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