摘要
目的
探究芬氏另枝菌(Alistipes finegoldii)对炎症性肠病(inflammatory bowel disease, IBD)的影响,并进一步阐明其潜在的相关致病机制。
方法
6周龄的雄性C57BL/6J小鼠在饮用链霉素3 d后,被随机分为3组:对照组(Control组)、PBS组和给菌组(AF组)。通过灌胃给予芬氏另枝菌(每只1×1
结果
AF组小鼠的体重下降分数、疾病活动度指数评分、结肠缩短程度及组织病理评分均高于PBS组。肠屏障的紧密连接蛋白Occludin、Claudin 5的mRNA表达相较于PBS组下降,而炎症因子IL-17A的mRNA表达则升高。AF组相较于PBS组的肠道菌群α多样性降低;β多样性分析显示两组间肠道菌群多样性存在显著性差异。线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)分析发现,给菌组与PBS组之间存在显著差异的细菌类群: 在纲水平上,芽孢杆菌纲 (Bacilli);在目水平上,丹毒丝菌目(Erysipelotrichales);在科水平上,丹毒丝菌科(Erysipelotrichaceae)、海生线状菌科(Marinifilaceae);在属水平上,气杆菌属(Odoribacter)、杜博斯氏菌属(Dubosiella);在种水平上,纽约杜博斯氏菌(Dubosiella newyorkensis),在给菌组中显著高于PBS组。
结论
芬氏另枝菌能够促进炎症因子的分泌,损害肠黏膜通透性,改变肠道菌群的结构和多样性,从而加剧肠炎的发展。
炎症性肠病(inflammatory bowel disease, IBD)是一种病因尚不明确的肠道慢性炎症性疾病,临床上最常见的2种亚型为克罗恩病(Crohn’s disease, CD)和溃疡性结肠炎(ulcerative colitis, UC)。尽管IBD的确切病因在很大程度上仍不清楚,但近年来的研究表明,个体遗传易感性、外部环境因素、肠道微生物菌群以及免疫反应均参与了IBD的发病机
肠道微生物菌群是一个与宿主共同进化的复杂群落,具有多种功能,包括协助代谢营养物质、调节免疫反应和抵御病原体入
肠道菌群失调与IBD之间的关系是动态的、复杂的,并且相互作用、互为因
芬氏另枝菌(Alistipes finegoldii)是另枝菌属的一种细菌,常定殖于人的消化道中。此外,Shkoporov
1 材料与方法
1.1 实验动物
选择6周龄、雄性野生型无特定病原体(specific-pathogen-free, SPF)级C57BL/6J小鼠(北京维通利华实验动物技术有限公司),饲养于军事医学研究院实验动物中心。动物实验通过伦理委员会审核,伦理编号:IACUC-DWZX-2024-012。
1.2 主要试剂
RNA提取试剂盒FastPure Cell/Tissue Total RNA Isolation Kit V2、逆转录试剂盒Taq Pro Universal SYBR qPCR Master Mix和HiScript III RT SuperMix for qPCR试剂盒均购自南京诺唯赞生物科技股份有限公司;链霉素和粪便隐血定性检测试剂盒均购自北京索莱宝科技有限公司;葡聚糖硫酸钠(dextran sulfate sodium salt, DSS)购自MP公司;哥伦比亚血平板购自Thermo Fisher Scientific公司。
1.3 实验分组
小鼠混养1周后,随机分为3组:Control组、PBS组和AF组,每组12只。先饲喂含有链霉素的饮用水3 d便于灌胃细菌定殖,之后AF组给予芬氏另枝菌灌胃(每只小鼠每天1×1
Score | Percentage of weight loss (%) | Faecal viscosity | Blood feces |
---|---|---|---|
0 | <1 | Dry, hard | Negative occult blood test |
1 | 1-5 | Wet, hard | |
2 | 5-10 | Soft, sticky | Positive occult blood test |
3 | 10-20 | Soft, loose | |
4 | >20 | Loose stool | Blood feces |
Score | Mucosal damage | Inflammatory cell infiltration |
---|---|---|
0 | Normal | No infiltration |
1 | Mild loss of crypts | Sporadic infiltration |
2 | Moderate loss of crypts | Submucosal infiltration increased |
3 | Severe loss of crypts | Submucosal nest-like infiltration |
4 | Full-layer erosion and ulceration | Transmural infiltration |
1.4 细菌培养
本研究所用芬氏另枝菌来自课题组前期从结肠癌患者粪便中分离,使用哥伦比亚血平板培养,放置在厌氧箱(80% N2+10% H2+10% CO2)中,37 ℃培养48 h。在血平板上加入适量PBS溶液,用涂布棒把菌落刮去,收集菌液,于4 ℃、5 000 r/min离心8 min后重悬,调整浓度为5×1
1.5 小鼠粪便收集
在实验起点和终点分别收集小鼠粪便于1.5 mL的无菌EP管中,之后迅速转移到-80 ℃冰箱中保存。实验中涉及的引物序列如
Primers name | Primer sequences (5′→3′) |
---|---|
Gapdh-F | ATCACTGCCACCCAGAAGACTG |
Gapdh-R Occludin-F |
ATGCCAGTGAGCTTCCCGTTCAG CTGCTGCTGATGAATATAATAG |
Occludin-R | CCTCTTGATGTGCGATAA |
Claudin 5-F | GCTCTCAGAGTCCGTTGACC |
Claudin 5-R | CTGCCCTTTCAGGTTAGCAG |
IL-17A-F | TTTAACTCCCTTGGCGCAAAA |
IL-17A-R | CTTTCCCTCCGCATTGACAC |
TNF-α-F | CCCTCACACTCAGATCATCTTCT |
TNF-α-R | GCTACGACGTGGGCTACAG |
1.6 统计学分析
连续变量的组间比较在满足正态性条件时,采用mena±SD表示,使用独立样本t检验;否则使用秩转换的非参数检验(Mann-Whitney U检验)。本研究的统计分析均采用双侧检验,P<0.05表示差异具有统计学意义。作图时,*P<0.05;**P<0.01;***P<0.001;****P<0.000 1。
2 结果与分析
2.1 芬氏另枝菌加重了DSS诱导的急性结肠炎
DSS诱发的结肠炎是模拟IBD的常用动物模型。使用DSS诱导的急性结肠炎小鼠模型,研究了Alistipes finegoldii对结肠炎的影响及其作用机制。用链霉素处理无特异性病原体的C57BL/6J小鼠,以促进A. finegoldii在肠道的定殖,然后用A. finegoldii灌胃(

图1 芬氏另枝菌加重了DSS诱导的急性结肠炎。A:给小鼠饲喂含有2 mg/mL链霉素的饮用水3 d,然后灌胃0.2 mL PBS或0.2 mL 1 ×1
Figure 1 Alistipes finegoldii exacerbates DSS-induced acute colitis. A: Mice were given 3 days of water containing 2 mg/mL streptomycin, followed by 2 weeks of gavage of 0.2 mL of PBS or 0.2 mL of 1×1
2.2 芬氏另枝菌损害了肠道黏膜屏障
肠黏膜的炎症和结构破坏是炎症性肠病的病理特征,对小鼠结肠进行HE染色后观察发现,PBS组和AF组肠组织黏膜均可见大范围溃疡,黏膜上皮及隐窝结构消失,并伴有大量淋巴细胞、粒细胞浸润。根据黏膜损伤和炎性细胞浸润情况的组织病理评分显示,AF组小鼠结肠炎症程度更高(P<0.05,图

图2 芬氏另枝菌损害肠道黏膜屏障、促进炎症因子的分泌。A:代表性的HE染色图像,比例尺:100 μm;B:HE染色的组织学评分;C、D:小鼠肠道组织中紧密连接蛋白(Occludin、Claudin 5)的表达;E、F:炎症因子TNF-α和IL-17A在小鼠肠道组织中的表达。
Figure 2 Alistipes finegoldii impairs the intestinal mucosal barrier and promotes the secretion of inflammatory factors. A: Representative image of HE staining, scale bar: 100 μm; B: Histological scoring of HE staining; C, D: Expression of tight junction proteins (Occludin, Claudin 5) in mouse intestinal tissue; E, F:Expression of inflammatory factors TNF-α and IL-17A in mouse intestinal tissues.
2.3 芬氏另枝菌改变了肠道菌群的结构
2.3.1 肠道菌群门和属水平丰度以及菌群多样性变化
肠道菌群在炎症性肠病的发病机制中发挥着重要作用。16S rRNA基因测序技术的进步使得对肠道菌群的研究更加便利。收集小鼠实验终点时的粪便进行16S rRNA基因测序,以探究A. finegoldii灌胃后小鼠肠道菌群的变化。在门水平上,相较于对照组,PBS组和AF组的拟杆菌门(Bacteroidota)、放线菌门(Actinobacteriota)丰度均有所下降,其中AF组下降的程度更大,而疣微菌门(Verrucomicrobia)、变形菌门(Proteobacteria)的丰度则有所上升,AF组上升的程度更大(

图3 芬氏另枝菌灌胃后改变了肠道菌群的组成。A:门水平相对丰度的前10个物种;B:属水平相对丰度最高的20个物种;C:Shannon指数;D:Chao1指数;E:PCoA分析。
Figure 3 Alistipes finegoldii altered the composition of the intestinal flora after gavage. A: Top 10 species in relative abundance at the phylum level; B: Top 20 species in relative abundance at the genus level; C: Shannon index; D: Chao1 index; E: PCoA analysis.
2.3.2 差异细菌分析
为进一步识别肠道菌群中的差异物种,进行了线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)分析。LEfSe分析能够基于LDA效应大小发现组间在不同菌群、不同种属水平上存在统计学差异的生物标志物,即确定组间具有显著差异的物种。结果显示(图

图4 差异细菌分析。A:对组间存在显著差异的物种进行LEfSe分析;B:系统发育树图。
Figure 4 Differences in bacterial analysis. A: LEfSe analysis of species with significant differences among groups; B: Phylogenetic tree.
3 讨论与结论
IBD的病因涉及多方面因素,包括宿主的遗传易感性、肠道微生物群、其他环境因素和宿主免疫系统。目前,越来越多的研究证明肠道微生物在IBD中发挥重要作
芬氏另枝菌是另枝菌属的一种,其在肠炎中的作用既往研究结果差异巨大。本研究通过DSS诱导的急性肠炎小鼠模型,研究芬氏另枝菌对肠炎的影响。结果表明,给予芬氏另枝菌灌胃后小鼠的体重下降程度和疾病活动度较PBS组明显升高,结肠长度较PBS组明显缩短(P=0.019),结肠组织病理评分也显示炎症程度更高,提示芬氏另枝菌加重了肠道炎症;检测肠道黏膜紧密连接蛋白的基因表达发现,芬氏另枝菌灌胃后,紧密连接蛋白Occludin和Claudin 5的表达较PBS组下降,表明肠道屏障功能受损;检测相关炎症因子的表达发现,芬氏另枝菌灌胃后IL-17A的表达升高,说明芬氏另枝菌促进了炎症因子的分泌。粪便16S rRNA基因测序结果显示,芬氏另枝菌灌胃后,菌群的组成发生改变,小鼠肠道菌群的丰富度和多样性较PBS组均出现下降,菌落结构存在明显差异。具体而言,芬氏另枝菌灌胃后乳杆菌属(Lactobacillus)、宿主关联乳杆菌属(Ligilactobacillus)、肠杆状菌属(Enterorhabdus)等益生菌的丰度下降,而罗姆布茨菌属(Romboutsia)、苏黎世杆菌属(Turicibacter)、拟杆菌属(Bacteroides)、杜博西氏菌属(Dubosiella)的丰度升高。比较芬氏另枝菌组和PBS组发现,主要的差异细菌包括芽孢杆菌纲(Bacilli)、丹毒丝菌目(Erysipelotrichales)、丹毒丝菌科(Erysipelotrichaceae)、臭气杆菌属(Odoribacter)、海生线状菌科(Marinifilaceae)、杜博斯氏菌属(Dubosiella)、纽约杜博斯氏菌(Dubosiella newyorkensis);PBS组的主要差异细菌为梭菌纲(Clostridia)、鼠杆状菌科(Muribaculaceae)、大肠杆菌-志贺氏菌(Escherichia-Shigella)、颤螺菌科(Oscillospiraceae)。这些差异细菌的作用并不完全相同。例如,Schaubeck
综上所述,芬氏另枝菌能够损害肠道屏障,促进肠道炎症因子的分泌,降低肠道菌群的多样性,从而加重小鼠的肠道炎症。然而,本研究尚未深入探讨芬氏另枝菌与宿主免疫系统之间的相互作用,相关问题仍需进一步研究。
作者贡献声明
郝自创:完成实验并编写论文;李东:参与协助实验操作;童江辉:参与协助实验操作;覃小铭:参与协助实验操作;张欢:参与协助实验操作、数据分析和论文讨论;王雅婧:参与协助实验操作;杨瑞馥:参与实验方案的设计并对论文撰写和修改提出相关意见;谭亚芳:参与实验方案的设计并对论文撰写和修改提出相关意见;毕玉晶:参与实验的研究构思和设计,并参与文章的修改;智发朝:参与实验的研究构思和设计,并参与文章的修改。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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