis of pyrimidine, synthesis of orotic acid is also suppressed.This may be the key to the suppression of RNA synthesis, but the exact mechanism is unclear, we suspect that may be the ammonium chloride can suppress the dihydroorate dehydrogenase which can catalyze the synthesis of orotic acid. So we hope to find out the reasons for this phenomenon through theexperiment,it is helpful for us to know the mechanism of liver failure further, offering a new theoretical basis for the treatment of liver failure.Objective: After dealing with the normal liver cells with ammonium chloride, to check the growing status and test the changes of the expression of the dihydroorate dehydrogenase relate to the orotic acid synthesis.Methords:1. Cultivate normal human liver cell line(changliver),CCK-8 method is used to check the proliferation of cells after stimulated for 12,24,48 hours with different concentrations of ammonium chloride(1.25、2.5、5、10、20、40 and 80mmol/L)2. Use the flow Cytometer to check the apoptosis of changliver after stimulated with ammonium chloride(5、10、20 mmol/L) for 24 hours.3. Realtime-PCR was used to detect the expression of DHODH mRNA after stimulated with ammonium chloride(5、10、20 mmol/L) for 12,24,48 hours.4.Western blotting was used to detect the expression of DHODH protein after stimulated with ammonium chloride(5、10、20 mmol/L) for 12,24,48 hours.5. Using SPSS17.0 statistical software for processing the data result,, Quantitative data are shown by using mean ± standard deviation. Normally distributed data comparison between the two groups using t test. Classification data using R * C contingency table chi-square test or Fisher exact probability test. Inspection level of alpha = 0.05Results1. CCK-8 method find the ammonium chloride can suppress the proliferation of changliver,and with the increase of concentration of NH4 CL, the inhibition rate increased gradually.2, Results from the flow cytometry instrument detection show that changliver appears obvious apoptosis phenomenon after stimulated with ammonium chloride(5、10、20 mmol/L) for 24 hours, and with increase of concentration of NH4 CL, the apoptosis is more obvious.3.Realtime-PCR results indicate that the expression of dihydroorate dehydrogenase m RNA was significantly reduced after stimulated with ammonium chloride(5、10、20 mmol/L) for 12,24,48 hours compared with control group(p<0.05)4, Western-blot results indicate that the expression of dihydroorate dehydrogenase m RNA was significantly reduced after stimulated with ammonium chloride(5、10、20 mmol/L) for 12,24,48 hours compared with control group(p<0.05)Conclusion1,The ammonium chloride can suppress the proliferation of changliver, and with the increase of concentration of NH4 CL, the inhibition rate increased gradually.2,The ammonium chloride can cause apoptosis of changliver with the concentration of NH4 CL increases, the apoptosis is more obvious3, The expression of DHODH in changliver cells can be suppressed by ammonium chloride and then lead to the decrease of orotic acid synthesis,which can disturb the synthesis of pyrimidine nucleotide. |
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