黄芩苷对胶质瘤细胞增殖及侵袭的影响及其机制

Effect of baicalin on proliferation and invasion of glioma cells in vitro and its mechanism

目的探讨黄芩苷对U-251胶质瘤细胞增殖和侵袭的影响及可能机制。方法人星形细胞瘤细胞(U-251胶质瘤细胞)购自中国科学院典型培养物保藏委员会细胞库。采用0、10、20、50、100和200 mg/L等浓度黄芩苷处理U-251胶质瘤细胞筛选出半数抑制浓度(IC50)。将U-251胶质瘤细胞分为对照组(未行任何处理)、黄芩苷组(50 mg/L黄芩苷处理)和肿瘤坏死因子-α(TNF-α)组(50 mg/L黄芩苷和10μg/L浓度TNF-α处理),分别采用克隆形成实验和Transwell实验检测细胞增殖和侵袭;酶联免疫吸附法检测炎症因子白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)水平;蛋白免疫印迹法检测增殖细胞核抗原(PCNA)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、核因子活化B细胞-κ轻链增强子(NF-κB)、磷酸化-NF-κB(p-NF-κB)、NF-κB抑制蛋白α(IκBα)和磷酸化-IκBα(p-IκBα)蛋白表达水平,组间两两比较采用SNK-q检验。结果黄芩苷处理U-251胶质瘤细胞48 h的IC50为47.91 mg/L。黄芩苷组U-251胶质瘤细胞克隆形成率、侵袭细胞数、IL-1β和IL-6水平、PCNA、MMP-2、MMP-9、p-NF-κB和p-IκBα蛋白表达水平低于TNF-α组和对照组[克隆形成率分别为(21.04±2.42)%、(49.73±5.04)%和(56.29±5.37)%,侵袭细胞数分别为(40.02±4.16)、(68.18±5.22)和(76.25±6.50)个,IL-1β分别为(19.26±2.04)、(35.18±3.82)和(42.08±4.12)ng/L,IL-6分别为(113.38±10.24)、(204.55±20.16)和(216.07±18.52)ng/L,PCNA分别为(0.28±0.03、0.56±0.05和0.64±0.06),MMP-2分别为(0.09±0.01、0.19±0.02和0.22±0.02),MMP-9分别为(0.14±0.01、0.28±0.02和0.31±0.03),p-NF-κB为(0.17±0.02、0.38±0.04和0.35±0.03),p-IκBα为(0.14±0.01、0.41±0.04和0.37±0.03)],差异均有统计学意义(F=157.904、112.532、103.507、100.032、137.829、139.004、158.786、102.137、77.871,P<0.05)。结论黄芩苷通过抑制NF-κB信号通路调控炎症反应抑制U-251胶质瘤细胞增殖和侵袭。

Objective To study the effect of baicalin on proliferation and invasion of U-251 glioma cells in vitroand possible mechanism.Methods U-251 glioma cells were divided into control group[not treated with baicalin and tumor necrosis factor-α(TNF-α)],BAI group(treated with 50 mg/L baicalin)and TNF-αgroup(treated with 50 mg/L baicalin and 10μg/L TNF-α).The viability of U-251 cells was detected by cell counting kit-8(CCK-8)assay.Cell proliferation and invasion ability was detected by clone formation test and Transwell test,respectively.Interleukin(IL)-1βand IL-6 were detected by enzyme-linked immunosorbent assay.The expression levels of proliferating cell nuclear antigen(PCNA),matrix metalloproteinase(MMP)-2,MMP-9,nuclear transcription factor-κB(NF-κB),phosphorylated-NF-κB(p-NF-κB),NF-κB inhibitory proteinα(IκBα),phosphorylated-IκBα(p-IκBα)protein were detected by Western blotting.Results The rate of clone formation,number of invasion cells,IL-1 and IL-6 levels,and levels of PCNA,MMP-2,MMP-9,p-NF-B,and p-I B protein expression in baicalin group were significantly reduced as compared with those in control group and TNF-αgroup[clone formation rate:(21.04±2.42)%,(49.73±5.04)%,and(56.29±5.37)%,respectively;the number of invasive cells:(40.02±4.16),(68.18±5.22),and(76.25±6.50)cells,respectively;IL-1β:(19.26±2.04),(35.18±3.82),and(42.08±4.12)ng/L,respectively;IL-6:(113.38±10.24),(204.55±20.16),and(216.07±18.52)ng/L,respectively;PCNA:(0.28±0.03,0.56±0.05,and 0.64±0.06),respectively;MMP-2:(0.09±0.01,0.19±0.02,and 0.22±0.02),respectively;MMP-9:(0.14±0.01,0.28±0.02,and 0.31±0.03),respectively;p-NF-κB:(0.17±0.02,0.38±0.04,and 0.35±0.03)respectively;p-IκBα:(0.14±0.01,0.41±0.04,and 0.37±0.03),respectively].The differences were statistically significant(F=157.904,112.532,103.507,100.032,137.829,139.004,158.786,102.137,77.871,P<0.05).Conclusion Baicalin could inhibit proliferation and invasion of U-251 glioma cells in vitro by regulating inflammatory response by inhibiting the NF-κB signaling pathway.