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中华消化病与影像杂志(电子版) ›› 2024, Vol. 14 ›› Issue (01) : 8 -15. doi: 10.3877/cma.j.issn.2095-2015.2024.01.002

论著

RNA测序研究抗菌肽KT2治疗溃疡性结肠炎的作用机制
蒋心怡1, 顾丹丹1, 叶艳1, 缪佳蓉1,()   
  1. 1. 650032 昆明医科大学第一附属医院消化内科,云南省消化系统疾病临床研究中心
  • 收稿日期:2023-05-20 出版日期:2024-02-01
  • 通信作者: 缪佳蓉
  • 基金资助:
    国家自然科学基金(82260107); 云南省兴滇英才支持计划-名医项目(RLMY20220010); 云南省高层次卫生技术人才培养经费资助(H-2018040)

Study on therapeutic mechanism of antimicrobial peptides KT2 on ulcerative colitis based on RNA sequencing

Xinyi Jiang1, Dandan Gu1, Yan Ye1, Jiarong Miao1,()   

  1. 1. Department of Gastroenterology, The First Affiliated Hospital of Kunming Medical University, Yunnan Province Clinical Research Center for Digestive Diseases, Kunming 650032, China
  • Received:2023-05-20 Published:2024-02-01
  • Corresponding author: Jiarong Miao
引用本文:

蒋心怡, 顾丹丹, 叶艳, 缪佳蓉. RNA测序研究抗菌肽KT2治疗溃疡性结肠炎的作用机制[J]. 中华消化病与影像杂志(电子版), 2024, 14(01): 8-15.

Xinyi Jiang, Dandan Gu, Yan Ye, Jiarong Miao. Study on therapeutic mechanism of antimicrobial peptides KT2 on ulcerative colitis based on RNA sequencing[J]. Chinese Journal of Digestion and Medical Imageology(Electronic Edition), 2024, 14(01): 8-15.

目的

通过RNA测序(RNA-seq)研究抗菌肽KT2治疗溃疡性结肠炎(UC)小鼠的分子机制。

方法

用葡聚糖硫酸钠(DSS)构造小鼠UC模型,将60只小鼠随机分为对照组、DSS组、KT2组、mCRAMP组和美沙拉嗪组5组,每组12只。评估小鼠结肠炎症程度。从对照组、DSS组和KT2组中每组随机选4只小鼠的结肠组织进行测序,鉴定差异表达基因(DEGs)并进行基因富集分析,随后用定量逆转录聚合酶链反应(qRT-PCR)进行验证。

结果

KT2组小鼠炎症程度比mCRAMP组和美沙拉嗪组更低;KT2组与DSS组相比,共得到113个DEGs,其中下调DEGs71个,上调DEGs42个。基因富集分析显示下调DEGs主要与胶原代谢有关,上调DEGs主要与生物氧化有关,筛选出差异较大且研究甚少的DEGs进行qRT-PCR验证,发现Foxp3、FUT4、IFRD1、VEGF在DSS干预后表达下调,经抗菌肽KT2治疗后表达上调;Arg2、FXR在DSS干预后表达上调,经抗菌肽KT2治疗后表达下调。

结论

抗菌肽KT2治疗UC小鼠效果优于mCRAMP和美沙拉嗪;抗菌肽KT2可能通过上调Foxp3、FUT4、IFRD1、VEGF以及下调Arg2、FXR的表达治疗UC小鼠,提示上述基因可能参与抗菌肽KT2治疗UC的分子机制。

Objective

To investigate the molecular mechanism of antimicrobial peptide KT2 in the treatment of ulcerative colitis (UC) mice using RNA sequencing (RNA-seq).

Methods

UC mice models were constructed with dextran sulfate sodium (DSS) and sixty mice were randomly divided into control group, DSS group, KT2 group, mCRAMP group and mesalazine group, with 12 mice in each group. The inflammation severity of mouse colon tissues was evaluated. Colon tissues of four mice from each group of control group, DSS group and KT2 group were randomly selected for RNA-seq, identification of differential genes (DEGs) and gene enrichment analysis, and then verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Results

The inflammation severity of mouse colon tissues of KT2 group was lower than that of mCRAMP group and mesalazine group; Compared with DSS group, 113 DEGs was identified including 71 down-regulated genes and 42 up-regulated in the KT2 group. Gene set enrichment analysis suggested the down-regulated and up-regulated DEGs were mainly related to collagen metabolism and biological oxidation separately. DEGs with significant difference and few studies were screened for qRT-PCR verification. The expressions of Foxp3, FUT4, IFRD1 and VEGF were down-regulated after DSS intervention and up-regulated after treatment of antimicrobial peptide KT2. The expressions of Arg2 and FXR were up-regulated after DSS intervention, and down-regulated after antimicrobial peptide KT2 treatment.

Conclusion

The therapeutic efficacy of antimicrobial peptide KT2 in UC mice is better than mCRAMP and mesalazine; Antimicrobial peptide KT2 may treat UC mice by up-regulating the expressions of Foxp3, FUT4, IFRD1, VEGF and down-regulating the expressions of Arg2 and FXR, indicating that the above-mentioned DEGs may involve in the molecular mechanism of antimicrobial peptide KT2 in UC treatment.

表1 引物序列
表2 引物序列
图1 抗菌肽KT2对溃疡性结肠炎小鼠的治疗效果注:1A各组小鼠DAI评分;1B各组小鼠结肠形态及长度;1C各组小鼠结肠长度;1D各组小鼠病理评分;1E HE染色评估各组小鼠结肠组织病理变化;1F ELISA检测各组小鼠结肠TGF-β和粪便钙卫蛋白表达;1G qPT-PCR检测各组小鼠结肠TNF-α、IL-6、IL-1β相对表达水平。*P<0.05,**P<0.01,***P<0.001,****P<0.0001。
图2 差异表达基因(DEGs)的表达注:A DEGs表达的聚类热图;B DEGs表达的火山图(显示差异性最为显著的top20基因)。
图3 差异表达基因(DEGs)的基因本体论(GO)分析和京都基因与基因组百科全书(KEGG)分析注:A DEGs的GO富集散点图;B DEGs的KEGG信号通路富集散点图。
图4 差异表达基因(DEGs)的蛋白质相互作用分析网络图
图5 各组小鼠结肠组织中靶基因验证情况注:A Foxp3;B FUT4;C IFRD1;D VEGF;E Arg2;F FXR;G SLC51A;H Gprc5a;I COL3A1;J PGE2;K Clca4a的mRNA表达情况。*P<0.05;**P<0.01;***P<0.001;****P<0.0001。
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