桂枝茯苓胶囊通过调控NF-κB通路抑制卵巢癌细胞的迁移和诱导卵巢癌细胞的凋亡

Guizhi Fuling Capsule inhibits migration and induces apoptosis of human ovarian cancer cells by regulating the NF-κB signaling pathway

目的研究桂枝茯苓胶囊(GFC)抑制人卵巢癌转移的作用和潜在机制。方法60只Wistar大鼠随机分为4组,15只/组:给药组分别灌胃给予4、8、16 g·kg^(-1)·d^(-1)剂量的GFC悬液5 d,分别制备GFC低、中、高剂量含药血清,空白组给予相同体积的生理盐水制备空白血清;卵巢癌耐顺铂SKOV3/DDP细胞分为空白血清组、不同剂量GFC含药血清组和NF-κB抑制剂SN50阳性对照组。采用划痕实验检测含药血清对迁移的影响,采用流式细胞术检测含药血清对凋亡的影响,采用RT-qPCR和Westernblot检测含药血清对细胞中上皮表型E-钙粘蛋白(CDH1)、间质表型N-钙黏蛋白(CDH2)、caspase 3和NF-κB mRNA和蛋白表达的影响。采用ATAC-seq检测染色质开放区域CDH1、CDH2、caspase 3和NF-κB基因表达差异。结果GFC含药血清可剂量依赖性地抑制SKOV3/DDP细胞迁移,增加细胞凋亡(P<0.05,P<0.01)。随着含药血清剂量增加,其对CDH2和NF-κB的抑制作用,及其对CDH1和caspase 3的诱导作用均逐渐增强(P<0.05,P<0.01)。高剂量含药血清抑制SKOV3/DDP细胞迁移,诱导凋亡、caspase 3 mRNA和蛋白以及CDH1 mRNA表达,抑制CDH2和NF-κB mRNA和蛋白表达的作用与SN50相当(P>0.05),但诱导CDH1蛋白表达的作用弱于SN50(P<0.01)。含药血清可减弱染色质开放区域NF-κB、CDH2基因表达,增强CDH1表达,但对caspase 3无影响。结论GFC含药血清抑制SKOV3/DDP迁移作用可能与其诱导凋亡、caspase 3和CDH1表达,抑制CDH2和NF-κB表达,调控染色质开放区域NF-κB、CDH2和CDH1表达有关。

Objective To study the inhibitory effect of Guizhi Fuling Capsule(GFC)on migration of human ovarian cancer cells and explore the possible mechanism.Methods Sixty Wistar rats were randomized into 4 groups for daily gavage of saline or 4,8,or 16 g/kg GFC suspension for 5 days to prepare blank and low-,medium-and high-dose GFC-medicated sera.Cisplatin resistant ovarian cancer SKOV3/DDP cells were treated with these sera with nuclear factor-κB(NF-κB)inhibitor SN50 as the positive control,and the changes in migration ability and apoptosis of the cells were examined using scratch assay and flow cytometry,respectively;the changes in the mRNA and protein expressions of CDH1,CDH2,caspase 3 and NFκB were detected using RT-qPCR and Western blotting.ATAC-seq was used to analyze the changes in expressions of CDH1,CDH2,caspase 3 and NF-κB genes in the open chromatin.Results Treatment with GFC-medicated sera dose-dependently inhibited the migration(P<0.05),increased apoptosis(P<0.01),inhibited CDH2 and NF-κB mRNA expression(P<0.05),and enhanced caspase 3 and CDH1 mRNA expressions(P<0.01)in SKOV3/DDP cells.The effects of high-dose GFC-medicated serum were comparable to those of SN50(P>0.05),but its effect for enhancing DH1 protein expression was weaker than that of SN50(P<0.01).GFC-medicated sera significantly lowered the expressions of NF-κB and CDH2 and increased CDH1 expression in the open chromatin without obviously affecting caspase 3 expression.Conclusion GFC-medicated sera inhibits the migration ability of SKOV3/DDP cells possibly by promoting cell apoptosis and caspase 3 and CDH1 expressions,inhibiting CDH2 and NF-κB expressions,and regulating the expressions of NF-κB,CDH2 and CDH1 in the open chromatin.