Synthesis And Anti-intestinal Oxidative Stress Research Of Z0206 Biogenic Nanoselenium Particles

Birth,weaning,and the change of diet and environment can lead to intestinal oxidative stress and subsequent intestinal barrier injury in piglets.The damaged intestinal barrier is responsible for diarrhea and decrease of nutrient digestion and absorption,which reduces growth performance of piglet and economic gain of swine industry.Therefore,how to ameliorate intestinal oxidative stress has become a big challenge in swine production.Selenium is one of the essential trace elements to animal with the most important function as antioxidant.The safety threshold of inorganic selenium is narrow,which also has toxic effects.In addition,the commonly known safe selenium-adding form,selenomethionine(SeMet),may lead to accumulated toxicity due to its nonspecific incorporation into proteins in place of methionine.However,recent research has revealed that elemental nanoselenium particles possessing efficient antioxidative and immunomodulating activity with high level of safety.However,no research has studied the effect of elemental nanoselenium particles on intestinal oxidative stress.Our previous study has isolated Enterobacter cloacae Z0206,which had high extracellular polysaccharide yield and selenite tolerating ability.This bacterium could efficiently synthesize selenium enriched polysaccharide and form biogenic nanoselenium particles(BNS).Therefore,in the present research,1)we optimized the conditions for E.cloacae Z0206 to synthesize BNS,investigated the mechanism of BNS synthesis,and characterized the BNS particles;2)we studied the antioxidative and barrier protecting effects of BNS using the mouse and pig jejunum epithelial cell oxidative stress models,which was attributed to the ability to activate Nrf2-ARE pathway.Furthermore,we compared the function of BNS with SeMet and chemically synthesized selenium nanoparticles(Nano-Se).The main results are described in the following section:1 Biosynthesis and characterization of BNS1.1 Synthesis of BNS by E.cloacae Z0206.(1)Supplementing sodium selenite to the broth significantly inhibited bacteria growth rate,and such inhibotory effect was selenite dose dependent.(2)Based on Michaelis-Menten kinetic equation analysis,the selenite reducing rate by E.cloacae Z0206 enhanced with the increase of selenite concentration,and the optimized dose of selenite and incubation time were 10 mM and 144 h,respectively.(3)SEM-EDX and TEM-EDX analysis revealed that E.cloacae Z0206 reduced selenite to elemental selenium and formed spherical and nano-sized particles.1.2 The mechanism of synthesis BNS by E.cloacae Z0206.(1)Analysis of the selenite reducing ability of different fractions of E.cloacae Z0206 cells,i.e.membrane proteins,cytoplasm proteins and extracellular proteins,indicated that only membrane proteins exhibited the selenite reducing activity.(2)iTRAQ proteomic analysis showed that selenite did not alter the expression of Painter-type reaction related proteins,but significantly elevated the expression of fumarate reductase,these results were verified at transcript(mRNA)level by real-time PCR analysis.(3)In order to verify the role of fumarate reductase in selenite reduction in E.cloacae Z0206,we found that adding fumarate significantly reduced the selenite reducing rate.Fumarate reductase gene knockout also markedly reduced the selenite reducing rate.These results indicated that E.cloacae Z0206 may reduce selenite to elemental selenium through membrane located fumarate reductase.1.3 Characterization of BNS.The BNS was synthesized based on the optimized conditions,followed by isolation.TEM-EDX and nano-particle size analyses showed that BNS were monodispersed and homogeneous spherical structure,sizing from 80 to 250 nm(average:139.43 ± 7.44 nm).XPS analysis confirmed that selenium in BNS was zero-valent.FT-IR analysis of BNS confirmed the presence of functional characteristics of proteins and carbohydrates.These results indicated that BNS were monodispersed spherical nanoparticles and capped with E.cloacae Z0206 synthesized proteins and polysaccharides.2 Antioxidative effect of BNS on mouse intestine under oxidative stress2.1 Establishment of the mouse model with intestinal oxidative stress.The C57BL/6 mice were intraperitoneally injected with various concentrations of diquat(0-100 mg/kg body weight),and mice accepted doses over 50 mg/kg were dead.Therefore,samples of mice in 0-25 mg/kg group were collected and analyzed.Compared with control group,25 mg/kg diquat significantly increased the activity of ALT,AST,DAO and the concentration of DLA in serum indicating that diquat treatment leads to liver and intestinal barrier injury.25 mg/kg diquat significantly elevated the contents of ROS and MDA in jejunum,and the ROS content was dose-dependent.The change of ROS in liver and other intestinal segments were irregular.In addition,25 mg/kg diquat significantly improved the activity of TrxR and GPx in jejunum,but not in liver,indicating that diquat treatment induced antioxidative defense in jejunum.These results suggest that intraperitoneally injection mice with 25 mg/kg diquat could successfully induce jejunun oxidative stress.In the follow-up experiments,we used this mouse intestinal oxidative stress model,and choose the jejunum as the targeted issue.2.2 Optimization of BNS dose for mouse.Orally gavage mice with various doses of BNS(0-2 mg/kg body weight)for 7 days did not altered the feed intake or body weight.The ALT and AST activity in serum and MDA concentration in liver and jejunum were not significantly changed indicating that supplementation of BNS inhibited to oxidative injury in live or jejunum caused by 25 mg/kg diquat.The activities of GPx and TrxR in jejunum were significantly higher in a dose-dependent manner,and the dose of 0.5 mg/kg could elevate the activities of these two enzymes to peak values.Therefore,the dose of 0.5 mg/kg BNS was chosen for the follow-up experiments.2.3 BNS inhibited oxidative stress induced injury in mouse intestine barrier(1)Intestinal oxidative stress significantly increased the amount of gut-to-circulation FD4,DAO activity,and DLA content in serum,indicating that the intestinal barrier was significantly damaged.BNS pretreatment significantly inhibited intestinal barrier oxidative injury,which had better effect than that of SeMet and Nano-Se.(2)Intestinal oxidative stress induced jejunum epithelial cell apoptosis,which resulted in the discontinuous and disarrayed epithelium.BNS strongly inhibited oxidative stress induced cell apoptosis and prevented villus morphological change,which was better than SeMet and Nano-Se.(3)BNS significantly inhibited the ROS and MDA generation in jejunum induced by diquat,which was stronger than SeMet and Nano-Se.(4)The activity of TrxR and GPx in jejunum of diquat and selenium compounds pretreated groups were significantly higher than that in control group,suggesting induced antioxidant response.The activity of jejunum GPx in BNS pretreated mice was higher than other groups indicating that BNS improved antioxidant enzyme activity more efficiently than SeMet and Nano-Se.These results indicate that BNS could efficiently inhibit oxidative stress induced mouse intestinal barrier injury and protect intestinal redox homeostasis,with higher activity than SeMet and Nano-Se.3 Antioxidative effect of BNS in pig intestinal epithelial cells3.1 BNS inhibited oxidative stress induced injury in IPEC-J2 cell.(1)According to the pilot experiment,the in vitro study was performed with 60 ?M Diquat,and 0.1 ?M of BNS,SeMet and Nano-Se,respectively.(2)The transwell was adopted to simulate the intestinal barrier in vitro.Diquat treatment decreased the TER value rapidly and completely destroyed the barrier at 7 h,and significantly increased the permeation of FD4.BNS pretreatment significantly inhibited the decrease of TER value and FD4 permeation,while SeMet and Nano-Se pretreatment did not reduce cell barrier oxidative injury.(3)Diquat significantly increased the protein expression of Bax and active caspase-3 and decreased the expression of Bcl-2,which strongly promoted cell apoptosis.BNS pretreatment completely inhibited the increase of Bax and active caspase-3 and the decrease of Bcl-2,and cell apoptosis,which had higher inhibitory activity than SeMet and Nano-Se.These results indicate that BNS pretreatment could significantly ameliorate oxidative stress induced apoptosis and cell barrier injury in IPEC-J2 cells,and higher activity was observed when compared with SeMet and Nano-Se.3.2 Effect of BNS on redox homeostasis of IPEC-J2 cells.Compared with control group,diquat markedly increased the contents of ROS,PSSG and MDA,decreased the concentration of GSH and increased the production of GSSG.Compared with diquat group,BNS pretreatment significantly inhibited the increase of ROS,PSSG and MDA,and increased GSH content and decrease GSSG production.SeMet pretreatment only significantly inhibited the increase of ROS and MDA induced by diquat.These indices in Nano-Se pretreated group were not significantly different from that of diquat group.These results indicate that BNS could protect the cell redox homeostasis,and prevent the occurrence of oxidative stress,and higher activity was observed when compared with SeMet and Nano-Se.4 The mechanism of the antioxidative effect of BNS in vivo4.1 The differences in uptakes of BNS and Nano-Se by IPEC-J2 cell.The BNS and Nano-Se were all labeled with coumarin-6.The cellular uptake of BNS increased in a time-dependent manner,whereas the cellular uptake of Nano-Se was extremely low.After 6 h incubation,the amount of intracellular Nano-Se was about 1/10 of BNS.These results indicate that BNS possesses a much better transmembrane capacity than Nano-Se.4.2 Effect of BNS on Nrf2-ARE pathway.(1)Western blot analysis showed that BNS significantly increased the expression of Nrf2 and the downstream proteins,i.e.TrxR-1,NQO-1,HO-1 and Trx,and the selenoenzyme GPx-1.SeMet significantly stimulated the expression of GPx-1,TrxR-1 and Trx,whereas Nano-Se only elevated the expression of TrxR-1.(2)Enzyme activity analysis revealed that BNS remarkably elevated the activities of NQO-1,HO-1,TrxR and GPx,whereas SeMet and Nano-Se only significantly elevated the activities of TrxR and GPx.These results indicate that BNS exerts antioxidative function through activating Nrf2-ARE pathway in addition to increasing selenoenzyme activity,whereas SeMet and Nano-Se only acted as selenium donors to increasing the activity of selenoenzymes.4.3 The indispensable role of the Nrf2-ARE pathway in the antioxidative effect of BNS.(1)Western blot and immunofluorescence analysis revealed that BNS activated Nrf2-ARE pathway in a dose-dependent manner,with saturated activity at 0.1?M BNS level.(2)Analysis of different treating time of BNS showed that the expression of Nrf2 downstream enzymes and proteins reached the highest amount at 6 h of treatment.When prolonging the treating time to 24 h,we found that the Nrf2 protein content reached peak value at 8 h,and started to decrease at 12 h,and was similar to that of control group at 24 h.(3)BNS stimulated the nuclear translocation of Nrf2 in a time-dependent manner,and reached highest amount at 6 h.(4)Knockdown the expression of Nrf2 via Nrf2-siRNA abolished the inhibiting effects of BNS on ROS generation and oxidative stress induced cell death.These results indicate that Nrf2-ARE pathway plays a pivotal role in the antioxidative effect of BNS.4.4 The mechanism of activation of Nrf2-ARE pathway by BNS.Western blot analysis revealed that BNS treatment significantly promoted the phosphorylation of Nrf2 and MAPKs(ERK1/2,JNK and p38)and PI3K/AKT.Inhibiting the activation of MAPKs and PI3 K/AKT using specific inhibitors showed that inhibiting the phosphorylation of ERK1/2,p38 and AKT significantly inhibited Nrf2 phosphorylation induced by BNS,whereas inhibiting JNK pathway did not alter the phosphorylation of Nrf2.These results indicate that BNS could activate ERK1/2,p38 and AKT,and further phosphorylate Nrf2,which stabilized Nrf2 and increased the content of Nrf2.Increased Nrf2 in the cytoplasm promoted nuclear translocation of Nrf2 to activate the Nrf2-ARE antioxidant pathway.