Research On The Mechanism Of Hepatocyte Injury And Apoptosis Caused By Hyper-ammonia

BackgroundThe liver is one of the most important organ within the human body which has synthesis, detoxification, metabolism, hormones, bioconversion and immune defense function. Normal liver accout for 30-40% of the blood flow and make the harmful substances in the blood detoxification or inactivated, and synthesis of all essential nutrients, is known as the "plant" in the human body. It plays an important role in our life activities.Blood ammonia(BA) is the main product of amino acid metabolism which include the intestinal ammonia production, the kidneys secrete ammonia, muscle produced ammonia, etc.Excess ammonia has very big toxicity to human body.The liver is main ammonia metabolism organ.It can transfer the ammonia into non-toxic or low toxicity substances and remove excess ammonia.Liver failure (hepatic failure, HF) was caused by a variety of reasons which cause liver cell damage and liver dysfunction. HF is one of the severe conditons in emergency. Serious patients can cause hepatic encephalopathy, hepatorenal syndrome, hemorrhage.the mortality rate is as high as 70%～80%. The high blood ammonia level may be one of the important causes a series of abnormal liver failure.From 2005 to 2011 in our hospital the patients with liver failure treated with ammonia-lowering drugs can significantly remission in liver damage, and lower patient mortality, improve patient prognosis.we establish chronic hyperammonemia attack rat model and found that the enzymatic indicators of liver damage were significantly increased.The pathology study showed that hyperammonemia can induce liver cell damage which not by inflammation necrosis but cell apoptosis.The excessive hyperammonemia may play a regulatory role in the development process of hepatocyte apoptosis and liver damage.However, the high blood ammonia toxicity to the cells of the body is not equal, in the same concentration of blood ammonia, the growth of 293, HDF, Vero and PQXB1/2 cell lines were less affected, while McCoy, MDCK, HeLa, BHK cell lines were inhibited obviously.These showed that the effect of ammonia on cell has obvious cell specific. Liver failure elevated blood ammonia can damage neurons or glial cells leads to hepatic encephalopathy.The damage and toxicity of high blood ammonia to liver cells is unknown.To further study the effect of high blood ammonia on liver cell injury and its mechanism has important significance in liver failure.ObjectiveBy establishing the rat model of ALF and giving early lowering ammonia treatment, the study focuses on the influence of early lowering blood ammonia’s on hepatocyte apoptosis and explore the possible mechanism of liver damage. We also plan to culture hepatocyte with hyper-ammonia and explore the mechanism of hepatocyte apoptosis caused by hyper-ammonia.MethodsAnimal modelEstablishing ALF model group by intra-abdominally injecting D-galactosamine (450mg/kg) and LPS (100μg/kg) to 48 female SD rats. Besides, the intervention group should be injected ornithine and aspartate (1.5g/kg,6 hrs’ interval). The intra-abdominal injection of physiological saline is taken as blank control. The rats are narcotized and put to death after 12 hrs and 24 hrs. Take blood from heart and liver tissue. Dry chemistry method is used to test blood ammonia, ELISA is used to detect serum levels of ALT, AST, TNF-α and IL-6. The liver tissue is fixed in formalin, embedded in paraffin, desiccated and cut into slices. To observe pathological changes, it needs to do HE staining. The apoptosis rate will be tested by TUNEL. Extract DNA of tissue by freezing, observe DNA Ladder in the method of electrophoresis and detect relative expression level of P53 and SPP1 by applying RT-PCR. The statistical software SPSS is used to do data analysis and compare group difference and correlation analysis.Cell study.We added NH4Cl during culturing rat liver cells to build the hyper ammonia cell model, and applied BAPTA-AM and EGTA to block calcium inside or outside the cells for construction. The tetrazolium dye (MTT) assay was used to determine cell viability. Flow cytometric analysis was performed to analyze the effect of hyper-ammonia on apoptosis. The expression of apoptosis-related proteins(Cyt c and PARP) was evaluated by western blot analysis. Real-time PCR was performed to mRNA expression levels of some Ca2+ dependent protein (CALM, CAMK), and CAMK expression levels also were estimated by ELISA methods.ResultsAnimal model1 Blood ammonia reached peak at 12h and then become to drop at 24h.12h,24h hepatic failure group and OA intervention group compared with blank control group was significantly increased, OA intervention group compared with hepatic failure group was significantly reducing, and there were statistical differences between them(P<0.05).2 With the time of administration, the liver damage related indicators (ALT and AST) was significantly increased. Except the blank control group,24h group were higher than the 12h group.12h OA intervention group mean compared with hepatic failure group was decrease, but there was no significant statistically difference between them (P> 0.05).24h OA intervention group compared with hepatic failure group was significantly reducing, there were statistical differences between them (P<0.05). And ammonia was positively correlated with those. The relevant indicators of liver enzymes 24h were higher than the 12h group with the increase of administration time.3 Pathological changes increase with the increase of administration time.There was no significant difference between 12h hepatic failure group and the OA intervention group. And compared with 24h hepatic failure group, changes of pathological in 24h OA intervention group was milder. Blank the control group did not see the obvious exception.4 Except the blank control group, other groups all can see DNA ladder, which was apoptotic cell-specific. TUNEL results showed that hepatocyte apoptosis rate increased with the increase of administration time (24h>12h). The AI of eihter 12h or 24h hepatic failure and OA intervention group compared with blank control group was significantly increased, OA intervention group compared with hepatic failure group was significantly reducing, there were statistical differences between them (P<0.05). And ammonia was positively correlated with that.5 With the administration time extending,The expression of IL-6, TNF-a, p53 were increased with time (24h>12h). The expression of hepatic failure and OA intervention group compared with blank control group was significantly increased, OA intervention group compared with hepatic failure group was significantly reducing, there were statistical differences between them (P<0.05). And ammonia was positively correlated with those.6 SPP1 gene was highly expressed in ALF rat model. With the administration time extending, SPP1 levels were increased with time (24h>12h). In hepatic failure and OA intervention group compared with blank control group was SPP1 significantly increased, OA intervention group compared with hepatic failure group was significantly reducing, there were statistical differences between them (P<0.05). And ammonia was positively correlated with that.Cell study1. MTT results shows that after added NH4Cl in the rat liver cells culture medium, with time and concentration, the inhibition rate was gradually increased. EGTA+ NH4C1 group shows the same result with NH4Cl group, and they have almostly similar inhibition rate (p<0.05).BAPTA-AM+NH4Cl group and NH4Cl group are different(p<0.05).2. Flow cytometry results shows that the apoptosis rate of rat liver cell is strongly depended on the concentration and the treatment time of NH4Cl.what’s more, BAPTA-AM apoptosis rate decreased statistically significant than NH4CI group when the cells are treated with high concentration NH4CI (p<0.05).3. Western blot results show that use higher than 5.0mMol NH4Cl treated the ratliver cells for 72h, the PARP protein of NH4Cl group and EGTA group appears to be cut, BAPTA-AM group did not change; Cyt C expression levels of NH4Cl group and EGTA group are concentration-dependent, but BAPTA-AM group shows no difference.4. The discovery of PTP hole shows that NH4CI could make the fluorescence peak shift left, and with time Elapsing, the peak continue to the left.5. Real-time PCR results are shown:there is no difference of the CAM and CAPN mRNA expression in different times and different concentrations of NH4CI treatment conditions; with NH4CI concentrations increasing, for the same time, mRNA of CaMK is reduced.And with the same concentration of NH4CI, but with time increasing,mRNA of CaMK is reduced.6. ELISA:for different time and different concentrations of NH4Cl treated, CAMKⅡ shows no difference.Conclusion1. Early lowing ammonia can reduce hepatocyte apoptosis and the liver injury.and reduce the expression of TNF-α, IL-6, SPP1 and apoptosis related gene P53;2. The expression of TNF-a, IL-6, SPP1 and P53 gene were related to blood ammonia level and the rate of apoptosis;3. High blood ammonia can cause calcium overload and activation of the mitochondrial pathway apoptosis, but do not activate the Ca2+-CaM-CaMKII signaling pathway;4. Intracellular calcium ion chelating agent can effectively protect liver cell apoptosis induced by high blood ammonia.