吴茱萸碱调节Shh/Gli1信号通路对弥漫性大B细胞淋巴瘤细胞利妥昔单抗耐药性的影响

Impacts of Evodiamine on Rituximab Resistance in Diffuse Large B-Cell Lymphoma Cells by Regulating the Shh/Gli1 Signaling Pathway

目的:研究吴茱萸碱调节超音刺猬蛋白(Shh)/Gli家族锌指蛋白1(Gli1)信号通路对弥漫性大B细胞淋巴瘤(DLBCL)细胞利妥昔单抗(RIT)耐药性的影响。方法:体外培养OCI-LY10细胞采用梯度加药法构建其RIT耐药细胞系OCI-LY10/RIT,均以0、1、5、10、20、30μmoL/L的吴茱萸碱处理,采用CCK-8法测定各组OCI-LY10及OCI-LY10/RIT细胞活力并筛选出吴茱萸碱最佳作用浓度。将OCI-LY10/RIT细胞随机分为对照组、RIT组、RIT+吴茱萸碱组、RIT+空载组、RIT+吴茱萸碱+Shh过表达组,分组处理后以实时荧光定量PCR和免疫印迹实验检测各组细胞Shh/Gli1通路相关mRNA和蛋白表达;以CCK-8法和Edu染色检测各组细胞增殖;以流式细胞实验检测各组细胞凋亡;以免疫印迹实验检测各组细胞凋亡蛋白(Cleaved Caspase-3、Bax)与耐药蛋白{多药耐药相关蛋白5(MRP5)、P-糖蛋白(P-gp)}表达。体外培养OCI-LY10/RIT细胞并随机分为对照组、吴茱萸碱组、空载组、吴茱萸碱+Shh过表达组,分组处理后以CCK-8法检测0、16、32、64、128、256、384μg/mL的RIT处理下各组OCI-LY10/RIT细胞存活率,算出其耐药指数。结果:与对照组相比,RIT+吴茱萸碱组细胞凋亡率、Cleaved Caspase-3与Bax蛋白表达升高(P<0.05),Shh、Gli1 mRNA与蛋白表达、存活率、Edu阳性率、MRP5与P-gp蛋白表达降低(P<0.05);RIT组、RIT+空载组细胞各指标无明显变化(P>0.05)。与RIT组相比,RIT+吴茱萸碱组细胞凋亡率、Cleaved Caspase-3与Bax蛋白表达升高(P<0.05),Shh、Gli1 mR-NA与蛋白表达、存活率、Edu阳性率、MRP5与P-gp表达降低(P<0.05);RIT+空载组细胞各指标无明显变化(P>0.05)。与RIT+吴茱萸碱组相比,RIT+吴茱萸碱+Shh过表达组细胞凋亡率、Cleaved Caspase-3与Bax蛋白表达降低(P<0.05),Shh、Gli1 mRNA与蛋白表达、存活率、Edu阳性率、MRP5与P-gp表达升高(P<0.05)。与对照组相比,吴茱萸碱组细胞耐药指数降低(P<0.05),空载组细胞耐药指数无明显变化(P>0.05);与吴茱萸碱组相比,吴茱萸碱+Shh过表达组细胞耐药指数升高(P<0.05)。结论:吴茱萸碱可下调Shh/Gli1通路相关蛋白表达,从而减轻DLBCL细胞的RIT耐药性,诱导RIT处理下的RIT耐药DLBCL细胞凋亡并抑制其增殖。

Objective:To investigate the impacts of evodiamine on rituximab(RIT)resistance in diffuse large B-cell lymphoma(DLBCL)cells by regulating the Shh/Gli family zinc finger protein 1(Gli1)signal pathway.Methods:OCI-LY10 cells were cultured in vitro and their RIT resistant cell line OCI-LY10/RIT was constructed using gradient drug addition method.All cells were treated with 0,1,5,10,20 and 30μmoL/L of evodiamine,CCK-8 method was applied to determine the activity of OCI-LY10 and OCI-LY10/RIT cells in each group and to screen the optimal concentration of evodiamine.OCI-LY10/RIT cells were ran-domly grouped into control group,RIT group,RIT+evodiamine group,RIT+empty group,and RIT+evodia-mine+Shh overexpression group,after grouping and processing,real-time fluorescence quantitative PCR and immunoblotting experiments were applied to detect the expression of Shh/Gli1 pathway related mRNA and pro-tein of cells in each group;CCK-8 method and Edu staining were applied to detect cell proliferation in each group;flow cytometry was applied to detect cell apoptosis in each group;Western blot was applied to detect the expression of apoptotic proteins(Cleaved Caspase-3,Bax)and drug resistance proteins[multiple drug resistance related protein 5(MRP5),P-glycoprotein(P-gp)]in each group.OCI-LY10/RIT cells were cultured in vitro and randomly grouped into a control group,a evodiamine group,an empty group,and a evo-diamine+Shh overexpression group,after grouping and processing,the CCK-8 method was applied to detect the survival rate of OCI-LY10/RIT cells in each group under 0,16,32,64,128,256 and 384μg/mL RIT treatment,and calculate their drug resistance index.Results:Compared with the control group,the apoptosis rate,the expression of Cleaved Caspase-3 and Bax proteins in the RIT+evodiamine group increased(P<0.05),the expression of Shh,Gli1 mRNAs and proteins,survival rate,Edu positive rate,and the expression of MRP5 and P-gp proteins decreased(P<0.05);there was no obvious change in all cell indicators in the RIT group and the RIT+empty group(P>0.05).Compared with the RIT group,the apoptosis rate,the expression of Cleaved Caspase-3 and Bax proteins in the RIT group and the RIT+empty group increased(P<0.05),the expression of Shh,Gli1 mRNAs and proteins,survival rate,Edu positive rate,and the expression of MRP5 and P-gp proteins decreased(P<0.05);there was no obvious change in all indicators of cells in the RIT+empty group(P>0.05).Compared with the RIT+evodiamine group,the apoptosis rate,the expression of Cleaved Caspase-3 and Bax proteins in the RIT+evodiamine+Shh overexpression group decreased(P<0.05),the expression of Shh,Gli1 mRNAs and proteins,survival rate,Edu positive rate,and the expression of MRP5 and P-gp proteins increased(P<0.05).Compared with the control group,the cell resistance index in the evodiamine group decreased(P<0.05),the drug resistance index of cells in the empty group showed no obvious change(P>0.05);compared with the Evodiamine group,the evodiamine+Shh overexpression group showed an increase in cell resistance index(P<0.05).Conclusion:Evodiamine can down-regulate the ex-pression of Shh/Gli1 pathway related proteins,thereby reducing RIT resistance in DLBCL cells,inducing ap-optosis and inhibiting proliferation of RIT resistant DLBCL cells under RIT treatment.