Inhibition of Salmonella-induced IL-8 synthesis and expression of Hsp70 in enterocyte-like Caco-2 cells after exposure to non-starter lactobacilli

Oral administration of lactobacilli as probiotics is gaining importance in the treatment of intestinal inflammations. We investigated the effect of non-starter lactobacilli Lactobacillus casei subsp casei 2756, Lactobacillus curvatus 2775, and Lactobacillus plantarum 2142 as well as their spent culture supernatants (SCS) on Salmonella enteritidis 857 growth, interleukin (IL)-8 and heat shock protein 70 (Hsp70) synthesis in undifferentiated crypt-like and differentiated villus-like Caco-2 cells. The cells were infected with graded numbers of non-starter lactobacilli or S. enteritidis 857 for 1 h and allowed to recover for 24 h or exposed to 200 bacteria/cell for 1 h and allowed to recover for different periods of time. In another experiment S. enteritidis 857 was first pre-treated with SCS-lactobacilli for 1 h before infecting the cells. The levels of IL-8 and Hsp70 were assessed using sandwich ELISA and immunostaining of Western blots, respectively. The effect of SCS-lactobacilli on S. enteritidis 857 growth was evaluated by agar plate diffusion test. The non-starter lactobacilli induced a significant increase in the levels of both IL-8 and Hsp70. However, compared with the S. enteritidis 857 induced IL-8 synthesis, the levels of IL-8 induced by the lactobacilli at any equivalent bacterial number were far lower. After exposure of Caco-2 cells to S. enteritidis 857 pre-treated with SCS-lactobacilli, it appeared that their SCS inhibited the S. enteritidis 857 growth and IL-8 synthesis and in addition induced the expression of Hsp70. The differences in response of crypt- and villus-like Caco-2 cells are merely a reflection of their differentiation status. Our data suggest that the beneficial effect of non-starter lactobacilli to the intestinal inflammations might be associated with a decrease of the IL-8 levels. This effect could be mediated, at least in part, by the bacteria themselves or via a secreted antimicrobial product(s) either directly against the pathogens or indirectly through the synthesis of Hsp70.     

柚叶总黄酮对LPS所致Caco-2结肠上皮细胞间高通透性的保护作用

探讨柚叶总黄酮(PLTFE)对脂多糖(LPS,2 μg/mL)诱发Caco-2高通透性细胞模型的保护作用。Caco-2模型细胞以不同浓度的PLTFE(0、10、50、100、150 μg/mL)处理培养24 h进行后续实验。MTT法测定细胞生存率,细胞乳酸脱氢酶(lactate dehydrogenase,LDH)水平依说明书使用试剂盒测定。酶联法(enzyme linked immunosorbent assay,ELISA)测定白介素(interlukin-1β,IL-1β)、IL-8和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)分泌水平。跨上皮细胞电阻(trans epithelial electrical resistance,TEER)值和异硫氰酸荧光素-右旋糖酐(FD40)透过度用于评估细胞通透性水平。实时定量PCR(Quantitative real-time PCR,qRT-PCR)检测细胞IL-1β、IL-8、TNF-α、闭锁蛋白(Occludin)、紧密连接蛋白-1(claudin-1)、封闭小带蛋白(ZO-1)和肌球蛋白轻链激酶(myosin light chain kinase,MLCK)的mRNA表达。结果表明,柚叶总黄酮能显著提高受损细胞生存率至86.1%,抑制受损细胞中LDH的溢出(p<0.05)。同时还能有效抑制受损细胞中炎性细胞因子(IL-1β、IL-8、TNF-α)的分泌及mRNA转录。此外,柚叶总黄酮可增强细胞紧密连接因子(Occludin、claudin-1、ZO-1)的mRNA转录,抑制MLCK的mRNA转录,改善细胞间高通透性qRT-PCR法检测细胞中相关因子的mRNA转录水平。结果提示,柚叶总黄酮具有较强的抗炎活性,能通过上调细胞内细胞紧密连接相关因子的mRNA转录显著改善LPS造成的Caco-2细胞间高通透性的发生(p<0.05)。     

The nutritional supplement Active Hexose Correlated Compound (AHCC) has direct immunomodulatory actions on intestinal epithelial cells and macrophages involving TLR/MyD88 and NF-κB/MAPK activation

Active Hexose Correlated Compound (AHCC) is an immunostimulatory nutritional supplement. AHCC effects and mechanism of action on intestinal epithelial cells or monocytes are poorly described. AHCC was added to the culture medium of intestinal epithelial cells (IEC18 and HT29 cells) and monocytes (THP-1 cells) and assessed the secretion of proinflammatory cytokines by ELISA. Inhibitors of NFκB and MAPKs were used to study signal transduction pathways while TLR4 and MyD88 were silenced in IEC18 cells using shRNA. It was found that AHCC induced GROα and MCP1 secretion in IEC18 and IL-8 in HT29 cells. These effects depended on NFκB activation, and partly on MAPKs activation and on the presence of MyD88 and TLR4. In THP-1 cells AHCC evoked IL-8, IL-1β and TNF-α secretion. The induction of IL-8 depended on JNK and NFκB activation. Therefore, AHCC exerts immunostimulatory effects on intestinal epithelial cells and monocytes involving TLR4/MyD88 and NFκB/MAPK signal transduction pathways.     

具有潜在改善炎症反应益生菌的筛选

目的:探讨供试菌株对脂多糖(LPS)诱导THP-1细胞分泌炎性细胞因子的调节作用,筛选出具有潜在改善炎症反应的益生菌。方法:通过佛波酯(PMA)诱导THP-1成为成熟巨噬细胞,脂多糖(LPS)刺激成熟巨噬细胞产生炎症反应,评价益生菌对这种炎症反应的改善作用,并对具有改善炎症反应潜力的益生菌株进行胃肠道耐受能力、粘附能力、抑菌能力及抗生素敏感性评价。结果:植物乳杆菌SS-9、SD-H9、鼠李糖乳杆菌SD-L8及罗伊氏乳杆菌WX-94具有改善机体炎症反应的潜力,其中SS-9和SD-H9能显著降低炎症模型细胞IL-1β的分泌(P<0.05),WX-94和SS-9能显著降低炎症模型细胞的IL-6的分泌(P<0.05);同时四株菌均具有较高的胃肠道耐受能力、粘附能力,具有安全通过肠道的潜力;并可以不同程度地抑制致病菌大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌及阴沟肠杆菌的生长,具有抑制肠道感染的潜力;抗生素敏感性研究表明,四株菌均未发现抗生素耐受的风险。结论:植物乳杆菌SS-9、SD-H9、鼠李糖乳杆菌SD-L8及罗伊氏乳杆菌WX-94均可作为具有潜在炎症调节能力的益生菌进行开发和应用。     

Mode of inactivation of probiotic bacteria affects interleukin 6 and interleukin 8 production in human intestinal epithelial-like Caco-2 cells

Five lactic acid bacteria and two bifidobacteria strains were heat or irradiation inactivated. Inactivated cultures were evaluated for their effects on cytokines interleukin (IL) 6 and IL-8 production in human intestinal-like Caco-2 cells. For both heat- and irradiation-inactivated cultures, production of IL-6 and IL-8 was dependent on the specific microorganism. However, with all of the cultures, both IL-6 and IL-8 production was significantly higher (P < 0.05) in Caco-2 cells that were treated with heat-inactivated probiotic bacteria compare to the irradiation-inactivated bacteria. In the majority of the cases, heat-inactivated bacteria induced IL-6 and IL-8 production, whereas irradiation-inactivated bacteria attenuated both cytokine production. Our results indicate that the same probiotic bacteria used in the same cell culture could provide opposite cytokine production and immune modulation results based on its mode of inactivation; therefore, it is important to describe inactivation methods and conditions in detail when characterizing probiotic effects.     

Antibacterial activity of bioconverted eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) against foodborne pathogenic bacteria

The antimicrobial activity of bioconversion extracts of EPA and DHA against a range of foodborne pathogenic bacteria was investigated. The bioconverted EPA and DHA exhibited antibacterial activities against four gram-positive bacteria, Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus (ATCC 6538) and S. aureus (KCTC 1916) and seven gram-negative bacteria, Enterobacter aerogenes, Escherichia coli, E. coli O157:H7, E. coli O157:H7 (human), Pseudomonas aeruginosa, Salmonella enteritidis and S. typhimurium. The growth inhibition by both bioconverted EPA and DHA was similar against gram-positive bacteria, while the bioconverted extract of DHA was more effective than EPA against gram-negative bacteria as determined by minimum inhibitory concentration.     

Interaction of bifidobacteria with Caco-2 cells-adhesion and impact on expression profiles

The aim of the present study was to study different strains of bifidobacteria for adhesion to Caco-2 intestinal epithelial cells (IECs) and to test for the mRNA response of these cells following interaction with bifidobacteria. Adhesion was tested at different pH conditions using model epithelia consisting of transwell cultures of fully differentiated Caco-2 cells. Microarrays were used to characterize changes in global expression profiles of Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) and challenged with non-pathogenic Escherichia coli D2241 or four different strains of bifidobacteria. Furthermore, cytokine mRNA of IECs in responses to challenge with Bifidobacterium bifidum S17 or E. coli D2241 was tested in PBMC-sensitised Caco-2 cells using RT-PCR. Bifidobacteria showed strain-specific adhesion to Caco-2. Shift of apical pH from 7 to 4.5 resulted in strain-specific changes of adhesion. Global expression profiles of PBMC-sensitised Caco-2 cells revealed differential expression of a significant number of genes only after challenge with E. coli D2241 while cells were essentially unresponsive to challenge with four strains of bifidobacteria showing different adhesion properties. Using a RT-PCR approach, in the same system a similar differential expression after challenge with E. coli D2241 or B. bifidum S17 was observed for various immune markers. The presented results suggest that Caco-2 cells might be specifically unresponsive to challenge with bifidobacteria irrespective of the level of adhesion.     

Formation of Succinyl Genistin and Succinyl Daidzin by Bacillus Species

ABSTRACT:  6"- O -Succinyl-4'-hydroxyisoflavone-7- O -β-D-glucopyranoside (succinyl-β-daidzin) and 6"- O -succinyl-6,4'-dihydroxyisoflavone-7- O -β-D-glucopyranoside (succinyl-β-genistin), 2 new isoflavone metabolites, are found in  cheonggukjang  or  natto , traditional soy-based foods fermented with  Bacillus  species. Standard isoflavones including daidzin, genistin, daidzein, and genistein, and mixtures of isoflavones extracted from roasted soybeans were added to the medium growing  Bacillus subtilis  or  B. subtilis  natto and formation of succinyl-β-daidzin and succinyl-β-genistin were monitored by high-performance liquid chromatography (HPLC). Samples containing  Bacillus  with daidzin and genistin produced succinyl-β-daidzin and succinyl-β-genistin, respectively, while those with daidzein and genistein did not produce succinyl derivatives. Daidzin in samples with  B. subtilis  and  B. subtilis  natto decreased by 39.7% and 10.7%, respectively, for 4 h incubation while genistin decreased by 66.8% and 17.6%, respectively. Genistein decreased faster than daidzein during incubation with  B. subtilis  or  B. subtilis  natto without formation of succinyl derivatives. In the case of mixture of isoflavones, succinyl derivatives increased and β-glucosides and aglycones of isoflavones decreased significantly for 8 h incubation ( P  < 0.05).     

Mucosal immunomodulation by the non-bacterial fraction of milk fermented by Lactobacillus helveticus R389

The health promoting effects ascribed to probiotic bacteria and fermented dairy products arise not only from bacteria themselves but also from metabolites derived from milk fermentation. Exopolysaccharides produced during milk fermentation and peptides derived from major milk proteins, when released during fermentation, are potential modulators of various regulatory processes in the body. The aim of this work was to increase the knowledge of the previously observed immunomodulating capacity of milk fermented by Lactobacillus helveticus R389 by the study of the mucosal immunomodulation exerted by the non-bacterial fraction of the milk fermented at a constant pH6 (NBFpH6). The effects on IL-6 production by small intestine epithelial cells, the profile of IgA+ and cytokine+ cells (IL-2, IL-6, IL-10, TNF-alpha and IFN-gamma) induced in the gut lamina propria, and the levels of total and specific secretory IgA in the lumen of BALB/c mice that received NBFpH6 for 2, 5 or 7 days were examined. There was an increase in the number of IgA+, IL-10+, IL-2+ and IL-6+ cells after all feeding periods. Total S-IgA in the small intestine lumen increased in mice that received NBFpH6 for 2 days. However, no specific antibodies against NBFpH6 were detected. Feeding of NBFpH6 for 7 days significantly (P<0.05) enhanced IL-6 secretion by small intestine epithelial cells. NBFpH6 induced a non-specific mucosal response that was down-regulated for protective immunity, enhancing IL-6 production by epithelial cells and IgA production in the small intestine. These events improve the immunological defenses at the intestinal level, increasing host protection against pathologies. Because mucosal immune responses induced by certain dietary antigens play a large part in the prevention of gastrointestinal diseases, the oral administration of a mucosal adjuvant such as NBFpH6 may positively affect the milieu of the intestinal lumen. The opportunity exists then to manipulate the constituents of the lumen of the intestine through dietary means, thereby enhancing the health condition of the host.     

Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part by increasing expression of zonula occludens-1 and myosin light-chain kinase in vivo

The effects of lactobacilli on impaired intestinal barrier function and paracellular permeability were evaluated in human epithelial Caco-2 cells treated with tumor necrosis factor-α and in mice with colitis induced by dextran sodium sulfate (DSS). Filter-grown Caco-2 monolayers were used as the intestinal epithelial model. Among the 4 lactobacilli studied, Lactobacillus rhamnosus OLL2838 most effectively suppressed barrier impairment and increased IL-8 secretion induced by tumor necrosis factor-α in Caco-2 cells; however, the conditioned medium from OLL2838 did not show any effect on barrier functions. The in vivo effects of OLL2838 on intestinal epithelial barrier function and colonic inflammation were assessed in DSS-induced colitis of BALB/c mice. Oral treatment with both live and heat-killed OLL2838 suppressed weight loss and recovered colon length. Additionally, barrier function was restored by the administration of live and heat-killed OLL2838 to the DSS-treated animals, which conferred protection against the increase in mucosal permeability associated with DSS-induced colitis. This may at least partially be because of the increased expression of zonula occludens-1 (4.8-fold) and myosin light-chain kinase (3.1-fold) in intestinal epithelial cells isolated from mice of the heat-killed OLL2838 group. Therefore, L. rhamnosus OLL2838 would be useful in the treatment of gastrointestinal diseases such as inflammatory bowel disease.     

金针菇蛋白聚糖对脂多糖诱导的Caco-2/RAW264.7细胞共培养模型炎症的抑制作用

随着生活节奏的加快和生活方式的改变,炎症性肠病的发病率不断增加,从饮食中寻找抑制炎症成分是防控炎性肠病的有效途径。本实验以金针菇蛋白聚糖（Flarnmulina velutipes proteoglycans,FPG）1-1为原料,建立脂多糖（lipopolysaccharide,LPS）诱导的人结肠腺癌细胞（Caco-2）、巨噬细胞（RAW264.7）共培养炎症模型,测定FPG1-1对一氧化氮（nitrite oxide,NO）、活性氧（reactive oxygen species,ROS）及细胞因子的影响,以分析其对炎症的作用。结果显示：与模型组相比,质量浓度为200 μg/mL FPG1-1可以极显著降低RAW264.7 NO、ROS的生成（P＜0.01）,同时可以上调白细胞介素（interleukin,IL）-10、下调肿瘤坏死因子（tumor necrosis factor,TNF-α）、IL-1β、IL-6的分泌。以上结果表明,FPG1-1能够有效抑制LPS诱导的Caco-2/RAW264.7共培养模型炎症反应。本研究结果可为金针菇功能成分利用和抑制炎性肠病功能食品开发提供理论依据。     

纳豆激酶基因工程菌的构建及酶活力分析

纳豆激酶由纳豆芽孢杆菌(Bacillus subtilis natto)的aprN基因编码,在体内外具有很强的溶解纤维蛋白活性。利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增B.subtilis natto的aprN基因,并依据B.subtilis的密码子偏好性优化了起始30个氨基酸的密码子,构建了重组表达质粒pHT01-aprN。经限制性酶酶切、PCR扩增和测序验证了其编码的正确性。通过电击法将含有强启动子的pHT01-aprN导入B.subtilis,利用氯霉素抗性筛选获得B.s 168/pHT01-aprN工程菌。经IPTG诱导表达,摇瓶发酵培养最高酶活力为(289.00±3.42)U/mL,是野生菌的3.9倍,酶活力表达稳定性良好。     

Immunomodulatory consequences of oral administration of Lactobacillus rhamnosus strain GG in healthy volunteers

Probiotic microorganisms, especially lactic acid bacteria, are effective in the treatment of infectious diarrhoeal diseases and experimental colitis. Although the mechanisms by which these organisms exert their anti-inflammatory effects are largely unknown, immunomodulating effects are suggested. The objective of this study was to examine the effect of a 5-week oral administration of Lactobacillus rhamnosus subspecies GG (Lb. GG) on the cellular immune response to intestinal microorganisms in ten healthy volunteers. Peripheral blood cells (PB) were stimulated with either 'self' or 'non-self' preparations of faecal samples and isolated Bacteroides fragilis group-organisms (Bfg) or Escherichia coli (Esch. coli), and pro- and anti-inflammatory cytokines (IL-10, IL-4, IL-6, IFN-gamma, TNF-alpha) were measured in the culture supernatant. CD4+ T-lymphocyte activation was determined by measurement of intracellular ATP following lysis of the cells. The activational response of CD4+ T-lymphocytes towards isolated and heat-inactivated intestinal organisms was increased after the probiotic treatment. Additionally, TNF-alpha, IL-6 and in part IFN-gamma cytokine secretion by PB cells following stimulation with whole stool preparations and single members of the flora was significantly decreased, whereas the IL-10 and in part IL-4 cytokine secretion was increased at the end of the study. In contrast, the activational response of CD4+ T-lymphocytes following stimulation with whole 'non-self' intestinal flora was higher than by 'self' intestinal flora, but both responses showed a trend towards a reduction at the end of the study. This study documents a direct effect by Lb. GG on the cellular immune system of healthy volunteers and offers a promising tool to investigate systemic immunomodulation due to oral administration of probiotic microorganisms.     

Growth characteristics of Bacillus subtilis (natto) in milk

In this paper, Bacillus subtilis (natto) was incubated to develop a possible functional ingredient in ice cream. A lab‐scale culture revealed that incubation in the sterilised milk without dilution and concentration at 37°C for 28 h could obtain ideal growth characteristics of Bacillus subtilis (natto), especially with continuous aeration. Following freezing operation of the cultured milk, survival content of Bacillus subtilis (natto) was at 49–92%, while nattokinase activity was conserved at 62–98% comparing with the initial contents, which indicating a potential for application of natto functional ingredient in frozen milk products.     

Effects of kefir supernatant and lactic acid bacteria isolated from kefir grain on cytokine production by macrophage

The objective of this study was to investigate the in vitro immuno-modulating capacity and mechanisms of lactic acid bacteria (LAB) isolated from kefir grains and their individual supernatants by cytokine profiles through a toll-like receptor pathway. Results demonstrated that kefir supernatants, obtained from kefir fermented more than 24 h, induced the production of pro-inflammatory cytokines in RAW 264.7 cells. Among four LAB isolated from kefir grains and their supernatants, Lactobacillus kefiranofaciens M1 and its supernatant had strong potential to induce in vitro production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12 in RAW 264.7 cells and murine peritoneal macrophages. Moreover, blocking toll-like receptor (TLR)-2 using anti-TLR-2 mAb and TLR-2^?/? mice showed a significant inhibition (p < 0.05) of IL-6 and TNF-α production. These findings indicated that kefir influenced the secretion of pro-inflammatory cytokines TNF-α and IL-6 through TLR-2, which would potentially have beneficial effects on promotion of cell-mediated immune responses against tumors and also against intracellular pathogenic infections. The putative immunomodulin in the kefir supernatant was also characterized and may be a protein with a molecular mass larger than 30 kDa.     

扁枝槲寄生提取物对金黄色葡萄球菌抑菌机理的研究

本论文主要研究了95%乙醇扁枝槲寄生提取物的抑菌效应,并研究了抑菌机理。研究发现,95%乙醇扁枝槲寄生提取物对大肠杆菌、金黄色葡萄球菌、李斯特杆菌、铜绿假单胞菌、鼠伤寒沙门氏菌及变异微球菌均有明显抑菌效果,对枯草芽孢杆菌和纳豆芽孢杆菌无明显作用,对金黄色葡萄球菌抑菌效果最好(最低抑菌量为0.1 mg/m L)。进一步研究发现,提取物可增加金黄色葡萄球菌的细胞壁及细胞膜的通透性,引起细胞内含物如可溶性糖类、蛋白质等外泄,导致电导率上升。光学显微镜、扫描电镜以及透射电镜观察证实了提取物不仅能作用于菌体的壁膜系统,破坏菌体细胞的正常结构,还能通过抑制细胞分裂等方式来杀灭菌体。本文研究结果表明扁枝槲寄生具有开发成天然食品添加剂的良好前景。     

Anti-inflammatory effect and modulation of cytochrome P450 activities by Artemisia annua tea infusions in human intestinal Caco-2 cells

In an attempt to understand the beneficial health effects of Artemisia annua other than its anti-malaria properties, extracts from different cultivars prepared as tea infusions were investigated using Caco-2 cells on the intestinal inflammation and cytochrome P450 (CYP) activities. The characterisation of their phenolic compound (PC) profile revealed rosmarinic and chlorogenic acids as the main PCs. The extracts, assayed on Caco-2 cells at a plausible intestinal concentration, significantly decreased the secretion of pro-inflammatory cytokines, IL-8 and IL-6. This effect could be attributable at least to their content in rosmarinic acid, detected as a potent anti-inflammatory compound. The extracts also inhibited the activity of CYP3A4, whose expression was induced by 1,25-dihydroxyvitamin D₃, and of CYP1A1, induced by benzo(a)pyrene. Our results highlight the advantage of drinking A. annua infusions for their potent anti-inflammatory effect, linked to PC content, which could synergise their antimalarial activity.     

纳豆激酶基因的克隆及其在大肠杆菌和枯草芽孢杆菌中的表达

以纳豆芽孢杆菌HL-1全基因组DNA为模板,PCR分别扩增编码信号肽、前导肽及成熟肽的前纳豆激酶原基因序列(pre-pro-NK),编码前导肽、成熟肽的纳豆激酶原基因序列(pro-NK)和只编码成熟肽的纳豆激酶基因序列(NK),构建了大肠杆菌表达质粒pET28a-NK表达前纳豆激酶原基因及大肠杆菌-枯草杆菌穿梭质粒pHT315-NK表达纳豆激酶原基因和纳豆激酶基因,实现了纳豆激酶基因在大肠杆菌及枯草芽孢杆菌中的表达,并进行了活性分析。