红芪多糖介导STAT3通路抑制卵巢癌小鼠肿瘤细胞免疫逃逸

Hedysarum polysaccharide mediates the STAT3 pathway to inhibit the immune escape of tumor cells in ovarian cancer mice

目的探讨红芪多糖(HPS)介导信号转导子与转录激活子3(STAT3)通路抑制卵巢癌小鼠肿瘤细胞免疫逃逸的作用。方法取40只Balb/c小鼠通过皮下注射卵巢癌SKOV3细胞建立卵巢癌小鼠模型,将建模成功的小鼠随机分为模型组、米非司酮(MIF)组、HPS组、HPS^(+)MIF联合组。HPS组灌胃给予HPS(200 mg·kg^(-1)·d^(-1)),MIF组灌胃给予MIF(40 mg·kg^(-1)·d^(-1)),HPS^(+)MIF联合组灌胃给予HPS(200 mg·kg^(-1)·d^(-1))和MIF(40 mg·kg^(-1)·d^(-1)),模型组灌胃给予等量无菌生理盐水,连续干预14 d。观察记录各组小鼠瘤体积、瘤质量并计算抑瘤率;酶联免疫吸附测定(ELISA)法检测小鼠外周血白介素-2(IL-2)、白介素-10(IL-10)、转化生长因子-β1(TGF-β1)水平;流式细胞术检测各组小鼠肿瘤组织中CD4^(+)、CD4^(+)CD25^(+)叉头框蛋白P3(^(+)Foxp3^(+))调节性T细胞(Treg)水平;实时定量聚合酶链反应(RT-qPCR)检测各组小鼠肿瘤组织STAT3、程序性死亡配体-1(PD-L1)、程序性死亡受体1(PD-1)信使核糖核糖酸(mRNA)表达;蛋白质免疫印迹法(Western blot)检测各组小鼠肿瘤组织STAT3、PD-L1、PD-1蛋白表达。结果与模型组比,MIF组、HPS组、HPS^(+)MIF联合组小鼠瘤体积和瘤质量、外周血IL-2、IL-10、TGF-β1水平以及肿瘤组织CD4^(+)CD25^(+)Foxp3^(+)Treg细胞水平降低,肿瘤组织中CD4^(+)细胞水平升高,差异均有统计学意义(P<0.05);与MIF组和HPS组比,HPS^(+)MIF联合组小鼠瘤体积和瘤质量、外周血IL-2、IL-10、TGF-β1水平以及肿瘤组织CD4^(+)CD25^(+)Foxp3^(+)Treg细胞水平降低,肿瘤组织中CD4^(+)细胞水平升高,差异均有统计学意义(P<0.05)。与模型组比,MIF组、HPS组、HPS^(+)MIF联合组小鼠肿瘤组织STAT3、PD-L1、PD-1 mRNA及蛋白表达降低,差异均有统计学意义(P<0.05);与MIF组和HPS组比,HPS^(+)MIF联合组小鼠肿瘤组织STAT3、PD-L1、PD-1 mRNA及蛋白表达降低,差异均有统计学意义(P<0.05)。结论HPS对卵巢癌小鼠肿瘤细胞免疫逃逸有一定抑制作用,推测可能是通过介导STAT3信号通路抑制STAT3、PD-L1、PD-1的表达实现上述作用的。

This study was performed to investigate the effect of hedysarum polysaccharide(HPS)-mediated signal transducer and activator of transcription 3(STAT3)pathway in inhibiting the immune escape of tumor cells in ovarian cancer mice.A mouse model of ovarian cancer was established by subcutaneous injection of ovarian cancer SKOV3 cells in 40 Balb/c mice,and the mice that were successfully modeled were randomly divided into model group,mifepristone(MIF)group,HPS group,HPS^(+)MIF joint group.The tumor volume and tumor mass of the mice in each group were observed and recorded,and the tumor inhibition rate was calculated.The levels of interleukin-2(IL-2),IL-10 and transforming growth factor-β1(TGF-β1)in peripheral blood of mice were detected by enzymelinked immunosorbent assay(ELISA).Flow cytometry was used to detect the levels of CD4^(+),CD4^(+)CD25^(+)forkhead box protein P3^(+)(Foxp3^(+))regulatory T cells(Treg)in tumor tissues of mice in each group.The expression of STAT3,programmed death ligand-1(PD-L1)and programmed death receptor 1(PD-1)messenger ribonucleic acid(mRNA)in mouse tumor tissue.Western blot was used to detect the expression of STAT3,PD-L1,PD-1 proteins in mouse of each group.Compared with the model group,the tumor volume and tumor mass,the levels of IL-2,IL-10,TGF-β1 in peripheral blood,and the levels of CD4^(+)CD25^(+)Foxp3^(+)Treg cells in tumor tissue of mice in MIF group,HPS group,and HPS^(+)MIF combination group decreased,and the level of CD4^(+)cells in tumor tissue increased,the differences were statistically significant(P<0.05).Compared with the MIF group and the HPS group,the tumor volume and tumor mass,the levels of IL-2,IL-10,TGF-β1 in peripheral blood and the levels of CD4^(+)CD25^(+)Foxp3^(+)Treg cells in the tumor tissue of the mice in the HPS^(+)MIF combination group decreased,the level of CD4^(+)cells in tumor tissues increased(all P<0.05).Compared with the model group,the mRNA and protein expressions of STAT3,PD-L1 and PD-1 in the tumor tissues of the mice in the MIF group,HPS group,and HPS^(+)MIF combination group decreased(P<0.05).Compared with MIF group and HPS group,the mRNA and protein expressions of STAT3,PD-L1,PD-1 in tumor tissues of HPS^(+)MIF combination group decreased(P<0.05).In conclusion,HPS has certain inhibitory effect on the immune escape of tumor cells in ovarian cancer mice,which is presumed to be achieved by mediating the STAT3 signaling pathway and inhibiting the expression of STAT3,PD-L1,and PD-1.