Effect of leptin pretreatment on hypoxia-reoxygenation induced apoptosis in human L02 liver cells

Abstract

Objective To investigate the effect of leptin (LEP) pretreatment on hypoxia-reoxygenation (H/R) induced apoptusis in human L02 liver cells. Methods Human L02 liver cells were obtained from pharmacology laboratory, Zhong-Shan University and cultured in DMEM liquid culture medium in an incubator filled with 5% CO_2 at 37℃. The cells were divided into 6 groups ( n = 6 each) : group control (group C) ; grouphypoxia-reoxygenation (group H/R); group Ⅰ-Ⅳ pretreatment with LEP 100, 200, 400 and 800 μg/L + H/R. In group H/R and group Ⅰ-Ⅳ L02 cells were exposed to 95% N_2-5% CO_2 for 12 h followed by 12 h reoxygenation. In group Ⅰ-Ⅳ the cells were pretreated with LEP 100, 200, 400, 800 μg/L respectively before H/R. At the end of 12 h of reoxygenation, the cells were centrifuged and the supematant was collected for determination of ALT and AST concentrations. Apoptosis in L02 cells was detected by Hoechst 33342/PI staining. Fluorescent quantitative PCR was used to detect Bax and Bcl-2 mRNA expression. Results (1) ALT and AST concentrations were significantly increased after H/R. The increase in ALT and AST concentrations was ameliorated by pretreatment with LEP. (2) The H/R-induced apoptotic changes of the cells were attenuated by pretreatment with LEP. (3) The Bax mRNA and Bcl-2 mRNA expression was significantly increased in group H/R as compared with group C. Leptin pretreatmcnt significantly reduced Bax mRNA expression and increased Bcl-2 mRNA expression as compared with group H/R. Conclusion LEP pretreatment can decrease H/R-indtwed apoptosis in the L02 liver cells by down-regulation of Bax mRNA expression and up-regulation of Bcl-2 mRNA expression. Key words: Leptin; Apoptosis; Hepatocytes; Repedusion injury