CN113509495B - 鼠李糖乳杆菌在缓解肠道炎症中的应用 - Google Patents
鼠李糖乳杆菌在缓解肠道炎症中的应用 Download PDFInfo
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Abstract
本发明提供了一种鼠李糖乳杆菌在缓解肠道炎症中的应用，属于益生菌功能领域。为了解决目前缓解肠道炎症类药物具有较严重的副作用的问题，本发明提供一种鼠李糖乳杆菌JL1可以缓解肠道炎症功能，对本发明鼠李糖乳杆菌JL1缓解肠道炎症功能，通过抑制LPS诱导的Caco‑2细胞中的促炎基因TNF‑α、IL‑1β和IL‑6的表达和小鼠临床状态以及回肠剖检观察，验证肠道炎症的减轻。
Description
技术领域
本发明属于益生菌功能领域，具体涉及鼠李糖乳杆菌在缓解肠道炎症中的应用。
背景技术
目前，在缓解肠道炎症和肠黏膜损伤方面，虽可以通过常规的药物治疗从而减少促炎因子的分泌来进而降低炎症和损伤，但是长期服用药物会给机体带来很难逆转的副作用。因此，寻找安全无副作用的治疗或缓解肠道炎症的药物，已成为当前各国学者研究的热点。近年来，由于乳酸菌的天然无副作用、成本低廉等优点，在食品和医疗等领域得到了广泛的应用。许多研究表明，乳酸菌对肠道炎症和肠黏膜损伤的治疗具有积极作用。
肠道是机体的第一道防线，不仅负责营养物质的选择性吸收，还需负责抵御炎症因子及肠道内有害微生物入侵。当机体处于不良状态或突然接触到大量致病菌和毒力因子时，会引起肠道炎症和肠黏膜损伤，进而导致全身炎症反应综合征及多器官功能衰竭，严重时可危及生命。而常用的药物治疗不仅价格昂贵还具有副作用，严重影响患者的生活质量，近年来，乳酸菌由于其安全、易获得、成本低廉和无副作用等特点，逐渐成为治疗肠道疾病的研究热点。目前一些体内和体外试验已经证明乳酸菌可以缓解肠道炎症。
发明内容
本发明的目的是为了解决目前缓解肠道炎症类药物具有较严重的副作用的问题。
本发明提供了一种鼠李糖乳杆菌在制备治疗肠道炎症的药物中的应用。
进一步地限定，所述肠道炎症的症状包括减少结肠中炎症因子的含量。
进一步地限定，所述炎症因子为TNF-α、IL-1β和IL-6。
本发明还提供了一种含有鼠李糖乳杆菌的微生物菌剂，所述微生物菌剂含有鼠李糖乳杆菌JL1含量为108-106cfu/mL。
进一步地限定，所述微生物菌剂的剂型为片剂、胶囊剂、颗粒剂、散剂、液体制剂中的任意一种。
有益效果：本发明提供了鼠李糖乳杆菌JL1可以降低炎症因子TNF-α、IL-1β和IL-6含量，达到缓解肠道验证的作用。
附图说明
图 1是鼠李糖乳杆菌JL1对LPS诱导的Caco-2细胞促炎基因TNF-α表达的影响，横坐标是分组，纵坐标是TNF-α表达情况。
图 2是鼠李糖乳杆菌JL1对LPS诱导的Caco-2细胞促炎基因IL-1β表达的影响，横坐标是分组，纵坐标是IL-1β表达情况。
图 3是鼠李糖乳杆菌JL1对LPS诱导的Caco-2细胞促炎基因IL-6表达的影响横坐标是分组，纵坐标是IL-6表达情况。
图4是小鼠的精神状态。
图5是小鼠的小肠的状态。
具体实施例
鼠李糖乳杆菌JL1记载在Protective effects of a novel Lactobacillusrhamnosus strain with probiotic characteristics againstlipopolysaccharide-induced intestinal inflammation in vitro and in vivo[J]. Food&Function。
实施例1.
1.培养鼠李糖乳杆菌JL1
（1）活化两代至对数生长期的L. rhamnosusJL1经离心后获得菌泥，采用PBS缓冲液洗涤3次后，重悬于不含双抗的DMEM培养液中，调至菌落数为108、107和106cfu/mL备用。将达到极化状态的Caco-2细胞接种于六孔板中，培养液选取不含双抗的DMEM培养液，在37℃、含5% CO2的厌氧培养箱中培养，隔天换液，直至细胞达到极化状态。分别取1mL浓度为108、107和106cfu/mL菌悬液加入上述的106个Caco-2细胞，于5% CO2培养箱中共培养4 h，后弃去培养液，使用LPS（脂多糖）浓度为1μg/m L的DMEM培养液刺激细胞(105-106个Caco-2细胞) 4 h，4 h后收集细胞进行RNA提取；
（2）按照Simply P总RNA提取试剂盒说明书提取Caco-2细胞的总RNA，注意全程避光操作，超净台采用氯仿擦拭，所用器具都要采用DEPC水处理，提取后的总RNA采用RNase-free离心管收集并在-80 ℃条件下保存；
（3）以步骤（2）提取的RNA为模板，按照反转录试剂盒说明书对经检验合格的RNA样品进行反转录获得cDNA，反应体系见表1。在37 ℃条件下反转录20 min，在98 ℃条件下进行5 min的酶失活反应。将反应产物在-20 ℃条件下保存。
反应体系表如下：
（4）选取TNF-α、IL-1β和IL-6等3种促炎基因用于实时荧光定量PCR研究，采用GAPDH作为内参基因。将为未加L. rhamnosusJL1预处理也未用LPS刺激的Caco-2细胞的cDNA作为阴性对照组，未加L. rhamnosusJL1预处理但使用LPS刺激的Caco-2细胞的cDNA作为阳性对照组。将为未加L. rhamnosus JL-1预处理也未用LPS刺激的Caco-2细胞的cDNA作为阴性对照组(CG)，未加L. rhamnosus JL-1预处理但使用LPS刺激的Caco-2细胞的cDNA作为阳性对照组(LPS)。L、M和H组小鼠以10 mL/kg的剂量分别灌胃107、108和109cfu/mL的L.rhamnosus JL-1菌悬液
调整获得cDNA的浓度至合适浓度，按照ExScriptTM RT-PCR试剂盒说明书配制反应体系，反应体系见表2。实时荧光定量PCR反应参数为：预变性95 ℃，30 s；变性95 ℃，5s，延伸60 ℃，30 s，共40个循环。利用ABI QuantStudio 3实时荧光定量PCR仪进行扩增。试验采用 2-△△Ct法对目的基因的相对表达量进行计算。
如图1所示，与CG组相比，LPS刺激Caco-2细胞4 h可以显著提高TNF-α mRNA的表达水平（p<0.001），TNF-α的mRNA表达量提高了19.26倍。与LPS组比较，L. rhamnosusJL-1浓度为106cfu/mL的低剂量组对TNF-α mRNA的表达没有明显的作用，但中、高剂量组（107、108cfu/mL）能极显著抑制LPS诱导的Caco-2细胞的TNF-α mRNA的表达量（p<0.01）。
如图2所示，与CG组相比，LPS刺激Caco-2细胞4 h可以显著提高IL-1β mRNA的表达水平（p<0.001）。与LPS组相比，L. rhamnosusJL-1浓度为106cfu/mL的低剂量组可以显著抑制LPS诱导的Caco-2细胞的IL-1β mRNA的表达量（p<0.05），中、高剂量组（107、108cfu/mL）能极显著抑制LPS诱导的Caco-2细胞的TNF-α mRNA 的表达量（p<0.01），并且呈现出剂量依赖性。此外，L. rhamnosusJL-1浓度在108cfu/mL的高剂量组的IL-1β mRNA的表达量和CG组无显著差异（p>0.05）。
如图3所示，相比于CG组，LPS刺激Caco-2细胞4 h可以显著提高IL-6 mRNA的表达水平（p<0.001）。与LPS组比较，低、中、高剂量组（106、107、108cfu/mL）均能显著抑制LPS诱导的Caco-2细胞的IL-6 mRNA 的表达量（p<0.01），并且呈现出剂量依赖性。
选用60只鼠龄为8周的雄性 BALB/c小鼠，小鼠体重为20±2g，将小鼠随机分为5组：正常组（CG）、脂多糖处理组（LPS）、低剂量组（L）、中剂量组（M）和高剂量组（H）。鼠房温度为22±2 ℃，湿度为55±5%，12小时昼夜交替，小鼠自由采食饮水和饲料。小鼠购买后，采用基础饲料饲养适应环境一周。从第二周开始，每天上午9:00，CG和LPS组小鼠以10 mL/kg的剂量灌胃生理盐水，L、M和H组小鼠以10 mL/kg的剂量分别灌胃107、108和109 cfu/mL的L.rhamnosus JL-1菌悬液，连续灌胃两周后，按10mg/kg的剂量腹腔注射LPS诱导炎症反应，正常组腹腔注射等量生理盐水，实验全程跟踪观察小鼠状态。采用酒精和DEPC水溶液处理各种杀鼠用具，麻醉后脱颈法处死小鼠，采用75%酒精对小鼠体表进行消毒后解剖小鼠，肉眼观察小肠状态并拍照记录。
由图4可见，A：CG组，B：LPS组，C：L 组，D：M 组。E：H组，CG组小鼠精神状态很好，眼部没有白色分泌物，肛周没有排泄物。与CG组相比，接受LPS腹腔注射的各组小鼠均有发抖，活动量减少，不思饮食，精神萎靡的表现。此外，LPS组小鼠眼部有较多白色黏性分泌物，肛周被毛湿润，排便不成形且有黄色排泄物，L组和M组小鼠眼部有部分白色黏性分泌物，小鼠尾部沾有不成形粪便。相比于LPS组，H组小鼠小鼠眼部仅有少量白色黏性分泌物，发抖、腹泻症状有所减轻。
如图5所示，A：CG组，B：LPS组，C：L 组，D：M 组。E：H组，CG组小肠组织无显著变化，肠道有少量内容物。LPS 组小肠组织出现明显的肠黏膜水肿，肠壁变薄的现象，肠道内容物为黄色黏性物质且有较多出血点。L组和M组小肠表面有少量出血点，肠道黄色黏液性物质相比于LPS组减少，肠黏膜水肿程度减轻。H组小肠水肠道中有少量黄色内容物，肠黏膜水肿程度最轻。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可以做各种改动和修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (2)
1.鼠李糖乳杆菌（Lactobacillus rhamnosus）在制备治疗肠道炎症的药物中的应用，其特征在于，所述药物为含有鼠李糖乳杆菌的微生物菌剂，所述微生物菌剂含有鼠李糖乳杆菌JL1含量为106-108cfu/mL。
2.根据权利要求1所述的应用，其特征在于，所述微生物菌剂的剂型为片剂、胶囊剂、颗粒剂、散剂、液体制剂中的任意一种。
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