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mucosal and systemic inflammatory reactions [1, 6, 7]. Introduction The secretion of pro-inflammatory cytokines during the Intestinal epithelial barrier, a structure of continuous inflammatory response is a major factor in triggering the monolayer enterocytes, is dominated by intercellular disruption of the intestinal barrier . In piglets, intesti- tight junction . Under a normal state, it acts as a selec- nal barrier dysfunction and accompanied by the enhance- tive filter that enables the absorption of nutrients and ment of intestinal permeability are often observed, with ensures an effective defense against exogenous patho - subsequent diarrhea and growth retardation [9, 10]. gens, luminal antigens, etc. . For early weaned mam- Therefore, effective and safe preventive approaches to mals, however, the gastrointestinal tract is immature and maintenance of intestinal barrier function are urgently vulnerable to multitudinous stresses , and intestinal needed for piglets. Notably, lipopolysaccharide (LPS), epithelial barrier is frequently defective in various patho- an intrinsic component of the cell wall of gram-negative logical status, especially in bacterial infections induced bacteria, has been shown to be a key molecule in induc- by pathogenic bacteria [4, 5]. The damaged intestinal ing the production of pro-inflammatory cytokines . It epithelial barrier mainly causes the increase of intestinal often contributes to inflammatory intestinal damage in permeability, promotes the transfer of antigens in lumen piglets. The molecular mechanism is manifested by the to the subepithelial tissues, and further exacerbates the Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 3 of 15 activation of inflammatory signaling pathway by LPS, Experimental animals, diet, and design which induces the expression of key proteins of pro- The animal procedures were reviewed and approved by inflammatory factors and thus leads to intestinal barrier the Animal Care and Use Committee at Sichuan Agri- damage . Previous studies have shown that LPS can cultural University with the approval number of SYXK be used to construct a well-established model of inflam - (Sichuan)-2019–187. A total of twenty-four weaned mation in pigs [12, 13]. piglets (21-day-old), with an average initial body weight Yeast (Saccharomyces cerevisiae) is widely distributed (BW) of 7.42 ± 0.34 kg, were randomly allotted to two in nature and has been inseparable from human life groups (12 replicates per treatment and one piglets per . Yeast hydrolysate (YH), also known as yeast autol- replicate) receiving either a basal diet or a basal diet with ysate, is obtained from Saccharomyces cerevisiae via YH (5 g/kg) for 21 d. All piglets were fed individually in protein hydrolysis enzyme [15, 16]. Several researches metabolic cages (1.5 m × 0.7 m × 1.0 m) and housed in suggested that the autolytic yeast fractions or peptides an environmentally controlled room. Piglets were fed from autolyzed yeasts revealed physiological effects on three times daily at 8:00, 14:00 and 20:00 and have free anti-obesity [17, 18], anti-fatigue , anti-stress [20, 21] accessed to water and feed throughout the experiment. and immuno-promotional activities [22, 23]. For these After 21-d feeding trial, immunological challenge was reasons, YH has attracted much attention as a functional applied to the half of piglets in each treatment (Fig. 1A). material supplement and it is generally recognized as That means 6 piglets in each treatment were intraperi - non-toxic, effective and safe . Recently, several stud - toneally injected with LPS at 150 μg/kg BW, and the ies were focused on improving intestinal health and other 6 piglets were injected with an equal volume of immune-potentiating activities with YH. Specifically, YH sterile physiological saline. The basal diet (Table 1) was a has multiple roles on promoting digestion and absorp- corn-soybean meal-fish meal diet and was formulated to tion of nutrients , improving intestinal microflora meet or exceed National Research Council (NRC 2012) structure  and decreasing diarrhea of young animals  requirements for piglets from 7–11 kg and 11–25 kg , while also acts as an immunomodifier to prevent stage. The YH diet was formulated by replacing soybean gut inflammation . However, the protective effects meal with 5 g/kg YH in equal amounts in the basal diet. of dietary supplementation with YH on intestinal bar- The molecular weight of YH was less than 50 kDa and rier are limited and inconclusive. It is widely known mostly clustered below 25 kDa (Fig. S1). YH mainly pro- that hyperinflammation in intestine is one of the most vided a rich source of crude protein (45.50%), and con- factors causing intestinal barrier dysfunction [1, 2, 10]. tributed less to crude ash (6.47%) and crude fat (2.17%) Consequently, considering the above, we postulated that (Table S1). YH has the potential to prevent LPS-induced intestinal barrier dysfunction in piglets by alleviating inflamma - LPS injection tion via the related signaling pathways. This study tested The challenged piglets were intraperitoneally injected these hypotheses by assessing the effects of YH on the with Escherichia coli LPS (E. coli serotype O55:B5, Sigma systemic inflammatory response, intestinal morphology, Chemical Inc., St. Louis, MO, USA) at 150 μg/kg BW, expression of tight junction-related proteins and gut anti- and unchallenged piglets were administrated the same inflammatory capacity in piglets. volume of sterile physiological saline. The dose and serotype of LPS used in this study was consistent with Materials and methods the previous reports [12, 13]. Previous experiments Chemical analysis of yeast hydrolysate have presented that LPS injection particularly caused Yeast hydrolysate was provided by Jiangmen Thealth Bio - dramatic inflammatory response and intestinal barrier engineering Co., Ltd. (Guangzhou, China). The contents dysfunction in pigs, rats and mice. And these negative of moisture, crude protein, crude fat and crude ash were effects generally occurred within 3–6 h after LPS injec - measured with the reference of AOAC . Gross energy tion [7, 32]. Therefore, blood and intestinal samples was determined by an oxygen bomb calorimeter (Parr in this study were collected 4 h following LPS or saline instruments, Moline, IL, USA). The soluble protein in injection. yeast hydrolysate was extracted according to the method of Wang et al.  and fractionated by sodium dodecyl Growth performance sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) Yeast hydrolysate treatment was a main factor prior to system according to the previous study . The gel was the LPS challenge. Piglets were weighted individually on st nd stained with Coomassie Brilliant Blue (Beyotime, Shang- 1 d and 22 d of the experiment. Daily feed consump- hai, China) for 40 min and de-stained with deionized tion was recorded for each piglet. Average daily gain and water for 8 h. the ratio of feed intake to gain were calculated as well. Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 4 of 15 Fig. 1 Dietary yeast hydrolysate supplementation improved the growth performance of weaned piglets before LPS challenge. A Schematic of the feeding experiment and LPS challenge. B–D Eec ff ts of dietary YH supplementation on average daily feed intake (ADFI), average daily gain (ADG) and the ratio of feed intake to gain (F/G) of weaned piglets. Control, piglets were fed with a basal diet; YH, piglets were fed with a YH containing diet, * ** 5 g/kg. P < 0.05, P < 0.01, compared with control group Blood sample collection and analysis analyzed and calculated by Image Pro Plus 6.0 software Four hours following LPS and saline injection, blood sam- (Media Cybernetics, Bethesda, MD, USA). Other sec- ples were collected in 10 mL vacutainer tubes via anterior tions were stained using immunofluorescence for TLR4 vena cava. Blood was centrifugated at 3500 × g for 10 min protein. Briefly, mouse anti-TLR4 monoclonal antibody at 4 ℃. Serum samples were stored at −20 ℃ until sub- (1:100, sc-293072, Santa Cruz, Dallas, TX, USA) was sequent analysis for inflammatory markers and diamine incubated overnight at 4 ℃. Corresponding secondary oxidase (DAO) concentrations. Commercially available antibody (Cy3 conjugated Goat Anti-mouse IgG, 1:300, porcine ELISA kits (Chenglin Biological Technology Co., GB21301 from Servicebio, Wuhan, China) was incu- Ltd., Beijing, China) were performed according to the bated for 50 min at room temperature. The slides were manufacturer’s instructions for the following indicators: washed three times with PBS, and then incubated with adrenocorticotropic hormone (ACTH), cortisol, C-reac- DAPI solution at room temperature for 10 min and tive protein (CRP), serum amyloid A (SAA), haptoglobin stored in the dark. After immunofluorescence, micro - (HP), tumor necrosis factor-α (TNF-α), interleukin-1β (IL- photographs were acquired with an inverted microscope 1β) and DAO in serum. (Leica DMI400B, Wetzlar, Germany). In addition, the inner wall of the middle jejunum was washed with ice- cold saline and the mucosal samples were then scraped Intestinal samples collection and analysis into a sterile tube. Mucosal samples were immediately Piglets were euthanized with pentobarbital sodium placed into liquid nitrogen and stored at −80℃ until the (200 mg/kg) in a separate sampling room away from analysis of genes and proteins expressions. About 0.5 g other animals. The intestine was immediately removed. of frozen jejunal mucosal scrapings were homogenized A 2-cm segment was removed from mid-jejunum and in ice-cold saline and prepared into a 10% homogenate, fixed with 4% paraformaldehyde solution. Paraffin crushed using an ultrasonic cell crushing system at 4 °C embedding was used to cut into cross sects (5 μm thick). and then centrifuged (3000 × g, 15 min, 4 °C). The col - The jejunal morphology was determined by hematoxylin lected supernatant was used to analyze TNF-α and IL-1β and eosin (H&E) stain. Intestinal morphological images contents by ELISA kits according to the manufacturer’s were photographed with a Nikon TS100 microscope instructions. (40 × and 100 ×). Villus height and crypt depth were Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 5 of 15 Table 1 Composition and nutrients levels of the basal diet (air- Table 2 Primer sequences used for real-time PCR dry basis, %) Gene Primer sequence (5’ →3’) Product Accession number length, bp Ingredients Content, % Nutrient level Content β-actinF: TCT GGC ACC ACA CCT TCT 114 DQ178122 Corn 31.42 Digestible energy, 14.73 MJ/kg R: TGA TCT GGG TCA TCT TCT CAC Extruded corn 30.00 Crude protein, % 18.50 ZO-1F: CAG CCC CCG TAC ATG 114 XM_005659811 Soybean meal 9.94 Ca, % 0.75 GAG A Extruded soybean 8.00 Total P, % 0.56 R: GCG CAG ACG GTG TTC Fish meal 4.00 Available P, % 0.37 ATA GTT Whey powder 5.00 Digestible Lys, % 1.30 OCCF: CTA CTC GTC CAA CGG 158 NM_001163647.2 Soybean protein 5.00 Digestible Met, % 0.38 GAA AG concentrate R: ACG CCT CCA AGT TAC Soybean oil 1.90 Digestible Thr, % 0.77 CAC TG Sucrose 2.00 Digestible Trp, % 0.21 ClDN-1F: TCT TAG TTG CCA CAG CAT 106 NM001244539 GG Limestone 0.85 R: CCA GTG AAG AGA GCC Dicalcium phosphate 0.45 TGA CC NaCl 0.20 MUC2F: GGT CAT GCT GGA GCT GGA 181 XM_003122394.1 L-Lys·HCl (78%) 0.33 CAGT DL-Met (99%) 0.07 R: TGC CTC CTC GGG GTC L-Thr (98.5%) 0.03GTC AC L-Trp (98%) 0.01 IL-1βF: CAG CTG CAA ATC TCT CAC 112 NM_214055.1 CA Chloride choline 0.15 1R: TCT TCA TCG GCT TCT CCA Vitamin premix 0.05 CT Mineral premix 0.30 TNF-αF: CGT GAA GCT GAA AGA CAA 121 NM_214022.1 Benzoic acid 0.30 CCAG Total 100.00 R: GAT GGT GTG AGT GAG GAA 1 AACG Vitamin premix provided the following per kg of diet: VA 9000 IU, VD 3000 IU, VE 20.0 IU, VK 3.0 mg, VB 1.5 mg, VB 4.0 mg, VB 3.0 mg, VB 0.02 mg, nicotinic 3 1 2 6 12R: CAG GCT TCC GTC ATC acid 30.0 mg, pantothenic acid 15.0 mg, folic acid 0.75 mg, biotin 0.1 mg TGG TT Mineral premix provided the following per kg of diet: Fe (FeSO ·H O) 100.0 mg, 4 2 CLDN-1 Claudin-1, OCC Occludin, MUC2 Mucin2, IL-1β interleukin-1β, TNF-α Cu (CuSO ·5H O) 6.0 mg, Zn (ZnSO ·H O) 100.0 mg, Mn (MnSO ·H O) 4.0 mg, I 4 2 4 2 4 2 Tumor necrosis factor-α, ZO-1 Zonula occludens-1 (KI) 0.14 mg, Se (Na SeO ) 0.3 mg 2 3 Nutrients levels were calculated values assay kit (Pierce, Rockford, IL, USA). Then, protein was transferred to polyvinylidene fluoride membranes using mRNA abundance analysis a wet Trans-Blot system (Bio-Rad). After blocking, mem- Total RNA was extracted from jejunal mucosa using the branes were incubated with primary antibodies: anti-TLR4 TRIZOL reagent (TaKaRa Biotechnology (Dalian) Co., Ltd., (sc-293072, Santa Cruz), anti-ZO-1 (61–7300, Invitrogen, Dalian, China). RNA integrity was verified by agarose gel MA, USA), anti-OCC (ab31721, abcam, Shanghai, China), electrophoresis. cDNA was synthesized with PrimeScript anti-TNF-α (ab6671, abcam), anti-IL-1β (sc-12742, Santa RT kit (TaKaRa). Real-time PCR was performed using Cruz), anti-NFκB-p65 (6956, CST, Cell signaling Tech- SYBR Premix Ex Taq reagents (TaKaRa) and CFX-96 RT- nology, Beverly, USA), anti-p-NFκB-p65 (3033, CST), qPCR Detection System (Bio-Rad, Hercules, CA, USA). The and anti-β-actin (sc-47778, Santa Cruz). After washing, genes of intestinal barrier and inflammatory markers related the corresponding secondary antibodies, goat anti-rab- primer pairs were synthesized by Sangon Biotech (Shanghai) bit/mouse IgG -HRP secondary antibody (sc-2030 and Co., Ltd. (Shanghai, China) and listed in Table 2. The mRNA sc-2031, Santa Cruz), were incubated at room temperature expression of target gene relative to housekeeping gene for 1 h. Visualization of membranes was performed with (β-actin) was calculated by the method of Arce et al. . the Clarity Western ECL substrate (Bio-Rad) and the ChemiDoc XRS imaging system (Bio-Rad). The β-actin Western blot analysis was applied as a controller for the mean of protein load. Western blot analysis was performed as previously described . Briefly, protein was extracted from jejunal Statistical analysis mucosa using the lysis buffer (Beyotime, Shanghai, China). Statistical analysis was performed using SAS soft- Protein concentration was measured with the BCA protein ware package (Version 9.4; S.A.S, Institute Inc., Cary, Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 6 of 15 NC, USA) . All data were expressed as mean val- caused dramatic intestinal hyperemia (Fig. 4A), induced ues with their standard error and checked for normal intestinal mucosal injury reflected by villous atrophy distribution using the Shapiro–Wilk test of SAS. Each and mucosal detachment (Fig. 4B), and decreased the piglet served as the statistical unit. Specifically, data on villus height (Fig. 4D) (P = 0.02). Compared with LPS growth performance prior to LPS challenge were ana- group, LPS + YH group attenuated the state of intestinal lyzed by two-tailed Student’s t-test. After LPS injection, hyperemia, improved the morphology and significantly data from serum and jejunum samples were statistically increased the villus height (P = 0.03). analyzed by two-way ANOVA using the PROC MIXED procedure of SAS with the following model: Eec ff ts of YH on the expression of intestinal barrier related genes in LPS‑challenged piglets y = μ + α + β + α × β + e ijk i j i j ijk Compared with saline treatment, LPS injection reduced the relative mRNA expressions of CLDN-1 (Fig. 5A), OCC where y is an observed trait, μ is the overall mean, α is ij i (Fig. 5C) and MUC2 (Fig. 5D) (P < 0.05), as well as the pro- the fixed effect of immunological challenge (i = saline or tein expressions of OCC (Fig. 5E and 5F; P = 0.01) and LPS), β is the fixed effect of dietary YH (j = 0 or 5 g/kg ZO-1 (Fig. 5E and G; P < 0.01) in jejunal mucosa. In com- YH), α × β is the interaction between LPS and YH and i j pared to LPS group, YH supplementation significantly e is the random error. Differences between the different ijk inhibited the down-regulation of mRNA expression of groups were analyzed by Duncan’s multiple comparison OCC (P = 0.02) and MUC2 (P < 0.01) and protein abun- method. P < 0.05 was considered statistically significant, dances of OCC (P < 0.01) and ZO-1 (P = 0.04) in jejunal and 0.05 < P < 0.10 indicated a trend. mucosa of LPS-challenged piglets. Eec ff ts of YH on the expression of TNF‑α and IL‑1β Results of jejunum in piglets challenged with LPS Eec ff ts of YH on growth performance in piglets prior to LPS As shown in Fig. 6, LPS injection enhanced the concen- injection trations of TNF-α (Fig. 6A; P = 0.01) in jejunal mucosa, As shown in Fig. 1, a 21-d feeding experiment was con- however, YH significantly reversed this change (P < 0.01). ducted to examine the effects of YH on growth per - A further analysis by RT-qPCR and western blot revealed formance of piglets under normal condition (Fig. 1A). that LPS challenge significantly increased the mRNA Compared with control group, dietary YH supplementa- expressions of TNF-α (Fig. 6C) and IL-1β (Fig. 6D) and the tion increased ADFI (P < 0.01) and ADG (P < 0.01) (Fig. 1B corresponding protein abundances (Fig. 6E–G; P < 0.01). and C), decreased F/G of piglets (P = 0.048) (Fig. 1D). Conversely, a down-regulation was observed in the mRNA expressions of IL-1β and the protein abundance of TNF-α and IL-1β in LPS + YH group (P < 0.01). Eec ff ts of YH on systemic inflammatory response and serum DAO concentration in piglets challenged Eec ff ts of YH on the TLR4/NF‑κB signaling pathway with LPS in LPS‑challenged piglets Results of serum acute phase protein, stress hormone and As shown in Fig. 7, we investigated the expression of TLR4, inflammatory cytokines concentrations in piglets chal - the major inflammation-associated receptor, by immuno - lenged with LPS were showed in Fig. 2 and Fig. 3. As fluorescence analysis and discovered that the distribution expected, LPS injection enhanced HP (Fig. 2B), cortisol of TLR4 in jejunum of piglets enhanced by LPS challenge (Fig. 2D), ACTH (Fig. 2E) and IL-1β (Fig. 3B) concentra- (Fig. 7A). Nevertheless, the variability was reversed with tions in serum (P < 0.01). However, LPS + YH group signifi - YH supplementation when compared to LPS group. More- cantly decreased the concentrations of HP, cortisol, ACTH over, we observed an up-regulation in the expressions of and IL-1β in serum compared with the LPS group (P < 0.05). TLR4 protein (Fig. 7B and C) and p-NFκB-p65 protein As shown in Fig. 4, compared with saline group, (Fig. 7B and D) in LPS group compared with saline group there was greater concentration of serum DAO in LPS (P < 0.01). LPS + YH group decreased the protein abun- group (P < 0.01), however, YH supplementation signifi- dances of TLR4 (P < 0.01) and p-NFκB-p65 (P = 0.01) com- cantly inhibited the increase of the serum DAO con- pared with LPS group. centration in LPS challenged piglets (P = 0.02). Discussion Eec ff ts of YH on intestinal morphology in LPS‑ challenged Yeast hydrolysate (YH), an autolysate of Saccharomyces piglets cerevisiae, has attracted much attention as a nutritional The effects of YH on intestinal morphology in LPS- additive, which involved in various biological activities challenged piglets were shown in Fig. 4. LPS challenge Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 7 of 15 Fig. 2 Dietary yeast hydrolysate supplementation inhibited the over-production of acute phase protein and stress hormone in piglets challenged with LPS. A–C Eec ff ts of dietary YH supplementation on the serum concentrations of serum amyloid A (SAA), haptoglobin (HP) and C-reactive protein (CRP) in piglets challenged with LPS. D and E Eec ff ts of dietary YH supplementation on the serum levels of stress hormones (D) cortisol and a,b,c (E) adrenocorticotropic hormone. Control, piglets were fed with a basal diet; YH, piglets were fed with a YH containing diet, 5 g/kg. Means with different superscript letters in a row were significantly different (P < 0.05) including growth promotion and immune regulation. (below 30 kDa) exhibits a low toxicity for rats . This Throughout the 21-d feeding experiment (pre-LPS-chal - hydrolysate is more accessible to animals due to its low lenge) in this study, we observed that dietary YH supple- molecular weight contributing to a higher solubility in mentation increased growth performance of piglets. This aqueous media and a better digestibility and absorptivity effect may be related to the molecular weight of YH. As . Previous studies have found that YH (below 10 kDa) shown in an animal model, the low molecular weight YH revealed physiological effects on anti-obesity [37, 38] and Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 8 of 15 Fig. 3 Dietary yeast hydrolysate supplementation attenuated the enhancement of serum concentrations of (A) tumor necrosis factor-α ( TNF-α) and (B) interleukin-1β (IL-1β) in piglets challenged with LPS. Control, piglets were fed with a basal diet; YH, piglets were fed with a YH containing a,b diet, 5 g/kg. Means with different superscript letters in a row were significantly different (P < 0.05) anti-stress [20, 21]. In this study, the molecular weight ACTH and cortisol, suggesting that YH attenuated LPS- of YH was less than 50 kDa and mostly clustered below induced stress response. To our knowledge, TNF-α and 25 kDa. Furthermore, the beneficial effects of YH on IL-1β were the critical inducers of APPs . And it is a growth performance have been characterized in rats , highly significant correlation between serum APPs and piglets , growing-finishing pigs , chickens  TNF-α levels in some infectious agents . In the pre- and fish [27, 42]. Similar results have been found in our sent investigation, LPS challenge resulted in an increase previous study that feeding YH significantly improved of TNF-α and IL-1β in serum. Undoubtedly, LPS caused growth performance for piglets under the normal physi- an excessive activation of immune system. Neverthe- ological condition and the reason might also be that YH less, YH supplementation decreased the concentrations could improve intestinal barrier function . Neverthe- of TNF-α and IL-1β in LPS-challenged piglets, which less, it remains to be studied whether YH can improve indicated dietary YH supplementation could decrease intestinal health under pathological states. In the present the systemic inflammation after LPS infection. Over- study, we focused on whether dietary YH attenuated the production of proinflammatory cytokines manufactured intestinal barrier impairment through inhibiting the mas- intestinal damage, such as villous atrophy, mucosal sive release of pro-inflammatory cytokines. Hence, we swelling, submucosal hemorrhage and exfoliation, and utilized an incontrovertible model for acute gut injure led to an increase of intestinal permeability . Several by injecting lipopolysaccharide (LPS). LPS is an intrinsic blood indictors have been used to evaluate the intestinal constituent of membranes in gram-negative bacteria and permeability. DAO, an enzyme found at high levels in it is a powerful endotoxin . It binds to TLR4 and sub- the mammalian intestinal mucosa, is a marker of matu- sequently activates downstream signaling pathways, trig- ration and integrity in response to intestinal epithelium gering an inflammatory response . . The levels of serum DAO are positively correlated Acute phase proteins (APPs) such as C-reactive pro- with intestinal permeability. In this study, YH supple- tein (CRP), haptoglobin (HP) and serum amyloid A mentation reduced DAO concentrations, indicating that (SAA) were secreted by hepatocytes and served as a YH had beneficial effects on attenuating the increase crucial role in the etiopathogenesis of immune diseases of intestinal permeability of piglets challenged with . In addition, some stress hormones (e.g., ACTH LPS. Meanwhile, intestinal permeability can usually be and cortisol) were frequently used in response to infec- assessed with intestinal epithelial barrier function . tious status in the body . With the infection and Therefore, it is possible that YH supplementation can injury of the host (such as LPS injection), the expres- mitigate systemic inflammatory associated with the sions or serum concentrations of APPs and stress hor- improvement of intestinal barrier function. mones will be dramatically enhanced [45, 46]. Indeed, Intestinal morphology is regarded as a visual reflection our results found that serum concentrations of HP, for the growth and development of gut and determined SAA, ACTH and cortisol in piglets were increased 4 h by villus height and crypt depth . This study showed after LPS-injection. However, supplementation of YH that LPS-injection induced intestinal damage including to the LPS-infected piglets reduced the levels of HP, villous atrophy, mucosal detachment, and a decrease of Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 9 of 15 Fig. 4 Dietary yeast hydrolysate supplementation attenuated the effects of LPS-injection on jejunal permeability and morphology. A Representative picture of the appearance of the intestinal tract of a piglet. B Hematoxylin and eosin section of jejunum at 40 times magnification (up) and 100 times magnification (down). C The concentrations of diamine oxidase (DAO) in serum of piglets. D–F The villus height, crypt depth and the villus height:crypt depth ratio of jejunum of piglets. Control, piglets were fed with a basal diet; YH, piglets were fed with a YH containing diet, a,b,c 5 g/kg. Means with different superscript letters in a row were significantly different (P < 0.05) villus height in jejunum of piglets. While dietary inclu- cells were analyzed in this study. Tight junctions are sion of YH could counteract the morphological changes multi-protein complexes including claudins, occludin of jejunum after LPS challenge, thereby maintaining the and ZOs, which defend against the passage of lumi- villus integrity and structure of intestinal mucosa. Con- nal antigens, pathogenic bacterium and their toxins . sidering the importance of intestinal epithelial barrier in Recently, the experimental results showed that YH inclu- intestinal function, the tight junctions between epithelial sion markedly attenuated the down-regulation mRNA Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 10 of 15 Fig. 5 Dietary yeast hydrolysate supplementation improved the barrier function of jejunal mucosa in piglets challenged with LPS. A–D The relative mRNA expression of Claudin-1 (CLDN-1), Zonula occludens-1 (ZO-1), Occludin (OCC) and mucin2 (MUC2) in the jejunal mucosa of piglets. E–G The protein abundance of OCC ZO-1 in the jejunal mucosa of piglets. Control, piglets were fed with a basal diet; YH, piglets were fed with a YH a,b,c containing diet, 5 g/kg. means with different superscript letters in a row were significantly different (P < 0.05) (See figure on next page.) Fig. 6 Dietary yeast hydrolysate supplementation inhibited the inflammatory response of jejunal mucosa in piglets challenged with LPS. A and B The concentrations of tumor necrosis factor-α ( TNF-α) and interleukin-1β (IL-1β) in the jejunal mucosa of piglets by ELISA method. C and D The mRNA expression of TNF-α and IL-1β in the jejunal mucosa of piglets. E–G The protein abundance of TNF-α and IL-1β in the jejunal mucosa of a,b,c piglets. Control, piglets were fed with a basal diet; YH, piglets were fed with a YH containing diet, 5 g/kg. Means with different superscript letters in a row were significantly different (P < 0.05) Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 11 of 15 Fig. 6 (See legend on previous page.) Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 12 of 15 Fig. 7 Dietary yeast hydrolysate supplementation decreased the protein abundance of TLR4 and p-NFκB-p65 in jejunal mucosa in piglets challenged with LPS. A Immunofluorescence staining (100 times magnification) of toll-like receptors 4 ( TLR4) in jejunum of piglets. B–D Relative protein abundance of B and C TLR4 and B and D phosphor-Nuclear factor-κB-p65 (p-NFκB-p65). Control, piglets were fed with a basal diet; YH, a,b,c piglets were fed with a YH containing diet, 5 g/kg. Means with different superscript letters in a row were significantly different (P < 0.05) Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 13 of 15 expressions of OCC, and the protein abundances of OCC which may represent a potential mechanism to deceler- and ZO-1 in jejunal mucosa of LPS-challenged piglets. ate the signaling of LPS, thereby enabling the inflamma - uTh s, dietary YH administration could maintain the tory response to be dampened. integrity of intestinal barrier by inhibiting the decrease of tight junction protein expression under immunologi- Conclusion cal stress, which might be the potential reasons that YH Dietary YH supplementation improves the growth mitigated the impairments of intestinal permeability and performance and attenuates LPS-induced intestinal morphology in LPS-injected piglets. inflammation and barrier injury. The underlying molec - It is generally accepted that cytokines are the critical ular mechanism is YH supplementation inhibits the modulators of intestinal inflammation . Over-pro - activation of TLR4/NF-κB signaling pathway induced duction of pro-inflammatory cytokines (e.g., TNF-α and by LPS, to prevent the over-production of inflamma - IL-1β) has been demonstrated to directly impair the tight tory cytokines, and thus improved the intestinal barrier junctional function of some epithelial and endocrine function. cells . Previous studies have indicated that alleviating intestinal inflammation might effectively prevent patho - Abbreviations genic bacteria and their toxins from disrupting intesti- ACTH Adrenocorticotropic hormone nal barrier function . Consequently, the reduction of ADFI Average daily feed intake intestinal pro-inflammatory cytokine concentrations ADG Average daily gain CLDN-1 Claudin-1 under stressful conditions is one of the major strategies CRP C-reactive protein to protect the intestinal barrier and mitigate intestinal DAO Diamine oxidase inflammation. In the present study, YH supplementation F/G The ratio of feed intake to gain HP Haptoglobin significantly reduced the mRNA expressions of TNF-α IL-1β Interleukin-1β and IL-1β, and the corresponding protein abundances in LPS Lipopolysaccharide jejunal mucosa in LPS challenged-piglets. Similarly, Wai-MUC2 Mucin2 NF-κB Nuclear factor-κB titu et al.  reported that piglets receiving a YH riched OCC Occludin in cell wall polysaccharides reduced TNF-α level in ileum SAA Serum amyloid A challenged by LPS. Hence, the protected effect of YH on TLR4 Toll-like receptors 4 TNF-α Tumor necrosis factor-α intestinal barrier appears to be achieved by suppressing YH Yeast hydrolysate the inflammatory response. This is also a possible reason ZO-1 Zonula occludens-1 that YH ameliorates the systemic inflammatory response. With a further view to investigating the molecular Supplementary Information mechanisms of YH in the alleviation of intestinal inflam - The online version contains supplementary material available at https:// doi. mation, we evaluated the activation of TLR4 signaling org/ 10. 1186/ s40104- 023- 00835-2. pathway. TLR4, a typical pattern recognition receptor Additional file 1: Fig. S1. SDS-PAGE analysis of yeast hydrolysate. in the TLR protein family, is widely distributed on the Additional file 2: Table S1. Chemical component of yeast hydrolysate. surface of various intestinal cells and plays an essen- tial role in LPS-mediated signaling . Mechanisti- cally, TLR4 activation triggered by LPS can induce the Acknowledgements We gratefully acknowledge our debt to the generous donation from Jiang- increased expression of downstream molecules (such as men Thealth Bioengineering Co., Ltd., and the help of Huifen Wang and NF-κB), and then enhance the expression of proinflam - Zhemin Gu in animal experiment and sample collection. matory cytokines-related genes, resulting in intesti- Authors’ contributions nal barrier damage [7, 53]. Currently, we observed that BY conceived and designed the experiment. RF and CL participated in the YH supplementation significantly decreased the pro - animal experiment, data analysis and drafting of the manuscript. DC, GT, PZ tein abundance and immunofluorescence intensity of and JH were responsible for animal care and sampling. JY and XM contributed technical support. YL and JL performed biochemical analysis. All authors have TLR4 in jejunal mucosa of piglet challenged with LPS, read and approved the final manuscript. as well as downregulated the protein levels of p-NF-κB. NF-κB, the master transcription factor for TLR4 signal- Funding This work was supported by the National Key Research and Development ing, is phosphorylated and translocated to the nucleus in Program of China (2018YFD0500605), the Key Research and Development the stimulated state, then promoting the release of pro- Program of Sichuan Province (2021YFYZ0008), and the Sichuan Pig Innova- inflammatory cytokines . Accordingly, YH supple - tion Team of National Modern Agricultural Industry Technology System of China (scsztd-2020–08-11). mentation inhibits the TLR4/NF-κB signaling pathway, Fu et al. Journal of Animal Science and Biotechnology (2023) 14:44 Page 14 of 15 15 Jung EY, Hong YH, Kim JH, Park Y, Bae SH, Chang UJ, et al. Eec ff ts of Declarations yeast hydrolysate on hepatic lipid metabolism in high-fat-diet-induced obese mice: Yeast hydrolysate suppresses body fat accumulation by Ethics approval and consent to participate attenuating fatty acid synthesis. Ann Nutr Metab. 2012;61(2):89–94. All procedures described in this study were approved by the Institutional Ani- https:// doi. org/ 10. 1159/ 00033 844. mal Care and Use Committee of Sichuan Agricultural University (No.20190129). 16. Mosser M, Chevalot I, Olmos E, Blanchard F, Kapel R, Oriol E, et al. Combination of yeast hydrolysates to improve CHO cell growth and Consent for publication IgG production. Cytotechnology. 2013;65(4):629–41. https:// doi. org/ 10. Not applicable. 1007/ s10616- 012- 9519-1. 17. Jung EY, Lee JW, Hong YH, Chang UJ, Suh HJ. Low dose yeast hydro- Competing interests lysate in treatment of obesity and weight loss. Prev Nutr Food Sci. 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Choose BMC and benefit from om: : 49 Luk GD, Bayless TM, Baylin SB. Diamine oxidase (histaminase). A circulat- ing marker for rat intestinal mucosal maturation and integrity. J Clin fast, convenient online submission Invest. 1980;66(1):66–70. https:// doi. org/ 10. 1172/ JCI10 9836. thorough peer review by experienced researchers in your ﬁeld 50. Chelakkot C, Ghim J, Ryu SH. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp Mol Med. 2018;50(8):1–9. rapid publication on acceptance https:// doi. org/ 10. 1038/ s12276- 018- 0126-x. support for research data, including large and complex data types 51. Capaldo CT, Nusrat A. Cytokine regulation of tight junctions. Biochim • gold Open Access which fosters wider collaboration and increased citations Biophys Acta. 2009;1788:864–71. https:// doi. org/ 10. 1016/j. bbamem. 2008. 08. 027. maximum visibility for your research: over 100M website views per year 52. Waititu SM, Yin F, Patterson R, Rodriguez-Lecompte JC, Nyachoti CM. Short-term effect of supplemental yeast extract without or with feed At BMC, research is always in progress. enzymes on growth performance, immune status and gut structure of Learn more biomedcentral.com/submissions weaned pigs challenged with Escherichia coli lipopolysaccharide. J Anim Sci Biotechn. 2016;7:64. https:// doi. org/ 10. 1186/ s40104- 016- 0125-5.
Journal of Animal Science and Biotechnology – Springer Journals
Published: Mar 17, 2023
Keywords: Inflammatory response; Intestinal barrier; Lipopolysaccharide; Piglets; Yeast hydrolysate
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