MUC2 Gene Expression Research Articles

Development of engineered IL-36γ-hypersecreting Lactococcus lactis to improve the intestinal environment

Interleukin (IL) 36 is a member of the IL-1-like proinflammatory cytokine family that has a protective role in mucosal immunity. We hypothesized that mucosal delivery of IL-36γ to the intestine would be a very effective way to prevent intestinal diseases. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse IL-36γ (rmIL-36γ). Western blotting and enzyme-linked immunosorbent assay results showed that the engineered strain (NZ-IL36γ) produced and hypersecreted the designed rmIL-36γ in the presence of nisin, which induces the expression of the recombinant gene. We administered NZ-IL36γ to mice via oral gavage, and collected the ruminal contents and rectal tissues. Colony PCR using primers specific for NZ-IL36γ, and enzyme-linked immunosorbent assay to measure the rmIL-36γ concentrations of the ruminal contents showed that NZ-IL36γ colonized the mouse intestines and secreted rmIL-36γ. A microbiota analysis revealed increased abundances of bacteria of the genera Acetatifactor, Eubacterium, Monoglobus, and Roseburia in the mouse intestines. Real-time quantitative PCR of the whole colon showed increased Muc2 expression. An in vitro assay using murine colorectal epithelial cells and human colonic cells showed that purified rmIL-36γ promoted Muc2 gene expression. Taken together, these data suggest that NZ-IL36γ may be an effective and attractive tool for delivering rmIL-36γ to improve the intestinal environment.

Evaluation of TNF-α and IFN-γ primed conditioned medium of mesenchymal stem cell in acetic acid-induced mouse model of acute colitis

IBD, an autoimmune-inflammatory disorder that affects people who are genetically prone to inflammation. There is a lot of interest in MSC-CM therapy, especially when primed with TNF-α + IFN-γ. Throughout the study, data were collected on the percentage of apoptotic cells, gene expression of ZO-1, Foxp3, GATA3, IDO-1, Muc2, T-bet, Notch1, TNFR2, and ROR-γt, colon weight and length, histopathological analysis, and DAI. TNF-α and IL-10 levels were assessed in addition to the NO level. The results suggest that primed MSC-CM improved DAI, mucosal deterioration, intestinal inflammation and NO concentration. The amount of TNF-α was decreased, but IL-10 and the colon’s percentage of apoptotic cells was increased. The mRNA expression of ZO-1, Foxp3, GATA3, IDO-1, and Muc2 genes increased greatly in the treatment groups, while the expression of T-bet, Notch1, TNFR2, and ROR-γt genes has decreased. These studies suggest that primed MSC-CM may combine with common treatments to improve responsiveness.

Enhancing pulmonary delivery and immunomodulation of respiratory diseases through virus-mimicking nanoparticles

This study introduces the nanobromhexine lipid particle (NBL) platform designed for effective pulmonary drug delivery. Inspired by respiratory virus transport mechanisms, NBL address challenges associated with mucus permeation and inflammation in pulmonary diseases. Composed of low molecular weight polyethylene glycol-coated lipid nanoparticles with bromhexine hydrochloride, NBL exhibit a size of 118 ± 24 nm, a neutral zeta potential, osmolarity of 358 ± 28 mOsmol/kg, and a pH of 6.5. Nebulizing without leakage and showing no toxicity to epithelial cells, NBL display mucoadhesive properties with a 60% mucin-binding efficiency. They effectively traverse the dense mucus layer of Calu-3 cultures in an air-liquid interface, as supported by a 55% decrease in MUC5AC density and a 29% increase in nanoparticles internalization compared to non-exposed cells. In assessing immunomodulatory effects, NBL treatment in SARS-CoV-2-infected lung cells leads to a 40-fold increase in anti-inflammatory MUC1 gene expression, a proportional reduction in pro-inflammatory IL-6 expression, and elevated anti-inflammatory IL-10 expression. These findings suggest a potential mechanism to regulate the excessive IL-6 expression triggered by virus infection. Therefore, the NBL platform demonstrates promising potential for efficient pulmonary drug delivery and immunomodulation, offering a novel approach to addressing mucus permeation and inflammation in pulmonary diseases.

Maternal Synbiotic Supplementation with B. breve M-16V and scGOS/lcFOS Shape Offspring Immune Development and Gut Microbiota at the End of Suckling.

Immune system development during gestation and suckling is significantly modulated by maternal environmental and dietary factors. Breastfeeding is widely recognized as the optimal source of nutrition for infant growth and immune maturation, and its composition can be modulated by the maternal diet. In the present work, we investigated whether oral supplementation with Bifidobacterium breve M-16V and short-chain galacto-oligosaccharide (scGOS) and long-chain fructo-oligosaccharide (lcFOS) to rat dams during gestation and lactation has an impact on the immune system and microbiota composition of the offspring at day 21 of life. On that day, blood, adipose tissue, small intestine (SI), mesenteric lymph nodes (MLN), salivary gland (SG), cecum, and spleen were collected. Synbiotic supplementation did not affect the overall body or organ growth of the pups. The gene expression of Tlr9, Muc2, IgA, and Blimp1 were upregulated in the SI, and the increase in IgA gene expression was further confirmed at the protein level in the gut wash. Synbiotic supplementation also positively impacted the microbiota composition in both the small and large intestines, resulting in higher proportions of Bifidobacterium genus, among others. In addition, there was an increase in butanoic, isobutanoic, and acetic acid concentrations in the cecum but a reduction in the small intestine. At the systemic level, synbiotic supplementation resulted in higher levels of immunoglobulin IgG2c in plasma, SG, and MLN, but it did not modify the main lymphocyte subsets in the spleen and MLN. Overall, synbiotic maternal supplementation is able to positively influence the immune system development and microbiota of the suckling offspring, particularly at the gastrointestinal level.

Insect live larvae as a new nutritional model in duck: effects on gut health

BackgroundThis study aimed to evaluate the effects of Hermetia illucens (Black soldier fly-BSF) and Tenebrio molitor (Yellow mealworm-YMW) live larvae as a new nutritional model on duck’s gut health, considering gut histomorphometry, mucin composition, cytokines transcription levels, and microbiota. A total of 126, 3-days-old, females Muscovy ducks were randomly allotted to three dietary treatments (6 replicates/treatment, 7 birds/pen): (i) C: basal diet; (ii) BSF: C + BSF live larvae; (iii) YMW: C + YMW live larvae. BSF and YMW live larvae were administered on top of the basal diet, based on the 5% of the expected daily feed intake. The live weight, average daily gain, average daily feed intake and feed conversion ratio were evaluated for the whole experimental period. On day 52, 12 ducks/treatment (2 birds/replicate) were slaughtered and samples of duodenum, jejunum, ileum, spleen, liver, thymus and bursa of Fabricius were collected for histomorphometry. Mucin composition was evaluated in the small intestine through histochemical staining while jejunal MUC-2 and cytokines transcription levels were evaluated by rt-qPCR. Cecal microbiota was also analyzed by means of 16 S rRNA gene sequencing.ResultsBirds’ growth performance and histomorphometry were not influenced by diet, with a proximo-distal decreasing gradient from duodenum to ileum (p < 0.001), respecting the physiological gut development. Mucin staining intensity and MUC-2 gene expression did not vary among dietary treatments, even though mucin intensity increased from duodenum to ileum, according to normal gut mucus physiology (p < 0.001). Regarding local immune response, IL-6 was higher in YMW group when compared to the other groups (p = 0.009). Insect live larvae did not affect cecal microbiota diversity, but BSF and YMW groups showed a higher presence of Helicobacter, Elusimicrobium, and Succinatimonas and a lower abundance of Coriobacteriaceae and Phascolarctobacterium compared to C birds (p < 0.05).ConclusionsThe use of BSF and YMW live larvae as new nutritional model did not impair gut development and mucin composition of Muscovy ducks, but slightly improved the intestinal immune status and the microbiota composition by enhancing regulatory cytokine IL-6 and by increasing minor Operational Taxonomic Units (OTUs) involved in short-chain fatty acids production.

Clinical Significance of MUC4 and Associated Proteins in Pancreatic and Periampullary Cancers.

This study primarily aimed to assess the expression of MUC4 in patients with pancreatic ductal adenocarcinoma (PDAC) as compared with controls and assess its clinical relevance. Serum MUC4 levels and MUC4 gene expression in snap-frozen tissue were analyzed through surface plasmon resonance and quantitative polymerase chain reaction, respectively. Tumor tissues and control tissues were analyzed for MUC4 and other mucins through immunohistochemistry. MUC4 expression in tumor tissue was found to be significantly elevated in PDAC patients as compared with chronic pancreatitis tissues and normal pancreatic tissues. Periampullary carcinoma and cholangiocarcinoma tissue also showed increased expression of MUC4 and other mucins. Differential expression of MUC4 in pancreatic tumor tissues can help to differentiate PDAC from benign conditions.

De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10µM. However, 10µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.

Resveratrol Alleviates Zearalenone-Induced Intestinal Dysfunction in Mice through the NF-κB/Nrf2/HO-1 Signalling Pathway.

Zearalenone (ZEA), a mycotoxin widely present in crops and food, poses a major threat to animal and human health. The consumption of ZEA-contaminated food or feed causes intestinal damage. Therefore, exploring how to mitigate the intestinal damage caused by its ZEA is becoming increasingly important. Resveratrol (RSV), a polyphenol compound, mainly exists in Vitis vinifera, Polygonum cuspidatum, Arachis hypogaea, and other plants. It has potent anti-inflammatory and antioxidant activity. The primary objective of this study was to assess the defensive effects of RSV and its molecular mechanism on the intestinal mucosal injury induced by ZEA exposure in mice. The results showed that RSV pretreatment significantly reduced serum DAO and that D-lactate levels altered intestinal morphology and markedly restored TJ protein levels, intestinal goblet cell number, and MUC-2 gene expression after ZEA challenge. In addition, RSV significantly reversed serum pro-inflammatory factor levels and abnormal changes in intestinal MDA, CAT, and T-SOD. Additional research demonstrated that RSV decreased inflammation by blocking the translocation of nuclear factor-kappaB (NF-κB) p65 and decreased oxidative stress by activating the nuclear factor E2-related factor 2 (Nrf2) pathway and its associated antioxidant genes, including NQO1, γ-GCS, and GSH-PX. In summary, RSV supplementation attenuates intestinal oxidative stress, inflammation, and intestinal barrier dysfunction induced by ZEA exposure by mediating the NF-κB and Nrf2/HO-1 pathways.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Effect of Ginger Root Extract on Intestinal Oxidative Status and Mucosal Morphometrics in Broiler Chickens.

This study was designed to assess the effect of ginger root extract (GRE) supplementation on the oxidative status and intestinal mucosal development in broiler chickens for 6 weeks. Day-old chicks (Ross 708 strain, n = 432) were distributed into six treatments with six replicate of twelve birds each: Negative CON (basal), MX (basal diet + bacitracin methylene disalicylate (BMD) 0.055 g/kg diet), GRE-1 (basal diet + 0.375% GRE), GRE-2 (basal diet + 0.75% GRE), GRE-3 (basal diet + 1.5% GRE), GRE-4 (basal diet + 3% GRE). Growth indices, goblets cell count, mucin (MUC2) in ileum tissue, antioxidant (SOD, CAT, and GPX) in ileum and liver, biological antioxidant potential (BAP), and reactive oxygen metabolite level in blood and intestinal villi measurement were determined. Body weight (BW) was highest (p < 0.05) in all groups except GRE-4, body weight gain (BWG) was best in GRE-1, while FCR was least in all groups except GRE-4. Optimum MUC2 gene expression, SOD, CAT, blood antioxidants, and intestinal morphometric values were observed in GRE-3. The inclusion of ginger root extract up to 1.5% improved growth and reduced oxidative stress while enhancing mucosal development in broiler chicks.

Effects of dietary pectin on the growth performance, intestinal barrier, and antioxidant status of juvenile rainbow trout (Oncorhynchus mykiss)

To evaluate the effects of dietary pectin on growth performance, intestinal barrier, and antioxidant status of juvenile rainbow trout (Oncorhynchus mykiss), 10-week feeding trial was conducted. Five isoproteic and isoenergetic diets were formulated to contain 0% (P0), 4% (P4), 8% (P8), and 16% (P16) pectin as well as 0.02% pectinase (PE) at the expense of wheat starch, respectively. Dietary inclusion of 416% pectin linearly promoted the feed intake and growth performance but depressed the feed efficiency, but significant differences were only observed between the P16 and P0 groups. Similarly, dietary inclusion of 816% pectin significantly increased the foregut trypsin and amylase activities but decreased the foregut and midgut sucrase activity. The midgut muc2 gene expression level was significantly down-regulated by the inclusion of 16% pectin. Dietary inclusion of 2% pectin increased the abundance of Proteobacteria and Stenotrophomonas but reduced the abundance of Actinobacteria, Bacteroides and Helicobacter; while the lowest values of observed species, Chao1 and ACE indices of intestinal flora were found in the P8 group. Fish fed with the P8 diet exhibited the lowest plasma catalase and midgut glutathione S-transferase activities as well as malondialdehyde content, but the highest midgut malondialdehyde content. Dietary inclusion of 816% pectin significantly depressed the plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and mechanistic target of rapamycin contents as well as alanine aminotransferase activity, the hepatic adenosine monophosphate deaminase and glutamate dehydrogenase (GDH) activities. Dietary treatment with 0.02% pectinase did not affect the growth performance, but depressed the foregut sucrase activity, the midgut amylase and sucrase activities, increased the plasma GH and IGF-1 contents as well as hepatic GDH and aspartate aminotransferase activities. In conclusion, high-dose of dietary pectin (816%) may promote the feed intake and intestinal digestive enzyme activities and thereby improve the growth performance of rainbow trout.

A45 ASSESSING THE ROLE OF MICROBIOTA DYSBIOSIS ON INTESTINAL TUFT CELL FUNCTIONS UPON GIARDIA INFECTION

Abstract Background Enteric tuft cells can detect and respond to intestinal parasitic infection by modulating host responses to aid parasite clearance, yet the extent of tuft cell functions upon other infections remains to be elucidated. Previous work in our lab showed that the enteric protozoan parasite Giardia induced tuft and goblet cell hyperplasia, yet Giardia colonization in Pou2f3-/- (tuft cell-deficient) mice was impaired. Not only can Giardia modulate the gut microbiota and lead to microbial dysbiosis, but the microbiota itself can influence Giardia colonization. Interestingly, certain microbiota-derived products can activate tuft cells in the gut. In this study, we investigated the role of microbiota dysbiosis in the crosstalk between Giardia and tuft cells. Aims We aim to explore the bidirectional effects between tuft cells and Giardia infection by investigating the role of 1) the host microbiota influenced by the presence or absence of tuft cells in altering Giardia infectivity, and 2) Giardia modified microbiota in contributing to tuft cell activity. Methods Fecal matter from 6–8-week-old C57Bl/6 and Pou2f3-/- mice were administered to polyethylene glycol (PEG) treated C57Bl/6 mice via oral gavage for 7 days, followed by gavage with 5x104Giardia muris trophozoites. Duodenal parasite burden was assessed 11 days after infection. Small intestinal (SI) microbiota was collected from 5–7-week-old C57Bl/6 and Pou2f3-/- mice following 11 days of G. muris infection and transplanted into PEG treated C57Bl/6 and Pou2f3-/- recipients. 11 days post-transfer, SI tuft and goblet cell hyperplasia was assessed in recipient mice by immunohistochemistry, and Dclk1, Atoh1, and Muc2 gene expression was quantified by RT-qPCR. Parameters of gut motility were evaluated. Results FMT with Pou2f3-/- mouse fecal matter prior to Giardia infection led to improved weight gain during infection compared to FMT from C57Bl/6 mice. Giardia modified microbiota (GMM) did not induce tuft or goblet cell hyperplasia 11 days after transfer. Jejunal Dclk1, Atoh1, and Muc2 were no different after GMM compared to control microbiota (CM) transfer. Ileal Atoh1 or Muc2 were upregulated in mice receiving GMM from C57Bl/6 or Pou2f3-/- mice, respectively. Intestinal transit time and stool water content was altered in mice which received GMM from Pou2f3-/- mice compared to CM. Conclusions The resident microbiota in tuft cell deficient mice may in part contribute to limiting Giardia infection. Giardia modified microbiota does not substantially contribute to tuft cell hyperplasia or related gene expression alterations at 11 days after microbiota transfer. The mechanistic contributors to tuft cell hyperplasia during Giardia infection and how a lack of tuft cells can lead to impaired colonization does not appear to be primarily driven by microbiota alterations and remains to be fully defined. Funding Agencies NSERC

PSVI-14 Mitigating the Negative Effects of Heat Stress in Grow-Finish Pigs Using Water Delivered Oregano Essential Oil

Abstract Grow-finish pigs (n = 192) were used in a 28-day experiment to determine if adding oregano essential oil to the water would mitigate the detrimental effects of heat stress. Pigs (initial BW = 51.9 kg) were blocked by sex and BW and randomly allotted to a 2x2 factorial arrangement of water treatment (oregano essential oil vs. control) and environment (heat stress vs. thermoneutral). There were 6 pigs/pen (4 barrows and 2 gilts/pen) and 8 pens/treatment. Pigs were given 15 d to acclimate to pens and water treatment under thermoneutral conditions (21.1℃). Following acclimation, one-half the pigs were subjected to a 3-day diurnal heat stress (12 h at 33.3℃ and 12 h at 26.7℃). On d 22 of the experiment (3 d post-heat stress), one barrow/pen was euthanized; jejunal mucosa scrapings were collected, flash frozen in liquid nitrogen, and stored at -80oC for subsequent RNA isolation and cDNA creation. Gene expression of CDKN1A, TP53, GPX1, SOD1, SLC2A2, SLC2A5, and MUC2 were determined by qPCR using SYBR green, normalized using housekeeper genes and expressed relative to the control treatment using the 2-ΔΔCT method. Data were analyzed as RCB using GLM procedure in SAS 9.4 with fixed effects of water treatment, environment, and the interaction. Heat-stressed pigs had reduced ADG (P &amp;lt; 0.003) and Gain:Feed (P &amp;lt; 0.008) during the 3d heat stress compared with pigs housed in thermoneutral conditions. Post-heat stress, ADG (P &amp;lt; 0.001) and Gain:Feed (P &amp;lt; 0.001) increased in previously heat-stressed pigs compared with thermoneutral pigs. There was no effect of oregano supplementation before or during heat stress. However, in the post-heat stress period, ADG was increased (P &amp;lt; 0.05) by oregano supplementation in the water for both heat stressed and non-heat stressed pigs. Oregano supplementation did not affect ADFI before heat stress. An interaction (P &amp;lt; 0.005) of heat stress and water treatment was observed for ADFI during heat stress and after heat stress. During heat stress, oregano supplementation resulted in reduced feed intake. Post-heat stress ADFI was lowest for non-heat stressed pigs with no oregano supplementation. Oregano essential oil increased water consumption before (P &amp;lt; 0.02), during (P &amp;lt; 0.03), and after heat stress (P &amp;lt; 0.02), with an overall increase in water consumption of 35% compared with control pigs (P &amp;lt; 0.04). No interactions of heat stress and oregano supplementation were observed for gene expression. SLC2A2 expression tended (P &amp;lt; 0.08) to be downregulated when oregano essential oils were added to the water and upregulated following an acute heat stress. SLC2A5 expression was increased in pigs given oregano compared with control pigs (P &amp;lt; 0.02), but was unaffected by heat stress. Water delivered oregano essential oil increased water intake and had a slight effect on intestinal gene expression but did not mitigate the effects of a short-term heat stress.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCβ3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

Evaluation the effect of dietary vitamin E, sesamin and thymoquinone bioactive compounds on immunological response, intestinal traits and MUC-2 gene expression in broiler Japanese quails (Coturnix japonica)

The current study was performed to determine the effect of dietary vitamin E, sesamin and thymoquinone bioactive lignans derived from sesame and black seed on immunological response, intestinal traits and Mucin2 gene expression in broiler quails. Three hundred and fifty (one days-old) quails were allotted to seven dietary treatments with five replicates as an experimental randomized design study. Treatments were basal diet as a control, control +100 and +200 mg of vitamin E, sesamin and thymoquinone per each kg of diet respectively. At 35 d of age, two quails from each pen were chosen, weighted, slaughtered, eviscerated and lymphoid organ relative weights were measured. Anti-body titers against Newcastle disease (ND), Sheep red blood cell (SRBC), and infectious bronchitis virus (IBV) and Avian influenza (AI) vaccination were determined. The serum activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and serum antioxidant activates such as superoxide dismutase (SOD),glutathione peroxidase(GPX), catalase (CAT) and total antioxidant capacity (TAC) were examined. The cell mediated immunity by dinitrochlorobenzene (DNCB) and phytohemagglutinin (PHA) challenges were assessed. The microflora populations of ileum, morphological traits of jejunum and mucin2 gene expression were analyzed. Data showed that the lymphoid organ (thymus, spleen and Bursa) relative weights and antibody titer against HI, AI, SRBC and IB vaccination were increased compared to the control (p ≤ 0.05). Serum activities of ALP, ALT and AST were decreased under influences of dietary treatments (p ≤ 0.05). The serum antioxidant activates of GPX,SOD,CAT and TAC were increased and Increasing in mean skin thickness after DNCB challenge and decrease wing web swelling response to PHA mitojen injection were observed (p ≤ 0.05). Salmonella enterica, E-coli and Coliforms colonies were decrease and Lactobacillus colonies increased instead (p ≤ 0.05). The villus height and surface, crypt depth and goblet cells density were increased compared to the control (p ≤ 0.05). The expression of MUC2 gene increased under influnces of vitamin E, sesamin and thymoquinone supplemented diets (p ≤ 0.05).

Evaluation of stimbiotic on growth performance and intestinal development of broilers fed corn- or wheat-based diets

In the antibiotics-free era, stimbiotic (STB) has been suggested as a new alternative of antibiotic growth promoters to modulate intestinal health via stimulating dietary fiber utilization in poultry production. The aim of this study was to evaluate the effects of STB supplementation in corn- or wheat-basal diet on growth performance, intestinal development, and function of broilers. A total of 512 one-day-old Arbor Acres(AA)broilers were randomly allocated 4 treatments, including corn group (CG), corn+100 g/t STB (CG+STB), wheat group (WG), wheat+100 g/t STB (WG+STB). The broilers were weighed at the days of 14, 28, and 42, of which 8 repetitions per treatment were randomly selected to determine the intestinal morphology, intestinal barrier, and cecal microbiota and metabolites. Our data showed that STB increased (P < 0.05) feed intake, body weight and reduced FCR for the overall period (0-42 d). At 28 d of age, significant increases in villus height and the villus height-to-crypt depth ratio (V/C) were found in the STB supplementation groups (P < 0.05). Addition of STB significantly increased intestinal mucosal DAO and AMPK enzyme activity and the gene expression of OCLN, CLDN1, ZO1, MUC2, SGLT1, PEPT1, FABP2, Ghrelin, and GCG in jejunum (P < 0.05), and significantly decreased the expression of the PYY gene. In addition, STB increased the relative abundance of beneficial bacteria, such as Akkermansia, Bifidobacterium, and Oscillospirales (P < 0.05). A significant increase in cecal short-chain fatty acid (SCFAs) concentration was also observed in the STB supplementation groups. At the cellular level, STB cannot directly increase the expression of small intestinal epithelial cells, and may indirectly improve intestinal barrier function by increasing the level of sodium butyrate. Overall, these results indicated that STB supplementation could improve the growth performance, intestinal development and barrier functions, and fiber fermentation in cecum of broiler chickens.

Nigella sativa-chitosan nanoparticles: Novel intestinal mucosal immunomodulator controls and protects against Salmonella enterica serovar Enteritidis infection in broilers

BackgroundSalmonella Enteritidis (SE) propagates in chickens' gastrointestinal surfaces and is transmitted to humans, causing food poisoning. Oral supplementation with natural nanoparticles can overcome the harsh gastrointestinal conditions facing oral vaccines and requires no antibiotic administration to protect against microbial infection. This study was designed to study Nigella sativa-chitosan nanoparticles (CNP-NS) prophylactic immunomodulatory efficacy against SE infection in broiler chicks. The CNP-NS was prepared and characterized, and its in vivo immunomodulatory activities against an avian virulent-MDR SE-induced challenge in chicks were investigated.ResultTo verify the immune-protective activities of the CNP-NS, colony forming units (CFU) in the liver and fecal droppings; intestinal histopathological alterations and immune cell recruitment; MUC-2, TLR-4, cecal cytokines, and specific IgA gene expression levels were assessed. On the 7th and 12th days after the SE challenge, the CNP-NS supplemented chicks showed complete clearance of SE CFU in livers and fecal droppings, as well as an improvement in food conversion rate compared to non-supplemented CNP-NS that revealed the presence of the challenge SE CFU on the same days. A prominent influx of antigen presenting cells and lymphoid aggregates into the intestinal wall, spleen, and liver was detected with improvements in the intestinal villi morphometry of the CNP-NS-supplemented chicks. The changes of INF-γ, IL-1β, and IL-4 cecal cytokines, as well as TLR-4, MUC-2, and IgA mRNA expression levels, confirm CNP-NS immunomodulatory activities and provide a mechanism(s) for its protective actions against the induced SE challenge of the tested chickens.ConclusionThese findings suggest promising useful insights into CNP-NS supplementation as a safe food additive for poultry meat consumers' and a protective immunomodulator of the chickens' mucosal immune systems. It could be recommended for epidemiological purposes to reduce the risk of SE food poisoning and transmission to humans.

Inhibitory activity of Limosilactobacillus reuteri isolated from camel milk against Helicobacter pylori effects in human gastric epithelial cells.

This study aimed to evaluate anti-Helicobacter pylori effects of Limosilactobacillus reuteri 2892 (L. reuteri 2892) isolated from camel milk in GC cell lines (AGS and MKN). From 15 camel milk samples, 132 microbial strains were isolated. Based on microbial and biochemical analysis, 11 potential probiotic candidates were selected. The potential probiotic candidates were assayed for anti-H. pylori activity, and the strain with the highest anti-H. pylori activity was identified genotypically. Based on 16S rDNA sequencing, the selected strain with the best activity against H. pylori (inhibition zone=15.5±0.8) belonged to the Lactobacillus reuteri strain 2892. Cell treatment with H. pylori HC-113 inhibits gene expression of Claudin-4, ZO-1, MUC5AC, and MUC2 in gastric cells, which are attenuated by L. reuteri 2892. The simulative effects of H. pylori HC-113 on the cell migration and invasion of gastric cells were lost when cells were cotreated with L. reuteri 2892. Cell treatment with H. pylori HC-113 promoted cell death, whereas cotreatment with L. reuteri 2892 markedly decreased necrotic and late apoptotic cells. The present study demonstrates that L. reuteri 2892 has potent anti-H. pylori effects and thus can be considered as an alternative protective agent against inflammatory effects of H. pylori in gastric cells.

Biosynthesized selenium nanoparticles to rescue coccidiosis-mediated oxidative stress, apoptosis and inflammation in the jejunum of mice.

One of the most crucial approaches for treating human diseases, particularly parasite infections, is nanomedicine. One of the most significant protozoan diseases that impact farm and domestic animals is coccidiosis. While, amprolium is one of the traditional anticoccidial medication, the advent of drug-resistant strains of Eimeria necessitates the development of novel treatments. The goal of the current investigation was to determine whether biosynthesized selenium nanoparticles (Bio-SeNPs) using Azadirachta indica leaves extract might treat mice with Eimeria papillata infection in the jejunal tissue. Five groups of seven mice each were used, as follows: Group 1: Non-infected-non-treated (negative control). Group 2: Non-infected treated group with Bio-SeNPs (0.5 mg/kg of body weight). Groups 3-5 were orally inoculated with 1×103 sporulated oocysts of E. papillata. Group 3: Infected-non-treated (positive control). Group 4: Infected and treated group with Bio-SeNPs (0.5 mg/kg). Group 5: Infected and treated group with the Amprolium. Groups 4 and 5 daily received oral administration (for 5 days) of Bio-SeNPs and anticoccidial medication, respectively, after infection. Bio-SeNPs caused a considerable reduction in oocyst output in mice feces (97.21%). This was also accompanied by a significant reduction in the number of developmental parasitic stages in the jejunal tissues. Glutathione reduced (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were dramatically reduced by the Eimeria parasite, whereas, nitric oxide (NO) and malonaldehyde (MDA) levels were markedly elevated. The amount of goblet cells and MUC2 gene expression were used as apoptotic indicators, and both were considerably downregulated by infection. However, infection markedly increased the expression of inflammatory cytokines (IL-6 and TNF-α) and the apoptotic genes (Caspase-3 and BCL2). Bio-SeNPs were administrated to mice to drastically lower body weight, oxidative stress, and inflammatory and apoptotic indicators in the jejunal tissue. Our research thus showed the involvement of Bio-SeNPs in protecting mice with E. papillata infections against jejunal damage.

Oral Exposure to Polystyrene Microplastics of Mice on a Normal or High-Fat Diet and Intestinal and Metabolic Outcomes.

Microplastics (MPs) are small particles of plastic ( in diameter). In recent years, oral exposure to MPs in living organisms has been a cause of concern. Leaky gut syndrome (LGS), associated with a high-fat diet (HFD) in mice, can increase the entry of foreign substances into the body through the intestinal mucosa. We aimed to evaluate the pathophysiology of intestinal outcomes associated with consuming a high-fat diet and simultaneous intake of MPs, focusing on endocrine and metabolic systems. C57BL6/J mice were fed a normal diet (ND) or HFD with or without polystyrene MP for 4 wk to investigate differences in glucose tolerance, intestinal permeability, gut microbiota, as well as metabolites in serum, feces, and liver. In comparison with HFD mice, mice fed the HFD with MPs had higher blood glucose, serum lipid concentrations, and nonalcoholic fatty liver disease (NAFLD) activity scores. Permeability and goblet cell count of the small intestine (SI) in HFD-fed mice were higher and lower, respectively, than in ND-fed mice. There was no obvious difference in the number of inflammatory cells in the SI lamina propria between mice fed the ND and mice fed the ND with MP, but there were more inflammatory cells and fewer anti-inflammatory cells in mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. The expression of genes related to inflammation, long-chain fatty acid transporter, and cotransporter was significantly higher in mice fed the HFD with MPs than in mice fed the HFD without MPs. Furthermore, the genus Desulfovibrio was significantly more abundant in the intestines of mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. Muc2 gene expression was decreased when palmitic acid and microplastics were added to the murine intestinal epithelial cell line MODE-K cells, and Muc2 gene expression was increased when IL-22 was added. Our findings suggest that in this study, MP induced metabolic disturbances, such as diabetes and NAFLD, only in mice fed a high-fat diet. These findings suggest that LGS might have been triggered by HFD, causing MPs to be deposited in the intestinal mucosa, resulting in inflammation of the intestinal mucosal intrinsic layer and thereby altering nutrient absorption. These results highlight the need for reducing oral exposure to MPs through remedial environmental measures to improve metabolic disturbance under high-fat diet conditions. https://doi.org/10.1289/EHP11072.