乌头碱调节C-C基序趋化因子配体2/C-C基序趋化因子受体2信号通路对膀胱癌细胞抗肿瘤活性及其作用机制研究

The anti-tumor activity and mechanism of aconitine on bladder cancer cells by regulating C-C-motif chemokine ligand 2/C-C-motif chemokine receptor 2 signaling pathway

目的 探讨乌头碱调节C-C基序趋化因子配体2(CCL2)/C-C基序趋化因子受体2(CCR2)信号通路对膀胱癌细胞抗肿瘤活性及其作用机制研究。方法 研究起止时间为2021年12月至2022年10月。采用细胞计数试剂盒(CCK-8)法检测2.5、5.0、10.0、20.0、30.0μmol/L乌头碱处理后的人膀胱癌5637细胞存活率,筛选出合适的乌头碱作用浓度。将5637细胞分为对照组、乌头碱低剂量(10μmol/L)组、乌头碱高剂量(20μmol/L)组、乌头碱高剂量(20μmol/L)+空载组、乌头碱高剂量(20μmol/L)+CCL2过表达组,分组处理后,采用CCK-8法、5-乙炔基-2’-脱氧尿苷(Edu)染色法及Hoechst 33258染色分别检测各组5637细胞存活率、增殖率、凋亡率;采用Transwell实验及划痕实验分别检测各组5637细胞侵袭数、迁移率;采用免疫荧光染色检测各组5637细胞凋亡相关蛋白BCL2相关X蛋白(Bax)和B淋巴细胞瘤-2(Bcl-2)表达比值(Bax/Bcl-2);采用免疫印迹法检测各组5637细胞CCL2/CCR2信号通路相关蛋白及上皮间质转化标志蛋白[神经钙黏素(N-cadherin)、锌指E盒结合同源盒1(ZEB1)、紧密连接蛋白1(ZO-1)、上皮钙黏素(E-cadherin)]表达。结果 与对照组相比,乌头碱低剂量组、乌头碱高剂量组、乌头碱高剂量+空载组细胞CCL2(0.69±0.09、0.20±0.03、0.19±0.04比1.21±0.13),CCR2(0.78±0.12、0.26±0.06、0.27±0.07比1.33±0.20)、Ncadherin(0.65±0.06、0.12±0.02、0.11±0.03比1.24±0.12)与ZEB1蛋白表达、存活率、增殖率、侵袭数、迁移率均降低(P<0.05),Bax/Bcl-2、细胞ZO-1与E-cadherin蛋白表达均升高(P<0.05);乌头碱高剂量组、乌头碱高剂量+空载组细胞CCL2、CCR2、N-cadherin与ZEB1蛋白表达、存活率、增殖率、侵袭数、迁移率相比乌头碱低剂量组进一步降低(P<0.05),Bax/Bcl-2、细胞ZO-1与Ecadherin蛋白表达进一步升高(P<0.05)。与乌头碱高剂量组相比,乌头碱高剂量+CCL2过表达组细胞CCL2、CCR2、N-cadherin与ZEB1蛋白表达、存活率、增殖率、侵袭数、迁移率升高(P<0.05),Bax/Bcl-2、细胞ZO-1与E-cadherin蛋白表达降低(P<0.05)。结论 乌头碱可通过CCL2/CCR2信号通路而抑制膀胱癌细胞存活、增殖及侵袭和迁移,促使其凋亡。

Objective To investigate the anti-tumor activity and mechanism of aconitine on bladder cancer cells by regulating the C-C-motif chemokine ligand 2(CCL2)/C-C-motif chemokine receptor 2(CCR2)signaling pathway.Methods The start and end period of this study was from December 2021 to October 2022.CCK-8 method was used to detect the survival rate of human bladder cancer 5637 cells treated with 2.5,5,10,20,30µmol/L aconitine,and the appropriate concentration of aconitine was selected.5637 cells were randomly assigned into control group,low-dose aconitine(10µmol/L)group,high-dose aconitine(20µmol/L)group,high-dose aconitine(20µmol/L)+no load group,high-dose aconitine(20µmol/L)+CCL2 overexpression group.After grouping and treatment,the survival rate,prolifer-ation rate and apoptosis rate of 5637 cells in each group were detected by CCK-8 method,5-Ethynyl-2'-deoxyuridine(Edu)staining method and Hoechst 33258 staining.Transwell test and scratch test were used to detect the invasion number and migration rate of 5637 cells in each group;immunofluorescence staining was used to detect the expression ratio of apoptosis related protein BCL2-associated X protein(Bax)and B-cell lymphoma-2(Bcl-2)(Bax/Bcl-2)of 5637 cells in each group;Western blotting was performed to detect the ex-pression of CCL2/CCR2 signaling pathway related proteins and epithelial mesenchymal transformation markers[N-cadherin(N-cad-herin),zinc finger e-box-binding homeobox 1(ZEB1),zonula occludens-1(ZO-1),and E-cadherin(E-cadherin)]in 5637 cells of each group.Results Compared with the control group,the expressions of CCL2(0.69±0.09,0.20±0.03,0.19±0.04 vs.1.21±0.13),CCR2(0.78±0.12,0.26±0.06,0.27±0.07 vs.1.33±0.20),N-cadherin(0.65±0.06,0.12±0.02,0.11±0.03 vs.1.24±0.12)and ZEB1 proteins,sur-vival rates,proliferation rates,invasion numbers and migration rates of cells in low-dose aconitine group,high-dose aconitine group and high-dose aconitine+no load group decreased(P<0.05),while the expressions of Bax/Bcl-2,ZO-1 and E-cadherin proteins in cells in-creased(P<0.05).Compared with the low-dose aconitine group,the expressions of CCL2,CCR2,N-cadherin and ZEB1 proteins,survival rates,proliferation rates,invasion numbers and migration rates of cells in the high-dose aconitine group,the high-dose aconitine+no load group further reduced(P<0.05),while the expressions of Bax/Bcl-2,ZO-1 and E-cadherin proteins in cells further increased(P<0.05).Compared with the high-dose aconitine group,the expressions of CCL2,CCR2,N-cadherin and ZEB1 proteins,survival rates,prolifera-tion rates,invasion numbers and migration rates of cells in the high-dose aconitine+CCL2 overexpression group increased(P<0.05),while the expressions of Bax/Bcl-2,ZO-1 and E-cadherin proteins decreased(P<0.05).Conclusion Aconitine can inhibit the survival,proliferation,invasion and migration of bladder cancer cells and promote their apoptosis through CCL2/CCR2 signaling pathway.