Effect of allicin on the expression of Bcl-2 and Bax protein in LM-8 cells
- VernacularTitle:大蒜素对LM-8细胞Bcl-2及Bax表达的影响
- Author:
Yu HE
;
Shuhua YANG
;
Gang ZHOU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2008;12(15):2997-3000
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Allicin has been proved to influence the proliferation and apoptosis of LM-8 cells.OBJECTIVE: To study the effects of allicin on the expression of apoptosis protein Bcl-2 and Bax in C3H mouse osteosarcoma cell line LM-8, and the correlation of the morphological changes of LM-8 cells with the changes of Bcl-2 and Bax.DESIGN: Randomized grouping, non-blind, cell level, in vitro study.SETTING: The experiment was carded out from September 2006 to May 2007 at the Center Laboratory of Union Hospital,Tongji Medical College, Huazhong University of Science & Technology.MATERIALS: Osteosarcoma cell line LM-8 was purchased from the Animal Center of the Fourth Military Medical University of Chinese PLA. Allicin presented by Wuhan Huihai Co., was a sulphide isolated from garlic corn. Cell Counting Kit-8 (CCK-8) from Dojindo Laboratories; Bcl-2 antibody, Bax antibody and SP immunohistochemistry kit from Fuzhou Maixin Biotech Co.METHODS: LM-8 cells were cultured in RPMI 1640 culture medium, then cell climbing films were made, and SP immunocyte-histochemistry was used to detect the expression of Bcl-2 and Bax protein. Inverted microscope was performed to observe the morphologic changes of LM-8 cells before and after adding with 5.0, 10.0 and 15.0 mg/L allicin. Serial subcultivated LM-8 cells were adjusted to 7.5× 107 L-1 in concentration, added to 96-pore culture plate, and treated with allicin at a serial concentration of 1.0, 5.0, 10.0 and 15.0 mg/L. Additionally blank culture medium pores without cells and allicin and pores without allicin were taken as control groups. The 96-pore culture plate was maintained for 24, 48 and 72 hours, and then fetched. CCK-8 was added to determine the optical density value. The inhibition rate of LM-8 cell growth was also calculated. LM-8 cells were treated with different concentrations of allicin (5.0, 10.0 and 15.0 rag/L) for 24, 48 and72 hours, and then we carried out an apoptosis analysis by flow cytometry.MAIN OUTCOME MEASURES: Expression of Bcl-2 and Bax protein in LM-8 cells; cell morphous, cell proliferation,and cell apoptosis.RESULTS: ①Expression of Bcl-2 and Bax protein: Allicin could lower the expression of Bcl-2 protein and enhance the expression of Bax. The positive expression of Bcl-2, Bax and Bcl-2/Bax expression all showed significant differences compared with control group (P ＜ 0.01).②LM-8 cell morphous: LM-8 Cells treated with allicin presented typical apoptosis changes under microscope.③LM-8 cell proliferation: Allicin could inhibit the growth of osteosarcoma cell line LM-8, when concentration of allicin added from 1.0 mg/L to 15.0 mg/L, the inhibition rate of LM-8 cells at 72 hours increased from (23.87±3.26)% to (58.32±5.38)%, and 50% inhibiting concentration was 11.09 mg/L.④LM-8 cell apoptosis: After 72 hours with treatment of allicin of 5.0, 10.0 and 15.0 mg/L in concentration, apoptosis rate of LM-8 cells revealed a dose-dependent relationship (P ＜ 0.05).CONCLUSION: ①Allicin can inhibit the proliferation of LM-8 cells in a dose-time-dependent manner.②Allicin can induce the apoptosis of LM-8 cells in a dose-dependent manner.③Allicin can inhibit LM-8 cell proliferation and induce LM-8 cell apoptosis, which is related to down-regulate Bcl-2 protein expression and up-regulate Bax protein expression.
