西瑞香素通过调控谷胱甘肽过氧化物酶4和铁蛋白重链1表达对786-O细胞铁死亡、增殖及侵袭的影响  

Daphnoretin induces ferroptosis in 786-O cells by suppressing glutathione peroxidase 4 and FTH1 expression,thereby inhibiting cell proliferation and invasion independently of the GSK/FoxO3a pathway

作　　者：王雪 水豪杰 张曙伟 胡潇威 黄帅帅 翁国斌 宋灵敏 Wang Xue;Shui Haojie;Zhang Shuwei;Hu Xiaowei;Huang Shuaishuai;Weng Guobin;Song Lingmin(Urology and Nephrology Institute,Ningbo Yinzhou No.2 Hospital,Ningbo 315000,China;Department of Outpatient,Ningbo Yinzhou No.2 Hospital,Ningbo 315000,China;Department of Urologic Surgery,Ningbo Yinzhou No.2 Hospital,Ningbo 315000,China)

机构地区：[1]宁波市鄞州区第二医院泌尿肾病研究所,宁波315000 [2]宁波市鄞州区第二医院门诊办公室,宁波315000 [3]宁波市鄞州区第二医院泌尿外科,宁波315000

出　　处：《中华实验外科杂志》2024年第11期2556-2560,共5页Chinese Journal of Experimental Surgery

基　　金：浙江省医药卫生项目(2024KY1609、2022KY1178);鄞州区科技项目(鄞科[2018]74号)。

摘　　要：目的:探讨西瑞香素(DAP)抑制肾细胞癌(RCC)细胞786-O的增殖、侵袭能力和诱导铁死亡的作用及分子机制。方法:采用不同浓度DAP(0、12.5、25.0、50.0、100.0μmol/L)处理786-O细胞后,细胞计数试剂盒(CCK-8)和Transwell检测细胞增殖和侵袭能力。12.5μmol/L DAP联合铁死亡诱导剂(0.2μmol/L Erastin或30 nmol/L RSL3)处理细胞24 h后,CCK-8检测细胞活力。12.5μmol/L DAP与10.0μmol/L铁螯合剂(DFO)或50 mg/L补铁剂(FAC)联合处理细胞24 h后,用总铁和Fe^(2+)检测试剂盒分析总铁和Fe^(2+)水平变化。用蛋白质印迹法(Western blot)检测各蛋白的表达。两组间比较用t检验,多组间比较用单因素方差分析。结果:CCK-8结果显示,0μmol/L DAP组吸光度值高于12.5、25、50、100μmol/L组(处理24 h:0.10±0.15比0.85±0.12、0.79±0.11、0.78±0.12、0.65±0.10;处理48 h:2.23±0.28比1.93±0.16、1.43±0.13、1.29±0.20、0.92±0.34)。Transwell结果显示,0μmol/L DAP组吸光度值高于12.5、25、50、100μmol/L组(2.93±0.36比2.24±0.40、1.90±0.44、1.18±0.29、0.97±0.37,均P<0.05)。HK-2细胞组的GPX4蛋白相对表达量低于786-O细胞组(100%比742%,F=40.48,P<0.01)。0μmol/L DAP组GPX4蛋白灰度值高于12.5、25、50、100μmol/L组(每组相对灰度值分别为1、0.84、0.81、0.73、0.49)。CCK-8结果显示,DAP联合Erastin组吸光度值低于DAP和Erastin单独处理组[(1.61±0.46)L/(g·cm)比(2.43±0.11)L/(g·cm)和(2.38±0.41)L/(g·cm),均P<0.05],同时,DAP联合RSL3组吸光度值低于DAP或RSL3单独处理组[(0.62±0.53)L/(g·cm)比(1.89±0.53)L/(g·cm)或(1.52±0.26)L/(g·cm),均P<0.05]。另外,DAP联合Erastin或RSL3组GPX4蛋白灰度值低于control、DAP、Erastin或RSL3组(每组相对灰度值分别为0.71、0.46、1、0.92、0.84、1.18)。786-O细胞组FTH1蛋白表达与HK-2细胞组间的差异无统计学意义(141.33%比100.00%,F=1.49,P>0.05)。但是,0μmol/L DAP组FTH1蛋白灰度值高于12.5、25、50、100μmol/L组(每组相对灰度值分别为1、0.9Objective This study examines the effects of daphnoretin(DAP)on the proliferation and invasion of 786-O cells and its ability to induce ferroptosis,exploring the underlying molecular mechanisms.Methods The impact of DAP on 786-O cell proliferation and invasion was measured using cell counting kit-8(CCK-8)and Transwell assays at various concentrations(0,12.5,25.0,50.0,100.0μmol/L).Additionally,cell viability was assessed with a CCK-8 assay following co-treatment with 0.2μmol/L Erastin or 30.0 nmol/L RSL3 and 12.5μmol/L DAP for 24 hours.Cells were also treated with 12.5μmol/L DAP alongside 10μmol/L deferoxamine(DFO)or 50 mg/L ferric ammonium citrate(FAC)for 24 hours,with subsequent measurement of total iron and Fe^(2+)concentrations using assay kits.Protein expressions were analyzed via western blot.Statistical analysis involved t-tests for two-group comparisons and one-way ANOVA for multiple groups.ResultsCCK-8 assays revealed that the absorbance values for the 0μmol/L DAP group exceeded those of the 12.5,25,50,and 100μmol/L groups after 24 and 48 h of treatment(0.10±0.15 vs.0.85±0.12,0.79±0.11,0.78±0.12,0.65±0.10,respectively;2.23±0.28 vs.1.93±0.16,1.43±0.13,1.29±0.20,0.92±0.34,respectively).Similarly,Transwell assays indicated higher absorbance values for the 0μmol/L DAP group compared to the 12.5,25,50,and 100μmol/L groups(2.93±0.36 vs.2.24±0.40,1.90±0.44,1.18±0.29,0.97±0.37,respectively;all P<0.05).GPX4 protein expression in HK-2 cells was significantly lower than in 786-O cells(100%vs.742%,F=40.48,P<0.01).The GPX4 protein expression in the 0μmol/L DAP group exhibited higher relative gray values compared to the 12.5,25,50,and 100μmol/L groups,with 1,0.84,0.81,0.73,and 0.49,respectively.The absorbance values for DAP combined with Erastin were lower than those observed with DAP or Erastin alone[(1.61±0.46)L/(g·cm)vs.(2.43±0.11)L/(g·cm)or(2.38±0.41)L/(g·cm),both P<0.05].The combination of DAP and RSL3 resulted in lower absorbance compared to DAP or RSL3 alone[(0.62±0.53)L/(g·cm)v

关 键 词：西瑞香素 肾细胞癌 谷胱甘肽过氧化物酶4/铁蛋白重链1 磷酸化糖原合成酶激酶-3β/叉头框转录因子3a 铁死亡 增殖和侵袭 

分 类 号：R737.11[医药卫生—肿瘤]