中华炎性肠病杂志(中英文)2024，Vol.8Issue(6) ：440-449.DOI:10.3760/cma.j.cn101480-20240219-00023

6-唾液酸乳糖减轻小鼠免疫检查点抑制剂相关肠炎的机制研究

Mechanism study of 6-sialyllactose alleviates immune checkpoint inhibitor-induced colitis in mouse

李可 董佳敏 杨心怡 莫锦灵 沈无名 蒋敬庭 张雯婷
中华炎性肠病杂志(中英文)2024，Vol.8Issue(6) ：440-449.DOI:10.3760/cma.j.cn101480-20240219-00023
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6-唾液酸乳糖减轻小鼠免疫检查点抑制剂相关肠炎的机制研究

Mechanism study of 6-sialyllactose alleviates immune checkpoint inhibitor-induced colitis in mouse

李可 1董佳敏 2杨心怡 2莫锦灵 2沈无名 1蒋敬庭 3张雯婷4
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作者信息

  • 1. 武进中医医院病理科,常州 213161
  • 2. 南通大学附属常州儿童医院儿科中心实验室,常州 213003
  • 3. 苏州大学附属第三医院肿瘤生物诊疗中心,常州 213003
  • 4. 南通大学附属常州儿童医院儿科中心实验室,常州 213003;苏州大学附属第三医院肿瘤生物诊疗中心,常州 213003;常州市气管炎研究所药学研究室,常州 213003
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摘要

目的 通过16SrDNA菌群测序探讨人乳6-唾液酸乳糖(6-SL)干预免疫检查点抑制剂相关肠炎(ICIC)的作用机制.方法 选用BALB/c小鼠,随机分为正常对照(NC)组(n=7)、葡聚糖硫酸钠(DSS)组(n=6)、ICIC组(n=6)、ICIC+6-SL组(n=6).DSS组以 3.5％DSS饮用水连续喂养7 d诱导结肠炎症;ICIC组在3.5％DSS饮用水喂养基础上于第0、4天分别腹腔注射细胞毒性T淋巴细胞相关抗原4(CTLA-4,剂量150 μig)构建小鼠ICIC模型;ICIC+6-SL组在ICIC造模基础上同时灌胃给予6-SL[150mg/(kg·d)].统计小鼠体质量和疾病活动性评分(DAI)变化.第7天处死所有小鼠,观察结肠大体和组织学形态变化,统计组织病理学评分;取新鲜结肠粪便进行16S rDNA测序,统计分析各组小鼠菌群多样性和物种差异.结果 ICIC组造模成功率100％,所有小鼠存活.在研究终点(第7天),与NC组和DSS组比较,ICIC组小鼠体质量更低(P＜0.05),DAI更高(P＜0.05),结肠黏膜组织形态完整性受损,并呈现典型的溃疡性病变;ICIC+6-SL组较ICIC组体质量下降显著缓解,DAI评分和病理评分也更低,差异均具有统计学意义(均P＜0.05).小鼠肠道粪便16S rDNA测序表明,ICIC组小鼠结肠菌落alpha多样性低于NC和DSS组(均P＜0.05),ICIC+6-SL组alpha多样性高于ICIC组(P＜0.05).在beta多样性分析中,各组结肠菌群PCoA分析的ANOSIM统计值R=0.376,P=0.001,而NMDS分析的Stress值=0.125,P=0.001,表明各组间小鼠结肠菌落构成存在差异,且NC组与ICIC组菌落差异最大,ICIC+6-SL组相较于ICIC组菌落组成接近于NC组.Lefse分析和基于Kruskal-Wallis检验的差异菌群分析表明,在门水平,与NC组相比,ICIC组拟杆菌门显著降低,Campilobacterota菌门丰度增加,给予6-SL可回调这2种菌门的丰度;在属水平,与其他组相比,ICIC组 unclassified_f_Lachnospiraceae、norank_f_Muribaculaceae 丰度最低,螺杆菌属、阿克曼菌属、大肠埃希菌属-志贺菌属富集;与 ICIC 组比较,ICIC+6-SL 组 unclassified_f_Lachnospiraceae、norank_f_Murinaculaceae丰度升高,同时螺杆菌属、大肠埃希菌属-志贺菌属丰度则受到显著抑制.结论 人乳寡糖单体6-SL可减轻ICIC小鼠肠道炎性损伤,降低疾病活动性.这一作用可能与6-SL调节肠道菌群结构、丰富菌群多样性、增加拟杆菌门丰度、抑制螺杆菌属和大肠埃希菌属-志贺菌属等致病菌的生长优势相关.

Abstract

Objective To investigate the mechanism of 6-sialyllactose(6-SL)in interfering the immune checkpoint inhibitor-induced colitis(ICIC)through the bacterial 16S rDNA sequencing.Methods BALB/c mice were randomly divided into the normal control(NC)group(n=7),dextran sulfate sodium(DSS)group(n=6),ICIC group(n=6),and ICIC+6-SL group(n=6).The DSS group was continuously fed with 3.5％DSS drinking water for 7 days to induce colonic inflammation;the ICIC group was administered cytotoxic T-lymphocyte-associated antigen 4(CTLA-4,150 µg)intraperitoneally on days 0 and 4 in addition to 3.5％DSS drinking water to establish the ICIC mouse model;the ICIC+6-SL group was given 6-SL[150 mg/(kg-d)]by gavage simultaneously with the establishment of the ICIC model.Changes in mouse body weight and disease activity index(DAI)were statistically analyzed,and all mice were sacrificed on day 7 to observe gross and histopathological morphological changes in the colon and to tally histopathological scores;the fresh colonic feces were collected for 16S rDNA sequencing to statistically analyze the diversity and species differences in the microbiota of mice of each group.Results The success rate of the ICIC model was 100％,with all mice surviving.At the endpoint of the study(day 7),compared with the NC and DSS groups,the ICIC group had lower mouse body weight(P＜0.05),higher DAI(P＜0.05),damaged integrity of colonic mucosal tissue,and typical ulcerative lesions;the ICIC+6-SL group showed significant alleviation of body weight loss,significantly lower DAI scores,and lower pathological scores compared to the ICIC group,with all differences being statistically significant(all P＜0.05).16S rDNA sequencing of mouse intestinal feces indicated that the alpha diversity of colonic microbiota in the ICIC group was lower than that in the NC and DSS groups(both P＜0.05),while the ICIC+6-SL group had higher alpha diversity than the ICIC group(P＜0.05).In beta diversity analysis,the ANOSIM statistical value R=0.376,P=0.001 for the PCoA analysis of colonic microbiota and a Stress value of 0.125,P=0.001 for the NMDS analysis indicated differences in the composition of colonic microbiota among the groups,with the greatest difference between the NC and ICIC groups,and the ICIC+6-SL group's microbiota composition was closer to that of the NC group compared to the ICIC group.Lefse analysis and Kruskal-Wallis test-based differential microbiota analysis showed that at the phylum level,compared to the NC group,the abundance of Bacteroidetes was significantly reduced in the ICIC group,while Campilobacterota was increased,and 6-SL administration could increase the abundance of Bacteroidetes and Campilobacterota in the ICIC group.At the genus level,compared to other groups,the abundance of unclassified_f_Lachnospiraceae and norank_f_Muribaculaceae was the lowest in the ICIC group,while Helicobacter,Akkermansia,and Escherichia-Shigella were enriched.Compared to the ICIC group,the abundance of unclassified_f_Lachnospiraceaeandnorank_f_Muribaculaceae was increased in the ICIC+6-SL group,while the abundance of Helicobacter and Escherichia-Shigella was significantly suppressed.Conclusions 6-SL,an oligosaccharide derived from human milk,alleviates intestinal inflammatory injury in ICIC mice,reducing disease activity.This beneficial effect may be related to its regulation of gut microbiota profiling,an increased diversity of microbiota,a restoration of Bacteroidetes,and an inhibition of the growth advantage of pathogenic bacteria such as Helicobacter and Escherichia-Shigella.

关键词

免疫检查点抑制剂相关肠炎/6-唾液酸乳糖/16SrDNA测序/人乳寡糖/肠道炎症

Key words

Immune checkpoint inhibitor-induced colitis/6-sialyllactose/16S rDNA sequencing/Human milk oligosaccharide/Intestinal inflammation

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出版年

2024
中华炎性肠病杂志(中英文)

中华炎性肠病杂志(中英文)

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