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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 investigate the inhibitory effect of total flavone of Litchi Semen (TFLS) on human colorectal cancer cell line HT29 in vitro and the effect of nuclear factor-kappa B (NF) -κB), Toll-like receptors (TLR4) and interleukin-1β (IL-1β) mRNA and protein expression were detected by Western blot. Methods: HT29 cells were cultured in vitro and treated with TFL (0,0.16,0.32,0.64,1.28,2.56 g · L -1) for 24 h, 48 h and 72 h, respectively. The effects of TFLS on the proliferation of HT29 were detected by MTT assay. The expressions of NF-κB, TLR4 and IL-1β mRNA in each group were detected by reverse transcription polymerase chain reaction (RT-PCR). The expressions of NF-κB , TLR4, IL ~ (-1) β protein expression. Results: The results of MTT assay showed that TFL had a good time-dependent and dose-dependent effect on proliferation of HT29, with the highest inhibition rate at 72 h and 2.56 g · L -1. Compared with the blank group, the expression of NF-κB and IL-1β gene and protein in HT29 cell line decreased after treated with TFL for 72 h, especially TFL 0.64 and 2.56 g · L -1 More obvious (P <0.05). TFL group TLR4 gene expression difference was significantly (P <0.05), protein expression was not significantly different. CONCLUSION: TFL can inhibit the proliferation of HT29 cell line in vitro. NF-κB may be the key factor that TFL inhibits the proliferation of HT29 cell line.