Bacillus amyloliquefaciens regulates oxidative damage through ssc-miR-10390 by targeting WT1 in the intestine of piglets

Abstract

Early weaning or enteric pathogen exposure frequently induces intestinal epithelial oxidative stress in piglets, which disrupts redox homeostasis, impairs tight junction integrity, and ultimately compromises intestinal barrier function. Bacillus amyloliquefaciens SC06 (SC06) was reported to regulate oxidative stress and intestinal barrier function in piglets, but the role of miRNA remains unclear in these processes. In this study, we aimed to investigate the effects of SC06 on miRNA profiles and its potential mechanisms of action. A total of 32 weaned piglets or IPEC-J2 cells were divided into four groups using a 2 × 2 factorial design (with or without SC06 treatment, and with or without diquat (DQ) treatment). Results showed that DQ increased the MDA content, reduced the activity of antioxidant enzymes and impaired the barrier function in intestine and IPEC-J2 cells. SC06 enhanced the antioxidant capacity and barrier function and also regulated the miRNA profiles, such as ssc-miR-10390 in piglets and IPEC-J2 cells. Further, the function of ssc-miR-10390 in ameliorating jejunal oxidative damage was confirmed. By using ssc-miR-10390 mimic, ssc-miR-10390 inhibitor and siRNA, we found that ssc-miR-10390 can enhance the antioxidant function and tight junction, and alleviate cellular inflammation in IPEC-J2 cells by targeting WT1. In conclusion, ssc-miR-10390-WT1 pathway plays an important role in SC06-mediated alleviation of oxidative damage in jejunal mucosa of piglets.

Supplementary Information

The online version contains supplementary material available at 10.1186/s42523-025-00505-w.

Introduction

During the production of piglets, stressors such as weaning, feed contamination and high-density feeding can lead to intestinal oxidative stress. Since the intestine plays important roles in digestion, absorption of nutrients, secretion of digestive enzymes and immunity, oxidative damage will decrease the feed utilization and increases the rate of dysbacteriosis and diarrhea rate and ultimately resulting in growth retardation [1]. The physical, chemical, immunological and microbiological barriers components contribute to the overall function of the intestinal barrier. As for the physical barrier, oxidative stress was reported to cause tight junction protein depolymerization, resulting in the entrance of macromolecular substances (such as bacteria and toxins) from the lumen into the submucosal tissue and ultimately into the systemic circulation [2]. Moreover, oxidative stress reduces the expression of MUC1 and MUC2 genes, leading to impaired mucosal barrier function [3]. Besides, the secretions of intestinal immune factors, such as the production of B lymphocyte immune globulin and mucosal surface secretory immunoglobulin A, were decreased by excessive level of ROS [4]. Additionally, oxidative stress is also closely associated with the microbiological barrier of piglets [5]. In oxidative stress, the abundance of facultative pathogenic bacteria within the genera Proteobacteria, Fusobacterium and Firmicutes was significantly elevated, while Bacteroidetes were less abundant, leading to a significant reduction in microbial diversity [6]. Thus, the dynamic interaction between the gut microbiota, immune system, and epithelial barrier function plays an important role in health. Under homeostasis, the expression of tight junction proteins, the formation of the mucus layer, and the differentiation of regulatory T cells can work together to maintain the immune tolerance. However, in a state of ecological imbalance, potential pathogens and their products dominate, triggering excessive pro-inflammatory signals that lead to barrier dysfunction and chronic inflammation [2–6].

MicroRNAs (miRNAs) are endogenous small non-coding RNA fragments of about 20 to 24 nt in length, which recognize target genes by base complementary pairing, degradation of target mRNA or inhibiting the translation of target mRNA, thereby affecting protein expression. Various studies have shown that miRNA can regulate the cellular redox status by binding oxidative stress- or antioxidation-related genes [7]. Xie et al. [8] suggested that ssc-miR-183 alleviated lipopolysaccharides (LPS)-induced oxidative stress in hippocampal neurons of weaned piglets by targeting the fibronectin 1 gene expression. Zhang et al. [9] showed that ssc-miR-192 alleviated oxidative damage in porcine granulosa cells. ssc-miR-132 can bind to genes related to oxidative stress such as Forkhead box O3 (FOXO3) [10].

Recent studies have established a link between probiotics and the regulation of host microRNAs (miRNAs), which in turn modulate intestinal barrier function and immunity. For instance, co-culture of Escherichia coli with T84 epithelial cells altered the expression of several miRNAs (e.g., hsa-miR-203), which promoted the expression of tight junction proteins [11]. Similarly, Bifidobacterium and Saccharomyces boulardii alleviated intestinal injury in rodent models by regulating miRNAs involved in barrier integrity and immune cell function [12, 13]. In piglets, supplementation with Saccharomyces boulardii was shown to affect the expression of ssc-miR-423-5p, influencing immune cell proliferation [14]. Collectively, these findings highlight the role of miRNAs as key mediators of probiotic effects. However, the specific miRNA-mediated mechanisms by which Bacillus amyloliquefaciens improves gut health in pigs remain largely unexplored. Previous studies have indicated that Bacillus amyloliquefaciens SC06 (SC06) can improve the intestinal integrity of piglets by improving intestinal morphology, tight junctions functionality, and the gut microbiota diversity [15]. SC06 also enhanced growth performance of piglets by elevating the activities of digestive and absorption enzymes, and increasing the intestinal antioxidant capacity, autophagy and immune function [16, 17]. However, whether SC06 can alleviate oxidative stress in piglets through miRNA remains unclear.

Diquat (DQ) acts by interfering with cyclic reduction-oxidation reactions. The formation of free radicals and aerobic autoxidation by diquat leads to superoxide production and oxidative damage [18]. In recent years, 10 mg/kg DQ had been used to construct an intestinal oxidative stress model in weaned piglets [19–21]. Therefore, in this study, by using DQ-induced oxidatively stressed piglets and porcine intestinal epithelial cells (IPEC-J2 cells), the effects of SC06 on the intestinal oxidative damage and miRNA expression was studied, and the role of SC06-mediated miRNA signaling pathway was also determined.

Results

SC06 decreased the jejunal oxidative stress in DQ-treated piglets

The DQ injection increased the contents of MDA and ROS (P < 0.01), and lowered the activities of CAT, SOD and GSH-Px (P < 0.01). The SC06 supplementation lowered the contents of MDA and ROS (P < 0.01), and elevated the activities of CAT (P = 0.02), SOD (P < 0.01) and GSH-Px (P < 0.01). Futhermore, in the DQ-treated piglets, the alterations in MDA (P < 0.01), ROS (P = 0.04), SOD (P < 0.01) and GSH-Px (P < 0.01) was reversed by the interaction between SC06 and DQ (SC06 × DQ) (Fig. 1).

Fig. 1.

Effects of SC06 on the jejunal oxidative stress of piglets. ^{a, b,c}Mean value within a row with no common superscript differ significantly (P < 0.05). Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

SC06 improved the jejunal morphology in DQ-treated piglets

HE staining results demonstrated that the DQ injection decreased the villus height and ratio of villus height to crypt depth (V/C) (P < 0.01). The SC06 elevated villus height (P < 0.01) and the V/C (P = 0.04). Moreover, SC06 × DQ increased the villus height (P < 0.01) in DQ-injected piglets (Fig. 2a). TEM analysis showed clear and complete tight junctions in the Con, SC06 and SC06 + DQ groups, but the tight junction was unclear and swollen in the DQ group (Fig. 2b).

Fig. 2.

Effect of SC06 on jejunal morphology in oxidatively stressed piglets. (a) HE staining, images were photographed under 50× magnification (b) TEM, images were photographed under 15k× magnification. ^{a, b}Mean value within a row with no common superscript differ significantly (P < 0.05). Red arrows indicate clear and intact tight junctions; Blue arrows indicate unclear, swollen tight junctions, Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

SC06 improved the jejunal physical, chemical and immunological barrier function in DQ-treated piglets

As for the physical barrier function, DQ decreased the gene expressions of ZO-1, Claudin and Occludin (P < 0.01). SC06 increased the gene expressions of ZO-1, Claudin and Occludin (P < 0.01). SC06 × DQ increased the gene expressions of ZO-1 (P < 0.01), Claudin (P = 0.01) and Occludin (P < 0.01) in DQ-injected piglets (Fig. 3a). As for the chemical barrier function, DQ decreased the gene expressions of MUC1 and MUC2 (P < 0.01). SC06 increased the gene expressions of MUC1 and MUC2 (P < 0.01). SC06 × DQ increased the gene expression of MUC2 in DQ-injected piglets (P < 0.01) (Fig. 3b). As for the immunological barrier function, DQ decreased the contents of IL-10 and sIgA and gene expression of IL-10 (P < 0.01) and increased the contents and gene expressions of IL-1β, IL-6, TNF-α and IFN-γ (P < 0.01). The SC06 treatment induced the opposite results (P < 0.01). SC06 × DQ decreased the contents of IL-6 (P < 0.01), IFN-γ (P < 0.01), IL-1β (P = 0.04) and lowered the gene expressions of IL-6 (P < 0.01) and IFN-γ (P < 0.01) in the DQ-injected piglets (Fig. 3c-d).

Fig. 3.

Effect of SC06 on intestinal mucosal barrier in weaned piglets. (a) tight junction gene expressions (b) mucin gene expressions (c) immune factors gene expressions (d) immune factors contents. ^{a, b,c}Mean value within a row with no common superscript differ significantly (P < 0.05). Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

SC06 improved the jejunal microbial barrier function in DQ-treated piglets

There were no significant differences in α-diversity and β-diversity among groups (P > 0.05) (Fig. 4). Nevertheless, at the phylum level, compared with the Con group, the abundance of Chlamydiae was increased in DQ group (P = 0.04). As for the top 20 abundant family, compared with the Con group, the abundance of Peptostreptococcaceae (P = 0.02) and Ruminococcaceae (P = 0.04) were decreased in the SC06 group. Compared with the DQ group, the abundance of Corynebacteriaceae (P = 0.03) and Ruminococcaceae (P = 0.04) were decreased in the SC06 + DQ group. As for the top 20 abundant genus, compared with the Con group, the abundance of Clostridium (P < 0.01) was decreased in the SC06 group. Compared with the DQ group, the abundance of SMB53 (P = 0.03) was decreased in the SC06 + DQ group (Tables 1, 2 and 3; Fig. 5).

Fig. 4.

Effect of SC06 on the diversity of jejunal mucosal microbiota in oxidatively stressed piglets. (a) α-diversity (b) β-diversity. Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

Table 1.

Significantly altered bacterial abundance between the SC06 and Con groups

bacteria	Con (%)	SC06 (%)	up/down	log2FC	P-value
f__Peptostreptococcaceae	49.72	4.47	down	-3.48	0.02
f__Lachnospiraceae	38.45	5.72	down	-2.75	0.02
f__unidentified_Clostridiales	53.20	6.50	down	-3.03	0.03
f__Rickettsiaceae	66.35	0.00	down	Inf	0.03
f__Vibrionaceae	0.00	64.27	up	Inf	0.03
f__Ruminococcaceae	64.60	4.83	down	-3.74	0.04
f__S24-7	78.64	0.72	down	-6.77	0.04
f__Corynebacteriaceae	19.16	4.44	down	-2.11	0.04
f__Halomonadaceae	0.00	36.76	up	Inf	0.04
f__[Weeksellaceae]	71.38	4.50	down	-3.99	0.04
g__Butyricicoccus	83.24	1.10	down	-7.26	< 0.01
g__Clostridium	39.45	5.63	down	-2.85	< 0.01
g__unidentified_Lachnospiraceae	40.17	4.33	down	-3.23	< 0.01
g__unidentified_Peptostreptococcaceae	56.10	3.76	down	-3.90	0.02
g__unidentified_Clostridiales	53.20	6.50	down	-2.95	0.03
g__Pediococcus	2.08	15.54	up	2.78	0.03
g__Oscillospira	84.50	3.91	down	-4.47	0.03
g__Wolbachia	66.35	0.00	down	Inf	0.03
g__Vibrio	0.00	38.56	up	Inf	0.03
g__Parabacteroides	70.32	2.72	down	-5.00	0.03
g__Corynebacterium	19.16	4.44	down	-2.18	0.04
g__unidentified_S24-7	78.64	0.72	down	-6.86	0.04
g__Halomonas	0.00	36.76	up	Inf	0.04
g__Dorea	63.15	6.07	down	-3.35	0.04

FC: fold change, Con group, basal diet; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06

Table 2.

Significantly altered bacterial abundance between the DQ and Con groups

Bacteria	Con (%)	DQ (%)	up/down	log2FC	P-value
p__Chlamydiae	0.00	56.07	up	Inf	0.04
g__Butyricicoccus	83.24	12.24	down	-2.77	< 0.01
g__Enhydrobacter	42.93	21.46	down	-1.00	0.02
g__unclassified_Enterococcaceae	0.74	40.65	up	5.78	0.03

FC: fold change, Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight

Table 3.

Significantly altered bacterial abundance between the SC06 + DQ and DQ groups

bacteria	DQ (%)	SC06 + DQ (%)	up/down	log2FC	P-value
f__Rickettsiaceae	33.65	0.00	down	Inf	0.03
f__Corynebacteriaceae	70.86	5.54	down	-3.68	0.03
f__Dermatophilaceae	2.65	44.25	up	4.06	0.03
f__Porphyromonadaceae	23.23	5.13	down	-2.18	0.03
f__S24-7	19.00	1.79	down	-3.40	0.03
f__unidentified_Clostridiales	33.73	6.58	down	-2.36	0.04
f__Ruminococcaceae	24.18	6.39	down	-1.92	0.04
f__unclassified_Lactobacillales	21.71	65.20	up	1.59	0.04
g__Aeromonas	0.00	65.60	up	Inf	< 0.01
g__unidentified_Peptostreptococcaceae	31.46	8.68	down	-1.99	0.02
g__Turicibacter	52.82	0.03	down	-4.44	0.02
g__SMB53	44.98	9.29	down	-2.35	0.03
g__unclassified_Ruminococcaceae	40.88	8.61	down	-2.20	0.04

FC: fold change, DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

Fig. 5.

Effect of SC06 on the abundance of jejunal mucosal microbiota at the phylum, family, and genus levels in oxidatively stressed piglets (top 20). Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

Functional analysis of the different bacteria

PICRUSt analysis was conducted to investigate the potential function of the gut microbiota and predict their classification based on the KEGG. Compared to the Con group, the tetracycline biosynthesis, lysosome and retinol metabolism were less enriched (P < 0.05), while arachidonic acid metabolism and other types of O-glycan biosynthesis were more enriched in the DQ group (P < 0.05). In comparison to the Con group, the pathogenic Escherichia coli infection, indole alkaloid biosynthesis, steroid biosynthesis were less enriched (P < 0.05), while renin-angiotensin system and spliceosome were more enriched in the SC06 group (P < 0.001). Additionally, compared to the DQ group, the ECM-receptor interaction was less enriched (P < 0.05), while the retinol metabolism (P < 0.05) was more enriched in the SC06 + DQ group (Fig. 6).

Fig. 6.

Effect of SC06 on KEGG enrichment of differential bacteria in jejunum mucosal oxidatively stressed piglets. Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

SC06 influenced the expression of jejunal MiRNA in DQ-treated piglets

The Venn diagram showed 1 common miRNA in all 4 groups, 12 common miRNAs in the Con and SC06 groups, 21 common miRNAs in the Con and DQ groups, and 15 common miRNAs in the DQ and SC06 + DQ groups (Fig. 7a). Among the differentially expressed miRNA (DEmiRNA), the DQ treatment up-regulated the expressions of ssc-miR-10390 (P < 0.01) and ssc-miR-138 (P < 0.01), and decreased the expression of ssc-miR-4331-5p (P = 0.02). The SC06 supplementation decreased the expression of ssc-miR-10,384 (P < 0.01) and increased the expressions of ssc-miR-7-5p (P < 0.01) and ssc-miR-138 (P = 0.03) in comparison with the Con group. Moreover, the SC06 + DQ treatment down-regulated the expressions of ssc-miR-10390 and ssc-miR-2483 (P < 0.01) in comparison with the DQ group (Tables 4, 5 and 6; Fig. 7b).

Fig. 7.

Effects of SC06 on the jejunal mucosal miRNA profiles of oxidatively stressed piglets. (a) Venn diagrams between groups (b) Differentially expressed miRNA between Con and DQ groups, Con and SC06 groups, and DQ and SC06 + DQ groups. Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

Table 4.

Differentially expressed MiRNA between the SC06 and Con groups

miRNA	Con (%)	SC06 (%)	up/down	log2FC	P-value
ssc-miR-10,384	32.97	17.29	down	-1.02	< 0.01
ssc-miR-7-5p	16.82	32.49	up	0.86	< 0.01
ssc-miR-138	12.59	24.22	up	0.87	0.03
ssc-miR-10a-3p	12.36	24.04	up	0.86	0.04

FC: fold change, Con group, basal diet; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06

Table 5.

Differentially expressed MiRNA between the DQ and Con groups (top 10)

miRNA	Con (%)	DQ (%)	up/down	log2FC	P-value
ssc-miR-10390	6.68	86.77	up	3.62	< 0.01
ssc-miR-708-3p	9.21	46.41	up	2.00	< 0.01
ssc-miR-195	12.21	47.89	up	1.59	0.01
ssc-miR-10a-3p	12.36	32.63	up	1.07	0.01
ssc-miR-138	12.59	30.50	up	0.98	0.02
ssc-miR-708-5p	19.89	44.35	up	1.95	0.02
ssc-miR-4331-5p	28.92	18.38	down	-0.92	0.02
ssc-miR-181c	14.87	41.24	up	1.12	0.02
ssc-miR-214-3p	14.32	39.17	up	1.10	0.03
ssc-miR-199b-5p	13.09	42.14	up	1.31	0.03

FC: fold change, Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight

Table 6.

Significantly altered bacterial abundance between the SC06 + DQ and DQ groups

miRNA	DQ (%)	SC06 + DQ (%)	up/down	log2FC	P-value
ssc-miR-10390	86.77	5.55	down	-3.99	< 0.01
ssc-miR-2483	56.09	7.11	down	-2.82	< 0.01
ssc-miR-10,386	58.82	11.26	down	-2.46	0.02
ssc-miR-411	37.08	4.43	down	-3.14	0.04
ssc-miR-6516	32.37	14.65	down	-1.15	0.04

FC: fold change, DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

Functional analysis of the DEmiRNA

According to the KEGG data, the mTOR and MAPK signaling pathways were the most enriched between the DQ and Con groups. The MAPK and Ras signaling pathways were the most abundant between the SC06 and Con groups. Furthermore, the Wnt, mTOR, and ErbB signaling pathways were the most enriched between the SC06 + DQ and DQ groups (Fig. 8).

Fig. 8.

Effect of SC06 on KEGG enrichment of differentially expressed miRNA in oxidatively stressed jejunal mucosal piglets. Con group, basal diet; DQ group, basal diet and intraperitoneal injection of DQ at 10 mg/kg body weight; SC06 group, basal diet containing 1 × 10⁸ cfu/g SC06; SC06 + DQ group, basal diet containing 1 × 10⁸ cfu/g SC06 and intraperitoneal injection of DQ at 10 mg/kg body weight

SC06 attenuated the jejunal mucosal oxidative damage and decreased the expression of ssc-miR-10390 in DQ-treated IPEC-J2 cells

Figure 9a illustrated that DQ increased the content of MDA (P < 0.01) and lowered the activities of CAT, SOD and GSH-Px (P < 0.01). The SC06 supplementation decreased the content of MDA (P < 0.01) and increased the activities of CAT (P = 0.01), SOD (P < 0.01) and GSH-Px (P < 0.01). In addition, SC06 × DQ decreased the MDA content (P = 0.02), and increased the activities of SOD (P < 0.01) and GSH-Px (P = 0.04) in DQ-injected IPEC-J2 cells. Moreover, DQ reduced the gene expressions of ZO-1, Claudin and Occludin (P < 0.01). SC06 also significantly increased the gene expressions of ZO-1, Claudin and Occludin (P < 0.01). SC06 × DQ increased the gene expressions of ZO-1, Claudin and Occludin in DQ-injected IPEC-J2 cells (P < 0.01) (Fig. 9b). In addition, DQ injection up-regulated the gene expressions of IL-1β, IL-6, TNF-α and IFN-γ (P < 0.01) and decreased the gene expression of IL-10 (P < 0.01). The SC06 induced the opposite results (P < 0.01). SC06 × DQ increased the gene expression of IL-10 and decreased the gene expressions of IL-1β, IL-6, TNF-α and IFN-γ in DQ-injected IPEC-J2 cells (P < 0.01) (Fig. 9c). Moreover, DQ increased the expression of ssc-miR-10390 (P < 0.01). SC06 decreased the expression of ssc-miR-10390 (P < 0.01). SC06 × DQ decreased the expression of ssc-miR-10390 in DQ-injected IPEC-J2 cells (P < 0.01) (Fig. 9d).

Fig. 9.

Effect of SC06 on oxidative damage in IPEC-J2 cells. (a) oxidative stress (b) tight junction gene expressions (c) immune factors gene expressions (d) the expression of ssc-miR-10390. ^{a, b,c}Mean value within a row with no common superscript differ significantly (P < 0.05). Con group, basal culture medium; DQ group, basal culture medium and addition of 950 µmol/mL DQ; SC06 group, basal culture medium containing 1 × 10⁸ cfu/mL SC06; SC06 + DQ group, basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ

SC06 alleviated oxidative damage through ssc-miR-10390 in the IPEC-J2 cell

Compared with the Con group, the expression of ssc-miR-10390 was not significantly influenced in the ssc-miR-10390-NC mimic (NC) group or ssc-miR-10390-NC inhibitor (NC inhibitor) group (P > 0.05). Compared with the NC group, the expression of ssc-miR-10390 was increased in the ssc-miR-10390 mimic (mimic) group (P < 0.01). Compared with the NC inhibitor group, the expression of ssc-miR-10390 was decreased in the ssc-miR-10390 inhibitor (inhibitor) group (P < 0.01) (Fig. S1).

Thereafter, the roles of ssc-miR-10390 in oxidative stress, physiological barrier function and inflammation in IPEC-J2 cells were further analyzed. As for the oxidative stress with the treatments of DQ and SC06, the transfection of ssc-miR-10390 mimic increased the content of MDA (P < 0.01) and decreased the activities of SOD, CAT and GSH-Px (P < 0.01). The transfection of ssc-miR-10390 inhibitor increased the activities of SOD, GSH-Px and CAT (P < 0.01), and decreased the content of MDA (P < 0.01) (Fig. 10a). As for physiological barrier function with the treatments of DQ and SC06, transfection of ssc-miR-10390 mimic reduced the gene expressions of ZO-1, Claudin and Occludin (P < 0.01). Transfection of ssc-miR-10390 inhibitor increased the gene expressions of ZO-1, Claudin and Occludin (P < 0.01) (Fig. 10b). As for the makers of inflammation affected by the treatments of DQ and SC06, the transfection of ssc-miR-10390 mimic increased the gene expressions of IL-1β, IL-6, TNF-α and IFN-γ (P < 0.01) and decreased the gene expression of IL-10 (P < 0.01). The transfection of ssc-miR-10390 inhibitor increased the expression gene of IL-10 (P < 0.01), and decreased the gene expressions of IL-1β, IL-6, TNF-α and IFN-γ (P < 0.01) (Fig. 10c).

Fig. 10.

SC06 alleviated oxidative damage in IPEC-J2 cells by ssc-miR-10390. (a) oxidative stress (b) tight junction gene expressions (c) immune factors gene expressions. ^{a, b,c, d}Mean value within a row with no common superscript differ significantly (P < 0.05). Con group, basal culture medium; DQ group, basal culture medium and addition of 950 µmol/mL DQ; SC06 + DQ group, basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; NC + SD group, transfection of ssc-miR-NC mimic and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; mimic + SD group, transfection of ssc-miR-10390 mimic and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; inhibitor NC + SD group, transfection of ssc-miR-NC inhibitor and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; inhibitor + SD group, transfection of ssc-miR-10390 inhibitor and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ

Prediction and validation of ssc-miR-10390 target gene

WT1 was predicted to be a target gene of ssc-miR-10390 by miRanda database (Table S4). Thus, dual luciferase reporter gene assay was performed for the validation of the target gene. Based on Fig. 11, compared with the wild transfected with NC, transfection with ssc-miR-10390 mimic reduced the ratio of firefly luciferin to renilla luciferin (P < 0.01). Compared with the mutant transfected with NC, transfection with ssc-miR-10390 mimic reduced the ratio of firefly luciferin to renilla luciferin (P < 0.01) (Fig. 11).

Fig. 11.

Dual-luciferase reporter gene assay. WT + NC group, wild type and transfection of ssc-miR-NC mimic (NC); WT + mimic group, wild type and transfection of ssc-miR-10390 mimic (mimic); MUT + NC group, mutant type and transfection of NC; MUT + mimic group, mutant type and transfection of mimic

SC06 alleviated oxidative damage through WT1 in the IPEC-J2 cell

As shown in Fig. S2, transfection of si-WT1-1099 had the best interference effect and significantly reduced the WT1 expression level (P < 0.01). Therefore, si-WT1-1099 was used in the subsequent experiments.

As for the oxidative stress with the treatments of DQ and SC06, transfection of si-WT1 increased the content of MDA (P < 0.01) and decreased the activities of SOD, GSH-Px and CAT (P < 0.01). Compared with the transfection of si-WT1, co-transfection of si-WT1 and ssc-miR-10390 inhibitor (si-WT1 + inhibitor) reduced the content of MDA (P < 0.01) and increased the activities of SOD and CAT (P < 0.01) (Fig. 12a). As for the physiological barrier function with the treatments of DQ and SC06, transfection of si-WT1 reduced the gene expressions of ZO-1, Claudin and Occludin (P < 0.01). Compared with the transfection of si-WT1, co-transfection of si-WT1 + inhibitor increased the gene expressions of Claudin and Occludin (P < 0.01), and decreased the gene expression of ZO-1 (P < 0.01) (Fig. 12b). As for the inflammation with the treatments of DQ and SC06, transfection of si-WT1 increased the gene expressions of IL-1β, IL-6, TNF-α and IFN-γ (P < 0.01), and decreased the gene expression of IL-10 (P < 0.01). Compared with the transfection of si-WT1, co-transfection of si-WT1 + inhibitor increased the gene expressions of IL-10 and IFN-γ (P < 0.01) and decreased the gene expressions of IL-1β, IL-6 and TNF-α (P < 0.01) (Fig. 12c).

Fig. 12.

SC06 alleviated oxidative damage in IPEC-J2 cells by WT1. (a) oxidative stress (b) tight junction gene expressions (c) immune factors gene expressions. ^{a, b,c, d}Mean value within a row with no common superscript differ significantly (P < 0.05). Con group, basal culture medium; DQ group, basal culture medium and addition of 950 µmol/mL DQ; SC06 + DQ group, basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; NC + SD group, transfection of siRNA negative control and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; si-WT1 + SD group, transfection of siRNA targeting WT1 (si-WT1) and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ; si-WT1 + inhibitor + SD group, co-transfection of si-WT1 and ssc-miR-10390 inhibitor (inhibitor) and basal culture medium containing 1 × 10⁸ cfu/mL and addition of 950 µmol/mL DQ

Discussion

In piglet production, a variety of factors can cause intestinal oxidative damage and lead to economic losses. Probiotics are able to regulate intestinal health and have antioxidant capacity. In the current study, the DQ injection was applied to induced jejunal mucosal oxidative stress by decreasing the activities of antioxidant enzymes and elevating the levels of ROS and MDA. The SC06 supplementation reversed these negative results. In addition, SC06 and DQ had significant interaction effects on these parameters, indicating the antioxidant capacity of SC06. Previous studies also showed that dietary supplementation with Bifidobacterium animalis JYBR-190 or Bacillus licheniformis can improve the jejunal antioxidant capacity in weaned piglets [22, 23]. Besides, it is reported that dietary probiotics supplementation can improve the jejunal morphology of weaned piglets [24]. Here, HE staining and TEM were also used to analyse the impacts of SC06 on the structure and morphology of jejunal mucosa. We found that DQ lowered the villus height, V/C and impaired the tight junction structure, while SC06 improved the jejunal mucosal morphology.

Intestines form a natural barrier to maintain the internal microbiome stability and prevent the invasion of pathogenic bacteria and toxins into the body. Intestinal epithelial cells form an effective barrier through a network of cellular junctions, such as tight junctions and adherens junctions, which effectively prevent the entry of bacteria, viruses, and endogenous toxins into the body. In the present study, DQ resulted in the down-regulation of ZO-1, Occludin and Claudin expressions, whereas SC06 resulted in the up-regulation of ZO-1, Occludin and Claudin expressions. SC06 and DQ had significant interaction effects on the gene expressions associated with these tight junction proteins. In accordance with our results, reports have also shown that Bacillus licheniformis increased the expressions of intestinal tight junctions of piglets [25]. Moreover, the Goblet cells can secrete mucus to form a chemical barrier. MUC1 is primarily located on the luminal surface of epithelial cells to protect the cells from extreme factors, and MUC2 is the main component of the mucus layer of the intestinal mucosa. Here, DQ reduced the transcriptions of MUC1 and MUC2. SC06 reversed this change, and SC06 and DQ had a significant interaction effect on the gene expression of MUC2. Furthermore, the intestinal mucosal tissue has an immunological barrier function by secreting immune factors such as TNF-α, interleukins and IFN-γ through the gut-associated lymphoid tissue and immune cells. We found that DQ reduced the contents of IL-10 and sIgA and the gene expression of IL-10, and increased the contents and gene expressions of IL-1β, IL-6, TNF-α and IFN-γ. However, SC06 reversed these changes. Meanwhile, there was a significant interaction between SC06 and DQ on the contents of IL-1β, IL-6 and IFN-γ and the gene expressions of IL-6 and IFN-γ. The above findings indicated that dietary SC06 supplementation effectively improved the jejunal barrier functions in DQ-induced oxidative stress in piglets. By using the in vitro study, we further confirmed that SC06 significantly decreased the oxidative stress, inflammation and improved the tight junction gene expressions in DQ-treated IPEC-J2 cells. Similarly, studies have also reported the beneficial effects of various probiotics, such as Bacillus subtilis DSM 32,540 and Lactobacillus johnelii BS15, on enhancing the intestinal barrier functions of piglets [26, 27].

Intestinal microbiota plays an essential role in host metabolism, immunity and development [28]. Previous study mainly investigated the fecal or cecal microbiota in piglets. However, the different parts of intestine have significant differences in structure, function, and microbial colonization, which directly lead to regional specificity in gene expression profiles and disease susceptibility across various intestinal segments [29–31]. Thus, the jejunal mucosal bacteria was detected in the present study. There were notable variations in mucosal microbiota at different taxonomic levels in the current investigation, although SC06 and DQ had no significant effects on α and β diversity between the groups. The relative abundance of Chlamydiae (phylum) bacteria in the DQ group was higher than that in the Con group. Chlamydiae is a kind of common pathogen in humans and pigs, causing different diseases [32]. In the jejunum, the Peptostreptococcaceae (family), Ruminococcaceae (family) and Clostridium (genus) in the SC06 group were less abundant than that in the Con group. Peptostreptococcaceae is generally considered to be the normal flora of the intestinal tract, but Jalanka et al. [33] reported that the abundance of Peptostreptococcaceae was increased in the colonic mucosa of children with ulcerative colitis. The increased abundance of Ruminococcaceae in the large intestines was positively correlated with Crohn’s disease, inflammatory bowel disease and irritable bowel syndrome [34]. Clostridium exists in the intestinal tract of humans and animals, most of which are non-pathogenic and a few are pathogenic. Lactobacillus plantarum ZLP001 has been shown to alleviate the increase in Clostridium abundance caused by inflammation in IPEC cells [35]. The relative abundance of Corynebacteriaceae (family), Ruminococcaceae (family) and SMB53 (genus) in the SC06 + DQ group was lower than that in the DQ group. Corynebacteriaceae are pathogenic bacteria of humans and animals, and closely related to inflammation [36]. SMB53 is a common opportunistic pathogen in human clinical practice. Yang et al. [37] found that SMB53 had a negative impact on the intestinal tract of pigs. KEGG enrichment analysis showed that the lysosome was less enriched in the DQ group, and spliceosome was more enriched in the SC06 group in comparison with the Con group. The SC06 + DQ treatment led to enriched retinol metabolism in comparison with the DQ treatment. In line with our findings, Li et al. [38] showed that administration of Lactobacillus sake JD10 promoted an increase in lysosomal pathway enrichment in RAW 264.7 cells. Increased enrichment of the spliceosome pathway was observed in Bacillus licheniformis-treated fish [39]. The enrichment of intestinal retinol metabolic pathways was increased in broilers treated with Bacillus subtilis [40]. Taken together, dietary SC06 supplementation improved the biological barrier function of DQ-injected piglets.

In addition, increasing evidence proved that intestinal oxidative damage is closely related to the regulation of miRNA. miRNA sequencing results showed that there were significant differences in the expressions of miRNA among groups. ssc-miR-10390 are core molecules for pigs in coping with various external stresses and pathogen infections. For example, the expression of ssc-miR-10390 significantly changed after infection with porcine epidemic diarrhea syndrome coronavirus and African swine fever virus [41–44], and it can influence cell survival by regulating target genes under cellular stress [45]. Similarly, ssc-miR-4331-5p has been shown to exacerbate virus-induced cell damage [46], while ssc-miR-7-5p has been reported to participate in forming a regulatory axis with cytoprotective functions [47]. Additionally, KEGG enrichment analysis showed that the DEmiRNAs were mainly enriched in mTOR and MAPK signaling pathways between DQ group and Con group. The DEmiRNAs were mainly enriched in Ras and MAPK signaling pathways between SC06 group and Con group. The DEmiRNAs were mainly enriched in Wnt, mTOR and ErbB signaling pathways between SC06 + DQ group and DQ group. The mTOR signaling pathway is an atypical protein kinase that plays an important role in cell growth, apoptosis, autophagy, and metabolism. Liao et al. [48] showed that dietary supplementation of baicalin alleviated deoxynivalenol-induced intestinal oxidative damage in piglets through the mTOR signaling pathway. Qin et al. [49] reported that glutamate alleviated LPS-induced oxidative damage in the intestine of weaned piglets through the mTOR signaling pathway. MAPK is one of the classical antioxidant signaling pathways. Lactobacillus rhamnosus GG alleviated LPS-induced intestinal inflammation and barrier impairment in piglets by regulating the MAPK/NF-κB signaling pathway [50]. Ras protein is an upstream signal molecule of ERK, a branch of MAPK signaling pathway. Dietary supplementation with resveratrol alleviated diarrhea and intestinal inflammation in intestine of weaned piglets by MAPK and Ras signaling pathways [51]. Wnt signaling pathway plays a key role in regulating gene expression and DNA damage response, and oxidative stress. Yousefi et al. [52] found that the expression of Nrf2ARE (Nrf2)-related antioxidant genes resulted in the inactivation of Wnt signaling pathway. The ErbB signaling pathway binds to a variety of signaling molecules to dimerize or heterodimerize ErbB members, which in turn initiates autophosphorylation and downstream signaling cascades [53]. Xie et al. [54] showed that crotonaldehyde induced vascular injury in human aortic endothelial cells through Wnt and ErbB signaling pathways. Taken together, dietary SC06 supplementation significantly regulated the expressions of jejunal miRNAs.

Based on the above findings, we found that ssc-miR-10390 was closely related to health of animals and ssc-miR-10390 was also a common DEmiRNA between DQ and Con groups and between SC06 + DQ and DQ groups, thus, the role of ssc-miR-10390 in the regulation of jejunal mucosal health was detected and verified. Consistent with the jejunal ssc-miR-10390 expression profiles, in the IPEC-J2 cells, the DQ treatment increased the expression of ssc-miR-10390, and SC06 decreased the expression of ssc-miR-10390. Furthermore, with the treatments of DQ and SC06, transfection of ssc-miR-10390 mimic increased the content of MDA, reduced the antioxidant enzymes activities, damaged the tight junction and aggravated the cellular inflammation, while transfection of ssc-miR-10390 inhibitor reversed these results. Therefore, ssc-miR-10390 played an important role in SC06-mediated alleviation of oxidative damage in IPEC-J2 cells.

Thereafter, the target gene of ssc-miR-10390 was analysed. miRanda database showed that WT1 was the target gene of ssc-miR-10390. In addition, WT1 has been reported to regulate oxidative stress, apoptosis and barrier function. For example, hyperacetylation induced by the histone deacetylase inhibitor sodium butyrate upregulated the gene expression of WT1 in porcine kidney fibroblasts, indicating that histone acetylation was involved in the transcriptional regulation of WT1 in porcine kidney cells [55]. During mouse embryonic development, WT1 contributes to gut and mesentery growth [56]. However, the role of WT1 in the intestinal oxidative stress of weaned piglets has not been reported. Here, by using the dual luciferase reporter gene assay, we verified that WT1 is the target gene of ssc-miR-10390. However, compared with the mutant transfected with NC group, transfection of ssc-miR-10390 mimic reduced the ratio of firefly luciferin to reniferin, indicating that the binding site of ssc-miR-10390 to the target gene WT1 was not the one we predicted. This may be due to the specificity of the species. In the existing databases, validation data from pigs account for less than 10%, causing the predicted results to deviate from the true binding sites of pig-derived WT1. Therefore, the binding site sequences of ssc-miR-1090 and WT1 need to be further verified.

Subsequently, we used RNA interference technology to further investigate the role of WT1. Results showed that with the treatments of DQ and SC06, transfection of si-WT1 increased the content of MDA and gene expressions of IL-1β, IL-6, TNF-α and IFN-γ and decreased the activities of SOD, GSH-Px and CAT and gene expressions of ZO-1, Claudin, Occludin and IL-10. The results of rescue experiments showed that co-transfection of ssc-miR-10390 inhibitor and si-WT1 could partially reverse the reduction of antioxidant capacity, the destruction of tight junction and the increase of inflammation level caused by transfecting si-WT1, and alleviated oxidative damage. Therefore, the ssc-miR-10390-WT1 pathway played a key role in SC06-mediated alleviation of oxidative damage in IPEC-J2 cells.

Conclusions

Our results showed that SC06 supplementation ameliorated the DQ-induced oxidative stress by elevating the barrier function and miRNA profiles, such as ssc-miR-10390 in piglet jejunum and IPEC-J2 cells. Further, by using ssc-miR-10390 mimic, ssc-miR-10390 inhibitor and siRNA, we found that ssc-miR-10390 can enhance the antioxidant function and tight junctions, and alleviate cellular inflammation in IPEC-J2 cells by targeting WT1 gene. These findings indicated that the ssc-miR-10390 plays an important role in SC06-mediated alleviation of oxidative damage in jejunal mucosa of piglets via WT1.

Materials and methods

Bacteria

The SC06 strain, which has been assigned the number (CCTCC No: M2012280) by the China Center for Type Culture Collection. The SC06 was cultured according to our previous study [57]. In brief, SC06 was cultivated in Luria-Bertani media, and shaken at 180 r/min (37 ℃, 24 h). The pure SC06 was collected by centrifuging at 3000 r/min (4 ℃, 10 min). Following that, sterile 0.85% sodium chloride solution was used to wash the SC06 twice. During the trial, the purity of bacteria was checked [58].

Animal experimental design

A 28-day experiment was performed in a 2 × 2 factorial design by using 2 levels of SC06 (0 and 1 × 10⁸ cfu/g) and 2 levels of DQ injection (0 and 10 mg/kg) [59], thus, the 4 groups were control group (Con), DQ group, SC06 group and SC06 + DQ group. Thirty-two weaned 28-d-old male piglets (Duroc × Landrace × Yorkshire) with similar initial body weights (7.51 ± 0.39 kg) were randomly assigned to the above 4 groups. Each group of piglets was housed in a pen. On the 22nd day of the experiment, piglets in the DQ and SC06 + DQ groups were intraperitoneally injected with DQ, and those in the Con and SC06 groups were treated with the same volume of saline. The basal diet was prepared in accordance with NRC 2012 (Table S1).

Animal samples collection

Piglets were sacrificed on the 29th day, and sodium pentobarbital (50 mg/kg body weight) was delivered by a rapid intracardial injection for euthanizing the piglets and the jugular exsanguination procedures were also initiated to ensure the death. The mid-segments of the jejunum were dissected and washed with sterilized saline. One part of the jejunal segment was fixed in 4% paraformaldehyde solution for Hematoxylin–eosin (HE) staining, and the other part was cut and fixed in electron microscope fixative. The jejunal mucosal bacteria were then collected by wiping the mucosa with cotton swabs. Besides, jejunal mucosa were also carefully scraped off. The swab tips and jejunal mucosa were frozen immediately by snap freezing in liquid N₂ and then kept at − 80 °C for storge.

Cell culture and treatments

IPEC-J2 cells were cultivated in cell incubator (37 ℃, 5% CO₂) in DMEM/F12 (HyClone, USA) with 10% Fetal Bovine Serum (FBS, Gibco, USA), 1% penicillin, and 1% streptomycin. According to our previous study, the in vitro trial was conducted in a 2 × 2 factorial design with two SC06 dosages (0 or 1 × 10⁸ cfu/mL) and two DQ dosages (0 or 950 µmol/mL) [60]. The 4 treatment groups were: control group (Con), DQ group, SC06 group, SC06 + DQ group. Cell suspension at a concentration of 5 × 10⁵ cells/mL was seeded into 12-well culture plates (1 mL per well) (Corning, USA) and grown in a CO₂ incubator for 24 h. Cells in SC06 and SC06 + DQ groups were first cultured with 1 × 10⁸ cfu/mL SC06 for 6 h, then 950 µmol/mL DQ was added to the cells in the SC06 + DQ group for another 6 h to induce oxidative stress, while the same amount of PBS (Servicebio, China) was added to the cells in the SC06 group for another 6 h. The cells in the Con and DQ groups were cultured in the incubator for 6 h, then the cells in the DQ group were treated with 950 µmol/mL DQ for another 6 h, while the cells in the Con group were treated with the same volume of PBS for another 6 h.

RNA interference and cell transfection

GenePharma Co, LTD (Suzhou, China) generated the siRNA targeting WT1 (si-WT1), siRNA negative control (si-NC), ssc-miR-10390 mimic, ssc-miR-NC mimic, ssc-miR-10390 inhibitor, ssc-miR-NC inhibitor (Table S2). Lipofectamine 3000 transfection reagent (Invitrogen, USA) was used for IPEC-2 transfection in Opti-MEM (Gibco, USA). Following a 12 h transfection period, the Opti-MEM was replaced with DMEM/F12 (HyClone, USA) supplemented with 10% FBS (Gibco, USA). Subsequently treated with the corresponding SC06 (0 or 1 × 10⁸ cfu/mL) or DQ (0 or 950 µmol/mL) for 6 h.

Target prediction and dual luciferase reporter assay

Human embryonic kidney 293T (HEK-293T) cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM, HyClone, USA) containing 10% FBS (Gibco, USA), 1% penicillin, and 1% streptomycin at 37 ℃ with 5% CO₂ in cell incubator. The target genes of ssc-miR-10390 were predicted by TargetScan (www.targetscan.org), miRBase (www.mirbase.org) and miRanda (www.microrna.org) databases. To conform that the ssc-miR-10390 can target WT1, a dual luciferase reporter experiment was conducted. In brief, wild and mutant 3’UTR of WT1 mRNA were cloned into the psi-Check2 (Promega, USA), which was then co-transfected with the ssc-miR-10390 mimic or ssc-miR-NC mimic into HEK-293T cells. Firefly luciferase activity served as the normalization control. The HEK-293T cells were harvested 48 h after transfection, and a Dual Luciferase Reporter Assay Kit (Beyotime, China) was used to detect the luciferase activity. The activities of Firefly and Renilla luciferase were measured with a Fluoroskan microplate reader (Thermo Scientific, USA).

Cell samples collections

At the end of the cells assay, the supernatant was collected in a 1.5 mL centrifuge tube and stored at − 20 ℃. Subsequently, the cells were washed three times with PBS. A total of 1 mL of Trizol reagent (Tiangen, China) was added to each well and the lysis were collected into a 1.5 mL centrifuge tube and stored at − 80 ℃.

HE staining and transmission electron microscope (TEM)

The fixed jejunal segments were embedded and then stanined by HE. The villus height and crypt depth were observed by OLYMPUS microscope (OLYMPUS, Japan) and measured according to our previous study [61]. The jejunum was cut (1 mm³) and fixed in electron microscope fixative (Xavier Biotechnology Co., Ltd., Wuhan, China). Uranyl acetate staining was applied after the ultramicrotome cutting the tissue into ultrathin sections (Leica EM UC7, Wetzlar, Germany). Electron micrographs of the samples were then captured by the TEM (HITACHI HT7700 120kv, Tokyo, Japan).

Biochemical analysis

After homogenizing in ice-cold 0.85% saline (1:10, w/v), jejunal mucosal proteins were collected by centrifuge (2000 × g,10 min, 4 °C). The protein concentration was measured by the BCA Protein Concentration Determination Kit (Beyotime, China). The contents of malondialdehyde (MDA), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), secretory immunoglobulin A (sIgA) and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were determined by using ELISA kits (Enzyme-Linked Co., Ltd., Shanghai, China). The amount of ROS in the jejunal mucosa was also determined by ELISA kit (Meimian Industrial Co., Ltd., Jiangsu, China). The manufacturer’s instructions were strictly followed. The SpectraMax iD3 (Molecular Devices, China) was used to measure the absorbance.

Real-time quantitative PCR (RT-qPCR)

RNA extraction and RT-qPCR were performed according to our previous study [62]. The gene primers are presented in Table S3. The miRNA expressions were determined by RT-qPCR using miRNA 1st strand cDNA synthesis kit (Vazyme, China) and BioRad CFX96™ Real-Time PCR system (Bio-Rad Laboratories, USA). β-actin and GAPDH were used to normalize mRNA transcript levels. U6 was used to normalize miRNA transcript levels. The 2^−ΔΔCq method was used to estimate target the gene expressions.

DNA extraction and analysis of jejunal mucosal bacteria

The jejunal mucosa bacteria DNA was extracted by the fecal genomic DNA extraction kit (Tiagen, China). DNA integrity was examined by electrophoresis on a 1% agarose gel and quantified with a UV spectrophotometer. The V3-V4 region was amplified by PCR using 338 F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) primers. Finally, the samples were submitted for sequencing by the Illumina MiSeq platform.

The original tags were filtered (QIIME2 2019.4) and integrated by FLASH program incorporates software. The amplicon sequence variantions were clustered by 97% similarity (Uclust 1.2.22). The R language was used for diversity analysis. Linear discriminant analysis effect size was performed to detect differentially abundant taxa across groups using the default parameters. Finally, PICRUSt2 was used to predict functional genes of intestinal mucosal microbes.

Construction and sequencing analysis of small RNA libraries from jejunal mucosa

Trizol reagent was used to extract the jejunal mucosa RNA. Small RNA sequencing libraries were prepared using the NEB Next Multiplex Small RNA Library Prep for Illumina kit. Agilent 2100 Bioanalyzer and Agilent High Sensitivity DNA Kit were used to detect the quality of the library, Pico Green was used to detect the total concentration of the library under the total volume, and qPCR was used to quantify the effective library concentration. These were performed in Illumina platform PE150 mode.

The original data were quality checked by the script independently developed by Panomic Biomedical Technology Co., LTD (Suzhou, China). These sequences were aligned by Bowtie to porcine miRNA precursor and mature body sequences in miRBase to annotate the detected miRNAs.

DESeq1.30.0 software was used to analyze the differential expressions of miRNAs between the two comparison combinations. miRanda database was used to predict the target gene of miRNAs. Subsequently, KEGG pathway enrichment analysis of the target genes of differential miRNAs were performed by the clusterProfiler3.4.4 software.

Statistical analysis

Analysis of variance was performed using SPSS 20.0 software, and significant differences between groups were identified using Tukey’s multiple comparison. Results were presented as means and standard errors, and P < 0.05 indicated a significant difference.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

J.Y. performed the experimental research and data analysis, and wrote and edited the manuscript. Y.L. and Y.S. contributed to the study design, data analy sis, and writing and editing of the manuscript. H.L., K.Z. and J.Z. designed the experiment, and wrote and revised the manuscript. W. L. and Y. W. contributed to the study design and editing of the manuscript. All authors have read and approved the final manuscript.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 32102586, 32072766), Shandong Provincial Natural Science Foundation (Grant No. ZR2020QC183), Postgraduate Education Reform Project of Shandong Province (Grant No. SDYJSJGC2023061).

Data availability

The bacterial and small RNA raw data were submitted to the NCBI database under Bioproject IDs PRJNA1085509 and PRJNA1012679, respectively.

Declarations

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Animal Care and Use Committee of Qingdao Agricultural University (protocol number 20221125374).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.