Effects Of Mitochondrial Impairment Caused By Ammonia To The Apoptosis Of Hepatocytes And Bilirubin Transport Related Protein MRP2

Research background:Liver failure(hepatic failure, HF) is a clinical symptoms of metabolic dysfunction under which liver cells are subject to extensive and serious damage. It often accompanied by massive necrosis of liver cells, malignant rise of blood ammonia, serious gastrointestinal symptoms, rapidly deepening of jaundice, hemorrhage, cerebral edema and pulmonary edema even hepatic encephalopathy. This syndrome is a liver disease endangering people’s health seriously, owing to its serious condition, complex clinical symptom and high mortality. The hepatic encephalopathy is one of the most important complications of decompensated liver cirrhosis and liver failure, a syndrome of central nervous system dysfunction based on metabolic disorders. Its main clinical manifestation is consciousness disorder, conduct disorder and coma. The ammonia poisoning theory caused by metabolic disorder is constantly at the core position in the pathogenesis of hepatic encephalopathy. At the same time, blood ammonia plays a very important role in the diagnosis、treatment of hepatic encephalopathy.Cell apoptosis and mitochondrial: Compared with necrosis, apoptosis is a kind of programmed death process performed by the cell itself.When in necrosis, many events followed: Cell swelling, the damage of the cell membrane, the change of permeability and the intervention of macrophage and organelles scattered into the Cytoplasm. The result is inflammation of the tissues. Compared to cell necrosis, cell apoptosis is a common form of cell death. Cells narrow during apoptosis and DNA is cut into small pieces of 180bp-200 bp by endonuclease. Cell apoptosis as well as cell proliferation is the basic phenomenon of life, the basic measures to maintain the dynamic balance of the number of cells in the body, the exact process regulated by gene. Generally speaking, there are two main ways of apoptosis according to whether the mitochondria-mediated: one is through the intracellular enzyme caspase activated by the extracellular signal; the other is the release of apoptosis protease activating factor through the mitochondrial. The activated caspase can cause apoptosis through degradation of important protein in cells.The release of cytochrome C from the mitochondria to the cytoplasm is one of the signals of apoptosis. Many previous studies generally believe that: Cytochrome C release into the Cell stress or apoptosis signal can cause the release of cytochrome C from mitochondrial. As inducer of apoptosis, cytochrome C can form apoptotic bodies together with Apaf-1, caspase-9 precursor, ATP/d ATP and then activate Caspase-3, triggering a cascade reaction of caspases, leading to cell apoptosis. Many previous studies generally believe that: Cytochrome C release into the cytoplasm by the mitochondrial MPT pore or mitochondrial transmembrane channel composed by members of the Bcl-2 family. The open of membrane channel significantly change the permeability of mitochondrial In cell apoptosis or necrosis. Thus, contents into the mitochondria are released into the cytoplasm through membrane channels. Excessive entry of Ca2+, oxidation of Reduced Glutathione Tablets(GSH) and increased levels of Reactive Oxygen Species(ROS) all these can be considered as a reason for the continuous opening of m PTP. Mitochondrial membrane potential disappeared and cytochrome C released as a result. The mitochondrial m PTP pore(mitochondrial permeability transition pore) is mainly composed of Adenine nucleotide translocator in intima(ANT) and Voltage dependent anion channel(VDAC).the opening of m PTP pore will cause a decrease in mitochondrial transmembrane potential and release of cytochrome C, leading to cell apoptosis. The mitochondrial permeability transition pore(m PTP), the synthesis of ATP which is closely related to the function of mitochondrial, is believed to be associated with elevated ammonia levels and the production of ROS [1].The recent research shows that when exposed to physiological concentrations of ammonia free radicals increased significantly in cultured astrocytes. Evidence also showed an increase of superoxide production and a decrease in the activity of various antioxidant enzymes in animal model of acute ammonia poisoning [3]. Norenberg use calcium-sensitive fluorescence probe fluo-3 to prove that: when exposed to physiological concentrations of ammonia, intracellular calcium levels were significantly increased in cultured astrocytes and oxidative stress exists in ammonia poisoning. One of the consequences of oxidative-stress is the opening of Ca2+ dependent m PTP pore in the mitochondrial. So the concentration changes of calcium ion may be important causes for the opening of m PTP pore.Oxidative stress can change the functions of biological membrane by membrane lipid peroxidation, or destroy enzyme activity by combined with biological macromolecules and cause different extent injury to liver cells under the help of cell factor(such as TNF-α, NF- κ B). So antioxidant treatment plays an important role for the prevention and the treatment of liver disease [4].Blirubin is an endogenous molecule that is a byproduct of heme. After its formation in the systemic circulation catalyzed by HO-1, UCB must first enter the hepatocytes via OATP1B1 and OATP1B3. Once in the hepatocytes, indirect bilirubin is extensively metabolized by UGT1A1 to DBIL which is then shunted into the bile by MRP2. Hyperammonemia and jaundice are the common clinical symptoms of hepatic failure(HF). The level of bilirubin decreased along with the descendent blood ammonia concentration in hepatic failure patients, found in clinical work. The liver is a major site for ammonia metabolism in vivo. Elevated blood ammonia and “hepatocyte anergy" phenomenon in chronic hepatitis or liver failure caused us great concern. But how elevated ammonia influence pathophysiological process of liver cell? Where ammonia induced apoptosis of liver cells? Through what channels ammonia affects the bilirubin metabolism or transport? There are much mechanism about increased ammonia caused damage to astrocyte, including direct toxicity, oxidative stress, mitochondrial permeability transition and glutamine theory. But few about the toxicity ammonia cause to hepatocytes. The metabolism of bilirubin is closely related to energy consumption. In many models of hepatic encephalopathy ammonia was demonstrated to have interference on mitochondrial oxidative damage and energy metabolism. The current evidence suggests that ammonia can be applied to the TCA cycle and apple acid- mitochondrial aspartate shuttle enzyme, resulting in the deficiency generation of ATP and the occurrence of oxidative stress [5]. Therefore we investigated the effects of ammonia, which is believed to cause mitochondrial damage and energy barrier, on the bilirubin metabolism related enzyme. Elevated bilirubin levels can also be considered signs of oxidative stress. But few about the toxicity ammonia cause to hepatocytes and the effect of ammonia on bilirubin. We found in the clinical treatment: on the basis treatment of acute liver failure and cirrhosis, combined application of lowering blood ammonia drugs early for the entire process, can significantly relieve the liver injury, reduce the mortality and improve the prognosis of the patients.Through the establishment of cell model of ammonia poisoning, specificity damage to hepatic mitochondrial function is proved. Ammonia appears to interfere with bilirubin metabolism by causing an energy disorder. This study may provide insight into the possible molecular mechanism of jaundice induced by hyperammonemia, as well astargets for the treatment or prevention of bilirubin metabolic disorders. Research purpose:1.To verify the the proliferation of hepatocytes toxicity by MTT experiment from concentration gradient of NH4CL;2.To reveal the role of ammonia in ipromoting liver cell apoptosis and injury of liver cell function;3.To reveal the damage of ammonia to mitochondrial function and the presence of oxidative stress in hepatic;4.To prove that the efflux process of DBIL which mediated by protein MRP2 was hindered by ammonia which inhibited the ATP synthesis. Research methods:1.Normal liver cell line chang liver was cultivated, and MTT method was used to determine the proliferation of toxicity by different concentrations of NH4CL(5、10、20 和 40 mmol/L);2.The integrity of apoptosis index PARP was detected by protein immunoblot technology after ammonia’s effect. The activation of caspase-3 was detected by cell immunofluorescence. To confirm the influence of ammonia induced liver cell apoptosis combined with TUNEL method;3.Detection of TNFR1 and p53 m RNA by fluorescence quantitative PCR, which ruled out the exogenous way possible of ammonia induced liver cell apoptosis;4.The change of active oxygen substances ROS affected by antioxidants SOD and GSH was observated by Flow cytometry technology;5.The change of mitochondria morphology was observed under TEM. The changes of m PTP hole were observed under a fluorescence microscope after ammonium chloride treatment;6. Liver function index of ALT, AST, COX, ATP and redox indicator of GSH were detected by ELISA; Compare the changes of intracellular and extracellular DBIL;7. The m RNA and protein expression of HO-1, UGT1A1 and MRP2 were by Quantitative PCR and Western Blotting. Ammonia blocked the extracellular excretion of DBIL by causing an energy disorder. Result:The inhibition rate of ammonium chloride on chang liver cells is in a steady rise, increased with time. Decreased cell density and vacuolization phenomenon were observed under light microscope;When liver cells were treated with 10 mmol/L ammonium ion for 48 h, PARP protein was cut, caspase-3 was activated and apoptosis was significant; when cells were treated with 40 mmol/L ammonium ion dead cells were too much. There was no significant difference in TNFR1 and p53 m RNA.When liver cells were treated with10 mmol/L ammonium ion for 48 h, the morphology change of mitochondria was observed: mitochondria grown swelled and round, mitochondrial cristae were disordered and vacuolar degeneration. when liver cells were treated with 10mmol/L ammonium ion for 12 h, the GSH and ALT was significantly higher than that of control group(p<0.0001 and p<0.0001), The increase of COX and decrease of ATP were detected 24 h after ammonia exposure(p<0.000 1 and p<0.05), suggesting oxidative damage. While the increase of AST was detected 48 h after ammonia exposure, relative lag.When liver cells were treated with 10mmol/L ammonium ion for 24 h, reactive oxygen ROS significantly increased, produce of ROS was decreased in a certain degree when treated with SOD and GSH; SOD and GSH inhibited apoptosis of liver cell by ammonia in a certain degree.In 10 mmol/L NH4 CL group, Synthesis of ATP decreased significantly compared with the control group; Accumulation of intracellular DBIL and decreased of extracellular DBIL levels were deteced at the same time.When liver cells were treated with 10mmol/L NH4 CL for 24 h, both m RNA and protein levels of UGT1A1 increased than the control group, however, the level of MRP2 has experienced a significant decline, whereas HO-1 hasn’t. The adding of ATP increased the m RNA and protein levels of MRP2 significantly. Conclusion:1.10 mmol/L ammonia induced liver cells apoptosis and caused significant damage to liver cells function;2.Ammonia caused the liver cells mitochondrial oxidative damage and the opening of m PTP hole;3.Ammonia induced liver cells apoptosis by endogenous mitochondrial oxidative damage;4.Metabolism and excretion of bilirubin in ammonia poisoning hepatocyte are affected;5.Ammonia affects excretion of bilirubin through MRP2 protein by blocking the energy supply.