Tight Junction Gene Expression Research Articles

Effects of pectin's degree of methyl esterification on TLR2-mediated IL-8 secretion and tight junction gene expression in intestinal epithelial cells: influence of soluble TLR2.

This study investigates the anti-inflammatory effects of pectins with different degrees of methyl esterification (DM) on intestinal epithelial cells (IECs) expressing low and high levels of TLR2. It also studies the influence of soluble TLR2 (sTLR2) which may be enhanced in patients with inflammatory bowel syndrome on the inflammation-attenuating effects of pectins. Also, it examines the impact of pectins on tight junction gene expression in IECs. Lemon pectins with DM18 and DM88 were characterized, and their effects on TLR2-1-induced IL8 gene expression and secretion were investigated in low-TLR2 expressing Caco-2 and high-TLR2 expressing DLD-1 cells. The results demonstrate that both DM18 and DM88 pectins can counteract TLR2-1-induced IL-8 expression and secretion, with more pronounced effects observed in DLD-1 cells expressing high levels of TLR2. Furthermore, the presence of sTLR2 does not interfere with the attenuating effects of low DM18 pectin and may even support its anti-inflammatory effects in Caco-2 cells. The impact of pectins and sTLR2 on tight junction gene expression also demonstrates cell-type-dependent effects. Overall, these findings suggest that low DM pectins possess potent anti-inflammatory properties and may influence tight junction gene expression in IECs, thereby contributing to the maintenance of gut homeostasis.

Aspirin eugenol ester affects ileal barrier function, inflammatory response and microbiota in broilers under lipopolysaccharide-induced immune stress conditions.

The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on ileal immune function in broilers under lipopolysaccharide (LPS)-induced immune stress. Two hundred and forty one-day-old male Arbor Acres chicks were randomly divided into four groups (saline, LPS, saline + AEE and LPS + AEE) with six replicates of ten broilers each. The saline group and LPS group were fed the normal diet, while the other two groups received normal diet plus 0.1 g/kg AEE. Broilers in the LPS and LPS + AEE groups were injected intraperitoneally with 0.5 mg/kg B.W LPS in saline for seven consecutive days beginning at 14 days of age, while broilers in the saline and saline + AEE groups were injected with saline only. The results showed that AEE improved the ileal morphology and increased the ratio of villus height to crypt depth of immune-stressed broilers. LPS-induced immune stress significantly reduced the expression of the genes for the tight junction proteins occludin, zonula occludens-1 (ZO-1), claudin-1 and claudin-2, in the ileum, while AEE significantly up-regulated the expression of these genes. Compared with the saline group, the LPS-treated chickens showed significantly increased mRNA expression of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), and microsomal Prostaglandin E Synthesase-1 (mPGES-1) in the ileum, while they were significantly decreased by AEE supplementation. In addition, analysis of the ileal bacterial composition showed that compared with saline and LPS + AEE groups, the proportion of Firmicutes and Lactobacillus in the LPS group was lower, while the proportion of Proteobacteria and Escherichia-Shigella was higher. Similarly, Line Discriminant Analysis Effect Size (LEfSe) analysis showed that compared with the LPS group, Brevibacillus was dominant in the saline group, while the LPS + AEE group was rich in Rhizobium, Lachnoclostridium, Ruminococcaceae, Faecalibacterium, Negativibacillus, Oscillospiraceae, and Flavonifractor. These results indicate that dietary supplementation with 0.1 g/kg AEE could protect the intestinal health by improving the intestinal villus morphology, enhancing the expression of tight junction genes and alleviating inflammation to resist the immune stress caused by LPS stimulation in broilers, and the mechanism may involve COX-2-related signal transduction and improved intestinal microbiota composition.

In vitro faecal fermentation metabolites of 2′-fucosyllactose protect against intestinal epithelial injury: Infant enterotype effects

In this study, we conducted in vitro fermentation with infant faecal inocula dominated by Bifidobacterium longum, Bifidobacterium breve, and Bacteroides enterotypes, and assessed the efficacy of the metabolome associated with 2′-fucosyllactose (2′-FL) fermentation on lipopolysaccharide-induced epithelial cell barrier damage on Caco-2 cells. The results revealed that propionate and butyrate were more prominent in the Bacteroides-dominated group, whereas the Bifidobacterium-dominated group was associated with an increased molar fraction of lactate (∼16 mm). Additionally, our study demonstrated that 2′-FL faecal metabolites (FMs) decreased the production of inflammatory cytokines, and promoted tight junction gene expression by inhibiting the TLR4/NF-κB/MLCK signalling pathway and activating the Nrf2/HO-1 pathway. The 2′-FL FMs from Bacteroides-dominated enterotypes had a unique advantage in regulating the TLR4/NF-κB/MLCK signalling pathway, which can be attributed to the effects of propionate and butyrate.

Methionine sources and genotype affect embryonic intestinal development, antioxidants, tight junctions, and growth-related gene expression in chickens

Methionine (Met) is an essential and first limiting amino acid in the poultry diet that plays a significant role in chicken embryonic development and growth. The present study examined the effect of in ovo injection of DL-Met and L-Met sources and genotypes on chicken embryonic-intestinal development and health. Fertilized eggs of the two genotypes, TETRA-SL layer hybrid (TSL) — commercial layer hybrid and Hungarian Partridge colored hen breed (HPC) — a native genotype, were randomly distributed into four treatments for each genotype. The treatment groups include the following: 1) control non-injected eggs (NoIn); 2) saline-injected (SaIn); 3) DL-Met injected (DLM); and 4) L-Met injected (LM). The in ovo injection was carried out on 17.5 d of embryonic development; after hatching, eight chicks per group were sacrificed, and the jejunum was extracted for analysis. The results showed that both DLM and LM groups had enhanced intestinal development as evidenced by increased villus width, villus height, and villus area (P < 0.05) compared to the control. The DLM group had significantly reduced crypt depth, glutathione (GSH) content, glutathione S-transferase 3 alpha (GST3), occludin (OCLN) gene expression and increased villus height to crypt depth ratio in the TSL genotype than the LM group (P < 0.05). The HPC genotype has overexpressed insulin-like growth factor 1 (IGF1) gene, tricellulin (MD2), occludin (OCLN), superoxide dismutase 1 (SOD1), and GST3 genes than the TSL genotype (P < 0.05). In conclusion, these findings showed that in ovo injection of Met enhanced intestinal development, and function, with genotypes responding differently under normal conditions. Genotypes also influenced the expression of intestinal antioxidants, tight junction, and growth-related genes.

Tight Junction Claudins and Occludin Are Differentially Regulated and Expressed in Genomically Defined Subsets of Colon Cancer.

Metastatic colon cancer remains incurable despite improvements in survival outcomes. New therapies based on the discovery of colon cancer genomic subsets could improve outcomes. Colon cancers from genomic studies with publicly available data were examined to define the expression and regulation of the major tight junction proteins claudins and occludin in genomic groups. Putative regulations of the promoters of tight junction genes by colon-cancer-deregulated pathways were evaluated in silico. The effect of claudin mRNA expression levels on survival of colon cancer patients was examined. Common mutations in colon-cancer-related genes showed variable prevalence in genomically identified groups. Claudin genes were rarely mutated in colon cancer patients. Genomically identified groups of colon cancer displayed distinct regulation of claudins and occludin at the mRNA level. Claudin gene promoters possessed clustered sites of binding sequences for transcription factors TCF4 and SMADs, consistent with a key regulatory role of the WNT and TGFβ pathways in their expression. Although an effect of claudin mRNA expression on survival of colon cancer patients as a whole was not prominent, survival of genomic subsets was significantly influenced by claudin mRNA expression. mRNA expression of the main tight junction genes showed differential regulation in various genomically defined subgroups of colon cancer. These data pinpoint a distinct role of claudins and pathways that regulate them in these subgroups and suggest that subgroups of colon cancer should be considered in future efforts to therapeutically target claudins.

Effect of Echinacea purpurea (L.) Moench and its extracts on the immunization outcome of avian influenza vaccine in broilers

Ethnopharmacological relevanceEchinacea purpurea (L.) Moench (EP) is a perennial herbaceous flowering plant with immunomodulatory effects. However, the immunomodulatory effects of EP on broilers after vaccination are still unclear. Aim of the studyThe aim is to study the effect of EP and Echinacea purpurea (L.) Moench extracts(EE) on avian influenza virus (AIV) immunity, and further explore the potential mechanism of immune regulation. Materials and methodsBroilers were fed with feed additives containing 2% EP or 0.5% EE, and vaccinated against avian influenza. The samples were collected on the 7th, 21st, and 35th day after vaccination, and the feed conversion ratio (FCR) was calculated. Blood antibody titer, jejunal sIgA content, tight junction protein, gene and protein expression of TLR4-MAPK signaling pathway were also detected. ResultsThe results showed that vaccination could cause immune stress, weight loss, increase sIgA content, and up-regulate the expression of tight junction proteins, including zonula occludens-1 (ZO-1), Occludin, and Claudin-1, as well as the genes of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), receptor-associated factor 6 (TRAF6), activator protein 1 (AP-1) protein gene expression on TLR4-mitogen-activated protein kinase (MAPK) signaling pathway, and the protein expression of MyD88, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK). EP and EE could increase the body weight of broilers, further improve antibody titers, decrease FCR, increase sIgA levels, up-regulate the expression of tight junction proteins, including ZO-1, Occludin, and Claudin-1, as well as the genes of TLR4, MyD88, TRAF6, and AP-1 and the protein expression of MyD88, ERK, and JNK in the TLR4-MAPK signaling pathway. ConclusionIn conclusion, EP and EE can increase the broiler's production performance and improve vaccine immune effect through the TLR4-MAPK signaling pathway.

Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17β-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17β-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

β(2 → 1)-β(2 → 6) and β(2 → 1) fructans protect from impairment of intestinal tight junction's gene expression and attenuate human dendritic cell responses in a fructan-dependent fashion

β(2 → 1)-β(2 → 6) branched graminan-type fructans (GTFs) and β(2 → 1) linear fructans (ITFs) possess immunomodulatory properties and protect human intestinal barrier function, however the mechanisms underlying these effects are not well studied. Herein, GTFs and ITFs effects with different degree of polymerization (DP) values on tight junctions (TJs) genes CLDN-1, -2 and -3, CDH1, OCLN and TJP1 were studied in Caco-2 gut epithelial cells, under homeostatic and inflammatory conditions. Also, cytokine production in dendritic cells (DCs) was studied. Higher DP fructans decreased the expression of the pore forming CLDN-2. Higher DP GTFs enhanced CLDN-3, OCLN, and TJP-1. Fructans prevented mRNA dysregulation of CLDN-1, -2 and -3 induced by the barrier disruptors A23187 and deoxynivalenol in a fructan-type dependent fashion. The production of pro-inflammatory cytokines MCP-1/CCL2, MIP-1α/CCL3 and TNFα by DCs was also attenuated in a fructan-type dependent manner and was strongly attenuated by DCs cultured with medium of Caco-2 cells which were pre-exposed to fructans. Our data show that specific fructans have TJs and DCs modulating effects and contribute to gut homeostasis. This might serve to design effective dietary means to prevent intestinal inflammation.

Panaxynol alleviates colorectal cancer in a murine model via suppressing macrophages and inflammation.

Currently available colorectal cancer (CRC) therapies have limited efficacy and severe adverse effects that may be overcome with the alternative use of natural compounds. We previously reported that panaxynol (PA), a bioactive component in American ginseng, possesses anticancer properties in vitro and suppresses murine colitis through its proapoptotic and anti-inflammatory properties. Because colitis is a predisposing factor of CRC and inflammation is a major driver of CRC, we sought to evaluate the therapeutic potential of PA in CRC. Azoxymethane-dextran sodium sulfate (AOM/DSS) mice (C57BL/6) were administered 2.5 mg/kg PA or vehicle 3 times/wk via oral gavage over 12 wk. PA improved clinical symptoms (P ≤ 0.05) and reduced tumorigenesis (P ≤ 0.05). This improvement may be reflective of PA's restorative effect on intestinal barrier function; PA upregulated the expression of essential tight junction and mucin genes (P ≤ 0.05) and increased the abundance of mucin-producing goblet cells (P ≤ 0.05). Given that macrophages play a substantial role in the pathogenesis of CRC and that we previously demonstrated that PA targets macrophages in colitis, we next assessed macrophages. We show that PA reduces the relative abundance of colonic macrophages within the lamina propria (P ≤ 0.05), and this was consistent with a reduction in the expression of important markers of macrophages and inflammation (P ≤ 0.05). We further confirmed PA's inhibitory effects on macrophages in vitro under CRC conditions (P ≤ 0.05). These results suggest that PA is a promising therapeutic compound to treat CRC and improve clinical symptoms given its ability to inhibit macrophages and modulate the inflammatory environment in the colon.NEW & NOTEWORTHY We report that panaxynol (PA) reduces colorectal cancer (CRC) by improving the colonic and tumor environment. Specifically, we demonstrate that PA improves crypt morphology, upregulates crucial tight junction and mucin genes, and promotes the abundance of mucin-producing goblet cells. Furthermore, PA reduces macrophages and associated inflammation, important drivers of CRC, in the colonic environment. This present study provides novel insights into the potential of PA as a therapeutic agent to ameliorate CRC tumorigenesis.

Effects of lactic acid bacteria isolated from Tibetan chickens on the growth performance and gut microbiota of broiler.

Lactic acid bacteria (LAB) are organic supplements that have several advantages for the health of the host. Tibetan chickens are an ancient breed, which evolve unique gut microbiota due to their adaptation to the hypoxic environment of high altitude. However, knowledge of LAB isolated from Tibetan chickens is very limited. Thus, the purpose of this study was to assess the probiotic properties of Lactobacillus Plantarum (LP1), Weissella criteria (WT1), and Pediococcus pentosaceus (PT2) isolated from Tibetan chickens and investigate their effects on growth performance, immunoregulation and intestinal microbiome in broiler chickens. Growth performance, serum biochemical analysis, real-time PCR, and 16S rRNA sequencing were performed to study the probiotic effects of LP1, WT1, and PT2 in broiler chickens. Results showed that LP1, WT1 and PT2 were excellent inhibitors against Escherichia coli (E. coli ATCC25922), meanwhile, LP1, WT1, and PT2 significantly increased weekly weight gain, villus height, antioxidant ability and gut microbiota diversity indexes in broilers. In addition, LP1 and PT2 increased the relative abundance of Lactobacillus and decreased Desulfovibrio in comparison with T1 (control group). Additionally, oral LAB can reduce cholesterol and regulate the expression of tight junction genes in broiler chickens, suggesting that LAB can improve the integrity of the cecal barrier and immune response. In conclusion, LAB improved the growth performance, gut barrier health, intestinal flora balance and immune protection of broiler chickens. Our findings revealed the uniqueness of LAB isolated from Tibetan chickens and its potential as a probiotic additive in poultry field.

Exercise prevents obesity by reducing gut-derived inflammatory signals to brown adipocytes in mice.

Gut dysbiosis impairs nonshivering thermogenesis (NST) in obesity. The antiobesogenic effects of exercise training might involve the modulation of gut microbiota and its inflammatory signals to the brown adipose tissue (BAT). This study evaluated whether high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) prevent overweight through reduced gut-derived inflammatory signals to BAT in high-fat-fed mice. Sixty male C57BL/6 mice (3 months old) comprised six experimental groups: control (C) diet group, C diet + HIIT (C-HIIT) group, C diet + MICT (C-MICT) group, high-fat (HF) diet group, HF diet + HIIT (HF-HIIT) group, and HF diet + MICT (HF-MICT) group. The protocols lasted for 10 weeks. HIIT and MICT restored body mass, mitigated glucose intolerance, and prevented hyperinsulinemia in HF-trained groups. A chronic HF diet caused dysbiosis, but HIIT and MICT prevented gut dysbiosis and preserved tight junction (TJ) gene expression. HF-HIIT and HF-MICT groups exhibited a similar pattern of goblet cell distribution, agreeing with the decreased plasma lipopolysaccharide concentrations and interscapular BAT (iBAT) Lbp-Cd14-Tlr4 expression. The lowered Nlrp3 and Il1β in the HF-HITT and HF-MICT groups complied with iBAT thermogenic capacity maintenance. This study shows reliable evidence that HIIT and MICT prevented overweight by restoring the diversity of the gut microbiota phyla and TJ gene expression, thereby reducing inflammatory signals to brown adipocytes with preserved thermogenic capacity. Both exercise modalities prevented overweight, but HIIT rescued Zo-1 and Jam-a gene expression, exerting more potent anti-inflammatory effects than MICT (reduced LPS concentrations), providing a sustained increase in thermogenesis with 78% less distance traveled.

Dietary high β-conglycinin reduces the growth through enhancing hepatic lipid peroxidation and impairing intestinal barrier function of orange-spotted grouper (Epinephelus coioides)

β-conglycinin is one of the major soy antigen proteins in soybean meal (SBM) and exhibits growth inhibition and intestinal health damage in grouper, but the underlying mechanisms are still poorly understood. In this study, orange-spotted grouper (Epinephelus coioides) was used to investigate whether the poor growth performance induced by dietary β-conglycinin levels is associated with intestinal structural integrity disruption, the intestinal apoptosis and intestinal microbiota. The basal diet (FM diet) was formulated to contained 48% protein and 12% fat without SBM supplementation. Fish meal protein in the FM diet was replaced by SBM to prepare a high SBM diet (SBM diet). β-conglycinin at 3% and 7% were added into FM diets to prepare two diets (B-3 and B-7). Triplicate groups of fish (20 fish/tank) were fed one of the experimental diets twice daily in a feeding period of 8 weeks. Compared with FM diet, fish fed diets SBM and B-7 had decreased the growth rate, hepatosomatic index, whole-body lipid and ash contents, and increased whole-body moisture content. However, the maximum growth was observed for diet B-3 and was not different from that of FM diet. The liver total antioxidant capacity and glutathione peroxidase activity, the muscle layer thickness of middle and distal intestine, and the mucosal fold length of distal intestine were lower, while liver malondialdehyde content, intestinal diamine oxidase activity, d-lactic acid and endotoxin contents, and the number of intestinal apoptosis were higher in SBM and B-7 groups than that in FM and/or B-3 groups. SBM and B-7 diets down-regulated the intestinal expression of tight junction genes (occludin, claudin-3 and ZO-1), apoptosis genes (bcl-2 and bcl-xL) and anti-inflammatory factor genes (IκBα, TGF-β1 and IL-10), but up-regulated the intestinal expression of apoptosis genes (caspase-3, caspase-8 and caspase-9) and pro-inflammatory factor genes (NF-κB1, RelA, TAK1, IKK, MyD88, TNF-α, IL-1β, and IL-8) vs FM and/or B-3 diets. The richness and diversity indexes of OTUs, Chao1, ACE, Shannon and Simpson were not affected by dietary treatments. The relative abundances of intestinal bacteria (phylum Proteobacteria and genus Vibrio) were generally higher, and relative abundance of phylum Tenericutes was lower in SBM and/or B-7 groups than that in B-3 group. The above results indicate that high dietary β-conglycinin level, rather than intermediate level could decrease liver antioxidant capacity, reshape the intestinal microbiota, and impair the intestinal normal morphology through disrupting the intestinal tight junction structure, increasing intestinal mucosal permeability, and promoting intestinal apoptosis, which in turn triggers intestinal inflammatory responses and the occurrence of enteritis, and ultimately leads to poor growth performance in fish.

Protective effects of melatonin on deoxynivalenol-induced oxidative stress and autophagy in IPEC-J2 cells.

This study explored protective effects of melatonin (MEL) on deoxynivalenol (DON)-induced toxicity in porcine jejunum epithelial cells (IPEC-J2). Cells were preexposed to MEL and then exposed to DON to detect cell viability, apoptosis, and oxidative stress indicators. Compared to DON treatment, pretreatment with MEL significantly increased cell proliferation. (P<0.01), intracellular catalase (CAT) and superoxide dismutase (SOD) levels (P<0.05), decreased apoptosis and oxidative stress, and significantly attenuated the inflammatory response. RNA-seq analysis revealed that MEL protects IPEC-J2 from the adverse effects of DON by affecting the expression of tight junction and autophagy pathway-related genes. Similarly, further experiments revealed that MEL partly prevented DON-induced disruption of intestinal barrier function and decreased autophagy induced by DON via activation of the AKT/mTOR pathway. In conclusion, these results demonstrated the preventive properties of MEL against DON-induced cell damage by activating the antioxidant system and Inhibition of autophagy.

Butyrate restores the fat/lean mass ratio balance and energy metabolism and reinforces the tight junction-mediated intestinal epithelial barrier in prediabetic mice independently of its anti-inflammatory and epigenetic actions

Tissue/cellular actions of butyrate on energy metabolism and intestinal barrier in normal metabolic conditions or prediabetes are still unclear. In this work, we investigated the beneficial effect of dietary supplementation with sodium butyrate on energy metabolism, body mass composition, and intestinal epithelial barrier mediated by tight junction (TJ) in chow diet-fed normal and high-fat diet (HF)-fed prediabetic mice, considering the well-known butyrate action as an epigenetic and inflammatory regulator. Butyrate significantly reduced the fat/lean mass ratio, slightly ameliorated dyslipidemia, restored oral glucose tolerance, and increased basal energy expenditure in prediabetic HF-fed mice but had no effect on control animals. Such effects were observed in the absence of significant alterations in the hypothalamic expression of orexigenic and anorexigenic genes and motor activity. Also, butyrate suppressed the whitening effect of HF on brown adipose tissue but did not affect cell bioenergetics in immortalized UCP1-positive adipocytes in vitro. Butyrate reinforced the intestinal epithelial barrier in HF-fed mice and in Caco-2 monolayers, which involved higher trafficking of TJ proteins to the cell-cell contact region of the intestinal epithelia, without affecting TJ gene expression or the acetylation level of histones H3 and H4 in vivo. All metabolic and intestinal effects of butyrate in prediabetic mice occurred in the absence of detectable changes in systemic or local inflammation, or alterations in endotoxemia markers. Butyrate has no effect on chow diet-fed mice but, in the context of HF-induced prediabetes, it prevents metabolic and intestinal dysfunctions independently of its anti-inflammatory and epigenetic actions.

Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infect Human Intestinal Epithelial Cells and Induce Moderate Inflammation.

Klebsiella pneumoniae is an opportunistic pathogen that can produce moderate and severe infections in immunosuppressed hosts. In recent years, an increase in the isolation of hypermucoviscous carbapenem-resistant K. pneumoniae with sequence type 25 (ST25) in hospitals in Norwest Argentina was observed. This work aimed to study the virulence and inflammatory potential of two K. pneumoniae ST25 strains (LABACER01 and LABACER27) in the intestinal mucosa. The human intestinal Caco-2 cells were infected with the K. pneumoniae ST25 strains, and their adhesion and invasion rates and changes in the expression of tight junction and inflammatory factors genes were evaluated. ST25 strains were able to adhere and invade Caco-2 cells, reducing their viability. Furthermore, both strains reduced the expression of tight junction proteins (occludin, ZO-1, and claudin-5), altered permeability, and increased the expression of TGF-β and TLL1 and the inflammatory factors (COX-2, iNOS, MCP-1, IL-6, IL-8, and TNF-α) in Caco-2 cells. The inflammatory response induced by LABACER01 and LABACER27 was significantly lower than the one produced by LPS or other intestinal pathogens, including K. pneumoniae NTUH-K2044. No differences in virulence and inflammatory potential were found between LABACER01 and LABACER27. In line with these findings, no major differences between the strains were found when the comparative genomic analysis of virulence factors associated with intestinal infection/colonization was performed. This work is the first to demonstrate that hypermucoviscous carbapenem-resistant K. pneumoniae ST25 infects human intestinal epithelial cells and induces moderate inflammation.

Effect of Dietary Organic Selenium on Growth Performance, Gut Health, and Coccidiosis Response in Broiler Chickens.

A total of 252 one-day-old Ross broilers were randomly allocated to one of six treatments in a 2 × 3 factorial arrangement with respective Eimeria challenges (non-infection and infection) and three different selenium (Se) diets. Dietary treatments were as follows: (1) Se un-supplemented control (CON), (2) inorganic Se treatment (SS; 0.3 mg/kg as sodium selenite), and (3) organic Se treatment (SY; 0.3 mg/kg as selenized yeast). Six replicate cages were allocated per treatment. Chickens in the respective Eimeria infection groups were infected with an E. acervulina, E. tenella, and E. maxima oocyst mixture (15,000 oocysts/chicken) on day 16. Growth performance was measured on days 16, 22, and 24. On day 22, intestinal samples were collected from randomly selected chickens to evaluate gut lesion scores, antioxidant enzymes, and tight junction gene expression. Blood, breast, and liver samples were collected to analyze the Se concentrations on day 24. Dietary SY supplementation improved (p < 0.05) the growth performance of the chickens regardless of the Eimeria challenge. Moreover, independent of Eimeria infection, Se supplementation elevated (p < 0.05) the heme oxygenase 1 (HMOX-1) expression in jejunal mucosa at 6 days post-infection (dpi). Duodenal junctional adhesion molecule 2 (JAM-2) expression and jejunal occludin (OCLN) were elevated (p < 0.05) with dietary SY supplementation at 6 dpi. Among Se sources, broiler chickens fed with the SY diet showed higher (p < 0.05) Se concentrations in breast muscle and serum on 8 dpi. These results confirmed the beneficial effects of dietary Se and the efficiency of organic Se compared with inorganic Se for growth improvement and muscle Se enrichment in broiler chickens regardless of coccidiosis infection.

Puerarin Alleviates H2O2-Induced Oxidative Stress and Blood-Milk Barrier Impairment in Dairy Cows.

During the perinatal period, the bovine mammary epithelial cells of dairy cows exhibit vigorous metabolism and produce large amounts of reactive oxygen species (ROS). The resulting redox balance disruption leads to oxidative stress, one of the main causes of mastitis. Puerarin (PUE) is a natural flavonoid in the root of PUE that has attracted extensive attention as a potential antioxidant. This study first investigated whether PUE could reduce oxidative damage and mastitis induced by hydrogen peroxide (H2O2) in bovine mammary epithelial cells in vitro and elucidated the molecular mechanism. In vitro, BMECs (Bovine mammary epithelial cells) were divided into four treatment groups: Control group (no treatment), H2O2 group (H2O2 stimulation), PUE + H2O2 group (H2O2 stimulation before PUE rescue) and PUE group (positive control). The growth of BMECs in each group was observed, and oxidative stress-related indices were detected. Fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of tightly linked genes, antioxidant genes, and inflammatory factors. The expression of p65 protein was detected by Western blot. In vivo, twenty cows with an average age of 5 years having given birth three times were divided into the normal dairy cow group, normal dairy cow group fed PUE, mastitis dairy cow group fed PUE, and mastitis dairy cow group fed PUE (n = 5). The contents of TNF-α, IL-6, and IL-1β in milk and serum were detected. In BMECs, the results showed that the PUE treatment increased the activities of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC); ROS and malondialdehyde (MDA) levels were reduced. Thus, PUE alleviated H2O2-induced oxidative stress in vitro. In addition, the PUE treatment eliminated the inhibition of H2O2 on the expression of oxidation genes and tight junction genes, and the enrichment degree of NRF-2, HO-1, xCT, and tight junctions (claudin4, occludin, ZO-1 and symplekin) increased. The PUE treatment also inhibited the expression of NF-κB-associated inflammatory factors (IL-6 and IL-8) and the chemokine CCL5 in H2O2-induced BMECs. In vivo experiments also confirmed that feeding PUE can reduce the expression of inflammatory factors in the milk and serum of lactating dairy cows. In conclusion, PUE can effectively reduce the oxidative stress of bovine mammary epithelial cells, enhance the tight junctions between cells, and play an anti-inflammatory role. This study provides a theoretical basis for PUE prevention and treatment of mastitis and oxidative stress. The use of PUE should be considered as a feed additive in future dairy farming.

Empagliflozin attenuates intestinal inflammation through suppression of nitric oxide synthesis and myeloperoxidase activity in in vitro and in vivo models of colitis

Inflammatory bowel diseases (IBD) are characterized by chronic and relapsing inflammation affecting the gastrointestinal (GI) tract. The incidence and prevalence of IBD are relatively high and still increasing. Additionally, current therapeutic strategies for IBD are not optimal. These facts urge todays’ medicine to find a novel way to treat IBD. Here, we focused on the group of anti-diabetic drugs called gliflozins, which inhibit sodium glucose co-transporter type 2 (SGLT-2). Numerous studies demonstrated that gliflozins exhibit pleiotropic effect, including anti-inflammatory properties. In this study, we tested the effect of three gliflozins; empagliflozin (EMPA), dapagliflozin (DAPA), and canagliflozin (CANA) in in vitro and in vivo models of intestinal inflammation. Our in vitro experiments revealed that EMPA and DAPA suppress the production of nitric oxide in LPS-treated murine RAW264.7 macrophages. In in vivo part of our study, we showed that EMPA alleviates acute DSS-induced colitis in mice. Treatment with EMPA reduced macro- and microscopic colonic damage, as well as partially prevented from decrease in tight junction gene expression. Moreover, EMPA attenuated biochemical inflammatory parameters including reduced activity of myeloperoxidase. We showed that SGLT-2 inhibitors act as anti-inflammatory agents independently from their hypoglycemic effects. Our observations suggest that gliflozins alleviate inflammation through their potent effects on innate immune cells.

Supplemental enzymes and probiotics on the gut health of broilers fed with a newly harvested corn diet

Gut health is important for digestion and absorption of nutrient for animals. The purpose of this study was to investigate the therapeutic effect of enzymes and probiotics alone or in combination on the gut health of broilers fed with newly harvested corn diets. A total of 624 Arbor Acres Plus male broiler chickens were randomly divided into 8 treatment groups (PC: normal corn diet, NC: newly harvested corn diet, DE: NC+glucoamylase, PT: NC+protease, XL: NC+xylanase, BCC: NC+Pediococcus acidilactici BCC-1, DE+PT: NC+glucoamylase+protease, XL+BCC: NC+xylanase+Pediococcus acidilactici BCC-1). Each group was divided into 6 replicates, with 13 birds each. On d 21, intestinal morphological, intestinal tight junction and aquaporins gene expression, cecal short-chain fatty acid concentrations, and microflora were measured. Compared with the newly harvested corn diets (NC), supplemental glucoamylase (DE) significantly increased the relative abundance of Lachnospiraceae (P < 0.05) and decreased the relative abundance of Moraxellaceae (P < 0.05). Supplemental protease (PT) significantly increased the relative abundance of Barnesiella (P < 0.05), but the relative abundance of Campylobacter decreased by 44.4%. Supplemental xylanase (XL) significantly increased the jejunal mRNA expressions of MUC2, Claudin-1, and Occludin (P < 0.01), as well as the cecal digesta contents of acetic acid, butyric acid, and valeric acid (P < 0.01). Supplemental DE combined with PT increased the ileal mRNA expressions of aquaporins (AQP) 2, AQP5, and AQP7 (P < 0.01). Supplemental BCC significantly increased the jejunal villus height and crypt depth (P < 0.01), the jejunal mRNA expressions of MUC2, Claudin-1 and Occludin (P < 0.01), and the relative abundance of Bacteroides (P < 0.05). Supplemental xylanase in combination with BCC significantly increased jejunal villus height and crypt depth (P < 0.01), the ileal mRNA expressions of AQP2, AQP5 and AQP7 (P < 0.01), and the cecal digesta contents of acetic acid, butyric acid, and valeric acid (P < 0.01). This suggests that inclusions of supplemental protease (12,000 U/kg), glucoamylase (60,000 U/kg), or Pediococcus acidilactici BCC-1 (109 cfu/kg) individually or in combination with xylanase (4,800 U/kg) in the newly harvested corn diets can alleviate diarrhea in broilers, and be beneficial for the gut health.

Network pharmacology-based analysis on geniposide, a component of gardenia jasminoides, beneficial effects to alleviate LPS-induced immune stress in piglets.

Geniposide is the main medicinal component of Gardenia jasminoides, and its content is approximately 3-8% depending on its origin. Geniposide is a class of cyclic enol ether terpene glucoside compounds with strong antioxidant, free radical quenching and cancer-inhibiting activities. Many studies have reported that geniposide has hepatoprotective, cholestatic, neuroprotective, blood sugar and blood lipid regulation, soft tissue damage treatment, antithrombotic, antitumor and other effects. As a traditional Chinese medicine, gardenia, whether used as gardenia alone, as the monomer geniposide or as the effective part of cyclic either terpenoids, has been reported to have anti-inflammatory effects when used in the right amounts. Recent studies have found that geniposide has important roles in pharmacological activities such as anti-inflammation activity, inhibition of the NF-κB/IκB pathway, and cell adhesion molecule production. In this study, we predicted the anti-inflammatory and antioxidant effects of geniposide in piglets through network pharmacology based on the LPS-induced inflammatory response-regulated signaling pathway. The effects of geniposide on changes in inflammatory pathways and cytokine levels in the lymphocytes of inflammation-stressed piglets were investigated using in vivo and in vitro models of piglet lipopolysaccharide-induced oxidative stress. Network pharmacology identified 23 target genes, of which the main pathways of action were lipid and atherosclerosis, fluid shear stress and atherosclerosis, and Yersinia infection. The main relevant target genes were VEGFA, ROCK2, NOS3, and CCL2. Validation experiments showed that the interventional effects of geniposide reduced the relative expression of NF-κB pathway proteins and genes, restored the expression of COX-2 genes to normal levels, and increased the relative expression of tight junction proteins and genes in IPEC-J2 cells. This indicates that the addition of geniposide can alleviate inflammation and improve the level of cellular tight junctions.