紫草素通过Keap1/Nrf2信号通路介导的自噬对卵巢癌SK-OV-3细胞的影响  

Effects of Shikonin on Ovarian Cancer SK-OV-3 Cells Through Autophagy Mediated by Keap1/Nrf2 Signaling Pathway

作　　者：方堃[1] 邱芳[1] 丁娅妮 邓丹妮 覃庆锋[1] FANG Kun;QIU Fang;DING Yani;DENG Danni;QIN Qingfeng(Department of Gynecology,Guizhou Provincial People's Hospital,Guiyang 550002 Guizhou,China)

机构地区：[1]贵州省人民医院妇科,贵州贵阳550002

出　　处：《中药新药与临床药理》2023年第8期1053-1060,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：贵州省科技计划项目(黔科合支撑[2021]一般040)。

摘　　要：目的探讨紫草素通过Keap1/Nrf2信号通路介导的自噬对卵巢癌SK-OV-3细胞的影响。方法采用MTT法检测细胞增殖情况,计算顺铂、紫草素及在紫草素作用下顺铂的半数抑制浓度(IC50),以确定后续干预浓度。采用pcDNA3-EGFP-C4-Nrf2质粒及其阴性对照对SK-OV-3细胞进行转染,分别记为OE-Nrf2组、NC-Nrf2组。将SK-OV-3细胞分为对照组、顺铂(14μmol·L^(-1))组、顺铂(14μmol·L^(-1))+紫草素(9μmol·L^(-1))组,以及将转染后细胞分为顺铂(14μmol·L^(-1))+紫草素(9μmol·L^(-1))+NC-Nrf2组、顺铂(14μmol·L^(-1))+紫草素(9μmol·L^(-1))+OE-Nrf2组;培养48 h后,采用MTT法检测细胞的增殖抑制情况;RT-qPCR法检测细胞Nrf2mRNA表达情况;Transwell实验检测细胞侵袭能力;AO染色法检测细胞自噬情况;Western Blot法检测细胞增殖、侵袭及自噬相关蛋白(CyclinD1、PCNA、E-cadherin、N-cadherin、Vimentin、Beclin-1、LC3II/I、ULK、Keap1、Nrf2、p62)表达情况。结果(1)与对照组比较,紫草素4~20μmol·L^(-1)浓度组的SK-OV-3细胞活力明显降低(P<0.05),紫草素对SK-OV-3细胞的IC50为9.04μmol·L^(-1);单用顺铂10~80μmol·L^(-1)浓度组及紫草素(9μmol·L^(-1))+顺铂10~80μmol·L^(-1)浓度组的SK-OV-3细胞活力均明显降低(P<0.05)。与单用顺铂组比较,紫草素(9μmol·L^(-1))+顺铂10~80μmol·L^(-1)浓度组的SK-OV-3细胞活力均明显降低(P<0.05)。单用顺铂对SK-OV-3细胞的IC50为28.49μmol·L^(-1);与紫草素(9μmol·L^(-1))联用时,顺铂对SK-OV-3细胞的IC50为14.00μmol·L^(-1)。(2)与NC-Nrf2组比较,OE-Nrf2组SK-OV-3细胞的Nrf2 mRNA表达水平明显升高(P<0.05)。与对照组比较,顺铂组SK-OV-3细胞的Nrf2 mRNA及蛋白表达水平明显升高(P<0.05),Keap1蛋白表达水平明显降低(P<0.05);顺铂组、顺铂+紫草素组SK-OV-3细胞的增殖抑制率明显升高(P<0.05),CyclinD1、PCNA蛋白表达明显下调(P<0.05);单个视野细胞侵袭数目及N-cadherin、Vimentin蛋白表达水平明显降低(P<0.05),E-cObjective To investigate the effect of shikonin on ovarian cancer SK-OV-3 cells through autophagy mediated by the Keap1/Nrf2 signaling pathway.Methods Cell proliferation was detected by MTT method and the halfinhibitory concentrations(IC50)of cisplatin,shikonin and cisplatin in the presence of shikonin were calculated to determine the concentration of subsequent intervention.SK-OV-3 cells were transfected with pcDNA3-EGFP-C4-Nrf2plasmid and its negative control,which were noted as OE-Nrf2 group and NC-Nrf2 group respectively.SK-OV-3 cells were divided into control group,cisplatin(14μmol·L^(-1))group,cisplatin(14μmol·L^(-1))+shikonin(9μmol·L^(-1))group,as well as dividing the transfected cells into cisplatin(14μmol·L^(-1))+shikonin(9μmol·L^(-1))+NC-Nrf2group,cisplatin(14μmol·L^(-1))+shikonin(9μmol·L^(-1))+OE-Nrf2 group;after 48 hours of incubation,cells were examined for proliferation inhibition by MTT;mRNA expression of Nrf2 was detected by RT-qPCR;cell invasion ability was detected by Transwell assay;cell autophagy was detected by AO staining;cell proliferation,invasion and autophagy-related proteins(CyclinD1,PCNA,E-cadherin,N-cadherin,Vimentin,Beclin-1,LC3II/I,ULK,Keap1,Nrf2,p62)were detected by Western Blot.Results(1)Compared with the control group,the viability of SK-OV-3 cells was significantly reduced in the group with shikonin concentrations of 4-20μmol·L^(-1)(P<0.05),and the IC50of shikonin on SK-OV-3 cells was 9.04μmol·L^(-1);the viability of SK-OV-3 cells was significantly reduced in the group with cisplatin alone at concentrations of 10-80μmol·L^(-1)and shikonin(9μmol·L^(-1))+cisplatin at concentrations of 10-80μmol·L^(-1)group(P<0.05).Compared with the cisplatin alone group,the viability of SKOV-3 cells was significantly reduced in all the groups with shikonin(9μmol·L^(-1))+cisplatin at concentrations of 10-80μmol·L^(-1)(P<0.05).The IC50of cisplatin alone on SK-OV-3 cells was 28.49μmol·L^(-1);when combined with shikonin(9μmol·L^(-1)),the IC50of cisplatin on SK-OV-3 cell

关 键 词：卵巢癌 SK-OV-3细胞 顺铂 紫草素 Keap1/Nrf2通路 自噬 

分 类 号：R285.5[医药卫生—中药学]