L-theanine attenuates H2O2-induced inflammation and apoptosis in IPEC-J2 cells via inhibiting p38 MAPK signaling pathway.

This study investigated the protective effects of L-theanine on hydrogen peroxide (H2O2)-induced intestinal barrier dysfunction in IPEC-J2 cells. Results showed that L-theanine reduced H2O2-induced IPEC-J2 cells inflammation and apoptosis, and decreased protein phosphorylation levels of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa-B (NF-κB). The p38 MAPK inhibitor (SB203580) decreased oxidative stress, the protein expression of phosphorylation of p38 MAPK and NF-κB, the H2O2-induced increase in mRNA expression of pro-apoptotic and pro-inflammatory related genes expression and secretion, and tight junction protein related genes expression, which was similar to the effect of L-theanine. In conclusion, L-theanine inhibited H2O2-induced oxidative damage and inflammatory reaction, eliminated apoptosis, and protected intestinal epithelial barrier damage by inhibiting the activation of p38 MAPK signaling pathway.

Effects of Dietary Glutamine Supplementation on the Modulation of Microbiota and Th17/Treg Immune Response Signaling Pathway in Piglets after Lipopolysaccharide Challenge.

BACKGROUND: Glutamine (Gln) has an important effect on the growth performance and immune function of piglets. However, the effect of Gln on intestinal immunity in piglets through modulating the signaling pathways of the helper T cells 17 (Th17)/regulatory T cells (Treg) immune response has not been reported. OBJECTIVE: This study aimed to determine the effect of Gln on piglet growth performance and immune stress response and its mechanism in piglets. METHODS: Twenty-four weaned piglets were randomly assigned to 4 treatments with 6 replicates each, using a 2 × 2 factorial arrangement: diet (basal diet or 1% Gln diet) and immunological challenge [saline or lipopolysaccharide (LPS)]. After 21 d, half of the piglets on the basal diet and 1% Gln diet received the intraperitoneal injection of LPS and the other half received the same volume of normal saline. RESULTS: The results showed that Gln increased average daily feed intake and average daily weight gain in comparison with the control group (P < 0.05). Dietary Gln increased the villus height, villus height-to-crypt depth ratio, and the abundance of Bacteroidetes, Lactobacillus sp., and Ruminococcus sp. while reducing the abundance of Firmicutes, Clostridium sensu stricto 1 sp., and Terrisporobacter sp. (P < 0.05). Furthermore, Gln increased the concentration of short-chain fatty acids in the colon and the expression of genes of interleukin (IL)-10, transforming growth factor-beta-1, forkhead box P3 while downregulating the expression of genes of IL-6, IL-8, IL-1ß, tumor necrosis factor-α, IL-17A, IL-21, signal transducer and activator of transcription 3, and rar-related orphan receptor c in ileum (P < 0.05). Correlation analysis demonstrated a strong association between colonic microbiota, short-chain fatty acids, and ileal inflammatory cytokines. CONCLUSIONS: These results suggest that dietary Gln could improve growth performance and attenuate LPS-challenged intestinal inflammation by modulating microbiota and the Th17/Treg immune response signaling pathway in piglets.

Effects of betaine on growth performance and intestinal health of rabbits fed different digestible energy diets.

An experiment was conducted to determine the effects of betaine on growth performance and intestinal health in rabbits fed diets with different levels of digestible energy. During a 36-d experiment, a total of 144 healthy 35-d-old weaned New Zealand white rabbits with a similar initial body weight (771.05 ±â€…41.79 g) were randomly distributed to a 2 × 3 factorial arrangement. Experimental treatments consisted of two levels of digestible energy (normal: 10.20 and low: 9.60 MJ/kg) and three levels of betaine (0, 500, and 1,000 mg/kg). Results indicated that rabbits fed the diet with low digestible energy (LDE) had reduced body gain/feed intake on days 1 to 14 and 1 to 36 (P < 0.05), increased the apparent total tract digestibility (ATTD) of neutral detergent fiber, acid detergent fiber (ADF), and n-free extract, and decreased the ATTD of gross energy (GE), crude fiber, and organic matter (OM; P < 0.05). The LDE diet upregulated the gene abundance levels of duodenum junctional adhesion molecule-3 (JAM-3) and downregulated the ileum toll-like receptor 4, myeloid differentiation factor 88, and interleukin-6 (IL-6; P < 0.05). Activities of amylase, lipase, trypsin, and the immunoglobulin M content in the jejunum were decreased in the LDE treatment group (P < 0.05). Dietary betaine supplementation increased the ATTD of GE, dry matter (DM), ADF, and n-free extract by LDE (P < 0.05). The villus height, crypt depth, and goblet cell numbers were decreased, and the villus-crypt ratio was increased in the duodenum (P < 0.05). The gene abundance levels of duodenum IL-2 were downregulated, and the duodenum JAM-2 and JAM-3 were upregulated (P < 0.05). Furthermore, the addition of betaine to the LDE diet increased the ATTD of GE, DM, and OM in rabbits (P < 0.05). Gene abundance levels of ileum IL-6 and duodenum JAM-3 were upregulated (P < 0.05). In summary, LDE diets can reduce the activity of intestinal digestive enzymes and decrease the ATTD of nutrients. However, the addition of betaine to LDE diets improved the intestinal barrier structure and nutrient ATTD in rabbits, with better results when betaine was added at an additive level of 500 mg/kg.

Insufficient dietary energy can cause many negative effects on animal production and cause intestinal diseases, which are one of the main causes of morbidity and mortality in rabbits. Results of some experiments demonstrated that betaine has various physiological functions such as improving energy utilization and intestinal health. Therefore, the aim of this study was to evaluate the effects of betaine supplementation on growth performance, intestinal function, and health in rabbits fed diets with different levels of digestible energy. The results showed that the addition of betaine to a low-digestible energy diet improved the gut barrier structure and nutrient digestibility in rabbits.

Zinc glycine chelate ameliorates DSS-induced intestinal barrier dysfunction via attenuating TLR4/NF-κB pathway in meat ducks.

BACKGROUND: Zinc glycine chelate (Zn-Gly) has anti-inflammation and growth-promoting properties; however, the mechanism of Zn-Gly contribution to gut barrier function in Cherry Valley ducks during intestinal inflammation is unknown. Three-hundred 1-day-old ducks were divided into 5 groups (6 replicates and 10 ducks per replicate) in a completely randomized design: the control and dextran sulfate sodium (DSS) groups were fed a corn-soybean meal basal diet, and experimental groups received supplements of 70, 120 or 170 mg/kg Zn in form of Zn-Gly. The DSS and treatment groups were given 2 mL of 0.45 g/mL DSS daily during d 15-21, and the control group received normal saline. The experiment lasted 21 d. RESULTS: Compared with DSS group, 70, 120 and 170 mg/kg Zn significantly increased body weight (BW), villus height and the ratio of villus to crypt, and significantly decreased the crypt depth of jejunum at 21 d. The number of goblet cells in jejunal villi in the Zn-Gly group was significantly increased by periodic acid-Schiff staining. Compared with control, the content of intestinal permeability marker D-lactic acid (D-LA) and fluxes of fluorescein isothiocyanate (FITC-D) in plasma of DSS group significantly increased, and 170 mg/kg Zn supplementation significantly decreased the D-LA content and FITC-D fluxes. Compared with control, contents of plasma, jejunum endotoxin and jejunum pro-inflammatory factors IL-1ß, IL-6 and TNF-α were significantly increased in DSS group, and were significantly decreased by 170 mg/kg Zn supplementation. Dietary Zn significantly increased the contents of anti-inflammatory factors IL-10, IL-22 and sIgA and IgG in jejunum. Real-time PCR and Western blot results showed that 170 mg/kg Zn supplementation significantly increased mRNA expression levels of CLDN-1 and expression of OCLN protein in jejunum, and decreased gene and protein expression of CLDN-2 compared with DSS group. The 120 mg/kg Zn significantly promoted the expressions of IL-22 and IgA. Dietary Zn-Gly supplementation significantly decreased pro-inflammatory genes IL-8 and TNF-α expression levels and TNF-α protein expression in jejunum. Additionally, Zn significantly reduced the gene and protein expression of TLR4, MYD88 and NF-κB p65. CONCLUSIONS: Zn-Gly improved duck BW and alleviated intestinal injury by regulating intestinal morphology, barrier function and gut inflammation-related signal pathways TLR4/MYD88/NF-κB p65.

Dietary Betaine improves the intestinal health and growth performance of heat-stressed growing rabbits in summer.

The main objective of this study is to explore how various amounts of Bet affect growth performance, nutritional digestibility, and intestinal health of growing rabbits under high-temperature environment in summer. A total of 150 healthy 35-d-old weaned QiXing meat rabbits (Germany White rabbit × Sichuan White rabbit) were individually assigned to five treatments, each with 30 replicates and one rabbit per replicate. The control group was fed with basal diet, while the experimental group received a basal diet supplemented with 0.75, 1.0, 1.5, and 2.0 g Bet/kg diet, respectively. During the whole experimental stage, all animals can eat and drink freely, and they were kept in the rabbit house with an average daily temperature of 30.11 ±â€…0.5 ℃ and a relative humidity of 71.02 ±â€…5.07%. The results showed that dietary supplementation with 1.5 g/kg Bet increased average daily gain and decreased feed to gain ratio from days 1 to 42 as compared to the control group (P < 0.05), adding 0.75, 1.0, 1.5, and 2.0 g/kg Bet increased average daily gain and average daily feed intake from days 22 to 42 (P < 0.05), and increased the nutritional digestibility of acid detergent fiber (P < 0.05). Furthermore, dietary supplementation with 1.0, 1.5, and 2.0 g/kg Bet reduced d-lactate content and diamine oxidase activity in the serum (P < 0.05). Compared to the control group, supplementation of 0.75 and 1.5 g/kg Bet improved glutathione peroxidase activities in the duodenum and ileum, adding 0.75, 1.0, 1.5, and 2.0 g/kg Bet decreased malonaldehyde content in the duodenum and jejunum (P < 0.05). Moreover, the supplement of 1.5 and 2.0 g/kg Bet upregulated JAM-2 and IL-10 levels in the jejunum (P < 0.05). In conclusion, supplementation with Bet in the diet improves the growth performance, nutrient digestibility, and intestinal health of growing rabbits under high-temperature environments, and the 1.5 g Bet/kg diet group has the best effect.

Due to the lack of functional sweat glands, rabbits find it difficult to release excess heat under high-temperature conditions, resulting in heat stress. This high-level temperature condition leads to substantial damage to growth performance and intestinal health resulting in significant financial losses for the meat rabbit industry. This study found that adding betaine (Bet) to the diet can improve the growth performance and intestinal barrier integrity of heat-stressed growing rabbits, which may be related to improving intestinal antioxidant capacity and immune status. 1.5 g Bet/kg diet group showed better effects than 0, 0.75, 1.0, and 2.0 g Bet/kg diet groups in improving growth performance and intestinal health of heat-stressed growing rabbits.

Caffeic acid improves intestinal barrier functions by regulating colonic bacteria and tight junction protein expression and alleviating inflammation in weaning piglets.

The experiment investigated the effect of caffeic acid on bacteria, short-chain fatty acids (SCFA), and the expression of tight junction protein and inflammation related genes in the colon of weaning piglets. Thirty-six weaning piglets were allocated to three treatment groups, which were fed with a basal diet, a basal diet supplemented with 250 mg/kg or 500 mg/kg caffeic acid for 28 days. The results showed that caffeic acid treatment increased the contents of acetate acid, propionate acid and total SCFA. Moreover, real-time quantitative PCR showed that the number of Bifidobacterium (p < 0.05) and Lactobacillus (p < 0.05) were increased and the number of Escherichia coli (p < 0.05) was decreased by caffeic acid in colonic mucosa. Real-time quantitative PCR also showed that the mRNA levels of zonula occludens-1 (p < 0.01), claudin-1 (p < 0.01), occludin (p < 0.01), mucin 1 (MUC1) (p < 0.01), MUC2 (p < 0.01), interleukin 4 (IL-4) (p < 0.01) and IL-10 (p < 0.05) were increased, while the mRNA expression levels of histone deacetylases (p < 0.01), IL-1 (p < 0.01), IL-6 (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.01) were decreased, by caffeic acid in colonic mucosa. These results suggested that caffeic acid could improve intestinal barrier function in weaned pigs, which might be mediated by regulating colonic bacteria and tight junction protein expression and alleviating inflammation.

Dietary glutamate enhances intestinal immunity by modulating microbiota and Th17/Treg balance-related immune signaling in piglets after lipopolysaccharide challenge.

The purpose of this study was to explore the effects of glutamate on piglet growth performance and intestinal immunity function, and to further elucidate its mechanism. In a 2 × 2 factorial design involving immunological challenge (lipopolysaccharide (LPS) or saline) and diet (with or without glutamate), twenty-four piglets were randomly assigned to four groups, each with 6 replicates. Piglets were fed with a basal or glutamate diet for 21 d before being injected intraperitoneally with LPS or saline. Piglet's intestinal samples were collected 4 h after injection. Results showed that glutamate increased daily feed intake, average daily gain, villus length, villus area, and villus length to crypt depth ratio (V/C), and decreased the crypt depth (P < 0.05). Furthermore, glutamate increased the mRNA expression of forkhead box P3 (FOXP3), a signal transducer and activator of transcription 5 (STAT5) and transforming growth factor beta, while decreasing the mRNA expression of RAR-related orphan receptor c and STAT3. Glutamate increased interleukin-10 (IL-10) mRNA expression while decreasing the mRNA expression of IL-1ß, IL-6, IL-8, IL-17, IL-21, and tumor necrosis factor-α. At the phylum level, glutamate increased the Actinobacteriota abundance and Firmicutes-to-Bacteroidetes ratio while decreasing Firmicutes abundance. At the genus level, glutamate improved the abundance of beneficial bacteria (e.g., Lactobacillus, Prevotellaceae-NK3B31-group, and UCG-005). Furthermore, glutamate increased the concentrations of short-chain fatty acids (SCFAs). Correlation analysis revealed that the intestinal microbiota is closely related to Th17/Treg balance-related index and SCFAs. Collectively, glutamate can improve piglet growth performance and intestinal immunity by modulating gut microbiota and Th17/Treg balance-related signaling pathways.

Dietary tryptophan supplementation enhances mitochondrial function and reduces pyroptosis in the spleen and thymus of piglets after lipopolysaccharide challenge.

The thymus and spleen, the main reservoirs for T lymphocytes, modulate the innate immune response. Oxidative stress, excessive inflammation and abnormal pyroptosis can cause dysfunction of these organs. This study aimed to examine whether tryptophan supplementation can improve growth performance and mitochondrial function via the adenosine 5'-monophosphate-activated protein kinase (AMPK)/sirtuin1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) signalling pathway and decrease pyroptosis via the nucleotidebinding oligomerisation domain-like receptor protein 3 (NLRP3)/caspase-1/gasderminD (GSDMD) signalling pathway in the spleen and thymus of piglets after lipopolysaccharide (LPS) challenge. Eighteen weaned piglets were allotted to three treatment groups: non-challenged control, LPS-challenged control and LPS + 0.2% tryptophan. On day 35, the pigs in the LPS and LPS + 0.2% tryptophan groups were injected with 100 µg/kg BW LPS, whereas those in the control group were administered with sterile saline. At 4 h postchallenge, the weaned piglets were sacrificed, and their thymuses and spleens were collected. Results showed that tryptophan enhanced growth performance and antioxidant status by increasing catalase, glutathione peroxidase and total superoxide dismutase activities and decreasing malondialdehyde and reactive oxygen species contents. Tryptophan also reduced the mRNA levels of proinflammatory cytokine genes and enhanced mitochondrial function by increasing the mRNA levels of mitochondrial transcription factor A, nuclear respiratory factor-1, mitochondria transcription factor B1, AMPKα1, AMPKα2, Sirt1 and PGC1α and the protein expression of phosphorylated AMPK, Sirt1 and PGC1α. It also reduced pyroptosis by decreasing the mRNA levels of NLRP3, apoptosis-associated speck-like protein containing CARD, caspase-1 and GSDMD and the protein expression of NLRP3, caspase-1 and GSDMD. These results indicate that tryptophan supplementation enhances growth performance and mitochondrial function via the AMPK/Sirt1/PGC1α signalling pathway and decreases pyroptosis via the NLRP3/caspase-1/GSDMD signalling pathway in the spleen and thymus of LPS-challenged piglets.

Naringin mitigates LPS-induced intestinal barrier injury in mice.

The aim of this study was to investigate the effect of naringin on lipopolysaccharide (LPS)-induced jejunal barrier function in mice. Forty-five 3-week-old healthy male Balb/c mice with similar body weights were randomly divided into control group, LPS group, LPS + naringin group, with 15 mice in each treatment group. The mice were intraperitoneally injected with the same dose of saline or LPS (10 mg per kg BW) at 43 d. The blood samples, liver and jejunal tissues were collected after 3 h of injection. The results showed that LPS significantly increased the serum diamine oxidase (DAO) activity, D-lactate (D-LA) concentration, and malondialdehyde (MDA) content in liver and jejunum, while decreased the activities of superoxide dismutase (SOD), glutathione peroxidase (Gpx) and catalase (CAT) in liver and jejunum. The LPS treatment caused an increase in the crypt depth and a decrease in the villus height and the ratio of villus height to crypt depth (V/C) of the jejunum. In addition, the LPS treatment significantly increased the mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, toll-like receptor 4 (TLR4), p38-mitogen-activated protein kinase (p38 MAPK), nuclear factor-κB (NF-κB) and kelch-like ECH-associated protein 1 (Keap1), while decreased mRNA expressions of zonula occludens 1 (ZO-1), occludin, claudin, mucin 2 (MUC2) and junctional adhesion molecule 2 (JAM2), Gpx, SOD1, GST, CAT and nuclear factor-erythroid 2-related factor 2 (Nrf2). However, the naringin treatment mitigated these effects induced by LPS. Taken together, our findings suggested that naringin attenuates LPS-induced intestinal barrier damage by inhibiting inflammatory factors and improving antioxidant function and intestinal tight junction, which might be mediated by activating the Nrf2 signaling and suppressing the TLR4/p38 MAPK/NF-κB signaling.

Calcium-sensing receptor protects intestinal integrity and alleviates the inflammatory response via the Rac1/PLCγ1 signaling pathway.

This study aimed to test the hypothesis that the calcium-sensing receptor (CaSR) can protect intestinal epithelial barrier integrity and decrease inflammatory response mediated by the Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. IPEC-J2 monolayers were treated without or with TNF-α in the absence or presence of CaSR antagonist (NPS 2143), CaSR overexpression, and Rac1 silencing, PLCγ1 silencing or spermine. Results showed that spermine increased transepithelial electrical resistance (TER), tight junction protein levels, the protein concentration of Rac1/PLC-γ1 signaling pathway, and decreased paracellular permeability in the presence of TNF-α. NPS2143 inhibited spermine-induced change in above-mentioned parameters. CaSR overexpression increased TER, the levels of tight junction proteins and the protein concentration of CaSR, phosphorylated PLCγ1, Rac1, and IP3, and decreased paracellular permeability and contents of interleukin-8 (IL-8) and TNF-α after TNF-α challenge. Rac1 and PLCγ1 silencing inhibited CaSR-induced increase in barrier function and the protein concentration of phosphorylated PLCγ1, Rac1, and IP3, and decrease in contents of IL-8 and TNF-α after TNF-α challenge. These results suggest that CaSR activation protects intestinal integrity and alleviates the inflammatory response by activating Rac1 and PLCγ1 signaling after TNF-α challenge, and spermine can maintain barrier function via CaSR/Rac1/PLC-γ1 pathway.

Effect of calcium-sensing receptor on the migration and proliferation of porcine intestinal epithelial cells.

The rapid healing of impaired intestinal surface plays a role in maintaining intestinal homeostasis. This study investigated the effect of calcium-sensing receptor (CaSR) on the migration and proliferation of intestinal porcine epithelial cells (IPEC-J2). Results showed that cell migration area and width were increased by R568 (CaSR activator) and decreased by NPS2143 (CaSR inhibitor). The protein level of GTP-rac1 and the phosphorylation of phospholipase C gamma 1 (PLCγ1) were increased by 2 µM R568. Furthermore, R568 + 120 µM NSC23766 (Rac1 inhibitor) and R568 + 1 µM U73122 (PLCγ1 inhibitor) decreased the protein level of GTP-rac1 and the phosphorylated PLCγ1, respectively, and both inhibited cell migration compared with R568. In addition, spermine increased the protein expression levels of CaSR and the levels of GTP-rac1 and the phosphorylated PLCγ1 and thereby promoted the migration of IPEC-J2 cells. Moreover, R568 improved the proliferation of the IPEC-J2 cells. Spermine increased cell proliferation, but NPS2143 incubated with spermine decreased cell proliferation compared with the spermine group. This study suggests that CaSR activation increased cell migration by activating Rac1 and PLCγ1 signaling and improved cell proliferation, and both effects were regulated by spermine by activating CaSR.

Effects of spermine on the proliferation and migration of porcine intestinal epithelial cells.

Whether spermine promotes the repair of porcine intestinal epithelium damage through Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase C-γ1 (PLC-γ1) signaling remains unclear. The current study investigated the effects of spermine addition on the proliferation and migration of IPEC-J2 cells and the effects of Rac1/PLC-γ1 signaling on cell migration. We showed that the inhibitors of Rac1 (NSC-23766) and PLC-γ1 (U73122) reduced cell migration and decreased the protein levels of Rac1 and PLC-γ1 in the cells. Moreover, spermine promoted the proliferation and migration of the IPEC-J2 cells, that is, 1 µM spermine exhibited the best effect, and spermine treatment increased the protein levels of Rac1 and PLC-γ1. Further experiments showed that spermine treatment increased cell migration and enhanced Rac1 and PLC-γ1 protein levels, compared with NSC-23766 and U73122 treatments with spermine. In conclusion, spermine treatment promoted the repair of damaged porcine intestinal epithelium by accelerating cell proliferation and migration mediated by Rac1/PLC-γ1 signaling.

Protective effects of selenized yeast on the combination of cadmium-, lead-, mercury-, and chromium-induced toxicity in laying hens.

The objective of this study was to investigate the toxic effects of a combination of cadmium (Cd), lead (Pb), mercury (Hg), and chromium (Cr) on laying performance, egg quality, serum biochemical parameters, and oxidative stress of laying hens, as well as the alleviating action of dietary supplementation of selenized yeast. A total of 160 Lohmann pink-shell laying hens (63-week-old) were randomly divided into four treatments with 10 replicates of four hens each. The treatments were the corn-soybean meal basal diet (control; CON), the CON diet supplemented with 0.4 mg selenium (Se)/kg from selenized yeast (Se); combined heavy metals group: the basal diet supplemented with 5 mg Cd/kg, 50 mg Pb/kg, 3 mg Hg/kg, and 5 mg Cr/kg (HEM), and the HEM diet supplemented with 0.4 mg Se/kg from selenized yeast (HEM+Se). The experimental period lasted for 12 weeks. The HEM diet decreased hen-day egg production, feed conversion ratio (FCR), and egg white quality (P < 0.05), but increased (P < 0.05) glutamic oxalacetic transaminase (AST) activity in the serum. HEM induced higher malondialdehyde (MDA) and reactive oxygen species (ROS) in the serum, liver, and ovary and significantly decreased (P < 0.05) the activity of total superoxide dismutase (SOD) and tended to decrease glutathione S-transferase (GST) (P = 0.09) in the serum. Meanwhile, HEM significantly decreased (P < 0.05) activity of SOD, GST, glutathione peroxidase (GPX), and glutathione (GSH) in the liver, and the activity of GPX and GSH in the ovary. Se addition of 0.4 mg/kg significantly (P < 0.05) improved hen-day egg production and FCR and decreased AST concentration and increased some enzyme activity in the serum, liver, and ovary. In conclusion, dietary HEM exposure depressed laying performance, and egg white quality was likely due to an impaired antioxidant capacity, disrupted hepatic function, and elevated HEM accumulation in the egg yolk and egg white of laying hens. Se addition of 0.4 mg/kg ameliorated toxic effects of HEM on laying performance, oxidative stress, and hepatic function.

Dietary licorice flavonoids powder improves serum antioxidant capacity and immune organ inflammatory responses in weaned piglets.

Weaning often induces oxidative stress and inflammatory response in piglets. This study investigated the effects of dietary licorice flavonoids powder (LFP) supplementation on antioxidant capacity and immunity in weaned piglets. Notably, 96 Landrace × Yorkshire × Duroc (DLY) weaned piglets were randomly allocated to four treatments with 6 replicates (4 animals per replicate) and fed with diet supplementation with 0, 50, 150, and 250 mg/kg LFP, respectively. The trial lasted for 5 weeks. The results showed that dietary LFP supplementation effectively increased the liver index (P < 0.05). In addition, dietary LFP supplementation reduced serum aspartate aminotransferase activity (P < 0.01). Piglets fed with 50 mg/kg LFP decreased total cholesterol and HDL-C content in serum (P < 0.05) and increased serum alkaline phosphatase activity (P < 0.01). Similarly, supplementation with 150 mg/kg LFP elevated the activity of total antioxidant capability (T-AOC) in serum (P < 0.01) and dietary with 150 and 250 mg/kg LFP increased T-AOC activity in spleen (P < 0.01). Moreover, dietary with 150 mg/kg LFP addition enhanced (P < 0.05) the serum IgG content of piglets. Additionally, compared with the control group, dietary 250 mg/kg LFP supplementation upregulated (P < 0.05) the mRNA abundance of Interleukin (IL)-1ß and monocyte chemoattractant protein 1 (MCP-1) in the spleen. Meanwhile, dietary 150 and 250 mg/kg LFP supplementation downregulated (P < 0.05) mRNA abundance of IL-10, and MCP-1 and 250 mg/kg LFP upregulated (P < 0.05) the expression of intercellular adhesion molecule 1 (ICAM-1), IL-1ß, IL-6, and tumor necrosis factor α (TNF-α) in the thymus. In conclusion, LFP supplementation improved the immune function of piglets by regulating the activity of serum biochemical enzymes, improving the antioxidant capacity, and alleviating inflammation of immune organs. This study indicated that LFP is potential alternative protection against early weaned stress in piglets.

Dietary L-theanine supplementation improves lipid metabolism and antioxidant capacity in weaning piglets.

The aim of this study was to explore the effects of dietary L-theanine (LT) supplementation on lipid metabolism and antioxidant capacity in weaned piglets. Twenty-one castrated DLY weaning piglets were randomly divided into three groups: a basal diet, a basal diet supplemented with 0.05% and 0.1% LT, respectively. Our data showed that dietary LT supplementation decreased T-CHO, TG, LDL-C and apoB levels and increased apoA and HDL-C levels in serum, but decreased the hepatic TG and T-CHO contents. Dietary LT supplementation increased the antioxidant capacity in serum and liver, and significantly increased the Nrf2 mRNA level and the nucleus Nrf2 protein level, but decreased the mRNA level of keap1 in the liver. In addition, dietary LT supplementation significantly increased HSL enzyme activity and the levels of CPT1 and TBA, while decreasing the enzyme activities of LPL and FAS in the liver. Furthermore, the mRNA levels HMG-CoAR, CPT-1a and PPARα and the protein levels of phosphorylated-AMPK and PGC-1α were increased by LT. Together, our data provide the first evidence that dietary supplementation of LT could improve lipid metabolism and antioxidant capacity in the liver of weaned piglets, and the effect might be mediated by activation of AMPK and Nrf2 signaling, respectively.

Dietary Tryptophan Supplementation Improves Antioxidant Status and Alleviates Inflammation, Endoplasmic Reticulum Stress, Apoptosis, and Pyroptosis in the Intestine of Piglets after Lipopolysaccharide Challenge.

Tryptophan can alleviate stress and improve intestinal health, but the precise mechanism has not been fully elucidated. This study aimed to examine the effects of tryptophan supplementation on antioxidant status, inflammation, endoplasmic reticulum (ER) stress, apoptosis, and pyroptosis signaling pathway in the intestine of piglets after Escherichia coli lipopolysaccharide (LPS) challenge. Thirty-two weaning piglets were allotted to four treatments including: non-challenged control, LPS-challenged control, LPS + 0.2% tryptophan and LPS + 0.4% tryptophan. On day 35 of feeding, piglets were injected intraperitoneally with 100 µg/kg of body weight LPS or saline. Among the LPS-challenged pigs, tryptophan supplementation improved intestinal morphology as indicated by greater villus height, villus area and smaller crypt depth, and antioxidant status, and decreased the mRNA expression and concentration of proinflammatory cytokines. Moreover, tryptophan downregulated the expression of ER stress (ER oxidoreductase-1α, ER oxidoreductase-1ß, glucose-regulated protein-78, activating transcription factor 6, C/EBP homologous protein), apoptosis (B-cell lymphoma-2, BCL2-associated X protein, caspase 3), and pyroptosis signaling pathway (nucleotide-binding oligomerization domain-like receptor protein 3, caspase 1, gasdermin-D, apoptosis-associated speck-like protein containing a CARD). Collectively, tryptophan supplementation can contribute to gut health by improving antioxidant status and alleviating inflammation, ER stress, apoptosis, and pyroptosis in the intestine of piglets after lipopolysaccharide challenge.

Effect of dietary L-theanine supplementation on skeletal muscle fiber type transformation in weaning piglets.

The aim of this study was to explore the effect of dietary L-theanine (LT) supplementation on skeletal muscle fiber type transformation in weaning piglets. Our data showed that LT significantly increased the slow-twitch fiber-related genes expression and the percentage of slow oxidative fiber, and decreased the MyHC IIb mRNA expression and the percentage of fast glycolytic fiber. In addition, LT significantly increased the succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities and increased the LDH activities. In addition, LT significantly affected mitochondrial biogenesis and function and antioxidative related genes expression, and increased the protein expression of p-adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear factor E2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) and decreased the Keap1 protein levels. Furthermore, our data indicated that LT significantly increased the mRNA and protein expression of prospero-related homeobox 1 (Prox1), calcineurin A (CnA), and NFATc1, suggesting that dietary LT supplementation promoted skeletal muscle fiber transition from types II to I might be via activation of calcineurin signaling pathway. Taken together, these findings suggested that LT promoted the transformation of muscle fiber types from slow oxidative to fast glycolytic by increasing antioxidant capacity and improving mitochondrial biogenesis and function and activation of calcineurin signaling pathway.

Spermine protects intestinal barrier integrity through ras-related C3 botulinum toxin substrate 1/phospholipase C-γ1 signaling pathway in piglets.

Weaning stress can cause tight junctions damage and intestinal permeability enhancement, which leads to intestinal imbalance and growth retardation, thereby causing damage to piglet growth and development. Spermine can reduce stress. However, the mechanism of spermine modulating the intestinal integrity in pigs remains largely unknown. This study aims to examine whether spermine protects the intestinal barrier integrity of piglets through ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase C-γ1 (PLC-γ1) signaling pathway. In vivo, 80 piglets were categorised into 4 control groups and 4 spermine groups (10 piglets per group). The piglets were fed with normal saline or spermine at 0.4 mmol/kg BW for 7 h and 3, 6 and 9 d. In vitro, we investigated whether spermine protects the intestinal barrier after a tumor necrosis factor α (TNF-α) challenge through Rac1/PLC-γ1 signaling pathway. The in vivo study found that spermine supplementation increased tight junction protein mRNA levels and Rac1/PLC-γ1 signaling pathway gene expression in the jejunum of piglets. The serum D-lactate content was significantly decreased after spermine supplementation (P < 0.05). The in vitro study found that 0.1 µmol/L spermine increased the levels of tight junction protein expression, Rac1/PLC-γ1 signaling pathway and transepithelial electrical resistance, and decreased paracellular permeability (P < 0.05). Further experiments demonstrated that spermine supplementation enhanced the levels of tight junction protein expression, Rac1/PLC-γ1 signaling pathway and transepithelial electrical resistance, and decreased paracellular permeability compared with the NSC-23766 and U73122 treatment with spermine after TNF-α challenge (P < 0.05). Collectively, spermine protects intestinal barrier integrity through Rac1/PLC-γ1 signaling pathway in piglets.

STIM1 promotes IPEC-J2 porcine epithelial cell restitution by TRPC1 signaling.

Intestinal epithelial restitution is partly dependent on cell migration, which reseals superficial wounding after injury. Here, we tested the hypothesis that stromal interaction molecule 1(STIM1) regulates porcine intestinal epithelial cell migration by activating transient receptor potential canonical 1 (TRPC1) signaling. Results showed that the knockdown of STIM1 repressed cell migration after wounding, reduced the protein concentration of STIM1 and TRPC1, and decreased the inositol trisphosphate (IP3) content in IPEC-J2 cells (p < 0.05). However, overexpression of STIM1 obtained opposite results (p < 0.05). The inhibition of TRPC1 activity by treatment with SKF96365 in cells overexpressing wild-type and mutant STIM1 attenuated the STIM1 overexpression-induced increase of cell migration, STIM1, TRPC1 and IP3 (p < 0.05). In addition, polyamine depletion caused by α-difluoromethylornithine (DFMO) resulted in the decrease of above-mentioned parameters, and exogenous polyamine could attenuate the negative effects of DFMO on IPEC-J2 cells (p < 0.05). Moreover, the overexpression of STIM1 could rescue cell migration, the protein level of STIM1 and TRPC1, and IP3 content in polyamine-deficient IPEC-J2 cells (p < 0.05). These results indicated that STIM1 could enhance porcine intestinal epithelial cell migration via the TRPC1 signaling pathway. Inhibition of cell migration by polyamine depletion resulted from the reduction of STIM1 activity.

Tryptophan Ameliorates Barrier Integrity and Alleviates the Inflammatory Response to Enterotoxigenic Escherichia coli K88 Through the CaSR/Rac1/PLC-γ1 Signaling Pathway in Porcine Intestinal Epithelial Cells.

Background: Impaired intestinal barrier integrity plays a crucial role in the development of many diseases such as obesity, inflammatory bowel disease, and type 2 diabetes. Thus, protecting the intestinal barrier from pathological disruption is of great significance. Tryptophan can increase gut barrier integrity, enhance intestinal absorption, and decrease intestinal inflammation. However, the mechanism of tryptophan in decreasing intestinal barrier damage and inflammatory response remains largely unknown. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. Methods: IPEC-J2 cells were treated with or without enterotoxigenic Escherichia coli (ETEC) K88 in the absence or presence of tryptophan, CaSR inhibitor (NPS-2143), wild-type CaSR overexpression (pcDNA3.1-CaSR-WT), Rac1-siRNA, and PLC-γ1-siRNA. Results: The results showed that ETEC K88 decreased the protein concentration of occludin, zonula occludens-1 (ZO-1), claudin-1, CaSR, total Rac1, Rho family member 1 of porcine GTP-binding protein (GTP-rac1), phosphorylated phospholipase Cγ1 (p-PLC-γ1), and inositol triphosphate (IP3); suppressed the transepithelial electrical resistance (TEER); and enhanced the permeability of FITC-dextran compared with the control group. Compared with the control group, 0.7 mM tryptophan increased the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; elevated the TEER; and decreased the permeability of FITC-dextran and contents of interleukin-8 (IL-8) and TNF-α. However, 0.7 mM tryptophan+ETEC K88 reversed the effects induced by 0.7 mM tryptophan alone. Rac1-siRNA+tryptophan+ETEC K88 or PLC-γ1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. NPS2143+tryptophan+ETEC K88 decreased the TEER and the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; increased the permeability of FITC-dextran; and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. pcDNA3.1-CaSR-WT+Rac1-siRNA+ETEC K88 and pcDNA3.1-CaSR-WT+PLC-γ1-siRNA+ETEC K88 decreased the TEER and enhanced the permeability in porcine intestine epithelial cells compared with pcDNA3.1-CaSR-WT+ETEC K88. Conclusion: Tryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-γ1 signaling pathway.