Weifen Li

Antioxidant Properties of Probiotic Bacteria

Oxidative stress defines a condition in which the prooxidant–antioxidant balance in the cell is disturbed, resulting in DNA hydroxylation, protein denaturation, lipid peroxidation, and apoptosis, ultimately compromising cells’ viability. Probiotics have been known for many beneficial health effects, and the consumption of probiotics alone or in food shows that strain-specific probiotics can present antioxidant activity and reduce damages caused by oxidation. However, the oxidation-resistant ability of probiotics, especially the underling mechanisms, is not properly understood. In this view, there is interest to figure out the antioxidant property of probiotics and summarize the mode of action of probiotic bacteria in antioxidation. Therefore, in the present paper, the antioxidant mechanisms of probiotics have been reviewed in terms of their ability to improve the antioxidant system and their ability to decrease radical generation. Since in recent years, oxidative stress has been associated with an altered gut microbiota, the effects of probiotics on intestinal flora composition are also elaborated.

Effects of Lead on Hepatic Antioxidant Status and Transcription of Superoxide Dismutase Gene in Pigs

Ninety-six castrated boars (Durocu2009×u2009Landraceu2009×u2009Yorkshire) were randomly divided into four groups, each of which was replicated three times with eight pigs. The groups received the same basal diet supplemented with 0, 5, 10, and 20xa0mg/kg lead, respectively. The malondialdehyde and glutathione levels, antioxidant enzymes activities, and zinc/copper superoxide dismutase (Zn/Cu SOD) mRNA content in the liver were determined to evaluate the lead hepatic intoxication caused by the lead. Results showed the increased lipid peroxides level and the reduced glutathione content, along with a concomitant decrease in the activities of superoxide dismutase, catalase, and glutathione peroxidase. Moreover, the level of hepatic Zn/Cu SOD mRNA was also significantly reduced. We suggest potential mechanism for lead intoxication in liver as follows: lead causes parallel decrease in Zn/Cu SOD mRNA and activities of antioxidant enzymes, leading to the declined ability of scavenging free radicals with excessive production of lipid peroxides, which seriously damages the hepatic structure and function.

Glycyrrhizic Acid Promotes M1 Macrophage Polarization in Murine Bone Marrow-Derived Macrophages Associated with the Activation of JNK and NF-κB

The roots and rhizomes of Glycyrrhiza species (licorice) have been widely used as natural sweeteners and herbal medicines. The aim of this study is to investigate the effect of glycyrrhizic acid (GA) from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GA were assessed. Our results showed that GA obviously increased the cell surface expression of CD80, CD86, and MHCII molecules. Meanwhile, GA upregulated the expression of CCR7 and the production of TNF-α, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Ym1, and Arg1 (the markers of alternatively activated (M2) macrophage). The functional tests showed that GA dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation are required for GA-induced NO and M1-related cytokines production, while ERK1/2 pathway exhibits a regulatory effect via induction of IL-10. Together, these findings indicated that GA promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GA in macrophage polarization.

Effect of dietary supplementation of Bacillus subtilis B10 on biochemical and molecular parameters in the serum and liver of high-fat diet-induced obese mice

While a high-fat diet (HFD) is assumed to be related to fat-mediated oxidative stress decreasing antioxidant enzyme activity, probiotics are believed to have positive effects on the regulation of HFD-induced obesity as well as lipid metabolism, energy homeostasis, and anti-oxidation. Because Bacillus subtilis B10 has beneficial effects on the abnormal lipid metabolism and the oxidative stress in HFD-induced obese mice, ICR mice were randomly assigned into an HFD group and the HFD was supplemented with 0.1% (w/w) Bacillus subtilis B10 (HFD+B10 group). Thereafter, 30-d treatments were run, and then hepatic lipid level and antioxidant status were measured. The expression of genes related to lipid metabolism and oxidative stress in the liver was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). We found that HFD-induced obese mice treated with B10 showed a decrease in weight gain, serum glucose activity as well as hepatic triglyceride (TG), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) activities. In addition, the gene expressions of antioxidant genes, glutathione reductase (GR), xanthine oxidase (XO), heat-shock protein 90 (Hsp90), and lipid synthesis gene 3β-hydroxysteroid-Δ24 reductase (DHCR24) in the HFD+B10 group were down-regulated, suggesting alleviation of oxidative stress, while the lipolysis gene 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), energy metabolism gene peroxisome proliferator-activated receptor α (PPARα) and the gene encoding tumor-suppressor protein p53 were up-regulated. The regulatory and positive effect of dietary supplementation of probiotic B10 suggests that it has a beneficial effect on the homeostasis of the lipid metabolism and on alleviating oxidative stress in HFD-induced obese mice.概要目的探究枯草芽孢杆菌B10 对高脂日粮诱导的小鼠脂肪代谢及氧化应激的改善作用, 并初步探讨其作用机制。创新点证明了枯草芽孢杆菌B10 可以有效改善高脂日粮诱导的小鼠脂肪代谢和氧化应激, 且发现此作用主要与B10 调节脂肪代谢基因 (PPARα 、DHCR24、HMGCS2) 及氧化应激基因 (XO、p53) 表达和谷胱甘肽过氧化物酶 (GSH-Px) 活力有关。方法将ICR 雄鼠分为对照组 (饲喂高脂日粮) 和实验组 (饲喂添加枯草芽孢杆菌菌粉的高脂日粮)。饲喂30 天后, 收集小鼠的血清及肝脏样品。采用试剂盒测定抗氧化及脂肪代谢相关指标和肝脏中8-羟基脱氧鸟苷 (8-OHdG) 含量。使用荧光定量聚合酶链式反应 (PCR) 测定小鼠肝脏中脂肪代谢和氧化应激相关基因的表达水平。结论饲喂含有枯草芽孢杆菌B10 的高脂日粮能够有效降低小鼠的体重 (表2), 降低血清中葡萄糖和甘油三酯含量及谷草转氨酶和谷丙转氨酶活力 (表3 和 4); 下调肝脏中脂肪合成相关基因表达量, 但上调脂肪分解相关基因表达量 (图1), 并提高肝脏中抗氧化相关基因表达量 (图2)。综上所述, 枯草芽孢杆菌B10 能有效调节小鼠脂肪代谢, 并改善其氧化应激。

Probiotic Bacillus amyloliquefaciens SC06 Induces Autophagy to Protect against Pathogens in Macrophages

Probiotics are increasingly applied in popularity in both humans and animals. Decades of research has revealed their beneficial effects, including the immune modulation in intestinal pathogens inhibition. Autophagy—a cellular process that involves the delivery of cytoplasmic proteins and organelles to the lysosome for degradation and recirculation—is essential to protect cells against bacterial pathogens. However, the mechanism of probiotics-mediated autophagy and its role in the elimination of pathogens are still unknown. Here, we evaluated Bacillus amyloliquefaciens SC06 (Ba)-induced autophagy and its antibacterial activity against Escherichia coli (E. coli) in murine macrophage cell line RAW264.7 cells. Western blotting and confocal laser scanning analysis showed that Ba activated autophagy in a dose- and time-dependent manner. Ba-induced autophagy was found to play a role in the elimination of intracellular bacteria when RAW264.7 cells were challenged with E. coli. Ba induced autophagy by increasing the expression of Beclin1 and Atg5-Atg12-Atg16 complex, but not the AKT/mTOR signaling pathway. Moreover, Ba pretreatment attenuated the activation of JNK in RAW264.7 cells during E. coli infection, further indicating a protective role for probiotics via modulating macrophage immunity. The above findings highlight a novel mechanism underlying the antibacterial activity of probiotics. This study enriches the current knowledge on probiotics-mediated autophagy, and provides a new perspective on the prevention of bacterial infection in intestine, which further the application of probiotics in food products.

Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production

Oxidative stress (OS) plays a major role in the gastrointestinal disorders. Although probiotics were reported to repress OS, few researches compared the antioxidant ability of different Bacillus strains and deciphered the mechanisms. To select a Bacillus strain with higher antioxidant capacity, we used H2O2 to induce intestinal porcine epithelial cell 1 (IPEC-1) OS model. The most suitable H2O2 concentration and incubation time were determined by the half lethal dose and methyl thiazolyl tetrazolium. Correlation analysis was performed to choose a sensitive indicator for OS. As for the comparison of Bacillus, cells were divided into control, Bacillus treatment, H2O2 treatment, and Bacillus pre-protection + H2O2 treatment. Bacillus were co-cultured with IPEC-1 for 3xa0h in Bacillus and Bacillus pre-protection + H2O2 treatments. Then, based on OS model, 300xa0μmol/L H2O2 was added into medium of H2O2 and Bacillus pre-protection + H2O2 treatments for another 12xa0h. Antioxidant and apoptosis gene expressions were detected to screen the target strain. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) pathway, reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψm), apoptosis, and necrosis were analyzed. Results revealed that heme oxygenase-1 (HO-1) gene expression had a positive correlation with H2O2 induction. Moreover, Bacillus amyloliquefaciens SC06 (SC06)-meditated IPEC-1 showed the best antioxidant capacity though modulating Nrf2 phosphorylation. Δψm was elevated, while ROS generation was reduced with SC06 pre-protection, resulting in decreased apoptosis and necrosis. Altogether, HO-1 expression could be regarded as an OS indicator. The regulation of Nrf2/Keap1 pathway and ROS production by SC06 are involved in alleviating OS of IPEC-1.

Protective immunity against Eimeria tenella infection in chickens following oral immunization with Bacillus subtilis expressing Eimeria tenella 3-1E protein

The current experiment was conducted to construct recombinant Bacillus subtilis WB600 expressing Eimeria tenella 3-1E protein to investigate the oral immunization protective effects against E. tenella. The merozoite surface antigen 3-1E gene of E. tenella was introduced into the pBS-H1 expression vector with a novel signal peptide sequence. After the electro-transformation, the expression of objective protein in B. subtilis WB600 was detected by Western blot. The results showed that the recombinant B. subtilis strain with the ability of high-level secretion of 3-1E was constructed successfully. Seven-day-old broiler chickens were orally vaccinated with B. subtilis WB600 harboring 3-1E (B.S-pBS-H1-3-1E) or B. subtilis WB600 with empty plasmid (B.S-pBS-H1) 10xa0days prior to challenge with sporulated E. tenella oocysts. The results showed the recombinant B. subtilis strain with the ability of high-level secretion of 3-1E was constructed successfully. Vaccination with B.S-pBS-H1-3-1E strain significantly increased the anti-coccidial index and reduced cecal lesion scores compared with the positive control group (chickens were challenged with sporulated E. tenella oocysts without oral administration of B.S-pBS-H1-3-1E strain) and B.S-pBS-H1 group. Ceca mucosal sIgA, secretion, and IL-2, IL-12, IFN-γ, and IL-10 level after challenge were greater in the B.S-pBS-H1-3-1E group than in the positive control group. Taken together, these results indicated that B. subtilis WB600 harboring 3-1E protein induces protective immunity against E. tenella.

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.

Echinacea pupurea extracts promote murine dendritic cell maturation by activation of JNK, p38 MAPK and NF-κB pathways

&NA; Dendritic cells (DCs) comprise a system of highly professional antigen presenting cells (APCs) which connect innate and adaptive immunity by undergoing dramatic shift in their maturation state. Phytomedicine Echinacea purpurea extracts (EE) could modulate murine dendritic cell fate and function. However, the underlying mechanism of EE on DCs development and maturation remains limited. In this study, immature DCs were induced phenotypic maturation with up‐regulated expression of key accessory molecules and the phagocytic activity was decreased after being treated with EE (400 &mgr;g/ml) for 48 h. We found that TLR1/2, JNK, p38‐MAPK and NF‐&kgr;B pathways were activated following EE exposure. Notably, JNK activation was demonstrated to be associated with increased IFN‐&ggr; response while p38‐MAPK pathway exhibited immuno‐regulatory effects via induction of IL‐10 and TGF‐&bgr;1. Furthermore, it was verified that NF‐&kgr;B signaling was responsible for EE‐induced synthesis of IFN‐&ggr;, IL‐12 and TGF‐&bgr;1, but not for IL‐10 induction. These results indicate that EE have the immunomodulatory potency to promote both phenotypic and functional maturation of BMDCs via modulating the activation of JNK, p38‐MAPK and NF‐&kgr;B pathways. Our findings contributed to the current understanding of the immunoregulatory function of EE and the mechanism of DCs maturation. Graphical abstract Figure. No caption available. HighlightsEchinacea purpurea extracts (EE) promoted phenotypic maturation while reduced the phagocytic activity of BMDCs.JNK activation was associated with EE‐induced IFN‐&ggr; response.p38‐MAPK exhibited immuno‐regulatory effects via induction of IL‐10 and TGF‐&bgr;1 following EE exposure.NF‐&kgr;B was responsible for EE‐induced synthesis of IFN‐&ggr;, IL‐12 and TGF‐&bgr;1, but not for IL‐10 induction.

A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis.

There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes.