CN112111430B - 一种抗氧化与抗衰老双效益生菌及其应用 - Google Patents
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Abstract
本发明公开了一种抗氧化与抗衰老双效益生菌，Lactobacillusplantarum H8，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.20168。该菌能够有效降低体内氧化应激标志物含量，调节抗氧化相关因子基因表达，治疗、改善或预防氧化引起的组织病变，预防或改善氧化引起的衰老。
Description
技术领域
本发明涉及微生物技术领域，更具体的说是涉及一种抗氧化与抗衰老双效益生菌及其应用。
背景技术
氧化应激与损伤能够导致机体系统性和代谢性疾病发生，例如阿尔茨海默病、帕金森病、肌萎缩性脊髓侧索硬化症、哮喘、过敏、慢性炎症、糖尿病等。
在氧化应激状态下，细胞中的促氧化物与抗氧化的平衡受到干扰，导致DNA羟化、蛋白质变性、脂质过氧化和细胞凋亡，最终损害细胞的生存能力。人体的抗氧化系统是一个可与免疫系统相比拟的、具有完善和复杂功能的系统，机体抗氧化的能力越强，就越健康，生命也越长。
因此，如何提高机体抗氧化能力，改善氧化应激引起的并发症或预防并发症的发生成为本领域亟待解决的技术问题。
发明内容
有鉴于此，本发明提供了一种抗氧化与抗衰老双效益生菌，可有效提高机体抗氧化能力，改善氧化应激引起的并发症或预防并发症的发生。
为了实现上述目的，本发明采用如下技术方案：
一种抗氧化与抗衰老双效益生菌，植物乳杆菌Lactobacillusplantarum H8，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC No.20168，保藏时间为2020年06月30日。
上述抗氧化与抗衰老双效益生菌在制备抗氧化产品中的应用，所述产品为食品或药品。
上述抗氧化与抗衰老双效益生菌在制备抗衰老产品中的应用，所述产品为食品或药品。
上述抗氧化与抗衰老双效益生菌在制备预防、改善或治疗氧化引起的组织损伤药物中的应用，所述组织包括肝组织、脾组织、脑组织或结肠组织。
一种抗氧化产品，包括上述抗氧化与抗衰老双效益生菌；所述产品为食品或药品。
一种抗衰老产品，包括上述抗氧化与抗衰老双效益生菌；所述产品为食品或药品。
一种预防、改善或治疗氧化引起的组织损伤的药物，包括上述抗氧化与抗衰老双效益生菌。
由上述技术方案可知，本发明公开的植物乳杆菌可有效降低体内氧化应激标志物含量，调节抗氧化相关因子基因表达，治疗、改善或预防氧化引起的组织病变，预防或改善氧化引起的衰老。
附图说明
图1所示为饲喂8周后各组小鼠肝、脾、脑、结肠切片H&E染色(200×)；
图2、3所示为各组小鼠肝脏中相关基因表达情况；
图4所示为各组小鼠肝脏中相关蛋白表达情况。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1植物乳杆菌Lactobacillusplantarum H8的分离及鉴定
于黑龙江地区从粘面子中分离得到H8菌株，活化2次后按2％接种量接种到MRS培养基中，37℃厌氧培养24h，用于后续实验。
经16S rRNA鉴定，H8菌株为植物乳杆菌。
实施例2动物饲养实验
32只雌雄各半的昆明小鼠(6周龄，20±2克)SPF级购自沈阳长生生物有限公司，涉及小鼠的程序严格按照国际动物护理标准进行，并经吉林农业大学动物保护机构和使用委员会批准。
在温度25℃，相对湿度60％，12h昼/夜循环照明条件下，自由进食、饮水适应一周后，随机分为4组。
各组分别饲喂：
(1)正常组(Normal)，腹腔每天注射生理盐水200mg/(kg/d)，灌胃生理盐水200mg/(kg/d)；
(2)氧化模型组(Control)，腹腔每天注射D-半乳糖200mg/(kg/d)，灌胃生理盐水200mg/(kg/d)；
(3)H8组，腹腔每天注射D-半乳糖200mg/(kg/d)，灌胃1.0×109cfu/(kg/d)植物乳杆菌H8；
(4)VC组，腹腔每天注射D-半乳糖200mg/(kg/d)，灌胃200mg/(kg/d)维生素C。
饲喂小鼠8周，观察小鼠在实验期间的健康状态和进食情况，实验结束前，将所有小鼠禁食24h，然后死于颈椎脱位。采集血液、大脑、心脏、肝脏、脾脏、肾脏、结肠样本进行后续实验。
实施例3
测定各组心、肝、脾、肾、脑的器官指数(动物器官的重量与其体重之比值，可以用于表征动物器官的功能状态，确定器官病变性质和程度，如器官发生萎缩，器官指数便随之降低)，结果如表1所示。
Control组心、肝、脾、肾、脑的器官指数均较Normal组显著下降，表明，腹腔注射D-半乳糖可引起各组织的萎缩。与Control组相比，H8、VC组器官指数显著增加，表明两组小鼠的组织萎缩程度均低于Control组。结果表明，H8组能有效抑制氧化衰老引起的器官指数下降，减轻D-半乳糖引起的机体组织萎缩。
实施例4血清和肝脏中NO、MDA、GSH、GSH-PX、SOD含量/活性测定
为了评估H8是否可以减轻体内氧化应激，对各组血清和肝脏中的NO、MDA含量以及SOD、GSH-Px、GSH活性进行测定。
取血于25℃、4000×g离心10分钟，收集上清液。根据试剂盒说明书(南京建城生物工程研究所，南京市，中国)测定小鼠血清中一氧化氮(NO)、丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)含量/活性。
制备10％小鼠肝匀浆，25℃、4000×g离心10分钟，根据试剂盒说明书采集上清液，测定肝组织中NO、MDA、SOD、GSH-Px和GSH的含量/活性。实验结果如表2所示。
表2
Control组血清和肝脏中SOD、GSH-Px、GSH活性最低(血清：82±10.26U/ml，148.33±31.45U/ml，14.51±1.84mg/L；肝脏：33.29±10.28U/ml，133.07±15.82U/ml，1.34±3.00mg/L)，NO和MDA含量最高(血清：44.27±0.04μmol/L，18.64±0.27nmol/ml；肝脏：2.43±0.79μmol/L，3.87±0.13nmol/ml)；与Control组相比，H8组处理后，氧化衰老小鼠NO和MDA含量显著降低(血清：36.44±1.46μmol/L，10.79±0.33nmol/ml；肝脏：0.97±0.09μmol/L，3.33±0.34nmol/ml)，SOD、GSH-Px、GSH活性显著升高(血清：122.43±13.29U/ml，210.23±12.8U/ml，17.65±4.14mg/L；肝脏：50.34±12.36U/ml，200.10±10.13U/ml，3.83±3.93mg/L)(所有p＜0.05)。
可见，H8菌株能有效的减轻体内的氧化应激标志物，具有潜在的抗衰老特性。
实施例5小鼠组织病理观察
收集小鼠脑、肝脏、脾脏和结肠组织，每个组织的面积约为0.5平方厘米，在10％福尔马林溶液中固定48小时。组织脱水，清除，嵌入蜡中，切割，然后进行苏木精和伊红(H&E)染色。在200倍光学显微镜(BX43，Olympus，Tokyo，Japan)下观察组织的形态学变化。
如图1所示，Normal组肝细胞组织良好，细胞质分布均匀，无炎症细胞浸润，未见明显病变。Control组小鼠肝细胞表现出明显病变，伴有大量出血，细胞排列紊乱，细胞呈不同程度肿胀，形态不规则，大量出现炎性浸润。与Control组相比，H8组肝细胞形态与Normal组接近，说明H8菌液可以减轻D-半乳糖诱导的肝组织病变。
Normal组脾组织结构完整，红髓和白髓的分布清晰可见。Control组红髓白髓分布混乱，红髓窦扩张，充满大量红细胞，白髓淋巴细胞减少，红髓索变窄。与Control组相比，H8组病理损伤较轻，红髓，白髓较为清晰，与Normal组接近，说明H8菌液组对D-半乳糖诱导的脾组织损伤具有一定的缓解作用。
Normal组脑组织皮质区呈锥形体，排列整齐致密，胞核饱满，核仁明显，边界清晰，Control组有部分呈现细胞核凋亡和固缩，脑组织结构疏松，间质水肿，排列紊乱，但是，用H8组处理后减轻了D-半乳糖诱导的脑组织病理，与Normal组接近。
Normal组结肠组织结构完整，隐窝结构良好，Control组结肠组织隐窝深度显著萎缩，杯状细胞丢失，结肠组织充血，出现炎性浸润。H8组的病理损伤较轻，隐窝萎缩程度减轻，与Normal组接近，说明H8组对结肠组织具有一定的保护作用。
可见，植物乳杆菌H8具有制备预防、改善或治疗氧化引起的组织损伤药物的潜力。
实施例6荧光定量PCR分析
取小鼠肝组织匀浆，用动物总RNA提取试剂盒提取组织总RNA，超微量分析仪确定纯度和浓度，按照逆转录试剂盒说明书进行反转录合成cDNA，将cDNA模板与SYBR Green混合物及上下游引物混合后，采用Stratagene Mx3000P检测相关基因的表达水平，引物序列如表3所示。循环参数如下：在95℃预变性15min，然后在95℃下40个循环10s，在60℃退火30s。以GAPDH为内参基因，Ct值通过荧光定量仪的分析软件给出，最后基因的表达水平用2－△△Ct表示，并将Control组中的基因表达量标准化为1。
表3
如图2、3所示，正常小鼠肝组织中Sirt1、NQO1、NOS1、NOS3、SOD1、HO-1、Cat、Nrf2、Sirt3、Trx、Sod2、Gclm的mRNA基因表达水平最强，分别是Control组的3.16-8.26倍；P53、NOS2的mRNA基因表达水平最弱，分别是Control组的0.19，0.35倍。D-半乳糖致衰老小鼠经H8处理后，肝脏中Sirt1、NQO1、NOS1、NOS3、SOD1、HO-1、Cat、Nrf2、Sirt3、Trx、Sod2、GclmmRNA基因水平与Control组相比显著升高，H8组分别是Control组的3.51-1.77倍，而P53、NOS2的mRNA基因表达水平显著降低，H8组分别是Control组的0.42，0.51。
可见，植物乳杆菌H8能够调节抗氧化相关因子的基因表达，从而在基因水平上减轻了D-gal诱导的衰老。
实施例6蛋白免疫印迹分析
取小鼠肝脏样品制备蛋白匀浆，BCA分析试剂盒测定蛋白质浓度，通过SDS-PAGE分离蛋白样品并转移到PVDF膜上，将膜在室温下用0.5％BSA封闭1.5小时，然后与一抗(Nrf2、HO-1、NQO1、Sirt1、Sirt3)在4℃下孵育过夜。洗涤三遍后，在TBST中与山羊抗兔辣根过氧化物酶偶联的二抗孵育1.5h。最后，使用ECL试剂观察免疫蛋白条带(美国AffinityBiosciences Inc.)，并通过NIH Image 1.63软件对免疫蛋白条带进行量化，用GAPDH作为内部对照将蛋白质水平标准化。重复该实验至少3次，每次都获得相似的结果。
如图4所示，正常小鼠肝脏中Sirt1，Nrf2，NQO1，HO-1，Sirt3的蛋白表达最强，D-半乳糖诱导肝组织蛋白表达最弱，经H8组处理后，与Control组相比蛋白表达显著升高。D-gal诱导衰老小鼠的Sirt1和Sirt3基因和蛋白水平显著降低，H8处理后显著逆转了Sirt1，Sirt3的抑制作用。H8显著上调Sirt1和Sirt3的基因和蛋白质表达，并显著促进Nrf2蛋白质的核易位。Nrf2的下游基因NQO1、NOS1、NOS3、SOD1、HO-1、Cat、Nrf2、Sirt3、Trx、Sod2、GclmmRNA的水平也显著提高，NOS2显著降低，抑制p53的转录活性。这表明H8能够调节Sirt1，Sirt3，从而防止D-gal诱导的衰老。Sirt1和Sirt3的激活促进了Nrf2-ARE的抗氧化途径，从而促进了II相代谢酶、抗氧化酶的转录活性,进而发挥抗氧化损伤的作用。Sirt1/Sirt3-Nrf2下游基因参与了H8对D-gal诱导的氧化应激的抗衰老过程。
可见，植物乳杆菌H8能够调节抗氧化相关蛋白，从而在蛋白质水平上防止了D-gal诱导的衰老。因此，可考虑使用植物乳杆菌H8预防、改善或治疗氧化引起的衰老。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (7)
1.一种抗氧化与抗衰老双效益生菌，其特征在于，所述益生菌为植物乳杆菌（Lactobacillus plantarum）H8，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.20168。
2.根据权利要求1所述的一种抗氧化与抗衰老双效益生菌在制备抗氧化产品中的应用，所述产品为食品或药品。
3.根据权利要求1所述的一种抗氧化与抗衰老双效益生菌在制备抗衰老产品中的应用，所述产品为药品。
4.根据权利要求1所述的一种抗氧化与抗衰老双效益生菌在制备预防、改善或治疗氧化引起的组织损伤药物中的应用，所述组织包括肝组织、脾组织、脑组织或结肠组织。
5.一种抗氧化产品，其特征在于，
所述产品包括权利要求1所述的一种抗氧化与抗衰老双效益生菌；所述产品为食品或药品。
6.一种抗衰老产品，其特征在于，
所述产品包括权利要求1所述的一种抗氧化与抗衰老双效益生菌；所述产品为药品。
7.一种预防、改善或治疗氧化引起的组织损伤的药物，其特征在于，
所述药物包括权利要求1所述的一种抗氧化与抗衰老双效益生菌。
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