Preparation, pharmacokinetics and anti-obesity effects on dogs of nuciferine liposomes.

BACKGROUND: Nuciferine (NUC), a natural compound extracted from lotus leaves, has been proven to have anti-obesity effects. However, the development and application of NUC as an anti-obesity drug in dogs are hindered due to its poor water solubility and low bioavailability. OBJECTIVE: To promote the development of NUC-related products for anti-obesity in dogs, this study prepared NUC into a liposome formulation and evaluated its characteristics, pharmacokinetics in dogs, and anti-obesity effects on high-fat diet dogs. METHODS: NUC liposomes were prepared by the ethanol injection method, using NUC, egg lecithin, and ß-sitosterol as raw materials. The characteristics and release rate in vitro of liposomes were evaluated by particle size analyser and dialysis method, respectively. The pharmacokinetics in dogs after oral administration of NUC-liposomes was carried out by the high-performance liquid chromatography (HPLC) method. Moreover, we investigated the anti-obesity effect of NUC-liposomes on obese dogs fed with a high-fat diet. RESULTS: NUC-liposome was successfully prepared, with an EE of (79.31 ± 1.06)%, a particle size of (81.25 ± 3.14) nm, a zeta potential of (-18.75 ± 0.23) mV, and a PDI of 0.175 ± 0.031. The cumulative release rate in vitro of NUC from NUC-liposomes was slower than that of NUC. The T1/2 and relative bioavailability of NUC-liposomes in dogs increased, and CL reduced compared with NUC. In addition, the preventive effect of NUC-liposomes on obesity in high-fat diet dogs is stronger than that of NUC. CONCLUSIONS: The liposome formulation of NUC was conducive to improve its relative bioavailability and anti-obesity effect in dogs.

L-arginine attenuates Streptococcus uberis-induced inflammation by decreasing miR155 level.

L-arginine, as an essential substance of the immune system, plays a vital role in innate immunity. MiR155, a multi-functional microRNA, has gained importance as a regulator of homeostasis in immune cells. However, the immunoregulatory mechanism between L-arginine and miR155 in bacterial infections is unknown. Here, we investigated the potential role of miR155 in inflammation and the molecular regulatory mechanisms of L-arginine in Streptococcus uberis (S. uberis) infections. And we observed that miR155 was up-regulated after infection, accompanying the depletion of L-arginine, leading to metabolic disorders of amino acids and severe tissue damage. Mechanically, the upregulated miR155 mediated by the p65 protein played a pro-inflammatory role by suppressing the suppressor of cytokine signaling 6 (SOCS6)-mediated p65 ubiquitination and degradation. This culminated in a violently inflammatory response and tissue damage. Interestingly, a significant anti-inflammatory effect was revealed in L-arginine supplementation by reducing miR155 production via inhibiting p65. This work firstly uncovers the pro-inflammatory role of miR155 and an anti-inflammatory mechanism of L-arginine in S.uberis infection with a mouse mastitis model. Collectively, we provide new insights and strategies for the prevention and control of this important pathogen, which is of great significance for ensuring human food health and safety.

Lnc-ANRIL modulates the immune response associated with NF-κB pathway in LPS-stimulated bovine mammary epithelial cells.

BACKGROUND: The antisense noncoding RNA in the INK4 locus (ANRIL) has been confirmed related to multiple disease progression, but the role and exact mechanisms of lnc-ANRIL in lipopolysaccharide (LPS)-induced inflammation of bovine mammary epithelial cells (MAC-T) remain unclear. AIMS: This manuscript focused on expounding the functional role of lnc-ANRIL through experiments performed in MAC-T. METHODS: At the in vitro level, we established a Bovine mammary epithelial cell (BMEC) cell model of mastitis by LPS treatment. Transfection of siRNA was examined by immunofluorescence localization and RT-qPCR. CCK8, clonogenic assay and EdU were used to detect the proliferation ability of the cells. Cell cycle and apoptosis were detected by flow cytometry and Western blot. The levels of inflammatory factors and oxidative stress markers were detected by ELISA kits. RESULTS: Cell Counting Kit-8, colony formation, and 5-ethynyl-20-deoxyuridine were adopted and the data illustrated that LPS could significantly suppress the cell proliferation, while knockdown of lnc-ANRIL expression obviously promoted MAC-T cell proliferation compared with LPS or LPS + si-NC group. Flow cytometry analysis demonstrated that lnc-ANRIL could induce MAC-T cell apoptosis. In addition, downregulation of lnc-ANRIL affected LPS-induced immune response by regulating inflammatory factor expressions and modulating the nuclear factor kappa B (NF-κB) axis in MAC-T cells. CONCLUSION: Our results suggest that lnc-ANRIL is involved in the regulation of cell proliferation, cell cycle, and cell apoptosis of MAC-T cells, and plays an important role in the inflammatory and immune response of MAC-T cells through the regulation of the NF-κB pathway, proposing new therapeutic strategies for the treatment of innate immune response-related disease such as bovine mastitis.

Mammary epithelial cell-derived exosomal miR-155-inhibitor played a key role in the treatment of mastitis via down-regulation of TLRs/NF-κB signaling pathway to inhibit inflammatory response.

Mastitis is a common disorder in women capable of altering the normal physiological function of the mammary gland. It has been reported that mammary epithelial cells (MECs) could be involved in treating mastitis by regulating the inflammatory response and miR-155 might participate in this process. However, the effects of MECs-derived exosomal miR-155-inhibitor in treating mastitis and the regarding mechanism are still unknown. In our study, mouse mammary epithelial cells (HC11) were applied to study the role of MECs-derived exosomal miR-155-inhibitor in the treatment of mastitis and explore the mechanism. Results in our study showed that specific markers including CD63 and Apo-A1 were expressed in blank exosomes and exosomes containing miR-155-inhibitor isolated from transfected HC11 cells. Results of immunofluorescence showed that the blank exosomes and exosomes (containing miR-155-inhibitor) labeled with PKH26 were absorbed in HC11 cells. The level of miR-155 was decreased obviously in Engineered exosomes with miR-155-inhibitor and HC11 cells Transfected with exosome containing miR-155-inhibitor. The level of miR-155 was increased and cell apoptosis was promoted obviously in HC11 cells induced by LPS, however, they were decreased obviously after transfecting with an exosome containing miR-155-inhibitor. The level of TLR2, TLR4, TLR6, NF-κB, TNF-α, and IL-1ß was increased obviously in LPS-induced HC11 cells, however, they were decreased obviously after transfecting with an exosome containing miR-155-inhibitor. The change in IL-10 level is opposite to the above genes. Taken together, exosomal miR-155-inhibitor could decrease the apoptosis of MECs and inhibit the inflammatory response to treat mastitis by down-regulation in the TLRs/NF-κB signaling pathway, which might be a new therapeutic target for mastitis.

Dietary supplementation with low and high polymerization inulin ameliorates adipose tissue inflammation via the TLR4/NF-κB pathway mediated by gut microbiota disturbance in obese dogs.

Inflammation induced by gut microbiota disorder plays an important role in promoting obesity. Inulin has beneficial effects on gut microflora and metabolic endotoxaemia. However, the chain length of inulin determines its different physiological effects. This study aimed to investigate the effect of low polymerization inulin (LPI) and high polymerization inulin (HPI) on inflammation in dogs with obesity induced by a high-fat diet and its potential mechanism. HPI, relative to LPI, significantly reduced the concentrations of LPS, IL-6 and TNF-α in serum and downregulated both the mRNA and protein expression of TLR4, NF-κB, TNF-α and IL-6 in adipose tissue. HPI and LPI intervention reduced adipose tissue fatty accumulation, which improved obesity. Supplementation with LPI and HPI increased gut microbiota diversity and altered specific bacterial populations at both the phylum and genus levels. The relative abundances of Prevotella, Fusobacterium and Enterobacter, which were positively correlated with the serum concentrations of LPS, IL-6 and TNF-α, were reduced. Our results demonstrate that both LPI and HPI can be used as an effective strategy for reducing inflammation and regulating gut microbiota, which can ameliorate obesity in dogs. Moreover, HPI exerts more positive regulation of the inflammatory response and gut microbiota dysfunction than LPI.

Gold as a coordinator of an imidazole conjugated dye of BODIPY derivatives for the identification of simple mercaptans.

Rapid detection of mercaptans in materials and the environment is of great help to material analysis and pollutant monitoring. Gold (Au) shows a high affinity to mercaptans. The coordination and steric effect of mercaptans to Au may be used for the development of new fluorescent sensors. It is possible to distinguish simple mercaptans (such as, HS-, thioglycolic acid) from glutathione (GSH) using Au as a coordinator of dye. Herein, a water-soluble fluorescent sensor of an imidazole conjugated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivative (BIM) was characterized by spectroscopic methods. BIM showed a large Stokes shift and high affinity to metals. Especially, Au-combined BIM produced a new complex BIMAu showing improved fluorescence emission, which can be quenched by thioglycolic acid and sodium hydrosulfide, but less affected by GSH. The detection limit of thioglycolic acid was 0.014 µM. Both NaSH and thioglycolic acid coordinated with BIMAu, while GSH took Au3+ away from BIMAu. These results indicate that the gold coordination competition between imidazole-substituted dyes and mercaptans is a good method for the development of new fluorescence chemosensors.

A dye-andrographolide assembly as a turn-on sensor for detection of phthalate in both cells and fish.

Phthalates can penetrate the environment and enrich various aquatic organisms through the food chain, which is involved in promoting the growth of breast cancer. It is of current interest to develop new sensors for phthalates. We herein reported a hydrogen-bond competing fluorescent sensor, BANP, for the detection of dibutyl phthalate (DBP). The BANP compound was synthesized by assembling andrographolide (Andro), nitro- and cyano-substituted BODIPY dye (BCN), and polyethylene glycol derivatives (DSPE-mPEG5000). BANP was found to be a turn-on fluorescent probe for DBP in water with a detection limit of 0.13 µg/g; the DBP-water system acts as a hydrogen bond switch to turn on the fluorescence. And BANP fluorescently detected DBP in contaminated fish meat. Moreover, BANP sensed the DBP-induced growth of human breast cancer MCF-7 cells, and the release of Andro in the DBP-cultivated cancer cells inhibited the proliferation of the MCF-7 cells. Taken together, BANP is a DBP-responsive probe for sensitive DBP detection in water, cells, and fish meats. The BANP sensor may be used in both in vitro fluorescence and cellular imaging analyses. Our results show that guest-induced reassembly brings forth significant fluorescence change, which is a promising way of designing new fluorescent probes for the analysis of phthalates in the environment and food.

miR-142-5p regulates lipopolysaccharide-induced bovine epithelial cell proliferation and apoptosis via targeting BAG5.

Bovine mastitis is a threat to the health of the dairy cow. MicroRNAs (miRs) serve an important role in the progression of bovine mastitis, regulating immune and defense responses. The present study aimed to investigate the possible effects and mechanisms of bovine mastitis underlying miR-142-5p and Bcl-2 associated athanogene 5 (BAG5) in in vitro lipopolysaccharide (LPS)-induced models. Reverse transcription-quantitative PCR and western blotting were performed to determine mRNA and protein expression levels, respectively. ELISAs were conducted to assess the levels of cytokines and an immunofluorescence assay was performed to determine the expression of BAG5. Cell Counting Kit-8, clone formation and 5-ethynyl-2'-deoxyuridine assays were conducted to determine cell viability and proliferation of bovine mammary epithelial MAC-T cells, respectively. Flow cytometry was performed to measure MAC-T cell cycle distribution and apoptosis, and a luciferase assay was conducted to verify whether BAG5 was a target of miR-142-5p. The results indicated that miR-142-5p was upregulated in MAC-T cells treated with LPS compared with the control group. miR-142-5p mimics transfection significantly activated the cytokines TNF-α, IL-1ß, IL-6 and IL-8, and significantly increased the expression levels of NF-κB signaling pathway-related proteins in LPS-treated cells. The luciferase activity of MAC-T cells treated with miR-142-5p mimics and BAG5 3'untranslated region wild type decreased, compared with mutant type. By contrast, BAG5 overexpression significantly downregulated the levels of cytokines, including TNF-α, IL-1ß, IL-6 and IL-8, in LPS-treated cells. BAG5 overexpression significantly promoted cell proliferation and viability, decreased apoptosis, and regulated Caspase-3, Caspase-9, Bcl-2 and Bax expression in LPS-treated MAC-T cells, which was significantly reversed by transfection with miR-142-5p mimics. In conclusion, the results of the present study suggested that miR-142-5p may promote the progression of bovine mastitis via targeting BAG5. Therefore, the present study provided the foundations for future investigations.

A Nano-BODIPY Encapsulated Zeolitic Imidazolate Framework As Photoresponsive Integrating Antibacterial Agent.

Drug-resistant bacteria challenge the antimicrobial agents and antibacterial strategy. To develop environmental friendly smart technology for treating pathogens, we report a kind of photoactivated nano-BODIPY (BCNBA@ZIF). First BODIPY compound (BC) was synthesized by coupling phenethyl caffeate (CAPE) with brominated BODIPY through B-O bonds. Next, BC was encapsulated in ZIF-8 together with 2-nitrobenzaldehyde (o-NBA) to form photoactivated BCNBA@ZIF nanoparticles. TEM confirm the structural change of BCNBA@ZIF after illumination. BCNBA@ZIF is less toxic to cells without illumination. Under illumination of blue LED light, the BCNBA@ZIF worked as a photoacid generator initiating the damage of ZIF shell with the release of BC, metal ions, and the production of singlet oxygen for achieving multifunctional antibacterial uses. Therefore, BCNBA@ZIF is a kind of photodriven smart "Domino" agent for bacterial inhibition.

The role of the ptsI gene on AI-2 internalization and pathogenesis of avian pathogenic Escherichia coli.

The LuxS/AI-2 quorum sensing mechanism can regulate the physiological functions of avian pathogenic Escherichia coli (APEC) through internalization of the small molecule autoinducer-2 (AI-2). The ptsI gene encodes enzyme I, which participates in the phosphotransferase system (PTS) that regulates the virulence and AI-2 internalization of bacteria. The aim of the present study was to determine the effect of ptsI on AI-2 internalization and other pathogenesis process in APEC using a ptsI mutant of the APEC strain DE17 (serotype O2), namely DE17ΔptsI. The results showed that deletion of the ptsI gene changed the rdar (red dry and rough) morphotype and decreased motility and biofilm formation in APEC (p < 0.05). Furthermore, scanning electron microscopy showed that the biofilm structure of DE17ΔptsI became sparse and more extracellular, as compared with the wild-type strain DE17. Moreover, AI-2 assay showed that AI-2 was internalized by DE17ΔptsI, while the recombinant PtsI protein had no AI-2 binding activity. Furthermore, deletion of the ptsI gene in APEC significantly increased adherence to DF-1 cells (p < 0.05). The 50% lethal dose of DE17ΔptsI was decreased by 17.8-fold and the bacterial loads of DE17ΔptsI were decreased by 13600-, 68.5-, 131-, and 3600-fold in the blood, liver, spleen, and kidney, respectively, as compared to the DE17. Moreover, histopathological analysis showed that the mutant DE17ΔptsI was associated with reduced pathological changes in the heart, liver, spleen, and kidney of ducklings, respectively, as compared to the wild-type strain DE17. The results of this study will benefit further studies on the functions of the ptsI in APEC.

The role of Ca(2+) mediated signaling pathways on the effect of taurine against Streptococcus uberis infection.

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-1ß, 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.

Variant innate immune responses of mammary epithelial cells to challenge by Staphylococcus aureus, Escherichia coli and the regulating effect of taurine on these bioprocesses.

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-1ß, 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.

High concentrate:forage ratio diet inhibiting omasal epithelium growth is associated with decreased cyclin D1 and CDK4 expression in growing goats.

The hypothesis that different concentrate : forage ratio diets alter omasal epithelium proliferation of growing goats via cyclins and regulation of the cell cycle was tested. Growing goats were fed with a high concentrate (HC, n = 8) or a low concentrate (LC, n = 8) diet for 42 days. The concentrate : forage ratio was 40:60 in the HC group and 0:100 in the LC group. In the HC group, the relative weight and DNA content of the omasal epithelium were lower, but the protein : DNA ratio was higher. Flow cytometry revealed that HC omasal cell numbers were smaller in S- and G2 /M-phases of the cell cycle and higher in the G0 /G1 -phases and were accompanied by reduced expression of cyclin D1 and CDK4 mRNA and protein. These data are consistent with morphologic observations in the HC that cell density decreased in the stratum spinosum (SS) plus stratum granulosum (SG) and stratum basale, and that cell density was lower in the SS plus SG. Thus, high-concentrate : forage ratio diet retards omasal epithelial growth by slowing the G1 to S phase transition of the cell cycle and is associated with decreased cyclin D1 and CDK4 expression in growing goats.