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目的:探讨二氢青蒿素对5-氟尿嘧啶治疗胃癌的辅助作用并研究其机制。方法:实验分为对照组、二氢青蒿素组、5-氟尿嘧啶组、5-氟尿嘧啶联合二氢青蒿素组和5-氟尿嘧啶+二氢青蒿素+SIRT1质粒组。MTT法检测胃癌细胞系BGC-823在5-氟尿嘧啶联合二氢青蒿素处理下的细胞活力。Western blot实验检测5-氟尿嘧啶联合二氢青蒿素对BGC-823细胞SIRT1和NADPH氧化酶表达水平,caspase-9和caspase-3活化水平及凋亡信号调节激酶1(ASK1)和c-Jun氨基末端激酶(JNK)蛋白磷酸化水平的影响。流式细胞术检测BGC-823细胞在5-氟尿嘧啶和二氢青蒿素联合处理下的活性氧簇(ROS)生成水平和细胞凋亡率。结果:二氢青蒿素处理能显著抑制BGC-823细胞SIRT1的表达并增加NADPH氧化酶的蛋白水平,明显提高BGC-823细胞对5-氟尿嘧啶的敏感性,降低5-氟尿嘧啶的半数抑制浓度;转染SIRT1表达质粒后,二氢青蒿素联合5-氟尿嘧啶对BGC-823细胞的杀伤活性受到显著抑制(P<0.05)。二氢青蒿素能明显促进5-氟尿嘧啶对BGC-823细胞生成ROS的诱导效应和ASK1及JNK的磷酸化(P<0.05)。用ROS清除剂N-乙酰半胱氨酸(NAC)或JNK特异性抑制剂SP600125处理后,二氢青蒿素联合5-氟尿嘧啶对BGC-823细胞的杀伤活性和caspase-9及caspase-3的活化均受到明显抑制(P<0.05)。另外,NAC能显著抑制二氢青蒿素联合5-氟尿嘧啶对JNK磷酸化的促进作用,而SP600125却不能影响BGC-823细胞ROS的产生,表明JNK是ROS的下游分子。结论:二氢青蒿素联合5-氟尿嘧啶通过SIRT1/NADPH氧化酶/ROS/JNK通路诱导胃癌细胞发生caspase依赖的凋亡。
Objective: To investigate the role of dihydroartemisinin in the treatment of gastric cancer with 5-fluorouracil and its mechanism. Methods: The experiments were divided into control group, dihydroartemisinin group, 5-fluorouracil group, 5-fluorouracil combined with dihydroartemisinin group and 5-fluorouracil + dihydroartemisinin + SIRT1 plasmid group. MTT assay was used to detect the cell viability of gastric cancer cell line BGC-823 treated with 5-fluorouracil and dihydroartemisinin. Western blot was used to detect the expression of SIRT1 and NADPH oxidase, the activation of caspase-9 and caspase-3 and the expression of apoptosis-regulatory kinase 1 (ASK1) and c-Jun amino acid in BGC-823 cells treated with 5-fluorouracil and dihydroartemisinin. The effect of phosphorylation level of the terminal kinase (JNK) protein. The reactive oxygen species (ROS) production and apoptosis of BGC-823 cells treated with 5-fluorouracil and dihydroartemisinin were detected by flow cytometry. RESULTS: Dihydroartemisinin treatment significantly inhibited the expression of SIRT1 in BGC-823 cells and increased the protein level of NADPH oxidase, significantly increased the sensitivity of BGC-823 cells to 5-fluorouracil, and decreased the half-inhibitory concentration of 5-fluorouracil. After transfection of SIRT1 expression plasmid, the killing activity of dihydroartemisinin combined with 5-fluorouracil on BGC-823 cells was significantly inhibited (P<0.05). Dihydroartemisinin can significantly promote the induction of ROS production by 5-fluorouracil on BGC-823 cells and the phosphorylation of ASK1 and JNK (P<0.05). The killing activity of dihydroartemisinin combined with 5-fluorouracil on BGC-823 cells after treatment with ROS scavenger N-acetylcysteine (NAC) or JNK specific inhibitor SP600125 and caspase-9 and caspase-3 Activation was significantly inhibited (P<0.05). In addition, NAC could significantly inhibit the promotion of phosphorylation of JNK by dihydroartemisinin combined with 5-fluorouracil, while SP600125 could not affect the production of ROS in BGC-823 cells, indicating that JNK is a downstream molecule of ROS. CONCLUSION: Dihydroartemisinin combined with 5-fluorouracil induces caspase-dependent apoptosis in gastric cancer cells through SIRT1/NADPH oxidase/ROS/JNK pathway.