Selenium-enriched Bacillus paralicheniformis SR14 attenuates H2O2-induced oxidative damage in porcine jejunum epithelial cells via the MAPK pathway.

Oxidative stress plays a detrimental role in gastrointestinal disorders. Although selenium-enriched probiotics have been shown to strengthen oxidation resistance and innate immunity, the potential mechanism remains unclear. Here, we focused on the biological function of our material, selenium-enriched Bacillus paralicheniformis SR14 (Se-BP), and investigated the antioxidative effects of Se-BP and its underlying molecular mechanism in porcine jejunum epithelial cells. First, we prepared Se-BP and quantified for its selenium and bacterial contents. Then, in vitro free radical scavenging activity was measured to evaluate the potential antioxidant effect of Se-BP. Third, to induce an appropriate oxidative stress model, we adopted different concentrations of H2O2 and determined the most suitable concentration by a methyl thiazolyl tetrazolium (MTT) assay. Regarding treatment with Se-BP and H2O2, we found that Se-BP increased cell viability and prevented lactate dehydrogenase release when administered prior to H2O2 exposure. Additionally, Se-BP markedly suppressed reactive oxygen species and malondialdehyde production in cells and effectively attenuated apoptosis. Compared with incubation with H2O2 alone, treatment with Se-BP significantly promoted phosphorylation of ERK and p38 MAPK signaling molecules. When administered with ERK and p38 MAPK inhibitors, Se-BP did not alleviate the decrease in cell viability. Our results suggest that Se-BP prevents H2O2-induced cell damage by activating the ERK/p38 MAPK signaling pathways.

Biogenic nanoselenium particles activate Nrf2-ARE pathway by phosphorylating p38, ERK1/2, and AKT on IPEC-J2 cells.

As the intestinal epithelium is vulnerable to oxidative stress because of frequent enterocyte renewal and continuous exposure to exogenous agents, it is meaningful to figure out how the epithelial cells exert antioxidant function. We previously synthesized a novel biogenic nanoselenium (BNS) particles and proved that BNS could effectively improve intestinal antioxidative function through activating Nrf2-ARE pathway. The objective of the present study was to investigate the mechanism by which BNS activate Nrf2-ARE pathway on the physiological function of intestinal epithelial cells. In the present study, we demonstrated that treatment of IPEC-J2 cells with BNS particles not only elevated the levels of downstream proteins of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) such as heme oxygenase-1 and NQO-1 in a time-dependent manner which started to weaken at 12 hr after treatment but also significantly activated Nrf2, mitogen-activated protein kinase (MAPK), and protein kinase B (AKT) pathway in a time-dependent manner within 24 hr. BNS particles significantly increased the content of phosphorylated-Nrf2, without evident influence on the level of Kelch-like ECH-associated protein 1 (Keap1). Moreover, BNS also induced the activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase, and AKT while phosphorylating Nrf2. Using specific protein kinase inhibitors, we found that the Nrf2-phosphorylating and antioxidative effects of BNS particles were abolished when p38, ERK1/2, and AKT were significantly inhibited. Overall, our data demonstrated that BNS particles activated Nrf2-ARE pathway through p38, ERK1/2, and AKT mediated-phosphorylation of Nrf2 to improve the antioxidant function of intestinal epithelial cells.

Characterization, antioxidant property and cytoprotection of exopolysaccharide-capped elemental selenium particles synthesized by Bacillus paralicheniformis SR14.

Instead of using existing methods to chemically synthesize elemental selenium particles (CheSePs), which first require separating and purifying polysaccharides or proteins and adding extra reducing agent, this study applied a novel method to directly assemble exopolysaccharide-capped biogenic elemental selenium particles (EPS-BioSePs) by Bacillus paralicheniformis SR14 during the metabolic process. Characterization by energy dispersive X-ray spectrometry (EDX), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), size measurement and chemical composition analysis verified that EPS-BioSePs exhibited a monodispersed and homogeneous spherical structure 293.73±4.03nm in size. Compared to a widely used form of CheSePs stabilized and coated by bovine serum albumin, EPS-BioSePs exhibited better antioxidant properties on scavenging DPPH, superoxide and ABTS free radicals, but not hydroxyl radical. In vitro experiments with porcine jejunum epithelial (IPEC-J2) cells also indicated a significant cytoprotection of EPS-BioSePs against hydrogen peroxide-induced oxidative stress, as exhibited by cell viability reduction and suppression of ROS generation. These results suggested that this new form of selenium possessed great antioxidant property and cytoprotection and exopolysaccharide-producing bacteria could gradually become an appropriate choice to synthesize biogenic elemental selenium particles with potential applications as antioxidants.

Aerobic biogenesis of selenium nanoparticles by Enterobacter cloacae Z0206 as a consequence of fumarate reductase mediated selenite reduction.

In the present study, we examined the ability of Enterobacter cloacae Z0206 to reduce toxic sodium selenite and mechanism of this process. E. cloacae Z0206 was found to completely reduce up to 10 mM selenite to elemental selenium (Se°) and form selenium nanoparticles (SeNPs) under aerobic conditions. The selenite reducing effector of E. cloacae Z0206 cell was to be a membrane-localized enzyme. iTRAQ proteomic analysis revealed that selenite induced a significant increase in the expression of fumarate reductase. Furthermore, the addition of fumarate to the broth and knockout of fumarate reductase (frd) both significantly decreased the selenite reduction rate, which revealed a previously unrecognized role of E. cloacae Z0206 fumarate reductase in selenite reduction. In contrast, glutathione-mediated Painter-type reactions were not the main pathway of selenite reducing. In conclusion, E. cloacae Z0206 effectively reduced selenite to Se° using fumarate reductase and formed SeNPs; this capability may be employed to develop a bioreactor for treating Se pollution and for the biosynthesis of SeNPs in the future.

Biogenic Nanoselenium Particles Effectively Attenuate Oxidative Stress-Induced Intestinal Epithelial Barrier Injury by Activating the Nrf2 Antioxidant Pathway.

In the present study, a new form of selenium nanoparticle (biogenic nanoselenium (BNS) particles) was synthesized using bacteria. The protection of BNS particles against oxidative stress-induced intestinal barrier dysfunction and the inherent mechanisms of this process were investigated, and selenomethionine (SeMet) and chemically synthesized nanoselenium (Nano-Se) particles were used for comparison. Characterization of BNS particles revealed that they were monodispersed and homogeneous spheres, with an average size of 139.43 ± 7.44 nm. In the mouse model of intestinal oxidative stress, BNS particles were found to protect the mouse intestinal barrier function and preserve intestinal redox homeostasis more efficiently than SeMet and Nano-Se. In vitro experiments with porcine jejunum epithelial (IPEC-J2) cells verified the stronger epithelial barrier-protecting effect of BNS particles against oxidative stress, with reduced cell apoptosis and an improved cell redox state. BNS activated the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and increased the expression of its downstream genes, including thioredoxin reductase (TXNRD)-1, NADPH dehydrogenase (NQO)-1, heme oxygenase (HO)-1, and thioredoxin (Trx), in dose- and time-dependent manners. In contrast, SeMet and Nano-Se merely enhanced the activity of the selenoenzymes TXNRD-1 and glutathione peroxidase (GPx)-1, indicating the role of selenium donors. Moreover, the knock down of Nrf2 significantly blocked the antioxidative effect of BNS, confirming that BNS protects the intestinal barrier from oxidative stress-induced damage by activating Nrf2 and its downstream genes. Our results suggest that BNS is a promising selenium species with potential application in treating oxidative stress-related intestinal diseases.

Butyrate upregulates endogenous host defense peptides to enhance disease resistance in piglets via histone deacetylase inhibition.

Butyrate has been used to treat different inflammatory disease with positive outcomes, the mechanisms by which butyrate exerts its anti-inflammatory effects remain largely undefined. Here we proposed a new mechanism that butyrate manipulate endogenous host defense peptides (HDPs) which contributes to the elimination of Escherichia coli O157:H7, and thus affects the alleviation of inflammation. An experiment in piglets treated with butyrate (0.2% of diets) 2 days before E. coli O157:H7 challenge was designed to investigate porcine HDP expression, inflammation and E. coli O157:H7 load in feces. The mechanisms underlying butyrate-induced HDP gene expression and the antibacterial activity and bacterial clearance of macrophage 3D4/2 cells in vitro were examined. Butyrate treatment (i) alleviated the clinical symptoms of E. coli O157:H7-induced hemolytic uremic syndrome (HUS) and the severity of intestinal inflammation; (ii) reduced the E. coli O157:H7 load in feces; (iii) significantly upregulated multiple, but not all, HDPs in vitro and in vivo via histone deacetylase (HDAC) inhibition; and (iv) enhanced the antibacterial activity and bacterial clearance of 3D4/2 cells. Our findings indicate that butyrate enhances disease resistance, promotes the clearance of E. coli O157:H7, and alleviates the clinical symptoms of HUS and inflammation, partially, by affecting HDP expression via HDAC inhibition.