Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jul 20;42(7):976-987.
doi: 10.12122/j.issn.1673-4254.2022.07.03.

[E.faecium QH06 alleviates TNBS-induced colonic mucosal injury in rats]

[Article in Chinese]
K Kuerbannaimu  1 , J Zhao  2 , A Mukaidaisi  2 , H Wang  3 , J Zhu  2 , W Pan  2 , A Kasimujiang  4
Affiliations

[E.faecium QH06 alleviates TNBS-induced colonic mucosal injury in rats]

[Article in Chinese]
K Kuerbannaimu et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the effect of Enterococcus faecium QH06 on TNBS-induced ulcerative colitis (UC) in rats and explore the mechanisms in light of intestinal flora and intestinal immunity.

Methods: Thirty-six male Wistar rats were randomized equally into control group, UC model group, and E.faecium QH06 intervention group. The rats in the latter two groups were subjected to colonic enema with 5% TNBS/ethanol to induce UC, followed by treatment with intragastric administration of distilled water or E.faecium QH06 at the dose of 0.21 g/kg. After 14 days of treatment, the rats were examined for colon pathologies with HE staining. The mRNA and protein expression levels of IL-4, IL-10, IL-12, and IFN-γ in the colon tissues were detected using RT-qPCR and ELISA, and the expression of TLR2 protein was detected with immunohistochemistry and ELISA. Illumina Miseq platform was used for sequencing analysis of the intestinal flora of the rats with bioinformatics analysis. The correlations of the parameters of the intestinal flora with the expression levels of TLR2 and cytokines were analyzed.

Results: The rats with TNBS- induced UC showed obvious weight loss (P < 0.01) and severe colon tissue injury with high pathological scores (P < 0.01). The protein expression levels of IFN-γ, IL-12, and TLR2 (P < 0.01) and the mRNA expression levels of IFN-γ, IL-12 and IL-10 (P < 0.05) were significantly increased in the colon tissues of the rats with UC. Illumina Miseq sequence analysis showed that in UC rats, the Shannon index (P < 0.05) ACE (P < 0.01)and Chao (P < 0.05) index for the diversity of intestinal flora both decreased with a significantly increased abundance of Enterobacteriaceae (P < 0.01) and a lowered abundance of Burkholderiaceae (P < 0.05). Compared with the UC rats, the rats treated with E. faecium QH06 showed obvious body weight gain (P < 0.05), lessened colon injuries, lowered pathological score of the colon tissue (P < 0.05), decreased protein expressions of IFN- γ, IL- 12, and TLR2 and mRNA expressions of IFN- γ and IL-12 (P < 0.01 or 0.05), and increased protein expressions of IL- 4 (P < 0.05). The Shannon index ACE (P < 0.05) and Chao (P < 0.05) index of intestinal microflora were significantly increased, the abundance of Enterobacteriaceae was lowered and that of Burkholderiaceae and Rikenellaceae was increased in E.faecium QH06- treated rats (P < 0.01 or 0.05). Correlation analysis showed that IFN-γ was positively correlated with the abundance of Enterobacteriaceae, and IFN-γ was negatively correlated with the abundance of Prevotellaceae, Desulfovibrionaceae, norank_o_Mollicutes_RF39 and Clostridiales_vadinBB60_group; TLR2 was negatively correlated with Clostridiales_vadinBB60_group, norank_o_Mollicutes_RF39 and Prevotellaceae.

Conclusion: E.faecium QH06 can alleviate TNBS-induced colonic mucosal injury in rats, and its effect is mediated possibly by increasing the abundance of SCFA-producing bacteria such as Prevotellaceae and inhibiting abnormal immune responses mediated by TLR2.

目的: 探讨屎肠球菌QH06对溃疡性结肠炎（UC）大鼠肠道菌群与肠道免疫的影响及其机制。

方法: 36只SPF级雄性Wistar大鼠随机分为正常对照组、UC模型组和屎肠球菌组（12只/组）。除正常对照组外，其余2组以5% TNBS/乙醇灌肠法建立UC模型。造模成功后屎肠球菌组用屎肠球菌QH06灌胃干预，剂量为0.21 g/kg，正常对照组和UC组用等体积的蒸馏水灌胃干预。干预14 d后用HE染色法观察各组大鼠结肠病理改变并评分，用RT-qPCR和ELISA法检测细胞因子IL-4、IL-10、IL-12和IFN-γ的mRNA和蛋白表达水平，用免疫组化和ELISA法检测TLR2蛋白表达量。利用IlluminaMiseq平台对各组大鼠肠道菌群测序同时进行生物信息学分析。最后肠道菌群和TLR2蛋白表达量、细胞因子等指标进行相关性分析。

结果: 与正常对照组相比，UC组大鼠体质量下降（P < 0.01），结肠组织损伤严重，病理学评分显著升高（P < 0.01）；ELISA和RT-qPCR结果显示，IFN-γ（P < 0.01），IL-12（P < 0.01），TLR2（P < 0.01）的蛋白表达水平和IFN-γ（P < 0.05），IL-12（P < 0.05），IL-10（P < 0.05）的mRNA表达水平上调。Illumina Miseq测序和分析显示，UC组大鼠肠道菌群多样性Shannon指数（P < 0.05）、Ace指数（P < 0.01）和Chao指数（P < 0.05）降低。Enterobacteriaceae（P < 0.01）增多，Burkholderiaceae（P < 0.05）减少。与UC组相比，屎肠球菌组大鼠体质量增加（P < 0.05），损伤减轻，病理学评分下降（P < 0.05），结肠组织中IFN-γ（P < 0.01），IL-12（P < 0.05）、TLR2蛋白表达量（P < 0.01）下调，IL-4蛋白表达量上调（P < 0.05），IFN-γ（P < 0.05）、IL-12（P < 0.05）的mRNA表达量下调，肠道菌群多样性Shannon指数（P < 0.05）、Ace指数（P < 0.05）和Chao指数（P < 0.05）升高。Enterobacteriaceae（P < 0.05）减少，Burkholderiaceae（P < 0.05）和Rikenellaceae（P < 0.01）增多；肠道菌群、细胞因子含量和TLR2蛋白表达量相关性分析结果显示，IFN-γ与Enterobacteriaceae呈正相关、而与Prevotellaceae，Desulfovibrionaceae、norank_o__Mollicutes _RF39，Clostridiales _vadinBB60 _group呈负相关；TLR2表达水平与Clostridiales_ vadinBB60_ group，norank_o__Mollicutes_RF39和Prevotellaceae呈负相关。

结论: 屎肠球菌QH06能减轻TNBS诱导的UC大鼠的结肠黏膜损伤，其机制可能与普氏菌科等短链脂肪酸产生菌丰度的升高，TLR2介导的异常免疫反应的抑制有关。

Keywords: Enterococcus faecium; Toll-like receptor 2; cytokines; intestinal flora; ulcerative colitis.

PubMed Disclaimer

Figures

图 1
图 1
大鼠体质量变化 Changes of body weight of the rats in the 3 groups. **P < 0.01 vs control group. #P < 0.05 vs UC group.
图 2
图 2
大鼠结肠组织病理变化 Pathological changes of the colon in the 3 groups (Original magnification: ×200). A: Control group. B: UC group. C: E.faecium group. D: Comparison of pathological scores of the colon among the 3 groups (n=8). **P < 0.01 vs control group. #P < 0.05 vs UC group.
图 3
图 3
各组大鼠结肠细胞因子蛋白表达水平比较 Expression levels of cytokines in the colon in the 3 groups (n=6). A: IFN-γ. B: IL-12. C: IL-10. D: IL-4. **P < 0.01 vs control group. #P < 0.05 and ##P < 0.01 vs UC group.
图 4
图 4
各组大鼠结肠组织TLR2表达 Expression level of TLR2 in the colon in the 3 groups (Scale bar=100 μm). A: control group. B: UC group. C: E. faecium group. D: Changes of TLR2 expression level in the 3 groups (n=6). **P < 0.01 vs control group. ##P < 0.01 vs UC group.
图 5
图 5
各组大鼠肠道菌群物种注释与评估 Annotation and evaluation of gut microbiota in the 3 groups (n=6). A: Relative abundance in OTU level. B: Pan analysis. C: Core analysis. D: Rarefaction curves in Sobs index. E: Shannon curves.
图 6
图 6
各组大鼠肠道菌群组成分析 Composition analysis of gut microbiota of rats in 3 groups (n=6). A: Venn diagram. B: Sunburst plot at phylum level. C: Bar plot at the family level. D: Pieplot at the genus level.
图 7
图 7
与UC发生相关菌属的差异分析 Analysis on the difference of bacteria correlated with UC occurrence (Genus level) (n=6).
图 8
图 8
物种差异分析(科水平) Species difference analysis (Family level). A: Analysis of the difference of intestinal microflora of rats in each group. Left: significant differences in the results of the comparison of bacteria; Right: the abundance of 15% of the comparison results; B: LEfSe analysis. The different color nodes in the left image represent the microbial groups that are significantly enriched in the corresponding groups and significantly influence the differences among groups. In contrast, the yellowish nodes show no significant difference among the groups or significantly affect the difference between groups. The image on the right shows LEfSe Bar. n=6.
图 9
图 9
细胞因子、TLR2与肠道菌群相关性 Correlation Heatmap of cytokines, TLR2 and gut microbiota at the family level. The x-axis was cytokine and TLR2, and the y-axis was bacterium. The correlation R and P values were obtained by calculation. The R-value was shown in different colors, P-value: * 0.01 < P≤0.05, **0.001 < P≤0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Segal JP, LeBlanc JF, Hart AL. Ulcerative colitis: an update. Clin Med (Lond) 2021;21(2):135–9. - PMC - PubMed
    1. Kobayashi T, Siegmund B, le Berre C, et al. Ulcerative colitis. Nat Rev Dis Primers. 2020;6(1):74. doi: 10.1038/s41572-020-0205-x. - DOI - PubMed
    1. Aximujiang K, Kaheman K, Wushouer X, et al. Lactobacillus acidophilus and HKL suspension alleviates ulcerative colitis in rats by regulating gut microbiota, suppressing TLR9, and promoting metabolism. Front Pharmacol. 2022;13:859628. doi: 10.3389/fphar.2022.859628. - DOI - PMC - PubMed
    1. Gao W, Howden BP, Stinear TP. Evolution of virulence in Enterococcus faecium, a hospital-adapted opportunistic pathogen. Curr Opin Microbiol. 2018;41:76–82. doi: 10.1016/j.mib.2017.11.030. - DOI - PubMed
    1. Choeisoongnern T, Sirilun S, Waditee-Sirisattha R, et al. Potential probiotic Enterococcus faecium OV3- 6 and its bioactive peptide as alternative bio-preservation. Foods. 2021;10(10):2264. doi: 10.3390/foods10102264. - DOI - PMC - PubMed