Effects of leptin on the expression of tumor necrosis factor-alpha in RAW264.7 cells

Youmei FENG; Qing CHEN; Jie ZHOU; Juan CHEN; Hesong ZENG

ABSTRACT

BACKGROUND:

Leptin is a kind of polypeptide hormone secreted by fatty tissue, previous studies have shown that leptin plays a certain role in the formation of atherosclerosis.

OBJECTIVE:

To investigate the effects of leptin on the expression of tumor necrosis factor-alpha (TNF-α) in RAW264.7 cells, and investigate the possible mechanism from the angle of the change of nuclear factor-κB (NF-κB) activity.

DESIGN:

A controlled observational experiment.

SETTING:

Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS The experiments were carried out in the Department of Biochemistry and Molecular Biology, Tongji Medical College, and the Department of General Surgery, Union Hospital affiliated to Huazhong University of Science and Technology between April 2005 and February 2006.The cultured RAW264.7 cells were divided into leptin treated groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. Each group had 3 sub-wells,and the experiments were repeated for 3 times.

METHODS:

Mouse macrophage RAW264.7 cells were incubated into a 6-well plate at the density of 109 cells L-1, cultured in RPMI-1640 culture medium containing bovine serum of 0.1 in volume fraction. When the cells grew to 80%, serum-free culture medium Opti-MEM was changed to culture for another 24 hours, and then the cells were divided into leptin groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. After the cells were incubated with leptin for 4 hours, the expression of TNF-α mRNA expression in RAW264.7 cells was detected with reverse transcription-polymerase chain reaction (RT-PCR). After the RAW264.7 cells were incubated with leptin for 1, 3, 6 and 9 hours, the expression of TNF-c protein expression was detected with double antibody sandwich enzyme-linked immunosorbent assay (ELISA). After the RAW264.7 cells were incubated with leptin for different times, the activity of NF-κB was detected analyzed with electrophoretic mobility shift assay. Another, the RAW264.7 cells were treated with or without 50 μmol/L leptin and/or 100 μmol/L PS1145 (I kappa B kinase specific inhibitor)divided into four groups blank control group, I kappa B kinase specific inhibitor PS1145 (10 μmol/L) treated group, leptin (50 μmol/L) treated group, leptin (50 μmol/L) + PS1145 (10 μmol/L) group. Aftere incubated for 6 hours, the activity of NF-κB and expression TNF-α mRNA were detected respectively.MAIN OUTCOME

MEASURES:

① Effect of leptin of different concentration on the expression of TNF-α mRNA and protein in RAW264.7cells; ② Effect of leptin of different concentration on the activity of NF-κB in RAW264.7 cells; ③ Influence of inhibition I kappa B kinase activity inhibition on expression of TNF-a induced by leptin in RAW264.7cells.

RESULTS:

① After the RAW264.7 cells were treated with leptin of different concentration, the TNF-α mRNA level was elevated in a dose-dependent manner, and it reached the peak value emerged in the 50 μg/L leptin treated group. ② The expression of TNF-α protein increased in dose-dependent and time-dependent manners, and it reached the peak val ue at 6 hours in the 50 μg/L leptin treated group. ③ The activity of NF-κB was also positively correlated with the leptin concentration, and it was the highest value at 6 hours treated with 50 μg/L leptin (P ＜ 0.05). ④ I kappa B kinase activity inhibition only partially suppressed the leptin induced elevation of TNF-α expression induced by leptin.

CONCLUSION:

Leptin can increase the expression and secretion of TNF-α in RAW264.7 cells directly in both dose-dependent and time-dependent manners, and the mechanism may be correlated with the activated NF-κB induced by leptin. It may be one of the mechanisms of atherosclerosis induced by leptin.