荔枝核总黄酮对结肠癌细胞株HT29的抑制作用及相关机制

Inhibitory Effect of Total Flavone of Litchi Semen on Colorectal Cancer Cell Lines HT29 and Expressions of NF-κB,TLR4,IL-1β and Its Mechanism

目的:探讨不同浓度的荔枝核总黄酮(total flavone of Litchi Semen,TFLS)对人的结直肠癌细胞株HT29的体外抑制作用及对该细胞系中核转录因子-κB(Nuclear factor-kappa B,NF-κB);Toll样受体4(Toll-like receptors,TLR4);白细胞介素-1β(interleukin-1β,IL^(-1)β)mRNA和蛋白表达的影响。方法:体外培养HT29细胞株,给予TFL(0,0.16,0.32,0.64,1.28,2.56 g·L^(-1))干预,给药后24,48,72 h,用四甲基偶氮唑蓝(MTT)比色法分别检测TFLS对HT29增殖的影响,采用逆转录PCR检测各组NF-κB,TLR4,IL^(-1)βmRNA表达;蛋白免疫印迹法(Western blot)检测NF-κB,TLR4,IL^(-1)β蛋白表达情况。结果:MTT检测结果显示TFL对HT29增殖呈良好的时间依赖性和剂量依赖性,以作用72 h,质量浓度为2.56 g·L^(-1)抑制率最高。与空白组比较,TFL组作用72 h后,HT29细胞系中NF-κB,IL^(-1)β基因和蛋白表达水平均降低,尤以TFL 0.64,2.56 g·L^(-1)组较明显(P<0.05)。TFL组TLR4基因表达较空白组差异明显(P<0.05),蛋白表达差异不明显。结论:TFL在体外对HT29细胞系的增殖具有抑制作用,NF-κB可能为TFL抑制HT29细胞系增殖的关键因子。

Objective： To study the inhibitory effect of total flavone of Litchi Semen （TFLS） on colorectal cancer cell line HT29 and its effect on mRNA and protein expressions of nuclear factor-kappaB （ NF- κB）, toll-like receptors 4 （TLR4） and interleukin-1β （IL-1β）. Method： Colorectal cancer cell line HT29 was cultured in vitro and treated with different concentrations of TFL （0, 0. 16, 0. 32, 0.64, 1.28, 2.56 g·L^-1 ）. After TFL treated for 24, 48, 72 hours, the inhibition effect on proliferation was detected by [ 3- （4, 5-dimethyl- 2-thiazolyl） -2, 5-diphenyl-2-H-tetrazolium bromide, MTT] analysis. Reverse transcription PCR was used to detect the mRNA expressions of NF-κB, TLR4, IL-1β. The protein expressions of NF-KB, TLR4, IL-1β were detected by Western blot analysis. Result： MTT method showed that TFL could inhibit HT29 cell proliferation in a time and dose dependent manner. The treatment for 72 h and the concentrations of 2.56 g-L-1 showed the highest inhibition rate. After treated with TFL for 72 hours, both of NF-κB, IL-1β mRNA and protein expressions decreased in the TFL group, compared with blank group, particularly in 0.64 g·L^-1 and 2.56 g·L^-1 concentration groups （P 〈 0.05 ）. TFL down-regulated TLR4 gene expression in HT29 cell line （P 〈 0.05） , but with insignificant differences in protein expressions. Conclusion ： TFL can significantly inhibit the proliferation of HT29 cells in vitro. NF-κB may be TFL＇s key factor in inhibiting HT29 cell line proliferation.