Effects And Mechanism Of Endotoxin On Liver Smac Apoptosis Channel

【Background and objective】Recent studies demonstrated that apoptosis is one of the major cellular machanismsleading the liver injury. Endotoxemia, as the second attack, plays an important role in itsdevelopment of serious hepatitis. Smac released from mitochondria is a pro-apoptoticmolecule discovered in 2000. Under normal conditions, it is lo-cated in intermembranespace but is released into the cytoplasm when being stimulated by apoptosis-inducingfactors. After binding with IAPs, it can increase the activity of cysteine-containingaspartate-specific proteases (caspase9 and caspase3). Though liver cells con-ain largeamount of smac, however, its roles in liver apoptosis, especially in the endotoxin-inducedliver apoptosis have not been well studied.This study, by using D-galactosamine(D-GalN)-induced rat model of hepatic failure and LPS-stimulated L02 hepatic cells,examined smac, a pro-apoptotic protein released from mitochondria, and its effects on theactivity of caspase9 and caspase3, with an attempt to explore a new approach for thecontrol of endotoxemia.【Methods】Liver Cell CultureLiver cells were cultured in the DMEM containing 10% calf serum at 37℃in a 5%CO2 in an incubator. Cell passaging was conducted each 3 to 4 days in a 6-mesh plate. Thecells I each group were stimulated with 10 mg/mL LPS for 4, 8, 16 and 24 h and harvestedfor further test. Animal ModelAcute hepatic failure was induced by i.p. injection of D-GaLN in rats. A total of 24healthy Wistar rats were divided into 4 groups at random: group 1 (blank control group, inwhich normal saline was given), group 2 (model group, in which 100 mg／kg D-GaLN wasi.p. administered), group 3 (model group, in which 200 mg/kg D-GaLN was i.p. injected)and group 4 (model group, in which 300 mg／kg D-GaLN was given).Sample CollectionAfter 24 h, 6% chloral hy-drate was i.p. injected for anesthesia. Femoral vein wasseparated under aseptic condition and severed. Then the whole blood was collected byemploying heparinized tube. The hepatic tissues were taken, put into liquid ni-trogen andstored at -70℃for later use.Experiment method1. The L02 cells were examined by Fluorescent Microscopy after Hoechst andacridine orange Staining.2. Flow Cytometric Examination of L02 Cell Apoptosis.3. Flow Cytometric Detection of Smac on L02 Liver Cell Membrane.4. Determination of Smac and Caspase9 By using immunohistochemical technique.5. Detection of Caspase3 Activity of on L02 Liver Cell.6. Separation of Mitochondria and Cell Plasma of L02 Liver Cell.7. Determination of Smac in mitochondria and Caspase9 in L02 Liver Cell by WesternBlotting.8. All rats's Blood sample were collected to examine alanine ALT,TBIL and TP.9. Determination of All rats's hepatic tissues by Hematoxytin (HE) staining. 10. Immunohistochemical Detection of Smac in All rats's hepatic tissues.11. The level of endotoxin in plasma detect by Limulus amebocyte lysate.12. Separation of Mitochondria and Cell Plasma of rats's hepatic tissues.13. Detection of Caspase3 Activity of rats's hepatic tissues.14. Determination of Smac in mitochondria and Caspase9 in rats's hepatic tissues.Statistics analysisSPSS version 13.0 was used to perfom statistics analysis.【Results】1.Under the fluorescent microscope, L02 apoptotic cells showed evenly distributed blueand green florescence within nucleus.2.The late-stage apoptosis rate of the normal control group was0.30±0.10%. The late-stageapoptosis rate after stimulation with LPS for 4,8,16 and 24h was2.30±0.45%,4.00±0.89%,6.20±1.17% and 18.20±2.26%, respectively. There were signifi-cantdifferences in the apoptosis rate (P＜0.05) between normal control and model groups(P＜0.01).3. The rate of smac expression was 0.77±0.14% in the con-trol group and the rates of smacexpression on liver cell membrane, after stimulation with 4, 8, 16 and 24h, were1.73±0.78%,2.84±0.56%,3.72±1.26%,8.66±1.93%, respectively. The rate increasedprogressively over time.4.The expression of smac protein and caspase9 were positive, and they were mainly locatedin cytoplasma, presenting as brown particles. The expression rates of smac and caspase9increased with the time of stimulation with LPS (P＜0.01). Compared to the normal controlgroup, the expressions of smac and caspase9 in the model groups were significantlystronger (P＜0.05).Spearman level analysis revealed that expression levels of smac and caspase9 in the different model groups were positively related.5.With the normal control group, there existed no sig-nificant difference in caspase3activity among different time points. At the 4th h, there was no difference in the caspase3activity between the model group and the normal control group. However, the caspase3activity increased significantly in the model group at the 8th h, 16th h and 24th hascompared with model group at the 4th and the normal control groups at all time points(P＜0.05).6.Western blot shows that The expession of smac expression in mitochondria weredecreased progressively over time after stimulation with 4, 8, 16 and 24h, there wasdifference between the model group and the normal control group(P＜0.05).and Theexpression of caspase9 in cells were increased progressively, there was difference betweenthe model group and the normal control group(P＜0.05).7.Smac protein was stained brown cytoplasma. With HE staining, no normal liver lobulewas found. And the cells were swelling and their contour was blurred, with some spottynecrosis. Around the portal area, there are serious inflammation and strips of massivenecrosis.8.Smac protein was stained brown cytoplasma in hepatic failure tissues.9.Blood serum ALT level in group 2 was increased slightly while it was substantiallyelevated in group 3 and group 4. Total blood serum bilirubin increased with the dosage ofD-GaLN in the model groups. Total blood serum protein in all the groups was normal withno significant difference found among them (P＞0.05).10. Rats in group 1 did not develop endotoxemia, while group 2,group 3 and group 4showed obvious symptoms of endotoxemia, which increased with the dosage of D-GaLN(P＜0.01).11. Activity of caspase3 in group 2 was increased slightly while it was substantiallyelevated in group 3 and group 4(P＜0.01).12. Western blot shows that The expession of smac expression in mitochondria were decreased progressively After the treatment with D-GaLN, there was difference betweenthe model group and the normal control group(P＜0.05).and The expression of caspase9 inHepatic Tissues were increased progressively, there was difference between the modelgroup and the normal control group(P＜0.05).【Conclusion】Smac were highly expessioned in hepatic cells, in vitro, endotoxin can ruducesapoptosis through Smac Apoptosis Channel. And Smac not only can be released into theCell Membrane but also can released into the Cell cytoplasm when being stimulated. Invivo, D-GalN alone was used for the establishment of hepatic failure model, and our resultsshowed that could induce obvious symptoms of endotoxemia and hepatic failure in rats,and demonstrated that endotoxin could induce apoptosis by promoting the release of smacfrom mitochondria and increasing the activity of start-up enzyme caspase9 and effectenzyme caspase3.Mitochondria signal channel plays an important role in hepatic apoptosisinduced by endotoxin. Mitochondria signal channel plays an important role in theendotoxin-induced apoptosis of hepatic cells by promoting the release of caspasee9 andcaspase3 from mitochondria to cytoplasma and Membrane.