雷公藤内酯醇对胰腺癌PANC1细胞Toll样受体4／核因子-κB信号通路的影响

Triptolide inhibited the invasion ability of pancreatic cancer cells through TLR4/NF-κB signaling pathway

目的探讨雷公藤内酯醇（TP）对人胰腺癌PANC1细胞株侵袭能力的影响及其与Toll样受体4／核因子．KB（TLR4／NF-KB）信号通路的关系。方法将PANC1细胞分为亲本细胞组、TP组、脂多糖（LPS）组和TP＋LPS组。TP组培养液中加入50ng／ml的TP，LPS组加入μg／ml的LPS，TP＋LPS组先用50ng／ml的TP处理2h，再加入μg／ml的LPS。各组细胞均常规培养24h。采用实时定量PCR和蛋白质印迹法检测TLR4、基质金属蛋白酶-9（MMP-9）mRNA和蛋白的表达，双荧光素酶报告基因系统检测NF-KB活性，Transwell小室检测细胞侵袭能力。结果亲本组、LPS组、TP组、TP＋LPS组的TLR4mRNA表达量分别为0．41±0．06、0．46±0．10、0．20±0．04和0．25±0．06，TLR4蛋白表达量分别为0．55±0．06、0．60±0．03、0．18±0．04和0．13±0．00；NF—KB活性分别为13．0±3．0、31．6±4．3、7．3±1．5和10．8±2．1；穿膜细胞数分别为（56．8±8．6）、（104．5±12．8）、（32．0±5．7）和（46．8±7．0）个；MMP-9mRNA表达量分别为0．36±0．05、0．58±0．07、0．18±0．03和0．30±0．004，MMP-9蛋白表达量分别为0．31±0．04、0．53±0．08、0．11±0．02和0．15±0．00。LPS组TLR4mRNA和蛋白表达量与亲本组差异无统计学意义，但NF-KB活性、穿膜细胞数、MMP-9mRNA和蛋白表达量显著高于亲本组（t值分别为8．654、7．593、6．655、4．982，P值均〈0．01）。rrP组TLR4mRNA和蛋白表达量、NF·KB活性、穿膜细胞数、MMP-9mRNA和蛋白表达量均显著低于亲本组（t值分别为-7．609、-9．948、-4．176、-5．915、-8．179、-9．948，P值均〈0．01）。TP＋LPS组TLR4mRNA和蛋白表达量、NF-KB活性、穿膜细胞数、MMP-9mRNA和蛋白表达量均显著低于LPS组（t值分别为-4．437、-14．805、-10．506、-9．700、-9．055、-8．932，P值均〈0．01）。结论TP具有抑制胰腺癌细胞侵袭的作用，其机制与抑制TLR4／NF-KB信号通路、下调MMP-9的表达有关。

Objective To investigate the role of TLR4/NF-κB signaling pathway in inhibited invasion ability of pancreatic cancer cells caused by triptolide （TP）. Methods PANC1 cells were divided into parental cells group, TP group, lipopolysaccharide （LPS） group and TP ＋ LPS group. 50 ng/ml of TP was added in culture medium in TP group, and 1 p,g/ml of LPS was added in culture medium in LPS group, while 50 ng/ml of TP was pretreated for 2 h and I μg/ml of LPS was added in culture medium in TP ＋ LPS group. All the cells were cultured for 24 h. The TLR4 and matrix metalloproteinase-9 （MMP-9） mRNA and protein expression were evaluated by real-time PCR and Western blot. The NF-κB activity was determined by dual-luciferase reporter assay system. The invasion ability of pancreatic cancer cells was evaluated by transwell invasion chamber assay. Results The TLR4 mRNA expressions in parental cells group, TP group, LPS group and TP ＋ LPS group were 0.41 ± 0.06,0.46 ± 0.10,0.20 ± 0.04, 0.25 ± 0.06 ; the TLR4 protein expressions were 0.55 ± 0.06,0.55 ±0.06,0.18 ±0.04,0.13 ±0.00; the activities of NF-KB were 13.0 ±3.0,31.6 ±4.3,7.3 ±1.5 and 10.8 ± 2.1, and the numbers of invasion cell were （56.8 ± 8.6） , （ 104.5 ± 12.8）, （ 32.0 ± 5.7） and （46.8 ± 7.0） ; the MMP-9 mRNA expressions were 0.36 ± 0. 05,0.58 ± 0.07,0.18 ± 0. 03,0.30 ± 0. 004 ; the MMP-9 protein expressions were 0.31 ±0.04,0.53 ±0.08,0.11 ±0.02,0. 15 ± 0.00. In LPS group, TLR4 mRNA and protein expressions were not statistic significant when compared with those in parental cells group, but the activities of NF-KB, the numbers of invasion cell, MMP 9 mRNA and protein expressions were statistically increased when compared with those in parental cells group （ t = 8. 654, 7. 593, 6. 655, 4.982, P 〈 0. 01 ）. TLR4 mRNA and protein expressions, activities of NF-KB, the numbers of invasion cell, MMP 9 mRNA and protein expressions in TP group were significantly lower than those in parental cells group （ t = -7.609, -9.948, -4.176, -5.915, -8.179, -9.948, P〈 0.01）. TLR4 mRNA and protein expressions, activities of NF-KB, the numbers of invasion cell, MMP 9 mRNA and protein expressions in TP ＋ LPS group were significantly lower than those in LPS group （ t = - 4.437, - 14. 805, - 10.506, - 9.700, -9. 055, -8. 932, P 〈 0.01 ）. Conclusions TP can inhibit pancreatic cancer cell invasion, and the mechanism is related to the inhibition of TLR4/NF-KB signaling pathway and down-regulation of MMP-9 expression.