穗花杉双黄酮对3T3-L1 细胞增殖、凋亡 及生物钟的影响

目的 探讨穗花杉双黄酮对3T3-L1 细胞增殖、凋亡及生物钟的影响。方法 采用不同浓度穗花 杉双黄酮处理正常和过表达昼夜节律运动输出周期故障蛋白（CLOCK）的3T3-L1 细胞，CCK-8 和EdU 检 测细胞增殖情况；流式细胞仪检测细胞凋亡情况；real-time RT-PCR 检测生物钟基因CLOCK 和BMAL1 的表 达；Western blotting 检测Cleaved caspase-3、Bcl-2、CLOCK 及BMAL1 蛋白的表达。结果 低浓度（2.5 ～ 10.0 mg/L）穗花杉双黄酮作用于3T3-L1 细胞后，3T3-L1 细胞增殖和凋亡无影响（P >0.05），但是当浓度达 20 和40 mg/L 反而抑制增殖（P <0.05）；通过上调Cleaved caspase-3 表达，下调Bcl-2 表达，促进凋亡的发生。 较低浓度穗花杉双黄酮对3T3-L1 细胞CLOCK、BMAL1 基因和蛋白的表达无影响（P >0.05），而浓度达20 和40 mg/L 则呈抑制作用（P <0.05）；过表达CLOCK 基因可减弱穗花杉双黄酮对3T3-L1 细胞的抑制作用 （P <0.05）。结论 3T3-L1 细胞在培养状态仍保持昼夜节律性，较高浓度穗花杉双黄酮可抑制3T3-L1 细胞 增殖并促进凋亡，其可作为抑制细胞增殖的药物，其作用机制与生物钟基因表达有关。

Effects of amentoflavone on proliferation, apoptosis and circadian clock of 3T3-L1 cells

Objective To explore the effects of amentoflavone on the proliferation, apoptosis and circadian clock of 3T3-L1 cells. Methods Normal and over-expressing 3T3-L1 cells were treated with different concentrations of amentoflavone. CCK8 and EdU kit were used to detect cell proliferation. Flow cytometry was used to detect apoptosis. RT-PCR was used to detect the expression of CLOCK and BMAL1 gene. And western blot was used to detect the expression of caspase-3, Bcl-2, CLOCK and BMAL1 protein. Result Lower concentration of amentoflavone (2.5, 10 mg/L) had a little effect on the proliferation (P > 0.05) and apoptosis (P > 0.05) of 3T3-L1 cells. But when the concentration ranged from 20 mg·L-1 to 40 mg L-1, it up-regulated the expression of caspase-3 (P < 0.05) and down-regulated Bcl-2 expression to inhibit proliferation (P < 0.05) and promote the occurrence of apoptosis (P < 0.05). Low concentration of amentoflavone has little effect on the expression of CLOCK and BMAL1 gene and protein in 3T3-L1 cells (P > 0.05), but the high concentration amentoflavone (20 mg/L, 40 mg/L) inhibited the expression of CLOCK and BMAL1 (P < 0.05). Over-expression of CLOCK gene could significantly recovery the inhibitory effect of amentoflavone on proliferation of 3T3-L1 cells (P < 0.05). Conclusions The 3T3-L1 cells still maintain circadian rhythms in the cultured state, and higher concentrations of amentoflavone can inhibit 3T3-L1 cell proliferation, suggesting that it may be as a drug that inhibits proliferation and the mechanism is related to CLOCK genes.