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Streptococcus uberis is a common cause of mastitis. The pathogenicity among different strains of S. uberis and the resultant host immune responses remain to be elucidated. Herein, we document immune responses among 3 strains of S. uberis, and preliminary explore whether and how intestinal immunity play a role in host anti-infection processes. Mice have been proved to be effective experimental animals for bovine mastitis, so utilizing a mouse intramammary infection model, we assay immune responses and gut flora changes of 3 S. uberis strains by histopathologic examination, RT-PCR, Western blot and 16s rDNA sequencing. We find that the immune responses among the 3 sequence type (ST) S. uberis strains may be linked to the hasA/B and lbp virulence genes, and the beta diversity of the intestinal may be independent of the ST type of S. uberis. Twenty phyla and 30 genera of intestinal flora are identified, Verrucomicrobia and Akkermansia being the most prominent phylum and genus respectively. These bacteria have strong anti-inflammatory and protective effects against S. uberis challenge. These datas provide a foundation for further studies to elucidate gut flora function and exploration of therapeutic targets for mastitis. This article is protected by copyright. All rights reserved.

Little is known about the regulatory effect of microbiota on the proliferation and regeneration of ISCs. Here, we found that L. reuteri stimulated the proliferation of intestinal epithelia by increasing the expression of R-spondins and thus activating the Wnt/β-catenin pathway. The proliferation-stimulating effect of Lactobacillus on repair is further enhanced under TNF -induced intestinal mucosal damage, and the number of Lgr5(+) cells is maintained. Moreover, compared to the effects of C. rodentium on the induction of intestinal inflammation and crypt hyperplasia in mice, L. reuteri protected the intestinal mucosal barrier integrity by moderately modulating the Wnt/β-catenin signaling pathway to avoid overactivation. L. reuteri had the ability to maintain the number of Lgr5(+) cells and stimulate intestinal epithelial proliferation to repair epithelial damage and reduce proinflammatory cytokine secretion in the intestine and the LPS concentration in serum. Moreover, activation of the Wnt/β-catenin pathway also induced differentiation toward Paneth cells and increased antimicrobial peptide expression to inhibit C. rodentium colonization. The protective effect of Lactobacillus against C. rodentium infection disappeared upon application of the Wnt antagonist Wnt-C59 in both mice and intestinal organoids. This study demonstrates that Lactobacillus is effective at maintaining intestinal epithelial regeneration and homeostasis as well as at repairing intestinal damage after pathological injury and is thus a promising alternative therapeutic method for intestinal inflammation.

Streptococcus uberis (S. uberis) is an important causative agent of mastitis, leading to significant economic losses to dairy industry. This research used a mouse mastitis model to investigate the protective effects of taurine on mammary inflammatory response and blood-milk barrier integrity in S. uberis challenge. The results showed that taurine attenuated S. uberis-induced mammary histopathological changes, especially neutrophil infiltration. The S. uberis-induced expression of pro-inflammatory mediators were decreased significantly by taurine. Further, we demonstrated that taurine limited the S. uberis-induced inflammatory responses via inhibiting the activation of NF-κB and MAPK signaling pathways. Inflammation usually disrupts the mammary barrier system. The recovery of claudin-3 and occludin expressions indicated that attenuation of inflammatory response by taurine can protect the integrity of blood-milk barrier in S. uberis infection. Taken together, our results reveal that the development of taurine as an effective prevention and control strategy for S. uberis-induced mastitis.

Mammary gland-derived Escherichia coli (E. coli) is an important pathogen causing dairy cow mastitis. Mammary gland mucosal immunity against infectious E. coli mainly depends on recognition of pathogen-associated molecular patterns by innate receptors. Stimulator of interferon (IFN) gene (STING) has recently been the dominant mediator in reacting to bacterial intrusion and preventing inflammatory disorders. In this study, we firstly proved that diguanylate cyclase YeaJ relieves mouse mammary gland pathological damage by changing E. coli phenotypic and host STING-dependent innate immunity response. YeaJ decreases mammary gland circular vacuoles, bleeding and degeneration in mice. In addition, YeaJ participates in STING-IRF3 signaling to regulate inflammation in vivo. While in vitro, YeaJ decreases damage to macrophages (RAW264.7) but not to mouse mammary epithelial cells (EpH4-Ev). Consistent with the results in mouse mammary gland, yeaJ significantly activates STING/TBK1/IRF3 pathway in RAW264.7 as well. In conclusion, the deletion of yeaJ gene facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study highlights a novel role for YeaJ in E. coli infection, which provides a better understanding of host-bacteria interactions and potential prophylactic strategies for infections. IMPORTANCE E. coli is the etiological agent of environmental mastitis in dairy cows, which cause massive financial losses worldwide. However, the pathophysiological role of yeaJ in the interaction between E. coli and host remains unclear. We found that YeaJ significantly influences various biological characteristics and suppresses severe inflammatory response as well as greater damage. YeaJ alleviates damage to macrophages (RAW264.7) and mouse mammary gland. Moreover, these effects of YeaJ are achieved at least partial by mediating the STING-IRF3 signaling pathway. In conclusion, the deletion of yeaJ gene facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study is the basis for further research to better understand host-bacteria interactions and provides potential prophylactic strategies for infections.

Streptococcus uberis (S. uberis) is an important pathogen causing mastitis, which causes continuous inflammation and dysfunction of mammary glands and leads to enormous economic losses. Most research on infection continues to be microbial metabolism-centric, and many overlook the fact that pathogens require energy from host. Mouse is a common animal model for studying bovine mastitis. In this perspective, we uncover metabolic reprogramming during host immune responses is associated with infection-driven inflammation, particularly when caused by intracellular bacteria. Taurine, a metabolic regulator, has been shown to effectively ameliorate metabolic diseases. We evaluated the role of taurine in the metabolic regulation of S. uberis-induced mastitis. Metabolic profiling indicates that S. uberis exposure triggers inflammation and metabolic dysfunction of mammary glands and mammary epithelial cells (the main functional cells in mammary glands). Challenge with S. uberis upregulates glycolysis and oxidative phosphorylation in MECs. Pretreatment with taurine restores metabolic homeostasis, reverses metabolic dysfunction by decrease of lipid, amino acid and especially energy disturbance in the infectious context, and alleviates excessive inflammatory responses. These outcomes depend on taurine-mediated activation of the AMPK–mTOR pathway, which inhibits the over activation of inflammatory responses and alleviates cellular damage. Thus, metabolic homeostasis is essential for reducing inflammation. Metabolic modulation can be used as a prophylactic strategy against mastitis.

Streptococcus uberis infection can cause serious inflammation and damage to mammary epithelial cells and tissues that can be significantly alleviated by taurine. Autophagy plays an important role in regulating immunity and clearing invasive pathogens and may be regulated by taurine. However, the relationships between taurine, autophagy, and S. uberis infection remain unclear. Herein, we demonstrate that taurine augments PTEN activity and inhibits Akt/mTOR signaling, which decreases phosphorylation of ULK1 and ATG13 by mTOR and activates autophagy. Activating autophagy accelerates the degradation of intracellular S. uberis, reduces intracellular bacterial load, inhibits over-activation of the NF-κB pathway, and alleviates the inflammation and damage caused by S. uberis infection. This study increases our understanding of the mechanism through which taurine regulates autophagy and is the first to demonstrate the role of autophagy in S. uberis infected MAC-T cells. Our study also provides a theoretical basis for employing nutritional elements (taurine) to regulate innate immunity and control S. uberis infection. It also provides theoretical support for the development of prophylactic strategies for this important pathogen.

A label-free electrochemical strategy is proposed combining equivalent substitution effect with AuNPs-assisted signal amplification. According to the differences of S1 protein in various infectious bronchitis virus (IBV) strains, a target DNA sequence that can specifically recognize H120 RNA forming a DNA-RNA hybridized double-strand structure has been designed. Then, the residual single-stranded target DNA is hydrolyzed by S1 nuclease. Therefore, the content of target DNA becomes equal to the content of virus RNA. After equivalent coronavirus, the target DNA is separated from DNA-RNA hybridized double strand by heating, which can partly hybridize with probe 2 modified on the electrode surface and probe 1 on AuNPs’ surface. Thus, AuNPs are pulled to the surface of the electrode and the abundant DNA on AuNPs’ surface could adsorb a large amount of hexaammineruthenium (III) chloride (RuHex) molecules, which produce a remarkably amplified electrochemical response. The voltammetric signal of RuHex with a peak near − 0.28 V vs. Ag/AgCl is used as the signal output. The proposed method shows a detection range of 1.56e−9 to 1.56e−6 μM with the detection limit of 2.96e−10 μM for IBV H120 strain selective quantification detection, exhibiting good accuracy, stability, and simplicity, which shows a great potential for IBV detection in vaccine research and avian infectious bronchitis diagnosis. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00604-020-04582-3) contains supplementary material, which is available to authorized users.

Due to its antioxidant properties, resveratrol may relieve the cellular oxidative injury induced by Streptococcus uberis (S. uberis) infection. However, the underlying molecular mechanisms remain unknown. Herein, we used S. uberis to challenge C57BL/6 mice or a mouse mammary epithelial cell line (EpH4-Ev), and the regulatory molecular mechanism of resveratrol on hosts' oxidative injury were investigated. The results showed that gavage of resveratrol alleviate the inflammatory responses and oxidative injury of mammary gland tissues induced by S. uberis infection via activating Nrf2 signaling pathways. To further understand the molecular mechanism, inhibitor of Nrf2 (ML385) and siRNA targeting p62 were used in mammary epithelial cells. The findings indicated that resveratrol mediates Keap1 degradation by activating p62, induces the expression of Nrf2 and its downstream antioxidant signaling pathways, and ameliorates oxidative damage during S. uberis infection. Collectively, these outcomes suggested that resveratrol can function as an activator of the p62-Keap1/Nrf2 signaling pathway to improve oxidative injury caused by S. uberis in mammary glands as well as in EpH4-Ev cells. Therefore, resveratrol may be useful to prevent and control S. uberis-induced bovine mastitis by relieving oxidative stress.

Mastitis caused by Streptococcus uberis (S. uberis) is a common and difficult-to-cure clinical disease in dairy cows. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2&minus, /&minus, and TLR4&minus, mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage, and bacterial count. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host in controlling the secretion of inflammatory factors and the elimination of intracellular S. uberis. Our results demonstrated that TLR2-mediated mROS has a significant effect on S. uberis-induced host defense responses in mammary glands as well as in MECs.

Chlortetracycline (CTC), one kind of common antibiotic for prevention and treatment of various diseases, also exhibits good performance in accelerating the growth of livestock. Macleaya cordata, a traditional Chinese medicine, is usually used as a natural additive in livestock because of its anti-microbial, anti-fungal, anti-inflammatory, and pesticidal activity. In this work, we studied whether M. cordata helps regulate the growth-promoting effect of CTC on broiler chickens. It is demonstrated that M. cordata improves the growth-promoting effect of CTC on growth performance indices of broiler chickens, such as survival rate, daily weight, and feed to weight rate. M. cordata also delays the maximum of CTC residues in plasma. It may depend on the higher values of operational taxonomic unit (OTU) and the indices of α diversity driven by simultaneous use of CTC and M. cordata.

Mammary gland-derived Escherichia coli (E. coli) is an important pathogen causing dairy cow mastitis. YdiV, with EAL-like domains, inhibits flagellum biogenesis and motility and affects c-di-GMP (eubacterial signaling molecule) concentration changes in bacteria. However, the pathophysiological role of ydiV in host-pathogen cross-talk still needs to be elucidated. In this study, firstly constructed the ydiV mutant (NJ17ΔydiV) and ydiV complementary (cNJ17ΔydiV) E. coli strains to infect mouse mammary epithelial cells (EpH4-Ev) and macrophages (RAW264.7), as well as mouse mammary glands, respectively. Then biological characteristics, adaptor molecules in related signaling pathways, proinflammatory cytokines and the extent of host cell damage was evaluated. Compared with E. coli NJ17 infected mice, the bacterial load in the mammary gland of NJ17ΔydiV was significantly lower and the extent of the damage was alleviated. Notably, the deletion of ydiV significantly aggravated cell damage in RAW264.7 cells and compared with the wild-type strain, NJ17ΔydiV significantly activated the STING/TBK1/IRF3 pathway in macrophages. In EpH4-Ev cells, although STING did not sense E. coli NJ17 invasion, IRF3 was activated by the NJ17ΔydiV strain. Taken together, ydiV deletion significantly affects a variety of biological characteristics and induces severe cell damage, while the STING/TBK1/IRF3 pathway actively participated in pathogen elimination in the host. This study highlights a new role for ydiV in E. coli infection and provides a foundation for further studies to better understand host-bacteria interactions and potential prophylactic strategies for infectious diseases.

There exists little available information on the mechanisms of lactation regulation until now. In order to explore the underlying mechanism, we injected IGF-I and GH recombinant vectors into the mammary gland, then RNA-seq analysis and nuclear proteomics were used for rapid high-throughput screening of DEGs and DEPs in the two groups linked to lactation regulation. KEGG analysis of 206 DEGs showed that the same 4 of top 10 enrichment pathways (ECM receptor interaction, protein digestion and absorption, focal adhesion and phagosome) involved in 4 co-expressed genes (IDO, BTG1, ITGB6 and keratin 83), the two groups enriched different metabolic pathways yet. Nuclear proteomics analysis showed 75 and 36 DEPs in the IGF-I and GH group respectively; Sixteen common proteins were identified between the IGF-I group and GH group, four of which (ALB, TPT1, CXXC-5 and ACTR2) significantly decreased and three of which (PRP1, PAG-9 and Hsp70) significantly increased. Similarly, DEPs in the two groups were enriched in same one of top 10 enrichment pathways (PI3K-Akt signaling pathway). Protein-protein interaction networks highlighted the contribution of glycosphingolipid biosynthesis, porphyrin and chlorophyll metabolism and the Jak-STAT signaling pathway to lactation regulation of GH and IGFI. GH and IGF-I improve milk yield, which may be linked to important nodal proteins (ALB and ACTB). Our research advances the understanding of the mammary gland transcriptome and nuclear proteomics during GH and IGF-I overexpression. Individual genes, proteins and pathways in this study point towards potential targets for lactation regulation.

Mastitis caused by Streptococcus uberis is a hazardous clinical disease in dairy animals. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2−/− and TLR4−/− mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage and bacterial count in mammary glands. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host control of secretion of inflammatory factors and elimination of intracellular S. uberis. Our results demonstrate that TLR2-mediated mROS have a significant effect on S. uberis-induced host defense responses in mammary glands as well as MECs.Author summaryS. uberis contributes significantly to global mastitis and remains a major obstacle for inflammation elimination due to its ability to form persistent infection in mammary tissue. The Toll-like receptor (TLR) family plays a significant role in identifying infections of intracellular bacteria and further triggering inflammatory reactions in immune cells. However, the detailed molecular mechanism by which TLR is regulated, and whether MECs, as the main cells in mammary gland, are tightly involved in these processes is poorly understood. Here, we used S. uberis to infect mammary glands of wild type, TLR2−/−, TLR4−/− mice and MECs to assess pathogenesis, proinflammatory cytokines, ROS as well as mROS levels during infection. We found that during S.uberis infection, it is TLR2 deficiency that induced more severe pathological changes through MyD88-mediated signaling pathways. In addition, our work demonstrates that mROS mediated by TLR2 has an important role in host defense response to combat S. uberis infection in mammary glands as well as MECs.

Mammary epithelial cells (MECs) play an important role against Streptococcus uberis infection which is one of the main causes of bovine mastitis and a potential threat to human health. Toll-like receptors (TLRs) and their mediated signaling pathways are critical in both innate and infection responses, yet their roles in anti-S. uberis infection in MECs remains poorly defined. In this work we investigated the regulatory mechanisms of TLR2 in inflammatory responses, where WT and TLR2−/− mice were euthanized at 15–18 days gestation, and mammary gland tissues were collected aseptically. The mouse MECs (MMECs) were isolated by combined digestion with type I collagenase, hyaluronidase and trypsin. We challenged MMECs with S. uberis and quantified antioxidant capacity as well as reactive oxygen species (ROS), proinflammatory cytokines and cell damage at different times. The loss of TLR2 function in MMECs results in more serious cell damage, increased cell adhesion, and significantly decreased ROS and mitochondrial ROS (mROS) with bactericidal function in response to S. uberis infection. Moreover, it was observed that the antioxidant capacity declined, and the production of TLR2-mediated cytokines (except CXC ligand 15) also were reduced. We demonstrated that TLR2 can mediate cellular anti-infective processes in MMECs by regulating the production of ROS and mROS and the secretion of cytokines. The results suggest an unpredicted role of TLR2 in MMECs in response to S. uberis infection.

Taurine may alleviate the inflammatory injury induced by Streptococcus uberis (S. uberis) infection by regulating intracellular Ca2+ levels. However, the underlying mechanisms remain unclear. Infection leads to subversion of phosphoinositides (PIs) which are closely related to Ca2+ signaling. In order to investigate whether taurine regulates inflammation by means of PIs/ Ca2+ systems, competitive inhibitors of taurine (β-alanine) siTauT, siPAT1, siPLC, siCaN, siPKC, and inhibitors of PLC (U73122), PKC (RO31-8220), and CaN (FK 506) were used. The results indicate that taurine transfers the extracellular nutrient signal for intercellular innate immunity to phosphoinositides without a need to enter the cytoplasm while regulating intracellular Ca2+ levels during inflammation. Both the Ca2+-PKCα-NF-κB, and Ca2+-CaM-CaN-NFAT signaling pathways of S. uberis infection and the regulatory roles of taurine follow activation of PIs/Ca2+ systems. These data increase our understanding on the mechanisms of multifunctional nutrient, taurine attenuated inflammatory responses caused by S. uberis infection, and provide theoretical support for the prevention of this disease.

Tryptophan (Trp) can produce bioactive compounds for appetite regulation, calcium mobilization, and mammary gland homeostasis via a serotonin pathway. This study evaluated the effects of Trp supplementation on the reproduction performance, milk yield, and composition of lactating sows, growth performance of their piglets, and the secretion function of porcine mammary epithelial cells (PMECs). The infrared emulsion analyzer and ELISA analyses revealed that feeding sows with a 0.12% Trp addition increased ( P < 0.05) sow average daily feed intake, milk yield, milk calcium concentration, average daily gain of piglets, fatty acid synthase (FAS) and lactose synthase (LS), β-casein secretion, intracellular Ca2+ level, the expression of calcium binding protein CaM, and the activity of CaMKII. In a cellular experiment of PMECs treated with Trp, ELISA and flow cytometry analyses revealed that the pretreatment of a Trp hydroxylase inhibitor reduced ( P < 0.05) FAS and LS synthesis, the intracellular Ca2+ level, and the activity of CaMKII. In conclusion, Trp supplementation at 0.12% increased sows' reproductive performance, milk yield, and calcium concentration and piglets' growth performance. Milk yield increased by Trp was linked to 5-hydroxytryptamine-mediated synthesis of FAS, LS, and β-casein in PMECs, while the increase in calcium concentration was attributed to increasing CaM expression and CAMKII activity.

An aptamer based assay is presented for the determination of the antibiotics oxytetracycline (OTC) and kanamycin (KAN). Magnetic beads were applied for separation, and gold nanoparticles (AuNPs) for signal amplification. DNA aptamers against OTC and KAN were firstly designed. After specific recognition events, the aptamer sequences were released from the surface of magnetic beads and the remaining DNA probes captured horseradish peroxidase (HRP) modified AuNPs. Subsequently, 3,3′,5,5′-tetramethylbenzidine and o-phenylenediamine are catalytically oxidized by HRP, and the generated colorimetric responses can reflect the concentrations of OTC (at 370 nm) and KAN (at 450 nm), respectively. Experimental results demonstrate that the method is highly sensitive with the detection limit as low as 1 ag mL−1 for OTC and KAN. An extremely wide linear range (over 11 orders of magnitude) is achieved. The high selectivity is attributed to the high affinity between aptamer and the substrate. The results of real sample tests also verify that the method is promising for antibiotics analysis in the applications of food monitoring and clinical diagnosis.

Mammary epithelial cells (MECs) play an important role in debating Streptococcus uberis (S. uberis) infection. Toll like receptor (TLR) engagement leads to the recruitment of phosphatidylinositol 3 kinases (PI3K). In order to investigate the relationship of TLRs/NF-κB and PI3K/Akt/mTOR signaling pathways in S. uberis infection in MECs, we challenged MECs (EpH4-Ev) with S. uberis 0140 J and quantified the adaptor molecules in these two signaling pathways, as-well-as proinflammatory cytokines and cell damage. The results indicate that the host’s responses to virulent S. uberis infection are complex. In MECs, both TLR2 and TLR4 are detecting S. uberis infection and TLR2 is the principal receptor. The role of the PI3K/Akt/mTOR pathway in inflammatory regulation is independent of the activation of TLRs/NF-κB. Cross-talk between PI3K/Akt/mTOR and TLRs/NF-κB signaling pathways promote inflammation. This study increases our understanding of the molecular defense mechanisms of MECs in S. uberis mastitis, and provides theoretical support for the prevention of this disease.

Aberrant mucin secretion and accumulation in the airway lumen are clinical hallmarks associated with various lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Mycoplasma pneumoniae , long appreciated as one of the triggers of acute exacerbations of chronic pulmonary diseases, has recently been reported to promote excessive mucus secretion. However, the mechanism of mucin overproduction induced by M. pneumoniae remains unclear. This study aimed to determine the mechanism by which M. pneumoniae induces mucus hypersecretion by using M. pneumoniae infection of mouse lungs, human primary bronchial epithelial (NHBE) cells cultured at the air-liquid interface, and the conventionally cultured airway epithelial NCI-H292 cell line. We demonstrated that M. pneumoniae induced the expression of mucins MUC5AC and MUC5B by activating the STAT6-STAT3 and epidermal growth factor receptor (EGFR) signal pathways, which in turn downregulated FOXA2, a transcriptional repressor of mucin biosynthesis. The upstream stimuli of these pathways, including interleukin-4 (IL-4), IL-6, and IL-13, increased dramatically upon exposure to M. pneumoniae . Inhibition of the STAT6, STAT3, and EGFR signaling pathways significantly restored the expression of FOXA2 and attenuated the expression of airway mucins MUC5AC and MUC5B. Collectively, these studies demonstrated that M. pneumoniae induces airway mucus hypersecretion by modulating the STAT/EGFR-FOXA2 signaling pathways.

Mammary epithelial cells (MECs), as part of the functional unit of the udder, are not only responsible for the synthesis of many components in milk that provide necessary nutritional and immunological support to the offspring, but also playing essential roles in the reaction to mastitis pathogens and the initiation of the immune signaling pathway. There are contributions of MECs to the signaling and production of pathogen associated molecular patterns (PAMPs) such as LPS, lipoteichoic acid (LTA), and β-glucans, but the crosstalk of different PAMPs induces signalings and productions in rat MEC that need further study. In the present study, we have demonstrated that β-glucan up-regulates Dectin1 and LPS up-regulates TLR4 directly, as confirmed by generation of siDectin1 and siTLR4 in rat MECs. Then our results have described that either β-glucan or LPS can activate RelB and/or p65 in rat MECs. Furthermore, the association of p65 and RelB has been analyzed that collaboration of β-glucan and LPS promotes p65/RelB heterodimers, producing inflammatory responses in rat MECs. In conclusion, summary of our present results suggests that β-glucan can be considered as a potential immuno-modulator, which s with TLR4 via NF-κB subunits to initiate and regulate the innate immunity in rat MECs.

In order to evaluate the role of taurine on the oxidative stress mediated by NADPH oxidase in Streptococcus uberis-induced (S. uberis) mastitis, rats were administered daily (per os) 100mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) from gestation day 14 until parturition. Seventy-two hours after parturition, approximately 100cfu of S. uberis was infused into each of 2 mammary glands. Pretreatment with taurine significantly decreased mRNA and protein expression of p47phox and p22phox in mammary tissues. The total anti-oxidation capability (T-AOC) levels and superoxide dismutase (SOD) activities decreased, while malondialdehyde (MDA) levels increased both in mammary tissues and serum of rats with intramammary S. uberis infusion. Gavage administration of taurine moderated this change. Concentrations of interleukin-1β (IL-1β) and IL-6 in mammary glands decreased as a result of taurine administration. Significant differences (P0.05) were present at 48 and 72 h post S. uberis-infusion (PI) for IL-1β and 72 h PI for IL-6. Our data indicate that, in S. uberis-induced mastitis, taurine has the ability of regulating redox conditions which leads to the suppression of oxidative stress and secretion of proinflammatory cytokines. This phenomenon may be ascribed to taurines's ability to inhibit the expression of NADPH oxidase.

Mastitis, caused by mammary pathogenic bacteria which are frequent implications of Escherichia coli, is an important disease affecting women and dairy animals worldwide. The β-glucan binding of dectin-1 can induce its own intracellular signaling and can mediate a variety of cellular responses. This work was to investigate the effect of β-glucan on the lipopolysaccharide (LPS)-induced inflammatory response and related innate immune signaling in primary rat mammary epithelial cells. Cells were treated with serum-free medium added with a DMSO solution containing β-glucans at concentrations of 0, 1, 5, 25 μmol/L for 12h, and then exposed to 10 μg/mL LPS for 40 min. Moreover, cells were pretreated with BAY 11-7082 to inhibit NF-κB and then successively exposed to 5 μmol/L β-glucan, 10 μg/mL LPS, 5 μmol/L β-glucan and 10 μg/mL LPS, according to the specific experimental design. Normal control cultures contained an equal volume of DMSO, which was collected at the same time. After incubating rat mammary epithelial cells for 40 min with 10 μg/mL LPS, TLR4, MyD88 and NF-κB expression all increased (P

This study investigated the putative protective effects of supplemental 2-aminoethane sulfonic acid (taurine) and reduced housing density on renal function in laying hens. We randomly assigned fifteen thousand green-shell laying hens into three groups: a free range group, a low-density caged group, and a high-density caged group. Each group was further divided equally into a control group (C) and a taurine treatment group (T). After 15 d, we analyzed histological changes in kidney cells, inflammatory mediator levels, oxidation and anti-oxidation levels. Experimental data revealed taurine supplementation, and rearing free range or in low-density housing can lessen morphological renal damage, inflammatory mediator levels, and oxidation levels and increase anti-oxidation levels. Our data demonstrate that taurine supplementation and a reduction in housing density can ameliorate renal impairment, increase productivity, enhance health, and promote welfare in laying hens.

This study was designed to investigate the putative protective effect of β-casomorphin-7 on diabetic nephropathy in a rat model, and to explore the possible mechanism of this effect. SD rats were randomly divided into the following three groups: control group, diabetes group and β-casomorphin-7-treatment group. All rats were euthanized after 30 days with or without β-casomorphin-7 treatment. Biochemical parameters including blood glucose and renal function were quantified. The concentration of plasma TGF-β1 was measured by ELISA. Histopathological changes to the kidney were studied by Masson and Sirius red staining. Expressions of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, cytokeratin19 and TGF-β1 mRNA in rat renal cortices were analyzed by real-time PCR. Changes in α-SMA and E-cadherin protein expression in rat renal cortices were quantified by Western blot. β-Casomorphin-7 treatment of diabetic rats reduced urinary glucose, urinary protein, serum creatinine, blood urinary nitrogen, plasma TGF-β1 and the ratio of kidney: body weight. Masson and Sirius red staining showed that β-casomorphin-7 treatment attenuated renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats had elevated expressions of α-SMA, vimentin and TGF-β1 mRNA and α -SMA protein and decreased expression of E-cadherin and cytokeratin19 mRNA, and E-cadherin protein. β-Casomorphin-7 treatment of diabetic rats partially normalized these changes. Our results suggest that administration of β-casomorphin-7 attenuates renal interstitial fibrosis caused by diabetes. This protective effect may be associated, in part, with down regulation of epithelial–mesenchymal transition of renal tubular epithelial cells.

The intent of this study was to evaluate the active defense reaction of mouse mammary epithelial cells and the cytoprotective and anti-inflammatory properties of taurine to lipopolysaccharide (LPS)-induced disfunction in mouse mammary epithelial cells. (1) Primary cultured mouse mammary epithelial cells were stimulated with LPS for 24 h (final concentration=0, 5, 10, 20 μg/mL). Western blotting demonstrated a significant decrease in the secretion of β-casein in the 20 μg/mL LPS treatment group (P

To investigate whether taurine ameliorates mammary damage in a rat model of S. uberis mastitis by suppressing inflammation related to the toll-like receptors/nuclear factor kappa B (TLRs/NF-κB) signaling pathway. Starting on gestation day 14 and continuing until parturition, 100 mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) was administered daily to rats. Seventy-two hours after parturition, rats were infused with 100 cfu of S. uberis into each of 2 mammary glands. The resultant inflammation, evidenced by swelling, degeneration of secretory epithelium, increased tissue loss and neutrophil (PMN) infiltration was observed. Pretreatment with taurine attenuated inflammatory changes and significantly decreased mRNA expression of TLR-2 (8 h post S. uberis-injection, PI), NF-κB p65 (16 h and 24 h PI), and NF-κB DNA binding activity (16 h PI). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) levels were also decreased. Significant differences (P

Taurine (Tau) is reported to have a key role in the regulation of the innate immune response and thus reduce tissue damage induced by bacterial infection. In this study, the effects of Tau on a rat model of mastitis induced by Streptococcus uberis (S. uberis) and the changes of T regulatory cells (Tregs) were assessed. Starting on gestation day 14 and continuing until parturition, 100 mg/kg of taurine (group TS) or an equal volume of physiological saline (group CS) was administered daily, per os. Seventy-two hours after parturition, rats were infused with approximately 100 cfu of S. uberis into each of two mammary glands. The results showed that the resultant inflammation, evidenced by swelling, secretory epithelial cell degeneration, increased adipose tissue and neutrophil (PMN) infiltration were evident in mammary tissue following injection with S. uberis. Pre-treatment with Tau attenuated these morphologic changes, the expression of interleukin (IL)-2, interferon (INF)-γ mRNA, myeloperoxidase (MPO) activity and N-acetyl-β-D-glucosaminidase (NAGase) in mammary tissue. The percentages of Foxp3+CD25+CD4+/lymphocytes (Tregs) were dramatically increased after the S. uberis challenge. Significant differences (P

The present study was aimed to investigate the possible effects of beta-casomorphin-7, against hyperglycemia and free radical-mediated oxidative stress in streptozotocin-induced diabetic rats by assaying the blood glucose level and the activity of plasma enzymatic antioxidants, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px). A significant increase in the levels of both blood glucose and oxidative stress with a predominant decrease in antioxidant status was observed in the diabetic rats when compared to control rats. After 15 days oral administration of beta-casomorphin-7 (7.5 x 10(-8) mol/day), the elevated blood glucose level was reduced. Oral administration of beta-CM-7 to diabetic rats showed an increase in the level of plasma insulin, the elevated plasma glucagon level was markedly reduced by the oral administration of beta-CM-7. Oral administration of beta-CM-7 to the diabetic group of rats also showed a significant elevation in the activity of SOD and catalase. Thus, the results of the present study suggest that beta-casomorphin-7 can protect rats from hyperglycemia and free radical-mediated oxidative stress in diabetic rats.

The retinoids, a group of natural or synthetic derivatives of vitamin A, exert various anti-neoplastic and immunomodulatory actions. Recent studies have demonstrated that retinoic acid protects rats against lipopolysaccharide (LPS)-induced mastitis, but the mechanism of action is unclear. In the present study, an LPS-induced rat mastitis model and primary cultures of rat mammary epithelial cells were used to investigate the effect of retinoic acid on the TLR4/NF-kappaB signaling pathway. The data indicated that toll-like receptor 4 (TLR4) gene expression reached its peak value earlier in retinoic acid-treated rats than in the control group, and that retinoic acid significantly decreased NF-kappaB DNA binding activity and the level of IL-1beta in the mammary gland. The animal study result was confirmed by an in vitro cell culture system trial. TLR4 protein expression and NF-kappaB DNA binding activity were significantly decreased in primary rat mammary epithelial cells pretreated with 1mumol/l retinoic acid at 1h post-LPS stimulation. IL-1beta gene expression was also significantly decreased at 2, 4 and 8h post-LPS stimulation. These findings demonstrate that direct action by retinoic acid leads to attenuation of the LPS-induced inflammatory response by suppression of the TLR4/NF-kappaB signalling system, thereby providing a novel explanation for the underlying effect proposed for retinoic acid in the protection of mammary tissue during LPS-induced acute mastitis.

The aim of this study was to evaluate, in rats, the changes in the T helper type 1 (Th1)/Th2 radio in mammary glands after an intramammary infusion of lipopolysaccharide (LPS) and to characterize the moderating effects of the polysaccharide nucleic acid of Bacillus Calmette Guerin (BCG-PSN) on the mammary gland. In the control group, the levels of IL-2 and INF-γmRNA expression increased, whereas IL-4 mRNA expression decreased after LPS challenge. As a consequence, the INF-[.gamma]/IL-4 mRNA ratio was significantly higher at 3, 6, and 9 h post-infusion (PI) compared to the control value (0 h; P< 0.01). BCG-PSN increased mRNA expression of both INF-γ and IL-4 before infusion of LPS. LPS challenge significantly the reduced Th1/Th2 cytokine ratio due to Th1 cytokine IFN-γ suppression and Th2 cytokine IL-4 upregulation compared with the control group. A significant reduction of N-acetyl-[.beta]-D-glucosaminidase (NAGase) was observed at 24 h PI in the BCG-PSN treatment group compared to the control group (P < 0.05). Thus, it was demonstrated that level of BCG-PSN might change the Th1/Th2 ratio mainly by enhancing the Th2 immune response. This is the first report of a Th1/Th2 change induced by coliform mastitis and characterization of the effect of BCG-PSN on mammary gland inflammation. This study makes a better understanding of the mechanisms of coliform mastitis and provides a putative novel strategy for the prevention and/or treatment of mastitis.

The effects of dehydroepiandrosterone (DHEA) on lipogenic gene mRNA expression were examined using cultured primary chicken hepatocytes. Cell samples were equilibrated to culture conditions for 24 h and then exposed to DHEA (1, 10 and 100 μM) dissolved in DMSO. The expression of sterol regulatory element-binding protein-1 (SREBP-1) mRNA in cultured primary hepatocytes did not vary with time, except for a significant decrease in gene expression when cells were treated with 10 or 100 μM DHEA for either 1 or 2 h. A similar tendency toward decreased gene expression was evident for acetyl CoA carboxylase. The expression of peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyl transferase I mRNA was dramatically enhanced subsequent to treatment with 10 or 100 μM DHEA for periods of time from 6 to 48 h, while there was a decrease in the expression of PPARα in the presence of 10 μM DHEA after 1 and 2 h of exposure, and in the presence of 100 μM DHEA after 2 h of exposure. Hepatocytes treated with 10 or 100 μM DHEA contained more mitochondria and mitochondria with a higher electron density than did untreated hepatocytes. Furthermore, cell survival was significantly inhibited by treatment with 100 μM DHEA at 24, 48 and 72 h. In contrast, 1 μM DHEA administration sig- nificantly increased cell survival after 72 h; however, 10 μM DHEA treatment had no pronounced effect on cell survival. Overall, the results reported here indicate that DHEA accelerates lipid catabolism by direct regulation of hepatic gene expression and by induction of changes in hepatocyte mitochondria.

To provide insight into the mechanisms of taurine attenuation of pro-inflammatory response in mouse mammary epithelial cell line (EpH4-Ev, purchased by ATCC, USA) after Streptococcus uberis (S. uberis, 0140J) challenge, we infected MECs with S. uberis (2.5×10(7)cfumL(-1), MOI=10) for 3h and quantified changes in TLR-2 and calcium (Ca(2+)) mediated signaling pathways. The results indicate that S. uberis infection significantly increases the expression of TLR-2, intracellular Ca(2+) levels, PLC-γ1 and PKC-α, the activities of transcription factors NF-κB and NFAT, and related cytokines (TNF-α, IL-1β, IL-6, G-CSF, IL-2, KC, IL-15, FasL, MCP-1, and LIX) in culture supernatants. Taurine administration downregulated all these indices, the activities of NF-κB and NFAT. Cytokine secretions were similar using special PKC inhibitor Go 6983 and NFAT inhibitor VIVIT. Our data indicate that S. uberis infection induces pro-inflammatory response of MECs through a TLR-2 mediated signaling pathway. In addition, taurine can prevent MEC damage by affecting both PLC-γ1-Ca(2+)-PKC-α-NF-κB and PLC-γ1-Ca(2+)-NFATs signaling pathways. This is the first report to demonstrate the mechanisms of taurine attenuated pro-inflammatory response in MECs after S. uberis challenge.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are important pathogens causing subclinical and clinical bovine mastitis, respectively. Taurine, an organic acid found in animal tissues, has been used for the treatment of various superficial infections and chronic inflammations. We challenged a bovine mammary epithelial cell (MEC) line (MAC-T) or a mouse mammary epithelial cell line (EpH4-Ev) with either E. coli or S. aureus and compared the responses of MECs to these 2 pathogens. We also examined the regulatory effects of taurine on these responses. Receptor analyses showed that both TLR2 and TLR4 are upregulated upon exposure to either E. coli or S. aureus. Taurine pre-treatment dampened upregulation to some extent. E. coli and S. aureus stimulated comparable levels of ROS, which could be inhibited by taurine pre-treatment. E. coli infection elicited a dramatic change in iNOS expression. Taurine significantly decreased iNOS expression in the S. aureus challenged group. Protein microarray demonstrated that 32/40 and 8/40 inflammatory molecules/mediators were increased after E. coli or S. aureus challenge, respectively. The fold changes of most molecules were higher in the E. coli infection group than that in the S. aureus infection group. Taurine negatively regulated the inflammatory profile in both bacterial infections. Pro-inflammatory cytokines (such as TNF-α) connected with TLR activation were down-regulated by taurine pre-treatment. The influence of TAK-242 and OxPAPC on cytokine/molecule expression profiles to E. coli challenge are different than to S. aureus. Some important factors (MyD88, TNF-α, IL-1β, iNOS and IL-6) mediated by TLR activation were suppressed either in protein microarray or special assay (PCR/kits) or both. TAK-242 restrained ROS production and NAGase activity similar to the effect of taurine in E. coli challenge groups. The detection of 3 indices (T-AOC, SOD and MDA) reflecting oxidative stress in vivo, showed that taurine improved the antioxidant ability of cells. We conclude that taurine can regulate the inflammatory response during infection with E. coli and prevent cell damage by affecting the signaling pathways mediated by TLRs and by improving the antioxidant ability of cells. In S. aureus infections, taurine's antioxidant ability may be the primary means of resistance to inflammation. This study provides a better understanding of the inflammatory mechanisms of E. coli and S. aureus mastitis, and it provides a putative strategy for the prevention of this disease.

Experiments were conducted to study the effects of dietary taurine and housing density on oviduct function in laying hens. Green-shell laying hens were randomly assigned to a free range group and two caged groups, one with low-density and the other with high-density housing. Each group was further divided into control (C) and taurine treatment (T) groups. All hens were fed the same basic diet except that the T groups’ diet was supplemented with 0.1% taurine. The experiment lasted 15 d. Survival rates, laying rates, daily feed consumption, and daily weight gain were recorded. Histological changes, inflammatory mediator levels, and oxidation and anti-oxidation levels were determined. The results show that dietary taurine supplementation and reduced housing density significantly attenuated pathophysiological changes in the oviduct. Nuclear factor-κB (NF-κB) DNA binding activity increased significantly in the high-density housing group compared with the two other housing groups and was reduced by taurine supplementation. Tumor necrosis factor-α (TNF-α) mRNA expression in the high-density and low-density C and T groups increased significantly. In the free range and low-density groups, dietary taurine significantly reduced the expression of TNF-α mRNA. Supplementation with taurine decreased interferon-γ (IFN-γ) mRNA expression significantly in the low-density groups. Interleukin 4 (IL-4) mRNA expression was significantly higher in caged hens. IL-10 mRNA expression was higher in the high-density C group than in the free range and low-density C groups. Supplementation with taurine decreased IL-10 mRNA expression significantly in the high-density group and increased superoxide dismutase (SOD) activity in the free range hens. We conclude that taurine has important protective effects against oviduct damage. Reducing housing density also results in less oxidative stress, less inflammatory cell infiltration, and lower levels of inflammatory mediators in the oviduct. Therefore, both dietary taurine and reduced housing density can ameliorate oviduct injury, enhance oviduct health, and promote egg production in laying hens.

Oxidative stress is implicated in the pathogenesis of diabetic nephropathy. The present study aimed to investigate the effect of β-casomorphin-7 (BCM7) on the oxidative stress occurring in kidney tissue in streptozotocin (STZ)-induced diabetic rats and proximal tubular epithelial cells (NRK-52E) exposure to high glucose (HG) by using biochemical methods. There is a significant decrease in plasma insulin and a significant increase in plasma glucagon in the rats of diabetic group. Oral administration of BCM7 for 30 days to rats with STZ-induced diabetes resulted in a significant increase in serum level of insulin, and a decrease in the level of glucagon. Moreover, rats with STZ-induced diabetes had lower levels of superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidative capacity (T-AOC), higher levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in the kidney than that in the control rats. The administration of BCM7 altered the changes of SOD, GPx, T-AOC, MDA and H2O2 in the kidney of diabetic rats. Furthermore, BCM7 alleviated high glucose-induced decreasement in SOD and GPx activity, increasement in MDA contents in the NRK-52E cells. BCM7 ameliorated the changes of angiotensin converting enzyme (ACE) and ACE2 levels in the kidney of diabetic rats and BCM7 lowered the levels of angiotensin (Ang)II in the kidney of diabetic rats and culture medium for cells. Moreover losartan (antagonist of angiotensin II type I receptor) lowered the high glucose-induced oxidative stress in the NRK-52E cells. Our results suggest that administration of BCM7 would alleviate high glucose-induced renal oxidative stress in vivo and in vitro, which may be associated with down regulation of the concentration of Ang II partly.

β-Casomorphin-7 (β-CM-7) is a milk biological active peptide. The present study is aimed to investigate the protective effects of β-CM-7, against oxidative stress in pancreas of streptozotocin-induced diabetic rats by assaying malondialdehyde (MDA), nitric oxide (NO) level, the activity of enzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), and NF-κB, inducible nitric oxide synthase (iNOS) gene expression. A significant increase in the level of oxidative stress was observed in pancreas of the diabetic rats when compared to control rats. After 15 d oral administration of β-CM-7 (7.5 × 10(-8) mol/d), the pancreas MDA level was markedly reduced. Oral administration of β-CM-7 to diabetic rats showed an obviously increase in the activity of catalase in pancreas, oral administration of β-CM-7 to the diabetic group of rats also showed a reduction of NF-κB and iNOS gene expression in pancreas. The elevated pancreas NO level was markedly reduced by the oral administration of β-CM-7. Thus, the results of the present study suggest that β-CM-7 may cause protective effects such as pronounced decreasing in oxidative stress and inhibiting NF-κB-iNOS-NO signal pathway in pancreas of diabetes rats.

Activated polymorphonuclear neutrophilic leukocytes (PMN) are able to produce large quantities of bactericidal molecules such as reactive oxygen species (ROS) that are associated with tissue damage in models of inflammatory mastitis. In this study, the putative protective effect of retinoid was evaluated in a lipopolysaccharide (LPS) induced mastitis model in rats. Commencing at 10 d of gestation, retinoid (dissolved in olive oil) or an equal volume of olive oil were administered daily by gavage to pregnant rats until parturition. LPS or pyrogen-free physiological saline were infused into the mammary gland 72 h after parturition. At pre-infusion (defined as 0 h) and at 2, 4, 8, 16 and 24 h post-infusion, six rats from each group were euthanized. Retinoid administration decreased PMN accumulation in mammary alveoli, significantly decreased the level of TNF-α in mammary tissues and IL-8 in serum at the different time points. ROS release was significantly increased after LPS infusion and was reduced by retinoid at 16 h PI. Retinoid reduced N -acetyl- β - d -glucosaminidase (NAGase) activity in both serum and mammary tissue at 8 h PI. Intercellular adhesion molecule 1 (ICAM-1) mRNA expression reached its peak value earlier in retinoid treated rats than in the control group. Overall, the results suggest that activated PMN play an important role in the pathogenesis of acute mastitis and retinoid administration may be an effective tool for protecting mammary tissue against PMN-induced oxidative stress during LPS-induced acute mastitis.