瑞士乳杆菌对小鼠肠道粘膜免疫应答及细胞因子的影响

探讨灌胃瑞士乳杆菌(Lactobacillus helveticus TS206)发酵制品对健康Balb/c 小鼠肠道黏膜免疫应答及细胞因子的影响。本实验将Balb/c 小鼠分为空白对照组、生理盐水组和瑞士乳杆菌组3 组,瑞士乳杆菌组的灌胃剂量为108CFU/mL,分别在灌胃后0 、1 、2、3 、4 、6 、8、12、24h 处死小鼠得到小肠组织样品,利用RIA 法和ELISA 法测得各组小鼠肠道黏膜SIgA 和细胞因子的含量,并作统计分析。结论：灌胃组小鼠肠道黏膜中SIgA 的含量与生理盐水对照组相比差异极显著(P ＜ 0.01);灌胃组小鼠肠道黏膜中IL-2 和IFN-γ的含量与生理盐水对照组相比具有显著性差异(P ＜ 0.05);灌胃组IL-4 与生理盐水对照组相比含量明显升高(P ＜ 0.05);而灌胃组和生理盐水对照组相比IL-6 的表达水平后期差异不显著(P ＞ 0.05);IFN-γ/IL-4 的值处于相互拮抗的状况,表明瑞士乳杆菌对健康小鼠具有维护肠黏膜免疫平衡的作用,也对保持Th1/Th2 平衡状态具有明显的作用,即证实瑞士乳杆菌作为益生菌具有调控小鼠肠道黏膜免疫应答的功效。     

瑞士乳杆菌对小鼠肠道黏膜和肠组织中细胞因子影响的研究

了解服用益生菌小鼠肠道黏膜和组织中细胞因子的变化情况,对阐明其对小鼠肠道黏膜免疫的影响具有重要的价值。本实验将60 只小鼠随机分成4 组：日食对照组、生理盐水对照组、瑞士乳杆菌灌喂组和大肠杆菌对照组,连续灌喂15d,采集小鼠小肠利用ELISA 法测得各组小鼠肠道黏膜和肠道组织中细胞因子的含量,并作分析。研究结果证实,与对照组相比,瑞士乳杆菌灌胃组小鼠肠黏膜和组织中4 种细胞因子明显升高,且存在显著性差异(P ＜ 0.05)：白介素-2IL-2 在3d 前达到最大值、白介素-4(IL-4)在5d 达到最高点、白介素-6(IL-6)在5d 达到顶峰、干扰素- γ(IFN- γ)在3d 前达到最大值;与对照组相比,瑞士乳杆菌灌胃组小鼠Th1 型和Th2 型T 细胞的不存在显著性差异(P ＞ 0.05),肠道免疫功能未出现异常,而大肠杆菌灌胃组Th1 型和Th2 型T 细胞平衡被打乱,免疫功能出现异常。     

黑灵芝多糖对免疫抑制小鼠肠道黏膜形态及肠道黏膜免疫的影响

目的：探究黑灵芝多糖对免疫抑制小鼠肠道黏膜形态及肠道黏膜免疫的影响。方法：腹腔注射环磷酰胺构建免疫抑制小鼠模型,将小鼠分为6 组：正常对照组、模型组、阳性对照组以及黑灵芝多糖低、中、高剂量（25、50、100 mg/（kg·d））组,灌胃期间每天记录小鼠体质量并观察其生活状态,空肠组织进行切片病理学观察,酶联免疫吸附实验测定肠道白细胞介素（interlrukin,IL）-6、IL-10、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）及干扰素-γ（interferon-γ,IFN-γ）的含量,反转录定量聚合酶链式反应分析T-bet、GATA-3 mRNA相对表达水平。结果：与模型组相比,低、中、高剂量黑灵芝多糖有助于体质量回升,改善肠道黏膜形态结构,提高IL-6、IL-10、TNF-α、IFN-γ细胞因子含量,增强T-bet、GATA-3 mRNA相对表达水平,使Th1/Th2平衡向Th1偏移。结论：黑灵芝多糖可改善免疫抑制小鼠的肠道黏膜形态,调节免疫抑制小鼠的肠道黏膜免疫。     

益生菌复合制剂对头孢曲松钠作用小鼠的抗氧化指标、细胞因子及肠道菌群的影响

目的:探究长双歧杆菌BB536和乳双歧杆菌HN019组成的益生菌复合制剂对头孢曲松钠引起的肠道菌群失调的改善效果。方法:头孢曲松钠（2 mg/g）连续灌胃小鼠5 d,构建肠道菌群失调模型小鼠,然后随机分为模型组,益生菌复合制剂低剂量组（2×105 CFU/g）、中剂量组（4×105 CFU/g）、高剂量组（1.2×106 CFU/g）,另设正常鼠为对照组,第6 d起各剂量组灌胃相应剂量的益生菌复合制剂,对照组和模型组灌胃等体积的生理盐水,连续灌胃30 d。灌胃结束后无菌收集小鼠粪便对肠道菌群计数,并进行16S rDNA高通量测序分析菌群的多样性以及结构,测定血清中白细胞介素2（Interleukin-2,IL-2）、白细胞介素6（Interleukin-6,IL-6）、白细胞介素1β（Interleukin-1β,IL-1β）和肿瘤坏死因子-α（tumor necrosis factor,TNF-α）水平,测定空肠和肝脏中丙二醛（Malondialdehyde,MDA）、总超氧化物歧化酶（Total Superoxide Dismutase,T-SOD）、谷胱甘肽（glutathione,GSH）和谷胱甘肽过氧化物酶（Glutathione peroxidase,GSH-PX）水平。结果:给药头孢曲松钠后,血清中IL-2、IL-6、IL-1β和TNF-α水平有上升趋势,空肠中MDA水平显著上升（P<0.05）且T-SOD水平显著下降（P<0.05）,在高剂量的益生菌复合制剂的干预下,IL-6和IL-1β水平显著降低（P<0.05）,IL-2和TNF-α水平极显著降低（P<0.01）,此外空肠和肝脏中的MDA水平均明显下降、T-SOD水平显著提高（P<0.05）,GSH-PX水平极显著提高（P<0.01）,空肠中GSH水平极显著提高（P<0.01）。在肠道微生物方面,抗生素造损后再灌胃益生菌复合制剂,与灌胃益生菌复合制剂前相比小鼠粪便中肠球菌和肠杆菌数量降低,乳杆菌和双歧杆菌数量提高,微生物多样性分析结果表明各剂量组与模型组相比微生物丰富度有所恢复,且中、高剂量组预测的肠道功能与对照组相比更为接近。结论:益生菌复合制剂可以促进抗氧化物质产生,降低细胞因子水平,促进有益菌的繁殖,提高肠道菌群的丰富度,改善了由头孢曲松钠引起的肠道菌群失调状态。     

合生元制剂对小鼠抗氧化能力和肠道菌群的影响

为研究合生元制剂对小鼠抗氧化能力、细胞因子和肠道菌群的影响,随机将48只BALB/c小鼠分为对照组、低剂量组（L）、中剂量组（M）和高剂量组（H）,并灌胃生理盐水和不同剂量合生元制剂,14 d后收集小鼠粪便进行菌落计数和16S rDNA高通量测序,测定小鼠肝脏、小肠组织中抗氧化指标,检测血清中细胞因子白介素-6（interleukin-6,IL-6）、白介素-1β（interleukin-1β,IL-1β）水平。结果表明：小肠和肝脏组织中,各个剂量组总超氧化物歧化酶（total superoxide dismutase,T-SOD）活性、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活性、谷胱甘肽（glutathione,GSH）含量上升,丙二醛（malondialdehyde,MDA）含量下降。H 组 IL-1β水平极显著降低（p＜0.05）。灌胃 14 d后,各剂量组与灌胃前相比,乳杆菌和双歧杆菌数量增加,16S rDNA测序表明给予合生元制剂可以增加Rikenella、Alistipes、Bifidobacterium和Lactobacillus等丰富度。灌胃上述合生元制剂可以提高小鼠抗氧化能力,降低细胞因子水平,促进乳杆菌和双歧杆菌繁殖,对肠道菌群具有调节作用。     

淡水鲈鱼小清蛋白致敏性及其对小鼠肠道菌群的影响

目的研究淡水鲈鱼小清蛋白(parvalbumin,PV)的致敏性,同时探索其对小鼠肠道菌群的影响。方法 36只C3H/HeJ小鼠按体重随机分为3组,分别为空白对照组(CK组)、卵清白蛋白(ovalbumin, OVA)致敏对照组(OVA组)和小清蛋白致敏组(PV组)。使用生理盐水、OVA溶液和PV溶液通过经口灌胃在6周时间内进行致敏,实验最后一天进行大剂量刺激,测定小鼠的体温变化、血清抗体水平和相关细胞因子水平,取腹腔灌洗液测定其中蛋白的含量以评价小鼠血管渗透性的变化,利用流式细胞术测定各组小鼠脾脏(SP)和肠系膜淋巴结(MLN)中T细胞亚群的水平。取小鼠粪便,对肠道菌群进行16S rRNA高通量测序,探索小清蛋白对小鼠肠道菌群的影响。结果与空白对照组相比,OVA小鼠血清抗体、细胞因子和免疫组织Th2相比水平均显著升高(P0.05),且表现出明显过敏症状。PV处理也使小鼠表现出明显的食物过敏症状,但仅显著升高了小鼠血清免疫球蛋白G1(Immunoglobulin G1, IgG1)和白细胞介素10(Interleukin 10, IL-10)的水平(P0.05)。16S rRNA高通量测序结果显示, OVA处理升高了小鼠肠道中属于拟杆菌门和变形菌门群落的水平,而PV处理升高了属于厚壁菌门菌群的丰度。结论淡水鲈鱼小清蛋白对小鼠具有一定的致敏性,但其致敏机理及对肠道菌群的影响作用与OVA不同。     

植物乳杆菌Sc52对脂多糖诱导小鼠肠道屏障损伤的保护作用

目的：研究植物乳杆菌（Lactobacillus plantarum）Sc52对脂多糖（lipopolysaccharide,LPS）诱导小 鼠肠道屏障损伤的保护作用。方法：选取6 周龄雄性ICR小鼠,随机分为3 组,实验组每天灌胃植物乳杆菌 Sc52（1010 CFU/d）,空白组与模型组灌胃等量生理盐水,连续两周后,除空白组外,一次性腹腔注射LPS（15 mg/kg） 建立小鼠肠屏障损伤模型。检测小鼠血清中炎症因子及与肠道通透性相关指标水平,苏木精-伊红染色观察回肠组 织病理学变化,气相色谱法测定小鼠肠道短链脂肪酸水平,平板计数法分析小鼠粪便中主要菌群数量,蛋白质印记 法分析小鼠回肠上皮细胞间紧密连接蛋白表达水平。结果：预先灌胃植物乳杆菌Sc52能够降低小鼠血清中肿瘤坏 死因子、白细胞介素-6、白细胞介素-12、一氧化氮等炎症相关因子水平。植物乳杆菌Sc52通过调节D-乳酸和二胺 氧化酶的水平,改善肠道通透性。灌胃植物乳杆菌Sc52能够提高小鼠肠道中乙酸和丁酸含量,能够增加小鼠肠道 中乳杆菌、双歧杆菌等有益微生物的数量,抑制有害微生物的生长;显著上调回肠组织中紧密连接蛋白Occludin和 Claudin-1的表达。结论：植物乳杆菌Sc52对小鼠肠道屏障功能具有保护作用。     

植物乳杆菌调节抗体和细胞因子水平缓解食物过敏的机制

食物过敏危害人体健康,利用益生菌特有的抗过敏作用来缓解食物过敏是治疗食物过敏的新方向,然而目前关于益生菌缓解过敏的机制研究还不够深入。本文以前期筛选出的具有抗过敏功能的4株植物乳杆菌为研究对象,利用Balb/c小鼠建立食物过敏模型,采用酶联免疫吸附实验(ELISA)、荧光定量PCR(RT-PCR)等方法研究植物乳杆菌对Th1/Th2相关细胞因子、抗体分泌的影响。ELISA结果表明,将4株植物乳杆菌与致敏小鼠脾细胞共培养可降低IL-4的分泌水平,增加IFN-γ的分泌水平,而荧光定量结果证实IL-4及其转录因子GATA-3 m RNA表达水平显著降低,IFN-γ及其转录因子T-bet mRNA表达水平显著升高,这一结果与细胞因子分泌水平结果一致。此外,4株植物乳杆菌均可下调致敏小鼠血清中IgE分泌水平,缓解由食物过敏造成的肠道黏膜及肺泡组织损伤。这些结果说明植物乳杆菌通过调节Th1/Th2平衡来缓解食物过敏。     

免疫低下和食物过敏小鼠外周血中细胞因子的变化

建立小鼠免疫低下模型和食物过敏模型,采用ELISA法检测外周血中细胞因子(IL-4、IL-6、IL-10、IL-12、IFN-γ、TGF-β)的变化。结果表明：与空白对照组比较,腹腔注射环磷酰胺所建立免疫低下小鼠胸腺指数和脾脏指数均有显著降低(P≤0.05),且在第3天达到最低;经卵清蛋白致敏小鼠外周血IgE水平极显著升高(P≤0.01)。免疫低下小鼠外周血中IFN-γ(除第3天外)、TGF-β含量(除第3天外)和IFN-γ/IL-4比值均显著降低,而IL-10水平也有下降趋势;除第2天外IL-6水平显著升高,但IL-4水平只在第3天显著性增高,IL-12先是下降随后呈现增多的趋势;食物过敏小鼠的IL-4、IL-6、IL-10和TGF-β水平均有显著性增高,IFN-γ/IL-4比值有显著性下降,而IFN-γ和IL-12没有明显的变化。因此,免疫低下和食物过敏小鼠的Th1/Th2细胞平衡均向Th2细胞偏移,免疫低下对Th1细胞的影响大,食物过敏对Th2细胞的影响更明显。     

Oral tolerance induction does not resolve gastrointestinal inflammation in a mouse model of food allergy

Scope : Oral immunotherapy (OIT) involving continuous oral administration of allergenic foods has gained attention as a therapy for food allergies. To study the influence of oral administration of allergenic foods on gastrointestinal symptoms including inflammation, we established a mouse model of food‐induced intestinal allergy. Methods and results : BALB/c mice were fed an egg white (EW) diet containing ovalbumin (OVA, a major EW allergen) after intraperitoneal sensitisation with OVA and Alum. The mice on the EW diet for one wk presented gastrointestinal symptoms (i.e. weight loss and soft stools) and inflammation in the small intestines (i.e. duodenum, jejunum and ileum). Further continuous EW diet resolved the weight loss but not the soft stools. Splenic CD4⁺ T‐cells of EW diet‐fed mice on the continuous diet showed less proliferation and cytokine production compared with those of control mice, suggesting tolerance induction by the diet. The continuous EW diet reduced levels of OVA‐specific IgE antibodies, but significantly aggravated the inflammation in the jejunum. Conclusion : Our mouse model would be useful to investigate inflammatory and regulatory mechanisms in food‐induced intestinal allergies. Our results suggest potential gastrointestinal inflammation in patients undergoing OIT as continuous administration of allergenic foods, even though the therapy may induce clinical tolerance.     

Citrus Tachibana Leaf Extract Mitigates Symptoms of Food Allergy by Inhibiting Th2‐Associated Responses

Although the incidence of food allergy continues to rise, there have been no effective therapeutic strategies. Citrus fruits contain a number of bioactive flavonoids with immune‐regulatory functions. The objective of this study was to determine whether Citrus tachibana (fruit body with peel, leaves, and branch) can protect against the development of food allergy and the mechanism behind it, and to identify the active compound(s) responsible. We found that C. tachibana leaf extract (CLE) mitigated ovalbumin (OVA)‐induced food allergy symptoms including increased rectal temperature, diarrhea, and anaphylaxis. This mitigation was likely due to CLE‐mediated decreases in cytokine release from T‐helper 2 cells (Th2 cells) in mesenteric lymph nodes. Moreover, higher levels of CLE attenuated systemic Th2 cell–mediated responses in mouse splenocytes sensitized with OVA+Alum. This was evidenced by CLE‐mediated reductions in Th2 cytokine release, including interleukin (IL)‐4, IL‐5, and IL‐13, but not the Th1 cytokines IL‐12 and interferon (IFN)‐γ, which was attributable to decreased gene expression levels. We also identified kaempferol as the most potent compound for reducing Th2‐associated responses in splenocytes. The findings of this study suggest that CLE suppresses Th2‐cell–mediated immune responses, contributing to alleviation of food allergy symptoms, and that kaempferol is a flavonoid with potential antiallergenic activity that targets Th2 cell–induced responses.     

Effects of enzymatic hydrolysis on the allergenicity of natural cow milk based on a BALB/c mouse model

Cow milk allergy is one of the most prevalent food allergies worldwide, particularly in infants and children. To the best of our knowledge, minimal research exists concerning the antigenicity of cow milk (CM). This study was performed to evaluate the allergenicity of enzymatically hydrolyzed cow milk (HM) in a BALB/c mouse model. The mice were randomly divided into 5 groups (n = 12/group), which were sensitized with phosphate-buffered saline, CM, and HM (Alcalase-, or Protamex-, or Flavorzyme-treated cow milk; Novo Nordisk; AT, PT, FT, respectively), respectively, using cholera toxin as adjuvant on d 0, 7, 14, 21. On d 28, the test mice were orally challenged with phosphate-buffered saline, CM, and HM (AT, PT, or FT) alone. Anaphylactic symptoms were monitored in the mice. Antibody, cytokine, histamine, and mouse mast cell protease-1 (mMCP-1) levels were measured using enzyme-linked immunosorbent assays. In addition, the numbers of T helper (Th)1 and Th2 cells, as well as the proportions of CD4⁺CD25⁺Foxp3⁺ Treg cells, in mouse spleens were detected using flow cytometry. Statistical significance was determined by one-way ANOVA. The results revealed significant differences between CM- and HM-challenged mice. Among these, the clinical scores of HM-challenged mice (AT, 1.50; PT, 2.00; FT, 1.92) were lower than those of CM-challenged mice (positive control, 2.83), but body weight and temperature of HM-challenged mice were higher than those of CM-challenged mice. In addition, significant reductions of allergen-specific IgE, IgG, histamine, and mMCP-1 were showed in HM-challenged mice, especially for histamine, ranging from 171.42 ng/mL to 214.94 ng/mL. Remarkable reductions of IL-4, IL-5, and IL-13 levels, as well as elevations of interferon-γ and IL-10 levels in the spleens of HM-challenged mice were also detected. Moreover, the number of Th2 cells decreased in the HM-challenged mice, to 2.36% (AT), 1.79% (PT), and 4.03% (FT), respectively, whereas the numbers of Th1 cells (AT, 6.30%; PT, 6.70%; FT, 6.56%) and the proportions of CD4⁺CD25⁺Foxp3⁺Tregs (AT, 8.86%; PT, 9.21%; FT, 9.16%) increased significantly. Our findings indicate that exposure to HM was sufficient to induce a shift toward a Th1 response, thereby reducing potential allergenicity. Importantly, these results will lay a theoretical foundation for the development of hypoallergenic CM products.     

Hydrolysate of β-lactoglobulin by Lactobacillus delbrueckii subsp. bulgaricus CRL 656 suppresses the immunoreactivity of β-lactoglobulin as revealed by in vivo assays

Cows' milk allergy, common in young infants, is often solved by the age of five; however, it can persist in some adults. The in vivo immunomodulatory potential of β-lactoglobulin (BLG) hydrolysates obtained with Lactobacillus delbrueckii subsp. bulgaricus CRL656 (H656) was studied. Sera from mice gavaged with H656 showed lower specific BLG-IgE values than those of allergic mice. Secretion of IL-10, INF-γ and IL-6 was increased and that of IL-4 decreased when allergic spleen cells were stimulated with H656. IL-4 secretion was significantly reduced and concentrations of IL-10, IL-17A and IL-6 were increased when H656 was orally administrated. Highest expression of IL-4 and IL-12 was observed for allergic mice and H656 gavaged mice, respectively. H656 immunisation had a positive effect on intestinal mucosa. Overall, H656 displayed an immunomodulating effect, balancing the allergic Th2 response by stimulating Th1 type cytokine secretion. The hydrolysate might promote oral tolerance towards BLG in infant foods.     

Anti-inflammatory activity of American yam Dioscorea trifida L.f. in food allergy induced by ovalbumin in mice

Tropical ecosystems are particularly rich in edible plant species with different bioactive substances. Among the plants with promising benefits for health are species from the genus Dioscorea (Dioscoreaceae), especially those named yam. Recent studies have shown the beneficial effects of different species of Dioscorea, and its main constituent, diosgenine, in the treatment of food allergy. In this study we evaluated the potential of D. trifida, the only yam native from South America, in the treatment of ovalbumin (OVA) induced food allergy in Balb/c mice. HPLC/DAD analysis showed the presence of three very distinctive groups of natural products in extracts and fractions: (I) very polar substances, including allantoin, (II) phenolic substances as flavonoids and phenolic acids and (III) diosgenin and derivatives. Sensitive mice received casein feed with supplementation of crude extract (CE) and fractions. The supplementation with all products from D. trifida reduced IGE, intestinal oedema and mucus production, parameters observed in OVA allergic mice. The results showed the potential of this food to prevent or treat this disease and the necessity to be better explored.     

虾制品致敏性诱发豚鼠相关细胞因子及Th1/Th2细胞平衡的变化

以消减致敏性南美白对虾的虾仁、虾肉和虾蛋白为样品,以pH 7.5的磷酸盐缓冲液为阴性对照,未处理的虾蛋白为阳性对照,建立豚鼠过敏模型,研究过敏豚鼠血清中相关细胞因子与食物过敏的相关性,并推断食物过敏对辅助性T（type 1/type 2 T-helper, Th1/Th2）细胞平衡的影响。收集过敏豚鼠的血清,采用酶联免疫吸附测定法检测血清中免疫球蛋白E（immunoglobulin E,IgE）、组胺（histamine,HIS）、相关细胞因子（白细胞介素（interleukin,IL）-1、IL-2、IL-3、IL-4、IL-6、IL-10、肿瘤坏死因子-α（tumor necrosis factor α,TNF-α）、干扰素-γ（interferon-γ,IFN-γ）质量浓度。结果表明,用未处理的虾蛋白致敏豚鼠,用致敏性消减程度不同的虾制品（虾蛋白、虾肉、虾仁）提取的蛋白激发,消减致敏性的虾蛋白、虾肉和虾仁提取蛋白激发后豚鼠血清中IgE含量分别为（3.905±0.120）、（4.813±0.188）、（5.199±0.327）U/mL,HIS质量浓度分别为（16.437±1.120）、（19.656±1.080）、（21.071±1.732）μg/mL,激发后血清中IgE和HIS质量浓度变化与致敏性程度呈正相关,致敏性越低,血清中IgE和HIS质量浓度越低。过敏豚鼠血清中IL-1、IL-2、IL-3、IL-4、IL-6、TNF-α质量浓度的变化与虾制品致敏程度呈正相关;同时,与IgE和HIS质量浓度变化规律具有一致性;血清中IL-10质量浓度变化与致敏性程度呈负相关;因此,这些细胞因子与食物过敏具有很好的相关性。过敏血清中IFN-γ没有呈现规律性变化,但IFN-γ/IL-4随着致敏性的增强而减小。因此,推测虾制品激发过敏豚鼠的Th1/Th2细胞平衡向Th2细胞偏移。