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目的观察CGI-58对5-FU诱导的巨噬细胞凋亡的影响,并探讨其作用及机制。方法用5-FU处理巨噬细胞(PLKO)与CGI-58敲低(knockdown)的巨噬细胞(CGI-58 KD)后分别用CCK-8法检测其细胞增殖能力;流式细胞术检测其细胞周期与凋亡;Western blot检测其凋亡蛋白(cleaved Caspase-3)的表达水平;Seahorse Assays检测其线粒体功能。结果 CCK-8检测发现CGI-58 KD+5-FU组细胞活力降低更明显(P<0.01);流式细胞术结果显示经5-FU处理后,CGI-58 KD组的细胞凋亡率明显高于PLKO组(P<0.01),Western blot检测发现CGI-58 KD+5-FU组cleaved Caspase-3的表达水平明显高于PLKO+5-FU组(P<0.01);Seahorse Assays检测发现CGI-58 KD组其基础耗氧率OCR明显降低(P<0.01),ATP生成减少(P<0.05),ROS表达增加(P<0.01);抗氧化剂NAC处理可拮抗巨噬细胞CGI-58缺失诱导的ROS增加(P<0.05)、5-FU杀伤作用(P<0.05)及cleaved Caspase-3表达水平。结论 CGI-58缺失可增强5-FU对巨噬细胞的杀伤作用并损伤巨噬细胞的线粒体功能,其作用机制可能是通过增加ROS的产生来实现的。
Objective To observe the effect of CGI-58 on 5-FU-induced apoptosis of macrophages and to explore its mechanism and mechanism. Methods The proliferation of macrophages (PLKO) and CGI-58 knockdown macrophages (CGI-58 KD) treated with 5-FU was detected by CCK-8 assay. The proliferation of macrophages Cell cycle and apoptosis. Western blot was used to detect the expression of cleaved Caspase-3. Seahorse Assays was used to detect the mitochondrial function. Results The cell viability of CGI-58 KD + 5-FU group was significantly lower than that of control group (P <0.01). The results of flow cytometry showed that the apoptosis rate of CGI-58 KD group was significantly higher after treatment with 5-FU The expression of cleaved Caspase-3 in CGI-58 KD + 5-FU group was significantly higher than that in PLKO + 5-FU group (P <0.01) by Western blot. The results of Seahorse Assays showed that CGI (P <0.01), decreased the production of ATP (P <0.05) and increased the expression of ROS (P <0.01) in the K-58 KD group. Antioxidant NAC treatment could antagonize the induction of CGI-58 in macrophages (P <0.05), the killing effect of 5-FU (P <0.05) and the expression of cleaved Caspase-3. Conclusions The deletion of CGI-58 enhances the killing effect of 5-FU on macrophages and damages the mitochondrial function of macrophages. The mechanism may be through increasing the production of ROS.