Effects of endotoxin on liver Smac apoptosis channel.
To study the effect of endotoxin on liver apoptosis, L02 liver cells were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver cells was detected by Western blotting. With in vitro study, the L02 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained by hoechst, the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on L02 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GalN. Hepatic mitochondria smac was reduced with dosage of D-GalN and, on the contrary, the activity of caspase3 was increased. D-GalN at 200 mg/kg increased Caspase9 while D-GalN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.
Chen
Miao
M
Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. chenmiao7910@163.com
Zhou
Jian
J
Li
Hui
H
Chen
Anqun
A
Zhang
Zhengang
Z
Tian
Deying
D
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D004731:Endotoxins / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D017093:Liver Failure / Q000139:chemically induced / Q000473:pathology; D024101:Mitochondrial Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C489013:DIABLO protein, rat; D004731:Endotoxins; D024101:Mitochondrial Proteins; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9