Xuefen Yang

Effect of Lactobacillus plantarum on diarrhea and intestinal barrier function of young piglets challenged with enterotoxigenic Escherichia coli K88

The present study was performed to investigate the preventative effect of Lactobacillus plantarum on diarrhea in relation to intestinal barrier function in young piglets challenged with enterotoxigenic Escherichia coli (ETEC) K88. Seventy-two male piglets (4 d old) were assigned to 2 diets (antibiotic-free basal diet with or without L. plantarum, 5 × 10(10) cfu/kg diet) and subsequently challenged or not with ETEC K88 (1 × 10(8) cfu per pig) on d 15 in a 2 × 2 factorial arrangement of treatments. Feed intake and BW were measured on d 15 and 18 (3 d after challenge) for determination of growth performance. On d 18, 1 piglet from each pen was slaughtered to evaluate small intestinal morphology and expression of tight junction proteins at the mRNA and protein levels while another piglet was used for the intestinal permeability test. Before and after ETEC K88 challenge, piglets fed L. plantarum had greater BW, ADG, and ADFI (P < 0.05) and marginally greater G:F (P < 0.10) compared to piglets fed the unsupplemented diet. After ETEC K88 challenge, the challenged piglets did not show an impaired growth performance but had greater incidence of diarrhea compared to the nonchallenged piglets. There was an interaction between dietary L. plantarum and ETEC K88 challenge (P < 0.05) as L. plantarum prevented the ETEC K88-induced diarrhea. Piglets challenged with ETEC K88 also had greater urinary lactulose:mannitol and plasma concentration of endotoxin, shorter villi, deeper crypt depth, and reduced villous height:crypt depth in the duodenum and jejunum and decreased zonula occludens-1 mRNA and occludin mRNA and protein expression in the jejunum (P < 0.05). These deleterious effects caused by ETEC K88 were inhibited by feeding L. plantarum (P < 0.05). There were no effects of either treatment on the morphology and expression of tight junction proteins in ileum. In conclusion, L. plantarum, given to piglets in early life, improved performance and effectively prevented the diarrhea in young piglets induced by ETEC K88 challenge by improving function of the intestinal barrier by protecting intestinal morphology and intestinal permeability and the expression of genes for tight junction proteins (zonula occludens-1 and occludin).

Protective effects of Lactobacillus plantarum on epithelial barrier disruption caused by enterotoxigenic Escherichia coli in intestinal porcine epithelial cells

Tight junctions (TJs) play an important role in maintaining the mucosal barrier function and gastrointestinal health of animals. Lactobacillus plantarum (L. plantarum) was reported to protect the intestinal barrier function of early-weaned piglets against enterotoxigenic Escherichia coli (ETEC) K88 challenge; however, the underlying cellular mechanism of this protection was unclear. Here, an established intestinal porcine epithelia cell (IPEC-J2) model was used to investigate the protective effects and related mechanisms of L. plantarum on epithelial barrier damages induced by ETEC K88. Epithelial permeability, expression of inflammatory cytokines, and abundance of TJ proteins, were determined. Pre-treatment with L. plantarum for 6h prevented the reduction in transepithelial electrical resistance (TEER) (P<0.05), inhibited the increased transcript abundances of interleukin-8 (IL-8) and tumor necrosis factor (TNF-α) (P<0.05), decreased expression of claudin-1, occludin and zonula occludens (ZO-1) (P<0.05) and protein expression of occludin (P<0.05) of IPEC-J2 cells caused by ETEC K88. Moreover, the mRNA expression of negative regulators of toll-like receptors (TLRs) [single Ig Il-1-related receptor (SIGIRR), B-cell CLL/lymphoma 3 (Bcl3), and mitogen-activated protein kinase phosphatase-1 (MKP-1)] in IPEC-J2 cells pre-treated with L. plantarum were higher (P<0.05) compared with those in cells just exposed to K88. Furthermore, L. plantarum was shown to regulate proteins of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. These results indicated that L. plantarum may improve epithelial barrier function by maintenance of TEER, inhibiting the reduction of TJ proteins, and reducing the expression of proinflammatory cytokines induced by ETEC K88, possibly through modulation of TLRs, NF-κB and MAPK pathways.

Dietary L-arginine supplementation affects the skeletal longissimus muscle proteome in finishing pigs.

Forty-eight Duroc x Landrace x Large White gilts were used to determine the relationship between proteome changes of longissimus muscle and intramuscular fat (IMF) content in arginine-supplemented pigs. Beginning at 60 kg BW, pigs were fed a corn- and soybean meal-based diet supplemented or not with 1% L-arginine until they reached a BW of 100 kg. Supplementation with 1% L-arginine did not affect the growth performance or carcass traits, while it increased IMF content by 32% (P < 0.01), it also decreased the drip loss at 48 h post-mortem and the b* meat color value at 24 h post-mortem; supplementation with 1% dietary L-arginine did not change the proportion of SFA and MUFA in muscle lipids. The proteome changes in longissimus muscle between the control and supplemented pigs showed that L-arginine significantly influenced the abundance of proteins related to energy metabolism, fiber type and structure. The increase in IMF content was positively correlated with the increased abundance of slow twitch troponin I (TNNI1) protein and negatively correlated with myosin heavy chain IIb (MyHC IIb) protein content. It is suggested that the proteome changes in longissimus muscle contributed to the greater IMF content in L-arginine supplemented pigs.

In Vitro Evaluation of Swine-Derived Lactobacillus reuteri: Probiotic Properties and Effects on Intestinal Porcine Epithelial Cells Challenged with Enterotoxigenic Escherichia coli K88.

Probiotics are considered as the best effective alternatives to antibiotics. The aim of this study was to characterize the probiotic potential of lactobacilli for use in swine farming by using in vitro evaluation methods. A total of 106 lactic acid bacterial isolates, originating from porcine feces, were first screened for the capacity to survive stresses considered important for putative probiotic strains. Sixteen isolates showed notable acid and bile resistance, antibacterial activity, and adherence to intestinal porcine epithelial cells (IPEC-1). One isolate, LR1, identified as Lactobacillus reuteri, was selected for extensive study of its probiotic and functional properties in IPEC-1 cell models. L. reuteri LR1 exhibited good adhesion to IPEC-1 cells and could inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells. L. reuteri LR1 could also modulate transcript and protein expression of cytokines involved in inflammation in IPEC-1 cells; the Lactobacillus strain inhibited the ETEC-induced expression of proinflammatory transcripts (IL-6 and TNF-α) and protein (IL-6), and increased the level of anti-inflammatory cytokine (IL-10). Measurement of the permeation of FD-4 showed that L. reuteri LR1 could maintain barrier integrity in monolayer IPEC-1 cells exposed to ETEC. Immunolocalization experiments showed L. reuteri LR1 could also prevent ETEC-induced tight junction ZO-1 disruption. Together, these results indicate that L. reuteri LR1 exhibits desirable probiotic properties and could be a potential probiotic for use in swine production.

Dietary soy isoflavone attenuated growth performance and intestinal barrier functions in weaned piglets challenged with lipopolysaccharide

This study was conducted to investigate the protective roles of soy isoflavone in weaned pigs challenged with lipopolysaccharide (LPS). A total of 72 weaned piglets (14 days of age) were randomly allotted into either 0 (control group) or 40 mg/kg soy isoflavone (ISO) supplementation group. On days 7 and 14, half of the pigs in each group were challenged with LPS. Soy isoflavone increased average daily gain (ADG) and average daily feed intake (ADFI) of piglets challenged with LPS at days 7-14 (P<0.05). The incidence of diarrhea and plasma concentrations of malondialdehyde (MDA) and endotoxin in piglets from LPS group were higher than those in control group (P<0.05). Soy isoflavone reduced the incidence of diarrhea and plasma concentrations of endotoxin in piglets challenged with LPS (P<0.05). LPS challenge decreased (P<0.05) mRNA abundances of β-defensin 2 (pBD-2), mucin (MUC-4), zona occludens 1 (ZO-1), and occludin in jejunal mucosa of piglets, and soy isoflavone upregulated (P<0.05) mRNA abundances of ZO-1 and occludin in jejunal mucosa of piglets challenged with LPS. The present results demonstrated that both p38 and TLR4 pathways in jejunal mucosa of piglets were activated by LPS challenge (P<0.05), and soy isoflavone reduced their activations (P<0.05). Collectively, our results suggested that supplementation of soy isoflavone could partly attenuate the barrier-damaged effects of LPS and improve the intestinal barrier function of weaned piglets, at least partially by inhibiting activations of p38 and TLR4 dependent pathways induced by LPS. This study provides a potential usage of soy isoflavone for alleviating intestinal barrier damages of neonates and piglets.

Effects of protein sources and levels in antibiotic-free diets on diarrhea, intestinal morphology, and expression of tight junctions in weaned piglets

This study examined effects of dietary protein sources and levels on intestinal health of 21 to 35 d-old weaned piglets fed antibiotics-free diets. A total of 150 weaned piglets (21 d of age) were allotted to 5 dietary treatment groups. Diets were formulated, based on corn-soybean meal, with different protein sources (fish meal and soy protein concentrate) to provide different dietary CP levels. Piglets within 5 dietary treatments were fed diets as follows, respectively: 1) control diet of 17% CP (control); 2) 19% CP diets formulated with more soy protein concentrate (SPC19); 3) fish meal (FM19); 4) 23.7% CP diets formulated with more soy protein concentrate (SPC23); 5) fish meal (FM23). The results showed that piglets from control group had higher ADG and lower incidence of diarrhea compared with those of other groups (P < 0.05). The incidence of diarrhea of piglets in FM19 group was lower than those from SPC23 group and FM23 group (P < 0.05). With the higher CP levels, villous height and villous height to crypt depth ratio of piglets in the duodenum and jejunum were decreased (P < 0.05), but crypt depth was increased (P < 0.05). Comparing control group and other groups, we found the expression of inflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFN-γ) were increased (P < 0.05) in the jejunum and colon of piglets, as did cystic fibrosis transmembrane conductance regulators (CFTR) in the distal colon. The relative transcript abundance of Zonula occludens-1 (ZO-1) in the jejunum, and occludin in the jejunum and ileum of piglets fed 23.7% CP diets were reduced compared with those fed control diet (P < 0.05). In conclusion, the 17% CP diet without in-feed antibiotics helped improve growth performance and relief of diarrhea of 21 to 35 d-old weaned piglets. Dietary CP level, rather than its source (either fish meal or soy protein concentrate), has more significant impacts on the growth performance and intestinal health of 21 to 35 d-old weaned piglets when fed antibiotics-free diets.

Lactobacillus plantarum Enhanced IL-22 Production in Natural Killer (NK) Cells That Protect the Integrity of Intestinal Epithelial Cell Barrier Damaged by Enterotoxigenic Escherichia coli

Interleukin (IL)-22-producing Natural Killer (NK) cells protect the gut epithelial cell barrier from pathogens. A strain of probiotics, Lactobacillus plantarum (L. plantarum, LP), was previously found by our laboratory to significantly improve the mucosal barrier integrity and function of the small intestine in pigs. However, it was unclear whether LP benefited the intestinal mucosal barrier via interactions with the intestinal NK cells. The present study, therefore, was focused on the therapeutic effect of NK cells that were stimulated by LP on attenuating enterotoxigenic Escherichia coli (ETEC)-induced the damage to the integrity of the epithelial cell barrier. The results showed that LP can efficiently increase protein levels of the natural cytotoxicity receptor (NCR) family, and the expression levels of IL-22 mRNA and protein in NK cells. Transfer of NK cells stimulated by LP conferred protection against ETEC K88-induced intestinal epithelial barrier damage in NCM460 cells. We found that NK cells stimulated by LP could partially offset the reduction in NCM460 cell monolayers transepithelial electrical resistance (TEER) caused by ETEC K88, and increase ZO-1 and occludin mRNA and protein expressions by ETEC K88-infected NCM460 cells. Furthermore, adding NK cells that were stimulated by LP to ETEC K88-infected NCM460cells, IL-22R1, p-Stat3, and p-Tyk2 expression by NCM460 cells was increased. Mechanistic experiment showed that NK cells stimulated by LP lost the function of maintaining TEER of NCM460 cells challenged with ETEC K88, when polyclonal anti-IL-22 antibody was used to block IL-22 production. Collectively, our results suggested that LP stimulation of NK could enhance IL-22 production, which might be able to provide defense against ETEC-induced damage to the integrity of intestinal epithelial barrier.

Effects of soybean isoflavone on intestinal antioxidant capacity and cytokines in young piglets fed oxidized fish oil

To investigate the effect of glycitein, a synthetic soybean isoflavone (ISF), on the intestinal antioxidant capacity, morphology, and cytokine content in young piglets fed oxidized fish oil, 72 4-d-old male piglets were assigned to three treatments. The control group was fed a basal diet containing fresh fish oil, and the other two groups received the same diet except for the substitution with the same dosage of oxidized fish oil alone or with ISF (oxidized fish oil plus ISF). After 21 d of feeding, supplementation of oxidized fish oil increased the levels of malondialdehyde (MDA), oxidized glutathione (GSSG), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), nuclear factor κ B (NF-κB), inducible nitric oxide synthase (iNOS), NO, and Caspase-3 in jejunal mucosa, and decreased the villous height in duodenum and the levels of secretory immunoglobulin A (sIgA) and IL-4 in the jejunal mucosa compared with supplementation with fresh oil. The addition of oxidized fish oil plus ISF partially alleviated this negative effect. The addition of oxidized fish oil plus ISF increased the villous height and levels of sIgA and IL-4 in jejunal mucosa, but decreased the levels of IL-1β and IL-2 in jejunal mucosa (P<0.05) compared with oxidized fish oil. Collectively, these results show that dietary supplementation of ISF could partly alleviate the negative effect of oxidized fish oil by improving the intestinal morphology as well as the antioxidant capacity and immune function in young piglets.中文概要目 的研究大豆异黄酮对氧化鱼油造成仔猪氧化应激的 缓解作用, 并初步探讨其作用机制。创新点首次在仔猪肠道氧化应激模型中证明大豆异黄酮 可缓解仔猪氧化应激, 且此作用与提高其免疫功 能相关。方 法将72 头平均体重约1.8 kg 的4 日龄杜×(长×大) 三元杂公猪随机分为3 组, 每组重复6 次, 每次 重复包括4头猪: 对照组(饲喂50 g/kg新鲜鱼油), 处理组1(添加50 g/kg 氧化鱼油)和处理组2(添 加50 g/kg 氧化鱼油+20 mg/kg 大豆异黄酮); 基础日粮根据NRC(2012)《猪的营养需要量》配 制, 每3 小时饲喂一次, 记录每天采食量; 实验 期21 天结束后, 称重, 计算平均日采食量、平 均日增重及料重比。用苏木精-伊红染色法观察 小肠形态结构, 并计算绒毛高度和隐窝深度, 应 用南京建成试剂盒测定肠道中抗氧化酶的活性, 应用酶联免疫吸附测定(ELISA)试剂盒测定细 胞炎症相关因子和细胞凋亡相关因子的表达。结 论饲喂氧化鱼油引起新生仔猪肠道产生氧化应激, 引起仔猪生长性能下降、肠道发育受损、细胞凋 亡增加及免疫功能失调。而添加大豆异黄酮能减 缓氧化鱼油造成的肠道损伤, 提高肠道粘膜完整 性, 提高肠道粘膜细胞抗氧化功能和免疫功能。

Influence of low protein diets on gene expression of digestive enzymes and hormone secretion in the gastrointestinal tract of young weaned piglets

To investigate dietary protein level effects on digestive mechanisms, weaned piglets were fed for 45 d with diets containing 20%, 17%, or 14% crude protein (CP) supplemented to meet requirements for essential amino acids. This article describes the influence of dietary protein on gastrointestinal hormones and expression of an array of digestive enzymes in the gastrointestinal tract and pancreas. Results indicated that there were no significant differences in expression of enzymes involved in carbohydrate digestion, except for maltase in the duodenum. In the jejunum, amylase expression in pigs fed 20% CP was much higher than that in pigs fed other diets (P<0.05) and maltase expression in those fed 17% CP was higher than that in other treatments (P<0.05). Although there were no remarkable differences in expression of aminopeptidase in the small intestine or carboxypeptidase in the pancreas (P>0.05), there was a trend towards higher expression of various proteases in pigs fed 17% CP. The duodenal expression of enteropeptidase in diets with 14% and 17% CP was significantly higher than that with 20% CP (P<0.05), but treatment differences did not existed in jejunum (P>0.05). The expression of GPR93 as a nutrient-responsive G protein-coupled receptor in 14% and 17% CP diets was significantly higher than that in 20% CP diet in the small intestine (P<0.05). The expressions of genes for pancreatic enzymes, lipase and elastase, were significantly higher in pigs fed diets with low CP, while similar trends occurred for carboxypeptidase, chymotrypsin and amylase. Conversely, the gastric expressions of pepsinogen A and progastricsin were lower with the 17% CP diet. Differences between treatments were found in the gastric antral contents of cholecystokinin and somatostatin: both increased in pigs fed 17% CP, accompanied by decreased content of motilin, which was also seen in plasma concentrations. These patterns were not reflected in duodenal contents. In general, 17% dietary CP was beneficial to the digestion of nutrient substance in the gastrointestinal tract.中文概要目 的研究蛋白日粮水平对断奶仔猪消化的影响机制。创新点从营养物质消化及激素变化方面研究低蛋白日粮对仔猪胃肠道消化吸收的影响,探究低蛋白日粮饲养的可行性。方 法18 头28 日龄断奶仔猪经过7 天适应期后平均体重为(9.57±0.64) kg。仔猪随机分为三组,每组6头,分别饲喂20%、17%和14%粗蛋白日粮并自由采食45 天。根据国家研究委员会(NRC)2012年的标准,通过添加赖氨酸、蛋氨酸、半胱氨酸、苏氨酸及色氨酸等理想蛋白模型满足仔猪生长需求。实验结束后,进行颈静脉采血并屠宰取样。结 论结果表明基于蛋白日粮水平对内分泌及消化酶影响,与14%及20%粗蛋白组相比,17%蛋白日粮更有利于胃肠道和胰腺等组织中三种主要营养物质的消化。因此,17%粗蛋白水平更适合断奶仔猪的营养需求,证实了低蛋白日粮对断奶仔猪饲养的可行性。

Lactobacillus reuteri LR1 Improved Expression of Genes of Tight Junction Proteins via the MLCK Pathway in IPEC-1 Cells during Infection with Enterotoxigenic Escherichia coli K88

Intestinal epithelial barrier damage disrupts immune homeostasis and leads to many intestinal disorders. Lactobacillus reuteri strains have probiotic functions in their modulation of the microbiota and immune system in intestines. In this study, the effects of L. reuteri LR1, a new strain isolated from the feces of weaning piglets, on intestinal epithelial barrier damage in IPEC-1 cells caused by challenge with enterotoxigenic Escherichia coli (ETEC) K88 were examined. It was found that L. reuteri LR1, in large part, offset the ETEC K88-induced increase in permeability of IPEC-1 cell monolayers and decreased the adhesion and invasion of the coliform in IPEC-1 cells. In addition, L. reuteri LR1 increased transcript abundance and protein contents of tight junction (TJ) proteins zonula occluden-1 (ZO-1) and occludin in ETEC K88-infected IPEC-1 cells, whereas it had no effects on claudin-1 and F-actin expression. Using colloidal gold immunoelectron microscopy, these effects of L. reuteri LR1 on ZO-1 and occludin content in IPEC-1 cells were confirmed. By using ML-7, a selective inhibitor of myosin light-chain kinase (MLCK), the beneficial effect of L. reuteri LR1 on contents of ZO-1 and occludin was shown to be dependent on the MLCK pathway. In conclusion, L. reuteri LR1 had beneficial effects on epithelial barrier function consistent with increasing ZO-1 and occludin expression via a MLCK-dependent manner in IPEC-1 cells during challenge with ETEC K88.