广谱抗生素对CT26荷瘤小鼠氟尿嘧啶化疗疗效的影响

Effects of broad-spectrum antibiotics on the efficacy of 5-fluorouracil treatment of CT26 tumor-bearing mice

目的研究结直肠癌荷瘤小鼠中广谱抗生素的使用及其诱导的抗生素耐药菌对氟尿嘧啶(5-fluorouracil,5-FU)化疗疗效的影响及其中的抗肿瘤免疫相关机制。方法结直肠癌细胞CT26皮下荷瘤小鼠随机分成4组:荷瘤对照组,氨苄青霉素、链霉素和多黏菌素处理的抗生素组,5-FU化疗组和抗生素^(+)5-FU组。监测记录小鼠肿瘤体积、体重。末次化疗后第7天,流式细胞术检测脾脏中免疫细胞亚群比例和与CT26共培养后增殖的CD8^(+)T细胞比例;16S rRNA测序分析肠道菌群组成,并分离培养各组小鼠肠系膜淋巴结中的细菌。用分离的细菌刺激骨髓来源的巨噬细胞,定量PCR法检测M1型和M2型巨噬细胞标志分子的表达,流式细胞术检测与细菌处理的巨噬细胞进行共培养的CD8^(+)T细胞的增殖。将分离自抗生素^(+)5-FU组的细菌和等体积PBS分别灌胃至进行5-FU化疗的CT26荷瘤小鼠,检测小鼠肿瘤大小、肠道菌群组成和与CT26共培养后增殖的CD8^(+)T细胞比例。结果抗生素^(+)5-FU组小鼠的肿瘤体积大于5-FU组,小鼠体重显著低于5-FU组。抗生素的使用对脾脏CD4^(+)T细胞、CD8^(+)T细胞、中性粒细胞的比例影响不显著,但促进单核细胞比例升高;抗生素^(+)5-FU组体外增殖的肿瘤特异性CD8^(+)T细胞比例下降,低于5-FU组。与对照组和5-FU组比较,抗生素的使用导致肠道菌群组成发生变化,菌群α多样性指数明显降低;从抗生素组、5-FU组和抗生素^(+)5-FU组小鼠的淋巴结中分别培养出大肠埃希菌、肺炎克雷伯菌和奇异变形杆菌。奇异变形杆菌处理的骨髓来源的巨噬细胞显著表达M2型巨噬细胞标志分子精氨酸酶,且与该组巨噬细胞共培养后增殖的CD8^(+)T细胞比例下降。奇异变形杆菌灌胃后小鼠肠道菌群中有变形杆菌属细菌富集,对肿瘤生长没有明显影响,但体外增殖的肿瘤特异性CD8^(+)T细胞比例下降。结论广谱抗生素抑制化疗疗效及肿瘤特异性CD8^(+)T细胞的增殖,抗生素耐药菌可能在其中发挥作用。

Objective To study the effects of broad-spectrum antibiotics and induced antibiotic-resistant bacteria on the efficacy of 5-fluorouracil(5-FU)chemotherapy for mice with colon cancer and to investigate the underlying mechanisms associated with anti-tumor immune responses.Methods BALB/c mice were subcutaneously injected with CT26 colon cancer cells and randomized into four groups:tumor-bearing control group,antibiotic group treated with ampicillin,streptomycin and colistin,5-FU group and anitibiotic^(+)5-FU group.Tumor volumes and body weights were measured and recorded.Seven days after the last 5-FU treatment,the percentages of splenic immune cell subpopulations and proliferated CD8^(+)T cells after co-culturing with CT26 were analyzed by flow cytometry.Gut microbiota composition was detected by 16S rRNA sequencing and the bacteria in mesenteric lymph nodes(mLN)were isolated and cultured.Bone marrow-derive macrophages were stimulated with identified bacteria and the expression of M1 and M2 polarization markers were assessed by quantitative PCR.The proliferation of CD8^(+)T cells co-cultured with bacteria-treated macrophages was analyzed by flow cytometry.In addition,tumor-bearing mice were treated with 5-FU and oral gavage of bacteria isolated from antibiotic^(+)5-FU group or PBS.Tumor volumes,gut microbiota composition and the percentages of proliferated CD8^(+)T cells co-cultured with CT26 were assessed.Results Tumor volumes were larger and body weights were lower in the antibiotic^(+)5-FU group than in the 5-FU group.The percentages of CD4^(+)T cells,CD8^(+)T cells and neutrophils did not varied significantly after using antibiotics,however,the percentage of monocytes was increased in the antibiotic group.The percentage of proliferated tumor-specific CD8^(+)T cells in the antibiotic^(+)5-FU group was decreased compared with that in the 5-FU group.Compared with the control group and 5-FU group,antibiotic usage was associated with the changes in gut microbiota composition with decreasedαdiversity indexes.Escherichia coli,Klebsiella pneumonia,and Proteus mirabilis were isolated from mLNs of the antibiotic group,5-FU group and antibiotic^(+)5-FU group,respectively.Bone marrow-derived macrophages stimulated with Proteus mirabilis expressed arginase at a high level,which was a M2 polarization marker of macrophage,and associated with the decreased percentage of proliferated CD8^(+)T cells after co-culturing.Bacteria of the genus Proteus were enriched in the gut microbiota of 5-FU-treated tumor-bearing mice with the oral gavage of Proteus mirabilis.Although no significant inhibitory effect on tumor growth was observed,the oral gavage of Proteus mirabilis was associated with the decreased percentage of proliferated tumor-specific CD8^(+)T cells in vitro.Conclusions Broad-spectrum antibiotics inhibited the efficacy of chemotherapy and the proliferation of tumor-specific CD8^(+)T cells,in which antibiotic-resistant bacteria might be involved.