JNK pathway promotes hepatocyte apoptosis by inhibiting Bcl-2 and upregulating ex pressions of Bim, caspase-3 and caspase-9 after cardiopulmonary bypass

摘要

Purpose: To study the effect of Jun N-terminal kinase (JNK) signaling pathway on hepatocyte apoptosis in vivo and in vitro, and to elucidate the mechanism of action. Methods: TdT-mediated dUTP Nick-End Labeling (TUNEL) method was used to determine apoptosis in control and cardiopulmonary bypass (CPB) groups at 0, 3 and 6 hours after rat surgery. The ex pressions of JNK and p-c-Jun in liver tissues at 0, 3 and 6 h after surgery, and the levels of p-c-Jun, Bcl-2 and Bim following overex pression of JNK, were determined using Western blot assay. Human liver cell line HL-7702 was cultured and transfected with over-expressed JNK plasmid and empty plasmid. Proliferation of HL-7702 cells after JNK over-ex pression was assessed by Cell Counting Kit-8 (CCK-8), while quantitative real-time polymerase chain reaction (RT-qPCR) was employed to evaluate mRNA ex pression levels of caspase-3 and caspase-9 mRNA after JNK over-ex pression. Apoptosis of the cells was determined by flow cytometry (FC) after JNK over-ex pression. Results: FC results showed that the number of apoptotic hepatocytes increased after JNK over-ex pression in hepatocytes while TUNEL assay results demonstrated that hepatocyte apoptosis increased in CPB group, when compared to control group; furthermore, the number of apoptotic cells gradually increased within 6 h after surgery. The ex pressions of JNK and p-c-Jun were higher in CPB group than in control group, and increased gradually in both groups within 6 h after surgery. Overex pression of JNK decreased the proliferation of hepatocytes, and also lowered protein ex pression levels of p-c-Jun and Bim; on the other hand, the protein ex pression levels of Bcl-2 fell, while mRNA ex pression levels of caspase-3 and caspase-9 mRNA increased. Conclusion: JNK pathway promotes hepatocyte apoptosis after cardiopulmonary bypass by inhibiting Bcl-2 pathway and promoting the ex pressions of Bim caspase-3 and caspase-9.