食欲素A对人肝癌细胞Hep G2增殖和凋亡的影响及机制

Effect and possible mechanism of Orexin A on proliferation and apoptosis of human liver cancer cells Hep G2

目的研究食欲素A(orexin A)对人肝癌细胞Hep G2增殖和凋亡的影响,及其可能作用机制。方法以不同浓度的orexin A干预Hep G2细胞24h,MTT法检测细胞的增殖能力,筛选最适的orexin A作用浓度。使用食欲素受体1选择性拮抗剂SB408124(10μM)预处理细胞1 h,然后用最适浓度的orexin A干预细胞24h,实验设置为:正常对照组、溶剂对照组、orexin A组、SB408124+orexin A组。MTT法检测细胞的增殖情况,流式细胞术检测细胞周期和细胞凋亡率,Hoechst染色观察细胞的形态变化,Western blot检测Bax、Bcl-2、Cytochrome c和Cleaved-caspase-3蛋白的表达。结果 Orexin A以剂量依赖性方式明显抑制Hep G2细胞的生长,最适宜浓度为0.5μM,细胞G0/G1期百分比(72.50%±1.32%)明显高于S期(19.6%9±1.15%)。Orexin A促进Hep G2细胞凋亡,细胞凋亡率显著升高(<0.01)。Orexin A显著上调Bax和Cleaved-caspase-3表达水平,下调Bcl-2和线粒体Cytochrome c表达水平。SB408124的干预能够减小orexin A对Hep G2细胞的影响。结论 Orexin A通过食欲素受体1抑制Hep G2细胞增殖并诱导其凋亡。

Objective To study the effect of orexin A on proliferation and apoptosis of human liver cancer cell Hep G2 in vitro and the possible mechanism. Methods Cell proliferation with different concentrations of orexin A intervention cells for 24 h was detected by MTT approach to screen the optimal concentration of orexin A. Cells were pretreated with orexin-1 receptor antagonist SB408124（10 M） for 1h, then treated with optimal concentration of orexin A for 24 h. Experiment was divided into normal control, solvent control, orexin A and SB408124＋orexin A groups. MTT assay was used to detect cell growth inhibitory rates. Cell cycle and apoptosis rates were determined by flow cytometry. Hoechst staining was used to observe the cellular morphology. Western blot was employed to detect the expression of Bax, Bcl-2, Cytochrome c and Cleaved-caspase 3. Results Orexin A inhibited the growth of Hep G2 cells in dose dependent manner, and the suitable concentration was 0.5 M, the percentage of G0/G1 phase cells（72.5%0±1.32%） was obviously higher than that of S phase cells（19.69%±1.15%）. Orexin A promoted the apoptosis of Hep G2 cells, the cell apoptosis rate of the cells reached（29.50±3.03） %）. Orexin A upregulated the expression levels of Bax and Cleaved-caspase-3, but downregulated the expression levels of Bcl-2 and mitochondrial Cytochrome c in Hep G2 cells（ 0.01）. SB408124 inhibited the mentioned effect of orexin A effectively. Conclusion Orexin A inhibits the proliferation of liver cancer cells Hep G2 and promotes the cells apoptosis through Orexin-1 receptor.