Resveratrol Alleviates Oxidative Stress Induced by Oxidized Soybean Oils and Improves Gut Function via Changing Gut Microbiota in Weaned Piglets

Abstract

Background: The objective of this study was to investigate the effects of dietary resveratrol supplementation on growth performance, redox status, inflammatory state, and intestinal function of weaned piglets fed oxidized soybean oils.

Methods: A total of twenty-eight castrated weaned male piglets with a similar body weight of 10.19 ±1 kg were randomly assigned to 4 dietary treatments for 28 days feeding trial with 7 replications per treatment and 1 piglet per replicate. Treatments were arranged as a 2×2 factorial with oil type [fresh soybean oils (FSOs) vs. oxidized soybean oils (OSOs)] and dietary resveratrol (RES) (0 vs. 300 mg/kg).

Result: Inclusion of OSOs decreased the villus/crypt ratio (VCR), while the villus height (VH) and VCR in the jejunum of weaned piglets was increased by dietary RES (P< 0.05). The activities of lipase, chymotrypsin, and lactase were decreased by OSOs, however dietary RES supplementation increased the activities of lipase, chymotrypsin, lactase, and α-amylase in the jejunum of weaned piglets (P< 0.05). Dietary RES increased the apparent digestibility of crude fat (EE). Dietary RES supplementation in the diets supplemented with OSOs decreased the level of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) in the plasma of weaned piglets, but failed to influence the IL-1β, IL-6, IL-8, and TNF-α level when diets supplemented with FSOs. Dietary RES alleviated the decrease of total-superoxide dismutase activity in the plasma of weaned piglets fed OSOs (P< 0.05). Dietary supplemented with OSOs and RES decreased the level of H₂O₂ in the plasma of weaned piglets (P< 0.05). RES alleviates the intestinal barrier damage fed OSOs in weaned piglets by increasing the mRNA expression of ZO-1 and Occludin. It is noteworthy that inclusion of OSOs in diets increased the abundance of Actinobacteria, and decreased the abundance of Tenercutes (P< 0.05). RES increased the abundance of Firmicutes, and decreased the abundance of Bacteroidetes (P< 0.05). At the genus level, RES decreased the abundance of Prevotella-1, Prevotellaceae UCG003, and Clostridium_sensu_stricto_6 in the colon. OSOs decreased the level of acetic acid, and dietary RES increased the level of acetic acid and butyric acid in the colon of weaned piglets.

Conclusions: Dietary RES supplementation improved the villus-crypt structure,  digestive enzyme activities and alleviated OSOs induced digestive absorption disorder. In addition, RES may alleviate OSOs immune status and energy metabolism of weaned piglets by affecting gut microbiota and its metabolite SCFAs. Notably, this positive effect of RES on OSOs may be related to decrease in the abundance of Prevotella_1 and Prevotellaceae_UCG-003.

Background

Dietary lipids give animals more time to digest and absorb other nutrients by slowing the rate of feed through the digestive tract¹. In addition, as a vitamin vehicle, dietary lipids can effectively alleviate heat stress and provide a concentrated energy source for animals². Soybean oil is a common source of dietary lipids added to animal diets. It is prone to lipid peroxidation, especially used for animal production in hot climates, because it contains a high concentration of unsaturated fatty acids³. It is well known that the presence of primary oxidation products will affect feed efficiency, which has a negative impact on animal performance⁴. Researchers have consistently demonstrated that the antioxidant status of swine⁵, broilers⁶, and rats⁷, which feed oxidized oil is significantly lower than animals that fed normal oil. In addition, the presence of oxidized oil in swine feeds leads to increased oxidative susceptibility of pork products during storage⁸. Equally, weaning induces oxidative stress in piglets, which may be led to diarrhea, anorexia, intestinal barrier dysfunction, disturbance of free radicals and antioxidant system^{9, 10, 11}. It has been stated that weaned piglets fed oxidized soybean oil will exacerbates intestinal redox imbalance, inflammation, and intestinal barrier injury ⁵. Similarly, a previous study demonstrated that inclusion of oxidized corn oil in diets decreased the average daily gain and average daily feed intake in finishing barrows¹⁰. Intestines are the primary site of pro-oxidation and anti-oxidation effects, exposure of animals to oxidized oil will increase the disruption of intestinal tissue structure, causing the imbalance of intestinal bacteria and increasing the likelihood of diarrhea and intestinal infections, and reducing the immunity and growth performance of animals^{7, 12, 13}. Previous study demonstrated that piglets fed oxidized oil had a lower abundance of Prevotella_9 and higher abundances of Bacteroides and Campylobacter than control diet-fed piglets, and the concentrations of butyric acid and total SCFAs in colonic chyme were decreased¹⁴. Therefore, we hypothesized that intestinal dysfunction induced by oxidized oil consumption may be associated with the changes of intestinal microbiota. However, there are few types of research on the influence of feeding oxidized soybean oil on the intestinal microbiota in weaned piglets, and the biological mechanism to explain these phenomena remains unclear.

Accumulating evidence suggested that supplementation of additives with antioxidant function (such as short-chain fructans-oligosaccharides¹⁴, pterostilbene¹⁵) in the feed allevates the oxidative stress and improves intestinal function of piglets. Phytogenic, a relatively new class of feed additives, comprises a wide variety of herbs, such as essential oils, polyphenols, etc^{15, 16}. In recent years, several studies have explored the relationship between polyphenolic compounds and gut bacteria as a new research perspective. Resveratrol (RES) is a C6-C2-C6 stilbene natural phenolic compound with three hydroxyl groups, several studies reported that RES has strong potential for promoting growth performance, antioxidative, anti-inflammatory by regulating the composition and content of the gut microbiota in animals undergoing stressful situations^{17, 18, 19}. It has been stated that supplementation of RES in the diet of weaned piglets partially increased the jejunum villus height and tended to decrease the number of apoptotic cells in jejunum¹⁵. In addition, it has been reported that RES alleviates the intestinal inflammatory and improves intestinal immune function of piglets by improving the proliferation of beneficial bacteria Lactobacillus and Bifidobacterium, and inhibiting the proliferation of gram-negative bacteria²⁰. Moreover, a previous study reported that dietary supplementation with RES alters the composition and metabolites of colonic microbiota in diquat-challenged piglets²¹. Hence, in the present study, we hypothesized that RES may ameliorate the oxidative stress induced by fed OSOs and improve intestinal health via modulating the gut microbiota of weaned piglets.

Materials And Methods

Performance and diarrhea incidence

After 28 days of feeding, piglets were fasted for 12 h before sacrifice. Body weights (BW) of piglets were individually evaluation on the mornings of day 1, and day 29 of the feeding trial. Deed intake per pen was recorded daily throughout the trial to calculate average daily feed intake (ADFI), average daily gain (ADG) and average daily feed intake/average daily gain (F/G). Clinical signs of diarrhea were visually assessed each morning by observers blinded to the treatments using a five-grade scoring system²³,1 = well-formed feces, 2 = slightly soft feces, 3 = soft and partially formed feces, 4 = loose and semiliquid feces, and 5 = watery feces. Then the average daily diarrhea index per replicating was calculated.

Sample collection and processing 

On the 29th day of the trial, blood samples (10 mL) were collected using heparin tubes from piglets by venous puncture and then centrifuged at 3 000 g for 15 min at 4°C. The plasma was immediately collected and stored at −20°C pending further analyses. After blood sampling, the piglets were sacrificed using electricity (250 V, 0.5 A, for 5 to 6 s), with subsequent jugular exsanguination. After opening the abdominal cavity, the gastrointestinal tract was removed to obtain portions of the jejunum, and the colon measuring approximately 20 cm was cut longitudinally and cleaned with ice-cold phosphate buffer solution (PBS). Mucosa samples were collected using scraping by sterile glass microscope slides. They were then snap-frozen in liquid nitrogen and stored at −80°C for subsequent analysis. Two continuous segments were carefully cut from the middle of the whole jejunum and colon for histological assay and mucosa collection. Sections of approximately 1.5 cm in length were fixed in fresh, chilled 4% paraformaldehyde for morphometric evaluation and histochemical staining.

Determination of apparent nutrient digestibility 

At the end of the experiment (day 24 to 27), feces from each group were collected for nutrient apparent digestibility determination, which was determined by the acid-insoluble ash method²⁴. All the feces of piglets were collected twice a day at 8 h and 20 h, put in plastic bags, weighed, and recorded. To prevent the loss of fecal ammonia, 10 mLH₂SO₄ were added and the feces was stored in a -20℃ refrigerator. Before the samples were determined, the feces samples were thawed from the refrigerator and mixed evenly for four days from each pig. About 200 g of the mixed feces samples were taken out and dried in an oven at 60± 5℃ for 48-72 h. Then the dried feces samples were crushed with a grinder and passed through a 40-mesh sieve for testing. Acid insoluble ash, dry matter (DM), crude protein (CP), crude fat (EE), gross energy (GE) in feces and feed were determined according to GB/T23742-2009 internal indicator method.

Determination of digestive enzymes in the jejunum

The sample preparation and the activities of jejunum digestive enzymes, including amylase (#C016-1-1), lactase(#A082-1-1), trypsin(#A080-3-1), and lipase(#A054-1-1), (#A080-2-2), maltase(#A082-3-1), sucrase(#A082-2-1) of the jejunum were determined using commercial kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China).

Intestinal oxidative stress status and plasma cytokines analyses

Based on the manufacturer’s instructions, commercial kits were used to determine total superoxide dismutase (T-SOD # A001-1-1), glutathione peroxidase (GSH-Px #A005–1-2), hydrogen peroxide (H₂O₂ # A064-1-1), as well as total antioxidant capacity (T-AOC # A015-1-2) and malondialdehyde (MDA #A003–1-2) were determined using commercial kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Inflammatory cytokines in the plasma were determined with an ELISA kit (Beijing Sino-UK Institute of Biological Technology.) according to the manufacturer’s instructions.

Determination of diamine oxidase (DAO) activity and D-lactate content in the plasma

Plasma D-lactate and the diamine oxidase (DAO, #A088-1-1) activity concentrations were determined using commercial ELISA kits provided by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). All procedures were performed with strict adherence to the manufacturer’s guidelines.

Mucosal morphometry and epithelial proliferation in jejunum and colon 

For intestinal morphological analysis, after being fixed in paraformaldehyde solution at room temperature for 24 h, jejunal tissue specimens were dehydrated using an upgraded series of ethanol and xylene and then processed into paraffin blocks. A cross-section with a thickness of 5µm was cut from each specimen and stained with hematoxylin and eosin. Three of each group were randomly selected well-oriented and intact villi and adjacent crypts were randomly selected for the measurements of villus height (VH) and crypt depth (CD) per slide by an assessor blinded to the treatments using optical microscopy (Nikon Eclipse 80i Nikon, Tokyo, Japan) and NIS-Elements 3.0 Imaging Software. Villus height needed to be evaluation in at least 10 villi from each pig and averaged. Data were analyzed using a digital microscope M8 and photographed at 40× magnification. The adjacent jejunum and colon were also fixed overnight in a 2.5.% glutaraldehyde solution at 4 ℃, and then, these samples were treated for observation by electron microscopy. Epithelial proliferation was assessed using immunofluorescence in combination with a standardized quantification pipeline in jejunum and colon mucosa. To determine the relative amount of proliferative epithelial cells in the jejunum and colon mucosa samples, tissues were stained with nuclei stain PCNA and Hoechst (n = 3 randomly selected piglets from each treatment group).

Immunofluorescence

The jejunum and colon with 5% blank goat serum wax block sample complete coverage, slice to be placed in the wet box, at 37 ℃ constant temperature and humidity incubator incubation for 30 min. The blocking solution was removed, and the primary antibody working solution (ABclonal ZO-1, Occludin, Bioss, BSM-33070m, Ki67) prepared with antibody diluent was directly added to the samples. The samples were completely covered, and the sections were placed in a wet box and incubated at 4℃ overnight. Rewarming: the samples were placed at room temperature, rewarming for a 15min, antibody working solution was removed, and buffer solution in TBST was washed once for 5 minutes. Buffer solution TBS was washed for 3 times, 5 minutes each time, and the second antibody was incubated: the fluorescent second antibody working solution corresponding to the primary antibody species was dropped on the samples. The samples should be completely covered, protected from light, and incubated at 37℃ for 1 hour. Remove the working solution of the secondary antibody and wash it with buffer TBST once for 5 minutes. Wash with buffer TBS 3 times, 5 minutes each time, dye the core: drop DAPI working solution on the sample, prepare with 0.01M pH7.2 TBS buffer solution, DAPI solution: 0.01M pH7.2 TBS buffer liquid volume ratio 1:500, avoid light, room temperature, incubated for 10 minutes; DAPI working solution was removed and washed with buffer TBST once for 5 minutes. Wash with buffer solution TBS for 3 times, 5 minutes each time, drop anti-fluorescence attenuation sealing tablets, then cover the cover glass sealing tablets, and observe under a fluorescence microscope and collect images.

RNA extraction and RT-PCR

The reaction system and the thermal cycling conditions used for RT-PCR were adjusted according to our previous study²⁵. After retrieving the snap-frozen jejunum mucosal samples, we isolated total RNA using TRIzol Reagent (#9109), as suggested in the manufacturer’s manual (TaKaRa Biotechnology, Dalian, Liaoning, China). Extracted RNA was removed in 50 µL of ultra-pure water. A NanoDrop ND-1000UV spectrophotometer (NanoDrop Technologies) was used to measure the purity and concentration of total RNA at 260 and 280 nm. Electrophoresis on a 1.5% agarose gel that had been stained Ultra GelRed™ (#GR501–01; Vazyme Biotech Co., Ltd.) was used to verify RNA integrity. One microgram of total RNA was then reverse-transcribed into complementary DNA using the Prime-Script™ RT Reagent Kit (#RR036A; TaKaRa Biotechnology). A real-time polymerase chain reaction (PCR) was performed on a QuantStudio 5 Real-Time PCR System (Applied Biosystems, Life Technologies, CA, USA) using the ChamQTM SYBR® qPCR Master Mix Kit (#Q311–02; Vazyme Biotech Co., Ltd.), as recommended in the manufacturer’s guidelines. The PCR process involved a 30-s pre-run at 95°C, 40 cycles of denaturation at 95°C for 5 s, and a 60°C annealing step for 30 s. For the melting curve conditions, one denaturation cycle was performed at 95°C for 10 s; the temperature was then increased from 65 to 95°C at a rate of 0.5°C/s. The primer sequences are shown in Supplementary Table 2. The average of β-actin and GAPDH was used as the internal control. The relative expression abundance of each target gene was calculated by the 2^{−Δ Δ} Ct method, as elucidated previously²⁶.

Gut microbiota analysis

Colonic digesta samples were collected from slaughtered piglets and immediately stored at −80 ℃. Bacterial genomic DNA was extracted from each sample (Qiagen DNA Stool Mini Kit, Duesseldorf, Germany). DNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA) and further assessed by running on 1% agarose gels. The V3–V4 hypervariable region of 16S rRNA genes was amplified using specific primer pairs (forward 50-ACTCCTACGGGAGGCAGCA-30 and reverse 50-GGACTACHVGGGTWTCTAAT-30) with barcodes to construct the sequencing libraries (TruSeq ® DNA PCR-Free Sample Prep Kit, Illumina, San Diego, CA, USA). The qualified DNA libraries were loaded in a NovaSeq platform with 2 × 250 bp paired-end sequencing. The paired-end reads were obtained and merged using FLASH software (V1.2.7, http://ccb.jhu.edu/software /FLASH/). Operational taxonomic units (OTUs) with a 97% identity were gathered with Unparsed (ver. 7.1, http://drive5.com/uparse /). Taxonomic annotation was performed using the Mothur algorithm (70% confidence) with the Silva Database (http://www.arb-silva.de/). The taxonomic composition of the bacterial community was then analyzed. RDA analyses were performed to identify the relationship among the identified differential genera of microbiota, growth performance, apparent digestibility, intestinal histomorphology, inflammatory cytokines, intestinal enzymatic activities, SCFAs, and treatments. Furthermore, according to the Pearson correlation analyses, several genera that were separately correlated with apparent digestibility, intestinal enzymatic activities and inflammatory cytokines, etc were also further identified.

Quantification of SCFAs 

Short-chain fatty acids (SCFAs), including acetate, propionate, butyrate, valerate, is butyrate, and isovalerate, were quantified by an external standard method using gas chromatography (GC) as described by a previous study. Briefly, 1.5 g colonic chyme samples were added to screw-capped tubes with 6 mL of distilled water. After mixing overnight at 4 ℃ and centrifugation at 1500 r/min for 10 min at 4 ℃, 2 mL of supernatant from each sample were transferred to another centrifuge tube, and 400 µL of meta-phosphoric acid (25% v/v) were added to remove the protein. The samples were then centrifuged at 12,000 r/min for 10 min at 4 ℃. The resulting supernatants (1 mL each) were transferred into gas chromatography sample bottles and analyzed using an Agilent 6890N GC (Palo Alto, CA, USA) coupled to a flame ionization detector with helium used as the carrier gas. An Agilent FFAP column (30 m × 0.53 mm i.d. × 1.00 µm (film thickness)) was installed for analysis, with a constant flow rate of 4.0 mL/min. Spitless injection volume was 0.2 µL of the sample. The injector and detector temperatures were 220 ℃ and 240 ℃, respectively. The GC oven temperature was held at 90 ℃ for 1 min and then increased to 190 ℃ at a rate of 20 ℃/min and held for 3 min. Samples were run in triplicate, with a coefficient of variation less than 15% within triplicate samples used for quality control.

Results

Dietary RES alleviates intestinal morphology abnormalities and nutrient absorption disorders caused by inclusion of OSOs in the diets of weaned piglets

As revealed by Table 1, inclusion of OSOs in the diet tended to decrease the average daily feed intake (ADFI) of weaned piglets (P= 0.060). But we observed that inclusion of OSOs or RES in diets failed to influence the initial weight (IW), average daily gain (ADG), average daily feed intake/the average daily gain(F/G), and final weight (FW). The diarrhea score of weaned piglets was evaluated as shown in Fig. 1A. We observed that inclusion of OSOs in diets increased the diarrhea index during days 13-24 (P< 0.05).

The integrity of the intestinal structure is the basic guarantee of intestinal digestion and absorption of nutrients. The villus height (VH), the crypt depth (CD), and the villus height: the crypt depth (VCR) of the intestinal are important indexes to measure intestinal digestion and absorption function²⁷. As shown in Fig. 3, inclusion of OSOs decreased the VCR in the jejunum of weaned piglets. However, the VCR in the jejunum of weaned piglets was increased by dietary RES (P< 0.05). We observed that dietary RES supplementation in the diets supplemented with OSOs increased the VH in the jejunum of weaned piglets (P< 0.044), but failed to influence the VH when diets supplemented with FSOs (interaction, P= 0.016). We observed that inclusion of OSOs or RES in diets failed to influence the CD in the jejunum or colon. Furthermore, we evaluation the apparent digestibility of weaned piglets (Table 2). We found that dietary RES supplementation increased the digestibility of EE of weaned piglets (P< 0.05). Inclusion of OSOs or RES in diets failed to affect the digestibility of DM, CP, GE, and CF of weaned piglets (P༞ 0.05). These findings indicated that dietary RES improved the intestinal structure and digestive absorption function of weaned piglets.

Then, changes in small intestinal digestive enzymatic activities were further analyzed (as shown in Fig. 1B). We observed that the activities of lipase, chymotrypsin, and lactase activities were decreased by inclusion of OSOs, however, dietary RES supplementation increased the activities of lipase, chymotrypsin, lactase, and α-amylase in the jejunum of weaned piglets (P< 0.05). We observed that dietary RES supplementation in the diets supplemented with OSOs increased the activity of trypsin in the jejunum of weaned piglets (P= 0.015), but failed to influence the activity of trypsin when diets supplemented with FSOs (interaction, P= 0.031). These results showed that dietary RES supplementation promotes the production of intestinal digestive enzymes and alleviates the digestive disorders induced by OSOs in weaned piglets. Considering the increased digestibility of fat, the genes related to fat transport in the intestine was evaluated. We found that the mRNA expression of jejunum absorption including FABP1 and the mRNA expression of jejunum absorption including FABP1, PPARγ, CD36 were increased by dietary RES (Fig. 2) (P< 0.05).

Dietary RES supplementation alleviates the oxidative stress state, inflammatory state, and intestinal barrier damage fed OSOs in weaned piglets

The results of antioxidant status in the plasma of weaned piglets are shown in Table 3. Inclusion of OSOs increased the activity of total superoxide dismutase (T-SOD) in jejunum, while the T-SOD activity in the jejunum of weaned piglets was increased by RES supplementation (P> 0.05). Dietary RES supplementation in the diets supplemented with OSOs tended to decrease the level of H₂O₂ in the plasma and increase the contents of H₂O₂ in weaned piglets when diets supplemented with FSOs (interaction, P= 0.015). The glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) level, and total antioxidant capacity (T-AOC) were not influenced by inclusion of OSOs or RES (P> 0.05). Furthermore, the genes related to antioxidant in the intestine were measure as shown in Fig. 2. We found that the mRNA expression of SOD2 in the jejunum and the mRNA expression of SOD2 and GPX1 in the colon were decreased by inclusion of OSOs, while dietary RES supplementation increased the mRNA expression of SOD2 in the jejunum and the mRNA expression of GPX1 in the colon of weaned piglets (P< 0.05), indicating that the oxidative stress state induced by OSOs was effectively alleviated by dietary RES.

Furthermore, considering that the oxidative stress of the organism could induces the production of inflammatory, we further evaluated the inflammatory state of piglets^{10, 11}. As shown in Fig. 4A, dietary RES supplementation in the diets supplemented with OSOs decreased the level of IL-1β, IL-6, IL-8, and TNF-α (P< 0.05) in the plasma of weaned piglets, but failed to influence the IL-1β, IL-6, IL-8, and TNF-α level when diets supplemented with FSOs (interaction, P< 0.01). As shown in Fig. 2, the mRNA expression of pro-inflammatory cytokines, including IL-1β and TNF-α in the jejunum of weaned piglets was increased by inclusion of OSOs in diets (P< 0.05). The mRNA expression of TNF-α and NF-κB were decreased by dietary RES supplementation (P< 0.05). Dietary RES supplementation increased the mRNA expression of IL-10 in the jejunum of weaned piglets induced by OSOs (P< 0.05). The mRNA expression of TLR4 in the colon was decreased by dietary RES supplementation (P< 0.05). There was an interaction between OSOs and RES on IL-1β in the colon of weaned piglets. Dietary RES supplementation in the diets supplemented with OSO tended to decrease the mRNA expression of IL-1β in the colon of weaned piglets but failed to influence the mRNA expression of IL-1β when diets supplemented with FSOs (interaction, P= 0.029).

Intestinal inflammatory factors affect the function of tight junction protein, thus damaging the integrity of intestinal mucosa, while DAO activity and D-lactic content in plasma are important indicators of intestinal mucosal injury and permeability changes^{28, 29}. Therefore, we further studied the effects of OSO on the DAO activity and D-lactate content in the plasma of weaned piglets. The results of intestinal permeability biomarkers in plasma of weaned piglets are shown in Fig. 4B. Inclusion of OSOs in the diets increased the DAO activity in the plasma of weaned piglets, the DAO activity and D-lactate content in plasma of weaned piglets were decreased by dietary RES (P> 0.05).

We further investigated the effects of dietary including OSOs on the intestinal barrier of weaned piglets. The expression of tight junction proteins in the jejunum and colon were detected (Fig. 2). The mRNA expression of ZO-1 in the jejunum and Occludin in the colon were decreased by dietary including OSOs of weaned piglets (P< 0.05). Dietary RES increased the mRNA expression of ZO-1and Occludin in jejunum and colon of weaned piglets (P< 0.05). But the mRNA expression of Claudin-5 and Claudin-6 in jejunum and colon were not influenced by inclusion of OSOs or RES (P> 0.05).

The results of immunofluorescence in jejunum and colon of weaned piglets are shown in Fig. 5. Inclusion of OSOs in the diets decreased the expression of Ki67 protein in the jejunum of piglets, the expression of ZO-1 in the jejunum of weaned piglets was increased by RES supplementation (P> 0.05). Inclusion of OSOs in the diet tended to decrease the expression of Occludin protein in the jejunum of weaned piglets (P= 0.055), the expression of Occludin in the jejunum of weaned piglets tended to increase by RES supplementation (P= 0.056). Dietary RES supplementation in the diets supplemented with OSOs failed to influence the expression of ZO-1 in the colon of weaned piglets but increased the expression of ZO-1 when diets supplemented with FSOs (interaction, P= 0.037).

Dietary RES affects the changes of gut microbiota and short-chain fatty acids (SCFAs) in the colon of inclusion of OSOs in the diets of weaned piglets

As is shown in Fig. 6A. We observe that inclusion of OSOs in diets decreased the abundance of Tenercutes (P< 0.05). Dietary RES supplementation increased the abundance of Firmicutes (P< 0.05), and decreased the abundance of Bacteroidetes (P< 0.05). In addition, there was an interaction between inclusion OSOs or RES on the abundance of Actinobacteria in the colon of weaned piglets. Dietary RES in the diets supplemented with OSOs increased the abundance of Actinobacteria (P= 0.018) in the colon of weaned piglets but failed to influence the abundance of Actinobacteria when diets supplemented with FSOs (interaction, P= 0.005). As shown in Fig. 6B, the gut microbiota composition at the genus level was further analyzed, the most dominant taxon was Prevotella_9 in all samples. Inclusion of OSOs in the diets increased the abundance of Parabacteroides in the colon of weaned piglets (P< 0.05). Dietary RES supplementation decreased the abundance of g_Clostridium_sensu_stricto_6 and PrevotellaceaeUCG003 in the colon of weaned piglets (P< 0.05). There was an interaction between OSOs and RES treatment for the abundance of Atopobiaceae_unclassified and Mollicutes_RF39_unclassified (P< 0.05). Dietary RES supplementation in the diets supplemented with OSOs tended to increase the abundance of Atopobiaceae_unclassified but tended to decrease the abundance of Atopobiaceae_unclassified in the colon of weaned piglets when diets supplemented with FSOs (interaction, P= 0.016). Dietary RES supplementation in the diets supplemented with OSOs failed to influence the abundance of Mollicutes_RF39_unclassified (P= 0.007) but increased the abundance of Mollicutes_RF39_unclassified when diets supplemented with FSOs (interaction, P= 0.027). The alpha-diversity results of weaned piglets were not influenced by inclusion of OSOs or RES (Supplementary Table 3) (P> 0.05).

One of the microbial metabolites is SCFAs, which play an important role in the intestinal health as an energy source for the host³⁰. Therefore, we further investigated the effects of dietary RES on SCFAs in colon contents of weaned piglets including OSOs in the diet. The level of SCFAs in the colon of weaned piglets are shown in Fig. 4C, inclusion of OSOs in the diet decreased the level of acetic acid and butyric acid in the colon of weaned piglets (P< 0.05). The level of acetic acid in the colon of weaned piglets was increased by dietary RES (P> 0.05). Dietary RES supplementation in the diets supplemented with OSOs increased the level of butyric acid in the colon of weaned piglets but failed to influence the level of butyric acid when diets supplemented with FSOs (interaction, P= 0.027). The mRNA expression of G-protein-coupled receptor-41 (GPR41) was increased by RES in the colon of the weaned piglets (P< 0.05), but the mRNA expression of G- protein-coupled receptor-43 (GPR43) was not influenced by inclusion of OSOs or RES (Fig. 2B) (P> 0.05).

Changes in gut microbiota mediate the apparent digestibility, intestinal histomorphology, antioxygenic properties, inflammatory cytokines, and intestinal enzymatic activities in weaned piglets

Diet is an important regulator of gut microbiome composition and diversity, which has beneficial effects on the host by regulating oxidative stress and metabolism³¹. RDA analyses were performed to identify the relationship among the identified differential genera of microbiota, weaned piglets’ performance, and treatments (Fig. 7). We found that weaned piglets without RES supplementation were all separately clustered in the diarrhea score, while the weaned piglets with RES supplementation were not well clustered, which indicated that dietary RES supplementation is beneficial to reduce the diarrhea score of weaned piglets. The weaned piglets RES supplementation was aggregated near the EE, lipase, α-amylase, chymotrypsin, while the aggregation was not well in no RES supplementation weaned piglets. Indicating that dietary RES supplementation is beneficial to increase the lipase and EE. The weaned piglets RES supplementation was aggregated near the T-SOD, H₂O₂, indicating that dietary RES supplementation is beneficial to the improvement of the intestinal antioxidant capacity. The weaned piglets RES supplementation was aggregated near the IL-1β, IL-6, IL-8, and TNF-α, indicating that dietary RES supplementation is beneficial to the recovery of the inflammatory state. The weaned piglets RES supplementation was aggregated near the acetic acid, butyric acid, and propionic acid. while the DAO and D-lactic aggregation were in weaned piglets NO RES. Furthermore, according to the Pearson correlation analyses, several key genera that were separately correlated with growth performance, nutrient apparent digestibility, intestinal histomorphology, antioxygenic properties, inflammatory cytokines, intestinal enzymatic activities, and SCFAs were also further identified and are listed in Table 4. Based on Pearson correlation analysis and RDA analysis, several different genera in the four treatment groups may affect the intestinal digestive enzyme activities, apparent digestibility, and pro-inflammatory factors. Specifically, of these genera, Prevotellaceae_UCG-003 and Prevotella_1 were negatively correlated with the increase of EE in weaned piglets. The abundances of Prevotellaceae_UCG_003 were negatively associated with the changes in α-amylase. The abundance of Prevotella_1 was positively associated with the changes in diarrhea rate13-16d. The abundance of Atopobiaceae_unclassified was negatively correlated with the increase of IL-6 in the plasma of weaned piglets.

Discussion

Intestinal morphology, an important indicator of intestinal health, is usually evaluated according to VH, CD, and VCR^{32, 33}. This study revealed that OSOs in the diet decreased the VCR in the colon and that dietary RES effectively alleviated gut morphology of weaning piglets, including VH and VCR in the jejunum of the weaned piglet. Previous studies have reported that RES restored damaged villus-crypt structure and improved gut absorption in pigs and broilers^{18, 34}. In this study, the lipase, chymotrypsin, and lactase activities were observed to decrease due to OSOs in the diets, and dietary RES increased the activities of lipase, chymotrypsin, and α-amylase in the jejunum of weaned piglets, indicating that supplementation of RES alleviated nutrient digestibility of weaned piglets fed diets including OSOs by restoring villus-crypt structure and enhancing digestive enzymes. In addition, as the gut is vulnerable to oxidative damage, lipid peroxidation products ingested by animals could often disrupt the redox balance and induce inflammation, resulting in a lack of antioxidant systems in the gut and other tissues^{35, 36}. According to this study, dietary RES further decreased the T-SOD activity in the plasma of weaned piglets induced by OSOs. SOD catalyzes the reduction of superoxide anion to hydrogen peroxide, which is further decomposed into water and oxygen by catalase, protecting cells from oxidative damage and scavenging free radicals in the body to maintain cell energy metabolism³⁷. Dietary RES with OSOs reduced the H₂O₂ in the plasma of weaned piglets, a major ROS synthesized by a mutational reaction catalyzed by SOD and reduced to water by GSH-Px or CAT. In addition, as a central redox signaling molecule, H₂O₂ induced oxidative stress in the gut epithelial^{38, 39}. Therefore, the inhibition of H₂O₂ level and the increase of T-SOD activity could be the reasons why dietary RES alleviated the beneficial effects of oxidative stress in weaned piglets fed diets with OSOs. Besides, dietary RES increased the SOD2 mRNA expression in the jejunum. Previous studies have also observed that RES improved the activities of various antioxidant enzymes^{39, 40}. In this study, OSOs increased the activity of DAO, while RES decreased the DAO activity and D-lactate content in the plasma of weaned piglets. D-lactic and DAO, chemical markers of gut permeability, were released into the bloodstream when the intestinal barrier function was impaired^{28, 29}. In addition, OSOs in the diet of weaned piglets decreased the mRNA expression of Occludin in the colon and ZO-1 in the jejunum and colon. The protein expression of Occludin in the jejunum of weaned piglets tended to decrease due to the inclusion of OSOs in the diet, while RES increased the protein expression of ZO-1 in the jejunum of weaned piglets. The protein expression of ZO-1 was in accord with the mRNA expression of ZO-1.

Similarly, previous studies demonstrated that the pretreatment of cultured cells with RES prevented hydrogen peroxide-induced epithelial barrier damage in vitro⁴¹. Involved in the assembly and stability of tight junctions, Occludin and ZO-1 were proteins with the best barrier function⁴². Ki-67 was a specific and reliable marker of cell proliferation⁴³, and OSOs decreased its protein expression in the jejunum of weaned piglets. Therefore, dietary RES with OSOs improved gut permeability, which might enhance ZO-1 and Ki-67 expression. These results further confirmed that RES improved the gut barrier damage of weaned piglets containing OSOs in the diets.

The gut microbiome largely affects the health and survival of the host by regulating nutrient metabolism, immune system maturation, and gut barrier establishment^{31, 44, 45}. Based on previous studies, body weight and gut microbial ecology were correlated. Obese subjects had an increased proportion of Firmicutes and a decreased proportion of Bacteroidetes⁴⁶, indicating that RES could promote the growth of weaned piglets by raising the Firmicutes/Bacteroides ratio. However, OSOs and RES failed to affect the growth performance of weaned piglets, probably because only 7 repeated sample sizes in this study (n= 7) were suitable for testing the effects of RES on gut barrier function and immune response in weaned piglets with diets including OSOs. Continuous testing should be conducted on more piglets to specify the growth performance. In our study, OSOs decreased the abundance of Tenericutes in the colon of piglets. Previous studies showed that oxidative stress reduced the abundance of Tenericutes in rats, which were associated with increased apparent digestibility of crude fiber in animals, and that healthy people had higher levels of Tenericutes than metabolically disturbed individuals^{47, 48, 49}. Moreover, dietary RES supplemented with OSOs raised the abundance of Actinobacteria but failed to work when FSOs were added to the diets. Actinobacteria as part of the normal gut microbiota were rarely observed in the gut microbiota of different gastrointestinal diseases⁵⁰. From these results, dietary RES supplementation alleviated some gastrointestinal diseases caused by the oxidative stress-induced decrease of Tenericutes abundance in weaned piglets via increasing the abundance of Actinobacteria. Interestingly, OSOs and RES exposure failed to affect the alpha-diversity of the gut microbiome of piglets, suggesting similar ecological diversity of the colonic microbiota of piglets after different treatments, which was consistent with the previous research⁴⁰.

In this study, dietary RES decreased colonic abundance of the Clostridium_sensu_stricto_6 of weaned piglets, identified as a common bacteria leading to diarrhea^{51, 52}. OSOs decreased the abundance of Parabacteroides in the intestines of piglets. The previous study demonstrated that Parabacteroides reduced the metabolic dysfunction of mice on a high-fat diet by converting bile acids and generating succinic acid⁵³. Furthermore, our study found that RES increased the apparent fat digestibility of EE in weaned piglets, which could be related to the increase of bile secretion⁵⁴. Therefore, we hypothesized that RES increased the apparent digestibility of EE in weaned piglets due to changes in the abundance of Paracteroides. Prevotella, an active hemicellulose-decomposing bacterium, is necessary for degrading plant non-fiber polysaccharides and proteins^{55, 56}. Based on our work, dietary RES decreased the abundance of Prevotella-1 and Prevotellaceae UCG003 in the colon of weaned piglets. However, increasing evidence suggested that Prevotella was also related to inflammatory conditions in the intestine^{57, 58}. Several studies reported that Prevotella could stimulate epithelial cell production of IL-8, IL-6, and chemokine ligand 20 (CCL20) and promote mucosal t-helper cell (Th17) immune response and neutrophil recruitment. Besides, Prevotella was positively correlated with IL-1β, IL-6, and TNF-α^{59, 60}. Previous studies concluded that dietary RES alleviated gut inflammation and decreased the concentration of IL-6 and TNF-α in the jejunum of piglets⁵⁵ while we observed that dietary RES supplementation with OSOs decreased the levels of IL-1β, IL-6, IL-8, and TNF-α in the plasma of weaned piglets. In addition, dietary RES supplementation in the diets including OSOs tended to increase the abundance of Atopobiaceae_unclassified, which was negatively correlated with IL-6 in the plasma of weaned piglets. Additionally, it was suggested that inhibition of C-C chemokine motif ligand 4 could increase intestinal Atopobiaceae abundance, inhibiting pro-inflammatory metabolites and reducing systemic inflammation⁶¹. These results demonstrated that RES might exert its anti-inflammatory effects by reducing the abundance of the colonic content species Prevotella-1, Prevotellaceae UCG003, and Atopobiaceae_unclassified to balance the cytokines in the plasma. Interestingly, the abundance of Prevotella_1 and Prevotellaceae_UCG-003 were negatively correlated with EE digestion. Meanwhile, the abundance of the former was positively correlated with the change of diarrhea rate from 13 to 16 days, while the latter's abundance was negatively correlated with the change of α-amylase in weaned piglets. It was also proved that hosts with Prevotella as the dominant gut flora were negatively correlated with feed efficiency and weight^{62, 63}. However, a positive correlation was found between the compromised Prevotella intestinal flora, body weight, and average daily gain of pigs⁶⁴. Therefore, we hypothesized that these conflicting studies might be different with strains associated with specific Prevotella spp. or positive and negative growth outcomes and that the nature of RES seemed to be different sometimes. In addition, we found that the dietary RES supplemented with OSOs failed to influence the abundance of Mollicutes_RF39_unclassified, which only increased when diets were supplemented with FSOs. According to reports, Mollicutes was associated with prostate cancer and bovine respiratory disease^{65, 66}. Conversely, several scholars reported RES and its analogs as an effective miRNA-mediated chemopreventive and therapeutic strategy in prostate cancer^{67, 68}. The contradictory results could be related to the dose of RES. RES modulate immune was reported to function in a dose-dependent manner, with low doses stimulating the immune system and high doses inducing immunosuppression, while faulty immune responses played a pathogenic role in a wide range of inflammatory diseases⁶⁹.

Recent years saw the emerging links between early gut flora imbalance and host disease risk. SCFAs were major final products of indigestible carbohydrates produced by microorganisms through fermentation, functioning as ligands for various immune responses, activating/targeting cell receptors expressed on specific host tissues and immune cell membranes⁷⁰. OSOs reduced acetic acid, and dietary RES increased acetic acid and butyric acid levels in the colon of weaned piglets. Past research demonstrated that dietary RES could increase butyric acid and isobutyric acid levels in the intestines of mice⁷¹, while butyric acid could alleviate gut inflammation by reducing IL-6 and IL-1 β levels and increasing IL-10 levels in broilers⁷². This study discovered that diets including OSOs enhanced the expression of mRNA pro-inflammatory cytokines such as IL-1β and TNF-α in the jejunum of weaned piglets, consistent with the previous results⁵⁸. Dietary RES decreased TNF-α and NF-κB mRNA expression in the jejunum and TLR4 mRNA expression in the colon. The mRNA expression of IL-1β and TNF-α was directly or indirectly related to the activation of the NF-κB pathway⁷³. SCFAs were used as histone deacetylase (HDAC) inhibitors to attenuate inflammatory responses by blocking the NF-κB signaling pathway^{74, 75}. Therefore, we hypothesized that RES might alleviate gut inflammation caused by OSOs-induced NF-κB pathway by regulating SCFAs. Besides, SCFAs played an important role in gut health, acted as an energy source for the host⁷⁶, and were proved to promote adipocyte differentiation in porcine adipose tissue⁷⁷. Nevertheless, our study noticed that dietary RES increased the expression of FABP1 in the jejunum and the expression of FABP1 PPARγ, and CD36 mRNA in the colon of weaned piglets. FABP1 was significant in the normal lipid metabolism of differentiated gut epithelial cells, especially the uptake and basolateral secretion of fatty acids⁷⁸. Meanwhile, the CD36 receptor, as a multifunctional membrane receptor, effectively promoted the ability of fatty acid uptake and improved lipid metabolism⁷⁹. Furthermore, a clinical study revealed that propionic acid and butyric acid could promote the formation of pig adipocytes and increase the expression of PPAR-γ, a transcriptional factor crucial for controlling lipid homeostasis⁸⁰, as well as CEBPA mRNA in a stromal vascular fraction⁷⁷. Our previous study demonstrated that maternal dietary RES increased fat content in longissimus Pectoris and milk^{36, 40}. The inclusion of RES in the diet was reported to promote the fatty acid oxidation rate and energy release⁸⁰. In addition, SCFAs participated in various host-signaling mechanisms and were crucial in inhibiting histone deacetylases and activating G-coupled-protein receptors⁸¹. This work observed that RES increased GPR41 protein levels in piglets, which agreed with the previous research results⁸². Therefore, we hypothesized that RES could affect the mRNA expression of FABP1, CD36, and PPARγ by increasing the level of short-chain fatty acids, thus alleviating the effect of OSOs on the energy metabolism of weaned piglets.

Conclusions

In conclusion, dietary RES improved the villus-crypt structure and digestive enzyme activities of jejunum, thus reducing intestinal barrier damage in weaned piglets with diets including OSOs. The dietary RES supplementation increased the antioxidant status in plasma, thereby increasing the antioxidant status of weaned piglets. In addition, RES may alleviate OSOs immune status and energy metabolism by affecting gut microbiota and its metabolite SCFAs. Notably, the positive effect of RES on OSOs was related to the decrease in the abundance of Prevotella_1 and Prevotellaceae_UCG-003.

Abbreviations

RES: resveratrol; FSOs: fresh soybean oils; OSOs: oxidized soybean oils; ADFI: average daily feed intake; ADG: average daily gain; F/G: average daily feed intake/average daily gain; DM: dry matter; CP: crude protein, EE: crude fat; GE: gross energy; VH: villus height; CD: crypt depth of the jejunum. VCR: villus/crypt ratio; DAO: diamine oxidase; T-SOD: total superoxide dismutase; GSH-Px: glutathione peroxidase; H2O2: hydrogen peroxide; T-AOC: total antioxidant capacity; MDA: malondialdehyde; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-8, interleukin 8; IL-10, interleukin 10; IL-17, interleukin 17; IL-22, interleukin 22; TNF-α, tumor necrosis factor α; NF-κB, nuclear factor κB; SCFAs: Short-chain fatty acids; ZO-1: Zonula occludens-1; GPR41: G-protein-coupled receptor-41; GPR43: G-protein-coupled receptor-41; PPARγ: peroxisome proliferator-activated receptor-γ; FABP1: fatty acid-binding protein-1; FABP4: fatty acid-binding protein-4; CD36: fatty acid transport protein.

Declarations

Acknowledgments

The authors thank all the participants and staff of this trial for their valuable contributions.

Funding

This work was supported by the National Natural Science Foundation of China (31872986).

Availability of data and material

The datasets produced and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

The author contributions are as follows: B. S., Q. M., and Y. G. conceived and designed the experimental plan. Y. G., Q. M., X. S. and Q. Z. were involved in the animal experiments, analysis and data collection.Y. G., Q. M., X. S. and Q. Z. analyzed the data and drafted the original manuscript. X. S. carried out the additional experiment and collected the data. Y. G. and Q. M. made a revision of this manuscript. All authors read and approved the final manuscript.

Competing interests

None of the authors had any personal or financial conflict of interest.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The protocols used in this experiment were approved by the Northeast Agricultural University Institutional Animal Care and Use Committee. All animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China.

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