黄连素靶向调控TLR4信号通路对恶性黑色素瘤生物学行为的影响及机制研究

Effect and mechanism of berberine-targeted regulation of TLR4 signaling pathway on the biological behaviour of malignant melanoma

目的探讨黄连素对恶性黑色素瘤Toll样受体4(TLR4)信号通路的调节作用,评估其对脂多糖(LPS)诱导的恶性黑色素瘤A375细胞增殖、迁移、凋亡的影响,为防治恶性黑色素瘤提供新的药物(黄连素)治疗依据。方法采用Western blotting法检测不同浓度黄连素对A375细胞TLR4、核因子κB(NF-κB)表达的干预作用。实验分组:A组为对照组;B组为LPS组;C组为TAK-242组;D组为LPS+TAK-242组;E组为LPS+TAK-242+80μmol/L黄连素组;F组为LPS+TAK-242+120μmol/L黄连素组。采用CCK-8、Transwell、流式细胞术检测黄连素靶向TLR4对A375增殖、迁移、凋亡的作用;采用酶联免疫吸附实验(ELISA)检测每组细胞上清中白介素-10(IL-10)和转化生长因子β(TGF-β)表达的影响。结果黄连素浓度升高,TLR4、NF-κB蛋白表达量下调越明显(均P<0.05)。与A组比较,B组增殖、迁移能力增强,凋亡无差异;C组增殖、迁移能力减弱,凋亡增加;D组增殖、迁移能力相较B组减弱,相较C组增强;与D组比较,E、F组增殖、迁移能力减弱,凋亡增加。黄连素在高浓度时细胞的增殖、迁移能力减弱更明显(均P<0.05)。B组IL-10与TGF-β的表达水平增加(均P<0.05);C组IL-10的表达水平降低,TGF-β的表达无显著变化;D组IL-10相较B组降低,相较C组增加;F组IL-10和TGF-β的表达水平显著降低(均P<0.05)。结论黄连素通过抑制TLR4/NF-κB信号通路抑制LPS诱导的恶性黑色素瘤细胞增殖、迁移,促进其凋亡。黄连素或可作为治疗恶性黑色素瘤的药物。

Objective To explore the regulatory effect of berberine on the TLR4 signaling pathway in malignant melanoma,to evaluate its effect on the proliferation,migration,and apoptosis of malignant melanoma A375 cells induced by lipopolysaccharide(LPS),and to provide a new drug(berberine)treatment basis for the prevention and treatment of malignant melanoma.Methods Western blotting was used to detect the intervention effects of different concentrations of berberine on the expression of TLR4 and NF-κB in A375 cells.The experimental groups were as follows:group A was control group,group B was LPS group,group C was TAK-242 group,group D was LPS+TAK-242 group,group E was LPS+TAK-242+80μmol/L berberine group,and group F was LPS+TAK-242+120μmol/L Berberine group.CCK-8,Transwell and flow cytometry assays were used to detect the effects of berberine targeting TLR4 on the proliferation,migration,and apoptosis of A375.We used enzyme-linked immunosorbent assay to detect the expression of IL-10 and TGF-βin the supernatant of each group of cell influences.Results With increased concentration of berberine,the expression of TLR4 and NF-κB protein was down-regulated more significantly(all P<0.05).Compared with group A,the proliferation and migration ability of group B was enhanced,but no difference existed in apoptosis.The proliferation and migration ability of group C was weakened,and apoptosis was increased.The proliferation and migration ability of group D was weaker than that of group B and stronger than that of group C.Compared with group D,the proliferation and migration ability of E and F groups weakened,and apoptosis increased.At high concentrations of berberine,the cell proliferation and migration ability weakened more obviously(all P<0.05).The expression levels of IL-10 and TGF-βin group B increased(all P<0.05),the expression level of IL-10 in group C decreased,and the expression of TGF-βdid not change significantly.The IL-10 of group D was lower than that of group B and increased compared with that of group C.The expression levels of IL-10 and TGF-βin group F were significantly reduced(all P<0.05).Conclusion Berberine inhibits the proliferation and migration of LPS-induced malignant melanoma cells and promotes their apoptosis by inhibiting the TLR4/NF-κB signaling pathway.Berberine may be used as a medicine to treat malignant melanoma.