青蒿素通过TLR4/NF-κB信号通路调节胃癌细胞的生长和炎症因子分泌的机制研究

Study on the mechanism of artemisinin regulating the growth of gastric cancer cells and the secretion of inflammatory factors through the TLR4/NF-κB signaling pathway

目的 研究青蒿素是否通过Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路调节胃癌细胞的增殖、凋亡和炎症因子分泌。方法 将胃癌AGS细胞分为对照(NC)组、50μmol/L青蒿素组、100μmol/L青蒿素组、200μmol/L青蒿素组和pcDNA+200μmol/L青蒿素组、pcDNA-TLR4+200μmol/L青蒿素组。给予青蒿素处理后,CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡,Western blot检测细胞周期蛋白D1(CyclinD1)、活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(Cleaved-caspase-3)、TLR4、p-p65、p65水平,酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-1β炎症因子的表达,RT-qPCR检测TLR4 mRNA表达。结果 与NC组比较,50μmol/L青蒿素组、100μmol/L青蒿素组、200μmol/L青蒿素组AGS细胞增殖活性、CyclinD1蛋白、TNF-α、IL-6、IL-1β、TLR4 mRNA、TLR4蛋白的表达水平和p-p65/p65比值均降低,凋亡率和Cleaved-caspase-3水平升高,并且不同浓度青蒿素组与NC组之间差异有统计学意义(P<0.05);200μmol/L青蒿素组AGS细胞中p-p65/p65比值比NC组低,差异有统计学意义(P<0.05)。与pcDNA+200μmol/L青蒿素组比较,pcDNA-TLR4+200μmol/L青蒿素组AGS细胞中TLR4 mRNA的表达水平、增殖活性、CyclinD1蛋白、TNF-α、IL-6、IL-1β和p-p65/p65比值均升高,凋亡率和Cleaved-caspase-3水平降低,并且两组之间差异有统计学意义(P<0.05)。结论 青蒿素通过下调TLR4/NF-κB信号通路,从而抑制胃癌细胞的增殖活性和炎症因子分泌,并促进凋亡。

Objective To investigate whether artemisinin regulates the proliferation,apoptosis and secretion of inflammatory factors in gastric cancer cells through the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway.Methods Gastric cancer AGS cells were divided into control(NC) group,50 μmol/L artemisinin group,100 μmol/L artemisinin group,200 μmol/L artemisinin group,pcDNA+200 μmol/L artemisinin group and pcDNA-TLR4+200 μmol/L artemisinin group.After the treatment,CCK-8 method was performed to detect cell proliferation activity,flow cytometry was used to measure cell apoptosis,cyclin D1(CyclinD1),activated cysteine-containing aspartate proteolytic enzyme-3(Cleaved-caspase-3),TLR4,p-p65,and p65 levels were detected by Western blot.The expression of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1β inflammatory factors were tested by enzyme-linked immunosorbent assay(ELISA),and the expression of TLR4 mRNA was detected by RT-qPCR.Results Compared with the NC group,the AGS cell proliferation activity,the expression levels of CyclinD1 protein,TNF-α,IL-6,IL-1β,TLR4 mRNA,TLR4 protein and p-p65/p65 ratio decreased in 50 μmol/L artemisinin group,100 μmol/L artemisinin group and 200 μmol/L artemisinin group.The apoptosis rate and Cleaved-caspase-3 level increased,and there was a statistically significant difference between the artemisinin group and the NC group at different concentrations(P<0.05).The ratio of p-p65/p65 in AGS cells in the 200 μmol/L artemisinin group was lower than that in the NC group,and the difference was statistically significant(P<0.05).Compared with the pcDNA+200 μmol/L artemisinin group,the expression level of TLR4 mRNA,proliferation activity,the expression level of CyclinD1 protein,TNF-α,IL-6,IL-1β and p-p65/p65 ratio in AGS cells in the pcDNA-TLR4+200 μmol/L artemisinin group increased,the apoptosis rate and the level of Cleaved-caspase-3 decreased,and the difference between the two groups was statistically significant(P<0.05).Conclusion Artemisinin inhibits the proliferation activity and inflammatory factor secretion of gastric cancer cells by down-regulating the TLR4/NF-κB signaling pathway,and promotes apoptosis.