目的 基于Kelch样ECH相关蛋白1/核因子E2相关因子2（Kelch-like ECH-associated protein-1/nuclear factor E2-related factor 2，Keap1/Nrf2）通路介导的自噬探讨紫草素对膀胱上皮癌BUC细胞化疗敏感性的影响。方法 体外培养人BUC细胞BIU-87，CCK-8法检测不同质量浓度（10、20、40、80、160 µg/mL）顺铂和不同浓度（2、4、8、16、32 µmol/L）紫草素＋顺铂（40 µg/mL）对BIU-87细胞增殖的影响。细胞转染实验，设置对照组、顺铂组（40 µg/mL）、紫草素组（40 µg/mL顺铂＋8 µmol/L紫草素）和NC组（转染negative control-Nrf2＋40 µg/mL顺铂＋8 µmol/L紫草素）和Nrf2组（转染hsa-Nrf2 mimic＋40 µg/mL顺铂＋8 µmol/L紫草素），实时荧光定量PCR（qRT-PCR）法检测转染后BIU-87细胞中Nrf2 mRNA表达情况；CCK-8法检测各组BIU-87细胞增殖情况；流式细胞仪检测各组BIU-87细胞凋亡情况；蛋白免疫印迹（Western blotting）法检测BIU-87细胞中Keap1、Nrf2、微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，LC3）І、LC3II蛋白表达情况。结果 与对照组相比，10、20、40、80、160 µg/mL顺铂可显著抑制BIU-87细胞增殖（P<0.05）；2、4、8、16、32 µmol/L紫草素联合顺铂可显著抑制BIU-87细胞增殖（P<0.05）。细胞转染实验中，与对照组相比，顺铂组BIU-87细胞增殖抑制率、凋亡率、细胞中Nrf2和LC3II蛋白表达水平显著升高，细胞中Keap1、LC3І蛋白表达水平显著降低（P<0.05）；与顺铂组相比，紫草素组BIU-87细胞增殖抑制率、凋亡率、细胞中Keap1和LC3І蛋白表达水平显著升高，Nrf2和LC3II蛋白表达水平显著降低（P<0.05）；紫草素组、NC组以上指标差异不具有统计学意义（P>0.05）；与NC组相比，Nrf2组BIU-87细胞增殖抑制率、凋亡率、细胞中Keap1、LC3І蛋白表达水平显著降低，Nrf2蛋白和mRNA及LC3II蛋白表达水平显著升高（P<0.05）。结论 紫草素可能通过抑制Keap1/Nrf2通路介导的自噬反应，增强BUC细胞BIU-87对顺铂的敏感性。

Objective To investigate the effect of shikonin on chemosensitivity of bladder urothelial carcinoma (BUC) cells based on autophagy mediated by Kelch-like ECH-associated protein 1/nuclear factor erythroid-2 related factor 2 (Keap1/Nrf2) pathway. Methods Human BUC cells BIU-87 were cultured in vitro, and the effects of cisplatin (10, 20, 40, 80, 160 μg/mL) and shikonin (2, 4, 8, 16, 32 μmol/L) on the proliferation of BIU-87 cells were detected by CCK-8 method. Control group, cisplatin group (40 μg/mL cisplatin), shikonin group (40 μg/mL cisplatin + 8 μmol/L shikonin group), NC group (transfected with negative control-Nrf2 + 40 μg/mL cisplatin + 8 μmol/L shikonin) and Nrf2 group (transfected with hsa-Nrf2 mimic + 40 μg/mL cisplatin + 8 μmol/L shikonin). The expression of Nrf2 mRNA in BIU-87 cells was detected by real-time fluorescence quantitative PCR (qRT-PCR), the proliferation of BIU-87 cells was detected by CCK-8 method, the apoptosis of BIU-87 cells was detected by flow cytometry, and Western blot was used to detect the expression of Keap1, Nrf2, microtubule-associated protein 1 light chain 3 (LC3) and LC3 Ⅱ in BIU-87 cells. Results Compared with control group, the proliferation inhibition rate of BIU-87 cells treated with 10, 20, 40, 80, and 160 μg/mL cisplatin was significantly higher (P < 0.05); The proliferation inhibition rate of BIU-87 cells treated with 2, 4, 8, 16, 32 μmol/L shikonin and cisplatin was significantly higher (P < 0.05). Compared with those in the control group, the proliferation inhibition rate and apoptosis rate of BIU-87 cells, the protein expression levels of Nrf2 and LC3 Ⅱ in cisplatin group were significantly higher, While the protein expression levels of Keap1 and LC3 І were significantly lower (P < 0.05). Compared with those in cisplatin group, the proliferation inhibition rate and apoptosis rate of BIU-87 cells, the protein expression levels of Keap1 and LC3 І in BIU-87 cells in shikonin group were significantly higher, while the protein expression levels of Nrf2 and LC3 Ⅱ were significantly lower (P < 0.05); There was no significant difference in the above indexes between shikonin group and NC group (P > 0.05). Compared with those in NC group, the proliferation inhibition rate and apoptosis rate of BIU-87 cells, the protein expression levels of Keap1 and LC3І in BIU-87 cells in Nrf2 group were significantly lower, while the protein expression levels of Nrf2 and LC3Ⅱ were significantly higher (P < 0.05). Conclusion Shikonin may enhance the cisplatin sensitivity of BUC cells BIU-87 by inhibiting the autophagy mediated by Keap1/Nrf2 pathway.

R285

国家自然科学基金项目（81973529）