Effects Of Bacillus Amyloliquefaciens On The Antioxidant Capacity Of Animals And Its Mechanism

Oxidative stress refers to a condition in which the redox balance in the cell is disturbed,leading to DNA hydroxylation,protein denaturation,lipid peroxidation,and apoptosis.In this study,we first established in vitro oxidative stress model with IPEC-1 and H2O2 and screened a strain of Bacillus(Bacillus amyloliquefaciens SC06)with potent antioxidant capacity based on this model.Then,we studied the effects of SC06 on growth performance,intestinal oxidative stress and autophagy of piglets.Thereafter,taking advantage of the IPEC-1 oxidative stress model,we determined the mechenisms of the decreased oxidative stress induced by SC06.Meanwhile,we also studied the impacts of SC06 on the oxidative stress,liver steotosis and psychological well-being of HFD-induced obese mice to elucidate the molecular mechanisms of SC06 to resist oxidation.This study contains 5 experiments which are listed as follows.Experiment 1.The establishment of oxidative stress model and the selection of Bacillus strains with potent antioxidant capacity(1)Establishment of IPEC-1 oxidative stress model.We first examined the cell viability of IPEC-1 using MTT method after treated with H2O2 at different concentrations for different time.Using the probit method,the median lethal dose(LD50)for H2O2 to IPEC-1 at 12 h was calculated to be 323.59 μmol/L.Moreover,with the increase of H2O2 concentration and induction time,cell viability witnessed a decline and the treatment of IPEC-1 with 300 μmol/L H2O2 for 12 h reduced cell viability to 54.04 ± 1.34%.Thus,300 μmol/L H2O2 was used to treat IPEC-1 for 12 h in order to induce the oxidative stress for the follow-up experiments.(2)Selection of an effective indicator for oxidative stress of IPEC-1 induced by H2O2.In order to choose an effective oxidative stress indicator,300 μmol/L H2O2 was used to treat IPEC-1 cells for various time periods(0,1.5,3,6,9,12,and 18 h),and the mRNA expressions of various antioxidant and apoptosis genes were measured.Results demonstrated that compared to other tested genes,heme oxygenase-1(HO-1)was extremely sensitive to H2O2 stimulation.After 12-h 300 μmol/L H2O2 incubation,HO-1 mRNA relative expression enhanded to 13.96 ± 1.31.Moreover,through the correlation analysis,we found that there was a good positive correlation under oxidative stress between HO-1 expression and the H2O2 treatment time(R2 = 0.9628).In previous study,it was confirmed that the induction of HO-1 could represent an adaptive response to oxidative injury with increasing cell resistande.Hence,in the study,we chose HO-1 as the primary indicator for oxidative stress in IPEC-1,and other antioxidant and apoptosis genes were also considered as secondary indicators.(3)Sceening of Bacillus strains with potent antioxidant ability.To determine the antioxidant effects of Bacillus,IPEC-1 cells were divided into four groups:control,Bacillus treatment,H2O2 treatment,and Bacillus pre-protection + H2O2 treatment groups.Cells were treated with B.subtillis SC02,B.amyloliquefaciens SC06,B.natto SC07,B.lichenifoemis SC08,B.polymyxa SC10,B.coagulans SC12,B.amyloliquefaciens SC33 and H2O2.Results showed that compared with the result in H2O2 group,SC06 pre-protection markedly decreased HO-1 transcription by 41%,indicating the most favorable effect among seven strains.Additionally,SC06 preprotection not only downregulated superoxidase-1(SOD-1),glutathione peroxidase-2(GPX-2),and thioredoxin-1(TRX-1)expressions but also upregulated glutathione S-transferase(GST)transcript level.Notably,SC06 significantly decreased caspase 3 expression,and the preprotection with SC06 remarkably upregulated anti-apoptotic gene expression and downregulated pro-apoptotic gene expressions.Thus,SC06 was chosen for further study to investigate the mechanisms of action of antioxidation.Experiment 2.Effects of SC06 as a substitute for antibiotics on antioxidant capacity and intestinal autophagy of pigletsPiglets are often exposed to oxidative stress due to weaning and environment factors.Here we used SC06 to replace aureomycin in the diets of piglets.Ninety piglets were divided into three groups:G1(containing 150 mg/Kg aureomycin in the diet);G2(containing 75 mg/Kg aureomycin and 1×108 cfu/Kg SC06 in the diet);G3(containing 2×108 cfu/Kg SC06 in the diet without any antibiotics).Results showed that SC06 replacement significantly increased the daily weight gain of piglets.Moreover,SC06 replacement elevated intestinal autophagy and activated Nrf2-Keap1 to improve antioxidant status of piglets.Meanwhile,c-Jun N-terminal kinase(JNK)phosphorylation was decreased while mammalian target of rapamycin(mTOR)expression was increased by SC06 replacement compared with antibiotic group.Experiment 3.Mehanism studies about the roles of SC06 in reducing IPEC-1 oxidative damageUsing the method mentioned in experiment 1,we analysed the mechanisms about the roles of SC06 in reducing IPEC-1 oxidative damage in terms of the antioxidation and ROS production.Our findings indicated that 3-h SC06 treatment activated Nrf2-Keapl signaling pathway to improve the activities of antioxidases.Further,SC06 pre-treatment also decreased NOX activity as well as the expression of p47Phox to reduce ROS level.Besides,the mitochondrial memberan potential(△ψm)was augemented by SC06.As a result,cell apoptosis and necrosis were improved.Experiment 4.The mechanisms of SC06 resistance against HFD-induced oxidative stress and liver injury in miceThe mechanisms of SC06 resistance against HFD-induced liver steotosis and oxidative stress were studied.60 male C57BL/6J mice were divided into 4 groups evenly.Mice were fed with NC(normal chew),NC+SC06,HFD(high fat diet)and HFD + SC06 respectively.Feeding period was 8 weeks.(1)Effects of SC06 on the obesity and insulin resistance of mice.Compared with NC,NC + SC06 did not alter body weight significantly.HFD-fed mice gained more weight from week 5 than NC-fed mice,but HFD + SC06 significantly relieved the HFD-induced body weight increases.Besides,the daily energy intake of mice in HFD group was increased compared with NC group,while HFD + SC06 administration decreased energy intake significantly.Perirenal and subcutaneous partial fat weights were not changed in NC + SC06-fed mice compared with NC-fed mice,but were higher in mice receiving HFD.Although weights of the perirenal,subcutaneous and epididymal fat were consistently decreased in HFD-fed mice received SC06,only subcutaneous fat weight reduced significantly.Further,histological analysis of perirenal,subcutaneous and epididymal adipose tissues showed that the size of adipocytes was increased in the HFD-fed mice versus the NC-fed mice but reduced in mice receiving HFD + SC06.In addition,HFD significantly increased insulin resistance,while HFD + SC06 improved insulin sensitivity obviously.(2)Effects of SC06 on liver injury.Here we found that SC06 effectively protected against HFD-induced fat accumulation in livers.Moreover,HFD significantly enhanced glutamic-pyruvic transaminase(GPT)activity,but HFD + SC06 only decreased GPT activity slightly.Additionally,TUNEL results indicated that HFD enhanced liver cell apoptosis,but SC06 treatments reduced the apoptosis.The mitochondrial TEM revealed that HFD seriously induced mitochondrial abnormalities.However,SC06 treatment effectively alleviated the mitochondrial injury.(3)Effects of SC06 on the secretion of inflammatory cytokines and adipokins.HFD significantly increased serum tumor necrosis facter-α(TNF-α),interleukin(IL)-1β,IL-6 and leptin levels,while HFD + SC06 decreased TNF-α,IL-6 and leptin markedly.(4)Effects of SC06 on the liver oxidative stress of mice.Mice fed with NC + SC06 exhibited higher CAT activity in liver.HFD significantly increased liver MDA.Compared to the HFD group,HFD + SC06 significantly lowered liver MDA content.By analyzing Nrf2-Keapl pathway,we found that that mice in NC + SC06 exhibited higher Nrf2 expression and phosphorylation levels.Meanwhile,the phosphorylation of Nrf2 was significantly up-regulated in HFD group compared to NC.Compared to HFD,mice receiving HFD + SC06 showed down-regulated p-Nrf2 expression.No significant differences were observed in Keapl expressions among groups.Additionaly,HFD obviously elevated ROS levels in liver,but HFD + SC06 inhibited the elevated ROS concentration induced by HFD.(5)Effects of SC06 on mice gut microbiota.Through 16S sequencing,we found that SC06 obviously changed the construction of gut microbiota.Compared with HFD group,HFD + SC06 enriched the relative abundance of Bacteroides,but decreased the relative abundance of Firmicutes as well as TM7.In week 8,compared to HFD group,HFD + SC06 reversed the abundance of species belonging to S24-7 family.Experiment 5.Effects of SC06 on the psychological welfare of HFD-induced mice with oxidative stressHFD-induced obesity and oxidative stress can cause sickness behavior that arise from changes in brain functions.Therefore,we hypothesize that SC06 may also improve obesity-related beha’vioral aberrations.Here,36 male C57BL/6J mice were divided into 3 groups evenly.Mice were fed with NC,HFD and HFD + SC06 respectively.Feeding period was 6 weeks.After 6-week feeding,the decreased mice body weight in mice feeding HFD + SC06 was accompanied by significant improvement of anxiety-like behavior and social interaction.Attenuation of sickness behavior was paralleled with up-regulated brain derived neurotrophic factor(BDNF)expression and down-regulated N-methyl-D-aspartic acid receptors(NR)expressions.Moreover,SC06 also reduced oxidative levels by activating Nrf2/Keapl pathway,decreasing ROS concentration and normalizing mitochondrial morphology.Meanwhile,reduced systemic inflammation and enhanced intestinal barrier function were also observed with SC06 supplementation.These findings indicate that dietary intake of SC06 can improve obesity-induced sickness behavior through modulating expressions of BDNF and NR,improving antioxidant capacity,reducing inflammation and enhancing intestinal barrier function.