The effect of 3'-meisoindigo on the function of splenocytes and thymocytes of C57BL/6 mouse

Hongbo GUO; Haiyan HU; Zhaoyang SONG; Lan DENG; Meixia ZHANG

ABSTRACT

BACKGROUND:

The effect of Chinese traditional medicine 3'-meisoindigo as well as indimbin derivatives on normal immunocytes is less reported while it is used for antitumor.

OBJECTIVE:

To investigate the effect of 3'-meisoindigo on the proliferation of the splenocytes and thymocytes of C56BL/6 mouse.DESIGN, TIME AND

SETTING:

The randomized cytopathology observation was performed between August 2007 and January 2008 at Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China.MATERIALS C57BL/6 mice, clean, male, 6-8 weeks old, weighing (20±2) g.

METHODS:

The thymus gland and the spleen of C57BL/6 mice were ground to get the single-cell suspension and cells were treated by 5, 10, 15, 20 and 25 μ mol/L 3'-meisoindigo,respectively. Cells without any drug treatment were used as blank control and cells treated by concanavalin A were used as positive control.MAIN OUTCOME

MEASURES:

Proliferation of splenocytes and thymocytes detected using MTT method; IL-12 activity in culture supematant detected using ELISA method; the cell cycle, apoptosis rate, cell death rate and intracellular reactive oxygen species level detected using flow cytometry; the mRNA level of bcl-2 and cdk2 detected using reverse transcription-polymerase chain reaction; the expression rate of Bcl-2, CDK2 and Bax detected using fluorescence microscope.

RESULTS:

After treating for 24 hours, 15, 20 and 25 μ mol/L 3'-meisoindigo can significantly inhibit the proliferation of thymocytes and splenocytes (P < 0.05) and the inhibition was dose-dependent and time-dependent. Cells resumed proliferation after removing the 3'-meisoindigo, although they had been treated by 25 μ mol/L 3'-meisoindigo for 72 hours. The secretion of IL-12 was markedly reduced in all 3'-meisoindigo groups versus control groups at each time point (P < 0.05). 15 μ mol/L 3'-meisoindigo could decrease the mRNA expression level of apoptosis-related protein bcl-2 and cyclin cdk2 gene, decrease the expression level of BCL-2 protein and CDK protein, increase Bax expression level, decrease Bcl-2/Bax ratio markedly and started apoptosis.15 μ mol/L 3'-meisoindigo arrested cells in the G2/M stage of the thymocytes and splenocytes, and intracellular reactive oxygen species level elevated dose-dependently and time-dependently.

CONCLUSION:

In certain concentration range, 3'-meisoindigo can reversibly inhibit the proliferation of thymocytes and splenocytes of C57BL/6 mouse and can inhibit IL-12 secretion in parallel, and start apoptosis.