23-羟基白桦酸通过抑制髓源性抑制细胞对小鼠结肠癌生长的影响

Effects of 23-hydroxybetulinic acid on the growth of mouse colon cancer by inhibiting myeloid-derived suppressor cells

目的探讨23-羟基白桦酸(23-HBA)通过干预髓源性抑制细胞(MDSCs)抑制小鼠结肠癌生长的作用机制。方法体内实验,建立小鼠结肠癌CT-26皮下移植瘤模型,阳性对照组5-FU腹腔注射3 d(2次),实验组23-HBA尾静脉每日注射,连续给药18 d。末次给药后处死小鼠,检测小鼠体质量、移植瘤质量及体积、脏器指数、血常规的变化,流式细胞术检测肿瘤组织MDSCs、CD8^(+)T细胞比例,RT-qPCR法检测肿瘤组织MDSCs细胞免疫抑制功能相关细胞因子精氨酸酶1(Arg1)、一氧化氮合酶(iNOS),以及CD8^(+)T细胞杀伤功能相关细胞因子颗粒酶B(GZMB)、穿孔素1(PRF1)、干扰素-γ(IFN-γ)mRNA的表达。体外实验,CCK8法检测骨髓(BM)细胞、MDSCs细胞存活率,确定23-HBA的安全作用浓度;采用40 ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF)和40 ng/mL白细胞介素-6(IL-6)诱导BM向MDSCs分化,同时加入不同浓度23-HBA(0、10.5、21、42μmoL/L)干预4 d,流式细胞术检测MDSCs细胞比例变化;BM体外诱导生成MDSCs后,将其分为对照组和23-HBA低、中、高剂量组(10.5、21、42μmoL/L),干预24 h,RT-qPCR法检测MDSCs细胞Arg1、iNOS mRNA表达;Western blot法检测MDSC细胞Arg1、iNOS蛋白表达。结果体内实验显示,与模型组比较,23-HBA干预后,小鼠移植瘤体积及质量均降低(P<0.05,P<0.01);肿瘤组织MDSCs细胞比例降低(P<0.05),CD8^(+)T细胞比例升高(P<0.05,P<0.01),Arg1 mRNA表达降低(P<0.05),GZMB、IFN-γmRNA表达升高(P<0.05,P<0.01)。体外实验显示,23-HBA剂量在10.5、21、42μmoL/L时,对BM、MDSCs细胞存活率没有显著影响(P>0.05);23-HBA高剂量可抑制BM向MDSCs分化(P<0.01);23-HBA呈剂量依赖性减少MDSCs细胞Arg1、iNOS mRNA表达(P<0.05,P<0.01),下调Arg1、iNOS蛋白表达(P<0.05,P<0.01)。结论23-HBA可通过干预MDSCs分化下调肿瘤内MDSCs细胞比例,减弱MDSCs细胞的免疫抑制功能,从而恢复CD8^(+)T细胞抗肿瘤活性,发挥抗结肠癌作用。

AIM To investigate the mechanism of 23-hydroxybetulinic acid(23-HBA)in inhibiting the growth of mouse colon cancer via myeloid-derived suppressor cells(MDSCs).METHODS The in vivo experiment of mouse colon cancer models establishment by subcutaneous CT-26 transplantation was followed by 18 days intraperitoneal injection of 5-FU at a frequency of twice every 3 days or daily injection of 23-HBA at the tail vein.After the last dose,the killed mice had their changes of body weight,tumor weight and volume,levels of organ index and blood routine test observed;their proportions of MDSCs and CD8^(+)T cells in tumor tissues detected by flow cytometry;and their expressions of arginase 1(Arg 1),nitric oxide synthase(iNOS)and cytokine granzyme B(GZMB),perforin 1(PRF 1)and interferon-γ(IFN-γ)mRNA related to the immunosuppression function of MDSCs cells in tumor tissues detected by RT-qPCR method.In the in vitro experiment,the survival rates of bone marrow(BM)cells and MDSCs cells were detected by CCK8 method to determine the safe concentration of 23-HBA.MDSCs derived from BM mediated by 40 ng/mL granulocyte-macrophage colony stimulating factor(GM-CSF)and 40 ng/mL interleukin-6(IL-6)were dosed with,different concentrations of 23-HBA(0,10.5,21,42μmol/L)for 4 days simultaneously,and had their ratio detected by flow cytometry afterwards.MDSCs inducted from BM in vitro were further divided into the control group and the low,medium and high dose 23-HBA groups(10.5,21 and 42μmoL/L)for 24 hrs corresponding intervention,and had their expressions of Arg 1 and iNOS mRNA detected by RT-qPCR,and their expressions of Arg1 and iNOS proteins detected by Western blot.RESULTS The result of the in vivo experiments revealed that in contrast to the model group,the 23-HBA groups displayed decreased volume and weight of transplanted mouse tumors(P<0.05,P<0.01);decreased proportion of MDSCs in tumor tissues(P<0.05);increased proportion of CD8^(+)T cells(P<0.05,P<0.01);decreased Arg 1 mRNA expression(P<0.05);and increased expressions of GZMB and IFN-γmRNA(P<0.05,P<0.01).It turned out that in the in vitro experiments,23-HBA at concentrations of 10.5,21,42μmoL/L had no significant effect on the survival rate of BM and MDSCs(P>0.05);23-HBA at high dose inhibited the differentiation of BM into MDSCs(P<0.01);and 23-HBA dose-dependently decreased the expressions of Arg 1 and iNOS mRNA(P<0.05,P<0.01),and downregulated the protein expressions of Arg1 and iNOS in MDSCs(P<0.05,P<0.01).CONCLUSION 23-HBA can regulate the proportion of MDSCs in tumor by intervening their differentiation,weaken their immunosuppressive function and thus restore the anti-tumor activity of CD8^(+)T cells in their role in anti-colon cancer action.