The Mechanisms Underlying The Apoptosis Of Hepatocytes In Mice With The Acute Hepatic Failure

【Background and objective】In China, acute hepatic failure is associated with high mortalityand low cure rate. The main pathological change is massive death ofhepatocytes which causes serious damage of liver function. The cliniccharacteristic is that the liver functions rapidly degenerate, leading tohepatic encephalopathy and coagulation disorders. The pathogeny of theacute hepatic failure is complicated. Due to the high incidence of viralhepatitis, infection by hepatitis viruses is still the first cause of the acutehepatic failure until now. Secondly, the acute hepatic failure can becaused by drugs, plants, chemicals and so on. Some results ofexperiments have indicated that the apoptosis plays an important role inthe onset of acute hepatic failure. But until now, the understanding of themechanism of apoptosis of hepatocytes is inadequate. Generally, there two mechanisms underlying apoptosis. One of the two mechanism is thedeath receptor pathway, in which, after the ligands carrying apoptosissignals bind to the relevant receptors on the cell membrane, the apoptosissignals are transmitted into the cells to activate biochemical reactions,which finally cause apoptosis. The other is the intrinsic or mitochondrialpathway, in which apoptosis can be induced by DNA damages in the cellnucleus. Whether there are DNA damages in the early acute hepaticfailure, and the relationship between DNA damages and apoptosis havenot been reported in literature. In order to research the pathogenesisunderlying the acute hepatic failure, this study will probe into themechanisms underlying apoptosis of hepatocytes in the animal model byusing comet assay, ELLISA, TUNEL and other methods, which isestablished by intraperitoneal injection with D-Galactosamine (D-gain)and lipopolysaccharide. The objectives are as fellows:1.To research the occurrence and development of DNA damages innucleus of hepatocytes of mice with the acute hepatic failure model.2. To study the relationship between variation of TNF-αconcentration ofmice blood serum and hepatocyte apoptosis in the acute hepatic failuremodel.3. To examine the changes in the damaged hepatocytes during the acutehepatic failure. 4. To research the effect of Diammonium Glycyrrhizinate Enteric-coatedCapsules on the damage of heaptocytes of mice with the acute hepaticfailure and the relevant mechanism.5. To study the effect of reduced glutathione on the damage and apoptosisof heaptocytes of mice with the acute hepatic failure and the relevantmechanisms.【Method】1. Inject intraperitoneally with D-Galactosamine (D-galn) andlipopolysaccharide to establish the mouse model of acute liver failure.2. Conduct comet assay to detect DNA damages in nucleus of hepatocytesof mice with the acute hepatic failure from earlier time point afterinjection with D-Galactosamine (D-galn) and lipopolysaccharide. TheOliver tail moment value is used to indicate the extent DNA damage.3. Adopt ELISA to measure the serum TNF-αconcentration of mice withthe acute hepatic failure.4. Employ HE staining method to observe the changes in cellularmorphology of mice hepatic tissue in the acute hepatic failure model.5. Use TUNEL method to detect hepatocyte apoptosis of mice in theacute hepatic failure model, and calculate the apoptosis index.6. Administer intragastrically with Diammonium GlycyrrhizinateEnteric-coated Capsules to the experimental group of mice, and three hours later inject intraperitoneally with D—galn and LPS, then use cometassay to detect DNA damages in nucleus of hepatocytes of theexperimental group of mice at different time points after injection, useTUNEL to detect hepatocyte apoptosis.7. Perform intramuscular injection with induced glutathione, and twohours later, inject intraperitoneally with D—galn and LPS, and then usecomet assay to detect DNA damages in nucleus of hepatocytes atdifferent time points after injection, use ELISA to measureTNF-αconcentration, adopt TUNEL to detect hepatocyte apoptosis.【Results】1. The DNA Olive tail moment values of the hepatocytes weresignificantly greater, 0.5 hour after intraperitoneal injection ofD-GaIN/LPS, and increased with time. Comparison between any twogroups showed that the differences were statistically significant (P＜0.05).The ratio of damaged hepatocytes was 100 percent at 1 hour time pointafter injection.2. 0.5 hour after intraperitoneal injection with D—galn and LPS, theTNF-αconcentration of mice in experimental group did not increase. Itincreased slightly till 1 hour. After two hours, the tendency of increasingis obvious, and the difference between experimental group and controlgroup is significant (P＜0.05). 3. 2 hours after intraperitoneal injection with D—galn and LPS, HEstaining results showed enhanced acidophilic staining of hepatocytes inexperimental group. Till 8 hours, the massive necrosis of hepatocytesoccured, and the hepatic sinusoid obviously expanded, became hyperemiaand hemorrhage.4. Until 6 hour time point after intraperitoneal injection with D—galn andLPS, the TUNEL results showed apoptotic cells in the experimentalgroup. Till 8h, the massive apoptosis occured.5. After intraperitoneal injection with D—galn and LPS, the Olive tailmoment values of the experimental group treated by DiammoniumGlycyrrhizinate Enteric-coated Capsules decreased significantly(P＜0.05).6. After intraperitoneal injection with D—galn and LPS, the Olive tailmoment values of the experimental group decreased significantly ,towhich induced glutathione was administered(P＜0.05), and the apoptosisindex of the administered group is significantly lower than that of thecontrol group, but the TNF-αconcentration of blood serum did not changesignificantly.【Conclusion】1. DNA damage in nucleus of hepatocytes of mice with acute hepaticfailure occurs earlier, and expands with time, which maybe induce apoptosis at the end. The toxicity of D—galn and LPS maybe cause theDNA damage. The necrosis showed by HE may also be related to DNAdamage.2. TNF-αconcentration in blood serum did not increase synchronouslywith DNA damage in nucleus of hepatocytes of mice with acute hepaticfailure, but the increase of TNF-αconcentration in blood serum mayfurther lead to the DNA damage.3. Diammonium Glycyrrhizinate Enteric-coated Capsules can reduceDNA damage in nucleus of hepatocytes mice with the acute hepaticfailure.4. Induced glutathione can reduce DNA damage in nucleus of hepatocytesand apoptosis in liver of mice with the acute hepatic failure, but this is notrelated to variation of serum TNF-αconcentration.