Experimental Study Of Gene Silencing On Apoptosis Of Liver Cells From Mice Of Acute Liver Failure With Endotoxemia

[Background/Objietive]Liver failure is a devastating clinical syndrome with a high mortality of80%,which appears the main performances with coagulation disorders, ascites, hepatic encephalopathy,and hepatorenal syndrome.There is currently no specific treatments.Hepatocyte apoptosis is an important event in the pathogenesis and progression of liver failure, Endotoxin(lipopolysaccharide [LPS]) syndrome is a particularly grave complication, the incidence is up to80.0%. LPS can mediate apoptosis of liver cells. Our previous study finds that second mitochondria-derived activator of caspases(SMAC) can medicate apoptosis of liver cells induced by LPS in vitro, the expression of SMAC is also closely related to the apoptosis rate of liver cells in acute liver failure rats with endotoxemia. Previous studies found that SMAC is necessary for apoptosis. RNA interference (RNAi) is the course of sequence-specific,post-transcriptional gene silencing.Consequently, Application of RNAi inhibiting the expression of SMAC,may reduce the apoptosis of liver cells of mouse with acute liver failure,which will provide a new treatment for acute liver failure,and also help to further clarify the role of SMAC for apoptosis. Finally,hepatitis E virus (HEV)-related acute liver failure serum LPS level was investigated,the affection of serum on human liver cell survival and apoptosis was observed,the protective effect of core polysaccharide antibody on acute liver failure serum-induced apoptosis was slao detected.[Method]1.Cellular levelSpecific interference sequences targeting human SMAC gene were designed and filtered out.The specific interference was transfected into liver cells, which was induced by LPS. Cells were collected at different time points after transfection(24h、36h、48h、60h),SMAC mRNA was measured by real time PCR.At48h post transfection,the expressions of SMAC protein in mitochondria and caspase-9protein in cytoplasms were assessed by western,caspase-3activity was measured by caspase-3activity assay kit,apoptosis rate was determined by flow cytometry.2.Animal levelSpecific sequence lentivirus targeting SMAC was constructed,which was measured by DNA sequencing,and the titer was also tested. Specific sequence lentivirus was imported into liver of mice using tail vein high-pressure injection technology.Mice were then given intraperitoneal D-Galactose(D-GaLN) and LPS. Signs of mice and the number of death mice were observed,blood and liver tissue were collected,serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) were tested using automatic biochemical analyzer,serum LPS level was measured by limulus assay kit,infection efficiency of lentivirus was tested by frozen sections,liver histopathology was assessed by HE staining,expressiones of SMAC mRNA were detected by real time PCR,SMAC protein in mitochondria and cytoplasm,caspase-9protein in cytoplasm were detected by western,Apoptosis was determined by the Tunel method3. Human level Serum from13patients with HEV-related acute liver failure were collected.Serum LPS level was measured by a quantitative chromogenic end-point tachypleus amebocyte lysate endotoxin detection.The serum was used to incubate with liver cells.The apoptosis rate of acute liver failure serum-induced cells was detected by flow cytometry,cells were later incubated with core polysaccharide antibody and the acute liver failure serum,and the apoptosis rate was also tested by flow cytometry.4.Statistical analysisThe results were expressed as mean±standard deviation(SD).a one-way ANOVA were used to determine significance. P values<0.05suggested significant.[RESULTS]1.Three siRNAs (siRNA1, siRNA2, siRNA3) had different inhibition effects on SMAC mRNA of human liver cells.The inhibition effects of siRNA2were the strongest,and about68.0%reduction of SMAC mRNA levels could be observed.2.1n LPS-induced human liver cells,SMAC protein in mitochondria,caspase-9protein and caspase-3activity in cytoplasms were significantly inhibited by siRNA2, LPS-induced apoptosis was also significantly suppressed.3. specific lentiviral targeting SMAC were constructed,which was verified correctly by DNA sequencing,the titer lentivirus was6E+8TU/ml.4. Under a fluorescence microscope,frozen section of liver tissue from mice injected with lentiviruses showed a large green fluorescence.5. Serum ALT and AST increased in model group,P<0.05compared with the control group.SMAC knockdown resulted in significant down-regulation of ALT and AST.6.After administration of D-GalN/LPS,Serum LPS Levels were greatly increased in mice of model group,the increase were attenuated significantly by specific sequence lentivirus. 7. Histological changes of liver tissues were observed.The control group showed normal lobular architecture with central veins,the morphology of the liver parenchyma was good with no congestion and inflammation.Histopathological change was more severe significantly in mice treated with D-GaLN/LPS only,as compared with control group.The extent of histopathological change was down-regulated by specific sequence lentivirus.Unrelated sequence group had a similar histopathological appearance,compared with the model group.8. In model mice with acute liver failure,SMAC mRNA levels significantly increased compared with normal mice, SMAC protein in mitochondria reduced,SMAC protein and caspase-9protein in cytoplasm increased significantly,the apoptosis rate of liver cells increased,the apoptotic index was33.0±5.6%.The mRNA and protein of SMAC,caspase-9protein were down-regulated in specific sequence group. The apoptotic index of apoptotic cells also reduced.9. The LPS level of serum from patients with acute liver failure was0.26Q0.02EU/ml, which was significant higher than the healthy serum.10. The apoptosis rate of acute liver failure serum-incubated cells was5.83±0,42%, the apoptosis rate was significantly affected by acute liver failure serum. Healthy serum had no effect on cell apoptosis. The apoptosis rate of core polysaccharide antibody and acute liver failure serum-incubated cells reduced, which was lower than that of single acute liver failure serum-stimulated cells unsignificantly.[conclusions]1. LPS-induced apoptosis of human liver cells and the apoptosis rate of liver cells from mice with acute liver failure can be effectively reduced by inhibiting the expression of SMAC,which provides a new tool and theoretical basis for the treatment of acute liver failure,and also shows that SMAC and its downstream pathway may be one of LPS-mediated pathway for apoptosis of liver cells.2.The serum from patients with HEV-related acute liver failure contain a high concentration of LPS,which can induce apoptosis of human liver cells.