基于微生物组和宿主转录组整合分析香砂六君子汤对ETEC诱导断奶腹泻仔猪回肠损伤的调控机制

doi:10.11843/j.issn.0366-6964.2024.02.036

摘要/Abstract

摘要： 本研究旨在探讨香砂六君子汤对产肠毒素大肠杆菌(entexotoxigenic Escherichia coli, ETEC)诱导的断奶仔猪腹泻的干预作用及其机制。将24头21日龄断奶仔猪随机分为空白组(CON)、模型组(MOD)和香砂六君子汤组(XS),连续14 d给XS组灌服香砂六君子汤(1 mL·kg^-1,1 g·mL^-1),其余组灌服等量无菌水,第15天给MOD组、XS组仔猪按1 mL·kg^-1连续3 d灌服10¹¹ CFU·mL^-1 ETEC菌液,XS组继续灌服香砂六君子汤。分别记录各组仔猪腹泻评分,HE法检测回肠组织病理学变化,实时荧光定量PCR检测回肠组织IL-1β、IL-6、IL-8 mRNA水平,转录组学分析回肠组织RNA差异表达情况,微生物宏基因组学分析回肠菌群变化情况,Western blot检测回肠组织p-p38/p38、p-ERK/ERK、p-JNK/JNK蛋白水平。结果表明,灌服ETEC后,MOD组腹泻评分极显著高于CON组(P＜0.01),回肠组织结构破坏,V/C值极显著下降(P＜0.01),且IL-1β、IL-6和IL-8 mRNA表达量极显著升高(P＜0.01),肠道菌群门水平上变形菌门极显著上调(P＜0.01),厚壁菌门极显著下调(P＜0.01),属水平上乳酸菌属下调,志贺菌属上调,回肠组织p-p38/p38和p-JNK/JNK比值显著或极显著升高(P＜0.05或P＜0.01)。与MOD组相比,XS组腹泻评分极显著下降(P＜0.01),回肠组织结构完整,V/C值极显著升高(P＜0.01),回肠组织IL-1β、IL-6和IL-8 mRNA表达量显著或极显著降低(P＜0.05或P＜0.01),肠道菌群门水平上变形菌门极显著下调(P＜0.01),厚壁菌门极显著上调(P＜0.01),属水平上乳酸菌属上调,志贺菌属下调,回肠组织p-p38/p38和p-ERK/ERK比值极显著降低(P＜0.01)。肠道组织基因转录组学筛选出与炎症免疫相关的差异显著的10个基因,分别为TNFAIP8L2、TRIM67、CXCL2、EGF、NOX1、CCL28、FABP2、FABP6、IL1RAP和CEBPB。微生物组学筛选出各组可能的标志物种分别为CON组的Lactobacillaceae,MOD组的Shigella,XS组的Deinococcus和Eubacterium。综上所述,香砂六君子汤可有效缓解ETEC诱导的断奶仔猪腹泻,提高肠道菌群中有益菌的丰度,逆转ETEC诱发的菌群结构的改变,并可通过抑制MAPK信号通路的激活,从而缓解ETEC诱导的肠道炎性损伤。

关键词: 香砂六君子汤, 产肠毒素大肠杆菌, 断奶仔猪, 腹泻, 肠道菌群, 转录组学, MAPK信号通路

Abstract: The aim of this study was to evaluate the effect and mechanism of Xiangsha Liujunzi decoction on ETEC-induced diarrhea in weaned piglets. Twenty-four 21-day-old weaned piglets were randomly divided into three groups: the control group (CON), the model group (MOD), and the Xiangsha Liujunzi decoction group (XS). The XS group was given Xiangsha Liujunzi decoction (1 mL·kg^-1, 1 g·mL^-1) for 14 consecutive days, and the other groups were given the same amount of sterile water. On the 15th day, piglets in groups MOD and XS were given 10¹¹ CFU·mL^-1 ETEC bacterial solution at a dose of 1 mL·kg^-1 for 3 consecutive days, and piglets in group XS were given Xiangsha Liujunzi decoction. The diarrheal degree of the piglets in each group was recorded by fecal score. Pathological changes of the ileum were observed by HE staining. By means of qRT-PCR, the mRNA expression levels of IL-1β, IL-6, and IL-8 in ileum tissue were detected. Transcriptomics and metagenomics were used to analyze the differential expression of RNA in intestinal tissues and changes in the intestinal flora, respectively. The protein levels of p-p38/p38, p-ERK/ERK, and p-JNK/JNK in ileum tissue were detected by Western blot. The results showed that, after challenging ETEC, the diarrheal score was significantly higher (P<0.01) in the MOD group compared to the CON group. The structure of ileum tissue was destroyed in the MOD group. The V/C value was significantly decreased (P<0.01), and the mRNA expression of IL-1β, IL-6, and IL-8 were significantly increased (P<0.01) in the MOD group compared to the CON group. At the phylum level, Proteobacteria was significantly increased (P<0.01), and Firmicutes was significantly decreased (P<0.01) in the MOD group compared to the CON group. At the genus level, Lactobacillus was decreased, and Shigella was increased in the MOD group compared to the CON group. The ratios of p-p38/p38 and p-JNK/JNK in ileum tissues were significantly increased (P<0.05, P<0.01) in the MOD group compared to the CON group. In comparison with the MOD group, the diarrhea score in the XS group was significantly lower (P<0.01). The structure of the ileum tissue was intact in the XS group. The V/C value was significantly increased (P<0.01), and the mRNA expression of IL-1β, IL-6, and IL-8 in the ileum tissue was significantly decreased (P<0.05, P<0.01) in the XS group compared to the MOD group. At the phylum level, Proteobacteria was significantly decreased (P<0.01), and Firmicutes was significantly increased (P<0.01) in the XS group compared to the MOD group. At the genus level, Lactobacillus was increased, and Shigella was decreased in the XS group compared to the MOD group. The ratios of p-p38/p38 and p-ERK/ERK in ileum tissues were significantly decreased (P<0.01) in the XS group compared to the MOD group. Ten genes with significant differences related to inflammatory immunity were screened by the intestinal tissue gene transcriptome, which were TNFAIP8L2, TRIM67, CXCL2, EGF, NOX1, CCL28, FABP2, FABP6, IL1RAP, and CEBPB. The possible marker species for each group were Lactobacillaceae in the CON group, Shigella in the MOD group, and Deinococcus and Eubacterium in the XS group. In conclusion, Xiangsha Liujunzi decoction can effectively alleviate ETEC-induced diarrhea in weanling piglets. Treatment with Xiangsha Liujunzi decoction increases the abundance of beneficial bacteria in the intestinal flora and reverses the changes in the structure of the intestinal flora induced by ETEC. Moreover, Xiangsha Liujunzi decoction inhibits the activation of the MAPK signaling pathway, thereby alleviating ETEC-induced intestinal inflammatory injury.

Key words: Xiangsha Liujunzi decoction, enterotoxigenic Escherichia coli, weaned piglets, diarrhea, gut microbiota, transcriptomics, MAPK signaling pathway

中图分类号:

S853.74

肖乐, 刘峻源, 曾雯玉, 汪芹, 韩雯珏, 刘彦泠, 范誉, 徐雨婷, 杨贝妮, 肖雄, 王自力. 基于微生物组和宿主转录组整合分析香砂六君子汤对ETEC诱导断奶腹泻仔猪回肠损伤的调控机制[J]. 畜牧兽医学报, 2024, 55(2): 797-808.

XIAO Le, LIU Junyuan, ZENG Wenyu, WANG Qin, HAN Wenjue, LIU Yanling, FAN Yu, XU Yuting, YANG Beini, XIAO Xiong, WANG Zili. Microbiome and Transcriptome Analyses Revealed the Regulatory Mechanism of Xiangsha Liujunzi Decoction on Ileal Injury Induced by ETEC in Weaned Piglets with Diarrhea[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 797-808.

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