CN117660229A - 一种缓解胰岛素抵抗的吉氏副拟杆菌及其应用 - Google Patents
一种缓解胰岛素抵抗的吉氏副拟杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种缓解胰岛素抵抗的吉氏副拟杆菌及其应用,属于微生物技术领域。本发明的吉氏副拟杆菌(Parabacteroides distasonis)NSP007能在不影响摄食量的前提下,抑制IR小鼠体重增加和体脂肪堆积;改善IR小鼠葡萄糖耐量、和胰岛素敏感性的同时,降低小鼠血清胰岛素和胰岛素抵抗指数;调节IR小鼠脂代谢紊乱和缓解肝脏组织损伤;改善IR小鼠肠屏障功能。本发明所述的吉氏副拟杆菌NSP007用于制备缓解IR的药物组合物与发酵食品,具有非常广泛的应用前景。
Description
技术领域
本发明涉及一种缓解胰岛素抵抗的吉氏副拟杆菌及其应用,属于微生物技术领域。
背景技术
胰岛素抵抗(Insulin resistance,IR)是胰岛素代偿性分泌增多的一种症状。IR是2型糖尿病(Type 2 diabetes mellitus,T2DM)的重要病理基础,T2DM及多种代谢性疾病的发生多与IR有关。T2DM患者将在2045年达7亿人,给人民的生活带来巨大负担。目前主要采用胰岛素增敏的药物来治疗IR,但长期服药具有诸多副作用。慢性炎症是导致IR的发生的重要因素之一。机制上,长期高脂饮食会导致肠道菌群失调,破坏肠道屏障功能,进一步致使肠道中的细菌内毒素(LPS)渗漏到血液中并激活toll样受体4(TLR-4),诱导肿瘤坏死因子α(TNF-α)的产生,引起炎症反应。TNF-α会直接影响胰岛素信号转导,进而阻碍细胞对葡萄糖的摄取,诱发IR。因此,通过改善肠屏障功能可从根源上减少LPS的渗漏,进而减缓IR的进程。有研究表明,多种肠道菌群(阿克曼氏菌、乳杆菌、双歧杆菌)会对宿主肠屏障功能和糖代谢产生有益作用。吉氏副拟杆菌是成人肠道中重要和常见菌种,具有抑制肠道致病菌的益生功能,是一种极具潜力的下一代“益生菌”。
公开号为CN108289917A的专利记载了一种含有吉氏副拟杆菌的的粪源细菌群落来治疗微生态失衡的系统和方法;公开号为CN116555096A的专利记载了一株吉氏副拟杆菌具有可将人参皂苷Re快速转化为人参皂苷Rg2和原人参三醇PPT的能力;公开号为CN116751699A的专利记载了一株具有抑菌作用的猪源狄氏副拟杆菌及其应用;公开号为CN113876815A的专利记载了一株狄氏副拟杆菌在治疗和预防雄性生殖毒性药物中的应用;公开号为CN111728986A的专利记载了一种含有吉氏副拟杆菌的复合菌剂能够有效降低肠道通透性和降低肠道炎症水平的作用;公开号为CN113651896A的专利记载了一种狄氏副拟杆菌胞外多糖及其提取方法与应用;公开号为CN110839693A的专利记载了一株吉氏副拟杆菌在预防或治疗肥胖或其相关疾病中的应用;公开号为CN107550942A的专利记载了一株吉氏副拟杆菌在治疗和预防代谢性疾病中的应用;公开号为CN112322545A的专利记载了一株狄氏副拟杆菌LCG-06及其与胆汁酸复配的复合菌剂与应用;公开号为CN113750121A的专利记载了一种狄氏副拟杆菌在制备阿尔茨海默病药物制剂中的应用;公开号为CN112410242A的专利记载了一株分离自直肠癌肿瘤组织的狄氏副拟杆菌株及其在抑制结直肠癌细胞系增殖中的应用;公开号为CN113143971A的专利记载了一种含有狄氏副拟杆菌株的胆汁酸复合菌剂在用于制备防治宠物泪痕的制剂中的应用;吉氏副拟杆菌具有多种益生作用,虽然有研究表明,吉氏副拟杆菌能够减轻肥胖或代谢性疾病的病症,但IR是肥胖、T2DM等多种代谢性疾病的重要病理基础,肠屏障受损是IR发生的重要诱因。目前尚未有报道表明吉氏副拟杆菌对IR和肠屏障功能受损的改善作用。
因此,急需一种对高脂饮食引起的肠屏障功能受损和IR等具有缓解作用的有益菌。
发明内容
为了缓解高脂饮食引起的肠屏障受损和IR,本发明从T2DM患者粪便中筛选出了一种吉氏副拟杆菌,并证明其具有全面改善肠屏障功能受损和IR的作用。本发明为糖尿病前期的干预提供了重要理论支撑和指导意义。
本发明提供了一株吉氏副拟杆菌(Parabacteroides distasonis)NSP007,已于2021年8月25日保藏于广东省科学院微生物研究所,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No:61888。
所述吉氏副拟杆菌(Parabacteroides distasonis)NSP007是从来源于江西地区的T2DM病人体粪便样本发酵液中分离得到的,该菌株经测序分析,其16S rDNA序列如SEQID NO.1所示,并将测序得到的序列在NCBI中进行核酸序列比对,结果显示与吉氏副拟杆菌的核酸序列相似度高达99%,将其命名为吉氏副拟杆菌(Parabacteroides distasonis)NSP007,同时,需要指出的是,生物材料保藏证明上的Parabacteroides distasonis PD26与吉氏副拟杆菌(Parabacteroides distasonis)NSP007是同一株菌的不同命名方式,本发明正文采用吉氏副拟杆菌(Parabacteroides distasonis)NSP007的命名。
所述的吉氏副拟杆菌NSP007具有下列性质:菌体特征:是一种革兰氏阴性杆状细菌,无孢子,直径在0.8-1.6×1.2-12μm,在厌氧血琼脂平板上菌落直径为1-2mm,正面形态圆形,中间凸起,边缘整齐,微白,不透明,表面湿润光滑。生长特性:该菌株为专性厌氧菌,最适生长温度是36℃-38℃,最适生长pH值为6.6-7.0,在含有葡萄糖的培养基中生长良好,12-24h可进入对数后期或稳定前期。
本发明还提供了一种微生物菌剂,所述微生物菌剂含有上述吉氏副拟杆菌(Parabacteroides distasonis)NSP007。
在本发明的一种实施方式中,所述微生物菌剂中,吉氏副拟杆菌(Parabacteroides distasonis)NSP007的活菌数不低于1×109CFU/mL或1×109CFU/g。
本发明还提供了一种产品,所述产品中含有上述吉氏副拟杆菌(Parabacteroidesdistasonis)NSP007。
在本发明的一种实施方式中,所述产品中,吉氏副拟杆菌(Parabacteroidesdistasonis)NSP007的活菌数不低于1×109CFU/mL或1×109CFU/g。
在本发明的一种实施方式中,所述产品为微生物菌剂、食品、药品、保健品或饲料添加剂。
在本发明的一种实施方式中,所述食品包括饮料、奶制品或其他含有上述吉氏副拟杆菌(Parabacteroides distasonis)NSP007的食品。
在本发明的一种实施方式中,所述药品包含吉氏副拟杆菌(Parabacteroidesdistasonis)NSP007、药物载体和/或药用辅料。
在本发明的一种实施方式中,所述药品或保健品的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液等剂型。
在本发明的一种实施方式中,所述药用辅料是药学上可接受的辅料。
在本发明的一种实施方式中,所述可接受的辅料包括一种或多种通常使用的增稠剂、抗氧化剂、酸碱调节剂、乳化剂、防腐剂、填充剂、粘合剂、润湿剂、崩解剂、润滑剂及矫味剂等。
本发明的一种实施方式中,所述填充剂为淀粉、蔗糖、乳糖、硫酸钙和/或微晶纤维素。
在本发明的一种实施方式中,所述粘合剂为纤维素衍生物、藻酸盐、明胶和/或聚乙烯吡咯烷酮。
在本发明的一种实施方式中,所述润湿剂为水、乙醇、淀粉和/或糖浆。
在本发明的一种实施方式中,所述崩解剂为羧甲基淀粉钠、羧丙纤维素、交联羧甲基纤维素、琼脂、碳酸钙和/或碳酸氢钠。
在本发明的一种实施方式中,所述润滑剂为滑石粉、硬脂酸钙、硬脂酸镁、微粉硅胶和/或聚乙二醇。
在本发明的一种实施方式中,所述矫味剂为单糖浆、蔗糖、卵磷脂、橙皮糖浆、樱桃糖浆、柠檬、茴香、薄荷油、海藻酸钠、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素钠、柠檬酸、酒石酸和/或碳酸氢钠。
本发明还提供了上述吉氏副拟杆菌(Parabacteroides distasonis)NSP007,或上述微生物菌剂在制备用于预防和/或治疗IR的药品的应用。
在本发明的一种实施方式中,所述药品中,吉氏副拟杆菌NSP007的活菌数不低于1×109CFU/mL或1×109CFU/g。
在本发明的一种实施方式中,所述药品包含吉氏副拟杆菌(Parabacteroidesdistasonis)NSP007、药物载体和/或药用辅料。
在本发明的一种实施方式中,所述药品的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液等剂型。
在本发明的一种实施方式中,所述药用辅料是药学上可接受的辅料。
在本发明的一种实施方式中,所述可接受的辅料包括一种或多种通常使用的增稠剂、抗氧化剂、酸碱调节剂、乳化剂、防腐剂、填充剂、粘合剂、润湿剂、崩解剂、润滑剂及矫味剂等。
本发明的一种实施方式中,所述填充剂为淀粉、蔗糖、乳糖、硫酸钙和/或微晶纤维素。
在本发明的一种实施方式中,所述粘合剂为纤维素衍生物、藻酸盐、明胶和/或聚乙烯吡咯烷酮。
在本发明的一种实施方式中,所述润湿剂为水、乙醇、淀粉和/或糖浆。
在本发明的一种实施方式中,所述崩解剂为羧甲基淀粉钠、羧丙纤维素、交联羧甲基纤维素、琼脂、碳酸钙和/或碳酸氢钠。
在本发明的一种实施方式中,所述润滑剂为滑石粉、硬脂酸钙、硬脂酸镁、微粉硅胶和/或聚乙二醇。
在本发明的一种实施方式中,所述矫味剂为单糖浆、蔗糖、卵磷脂、橙皮糖浆、樱桃糖浆、柠檬、茴香、薄荷油、海藻酸钠、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素钠、柠檬酸、酒石酸和/或碳酸氢钠。
本发明还提供了上述吉氏副拟杆菌(Parabacteroides distasonis)NSP007,或上述微生物菌剂在制备维持血糖健康水平的保健品中的应用。
有益效果
1、本发明筛选出了一株吉氏副拟杆菌(Parabacteroides distasonis)NSP007,具有缓解IR的作用,具体体现在:
1)能够在不影响小鼠摄食量的前提下,降低IR小鼠的体脂,抑制小鼠体重的增加;
2)能够改善高脂诱导的IR小鼠的葡萄糖耐量,胰岛素耐量,高胰岛素血症和胰岛素抵抗;
3)能够减轻高脂饮食诱导的IR小鼠肝脏纤维化和脂质沉积,降低血清中游离脂肪酸和血脂水平;
4)能够降低高脂饮食诱导的IR小鼠血清中促炎因子IL-10和IL-1β的含量、血清中LPS的水平。
5)能够提高高脂饮食诱导的IR小鼠肠道屏障功能相关基因和蛋白的表达。
2、吉氏副拟杆菌(Parabacteroides distasonis)是一种极具研究价值的潜在益生菌,其多种益生作用已被大量报道。本发明的发明人通过大量创造性实验研究发现,本发明的吉氏副拟杆菌NSP007,能有效缓解高脂饮食所引起的肠屏障功能受损和IR,可应用于预防T2DM和治疗IR及相关疾病的药物、食品或保健品中。
生物材料保藏
一株吉氏副拟杆菌(Parabacteroides distasonis)NSP007,已于2021年8月25日保藏于广东省科学院微生物研究所,分类学命名为:Parabacteroides distasonis,保藏编号为GDMCCNo:61888,保藏地址为广州市先烈中路100号大院59号楼5楼,广东省科学院微生物研究所。
附图说明
图1:吉氏副拟杆菌NSP007干预IR小鼠后肝脏照片(左)、肝组织H&E染色图(中)、肝组织油红O染色图(右),标尺为50μm。
图2:吉氏副拟杆菌NSP007干预IR小鼠后肝脏中F4/80的免疫组织化学染色,标尺为50μm.
图3:吉氏副拟杆菌NSP007干预IR小鼠后肠组织中Claudin-1,Muc-2,Occludin和ZO-1的免疫组织化学染色,标尺为:50μm。
具体实施方式
下述实施例中涉及到的小鼠购自湖南斯莱克景达实验动物有限公司,饲养于25±2℃、恒湿50±5%、光照12小时(8:00-20:00),隔音,自由摄食、饮水,适应性喂养一周后开始实验。下述实施例中所涉及的胰岛素试剂盒(购自crystal chem,货号:90080)、血糖试纸和血糖仪购自罗氏公司;所涉及的TNF-α炎症因子试剂盒(货号:FMS-ELM028)、IL-1β炎症因子试剂盒(货号:FMS-ELM002)购自南京福麦斯生物技术有限公司;所涉及的内毒素(LPS)检测试剂盒(货号:F10621-B)购自武汉华美生物工程有限公司;所涉及的游离脂肪酸(FFA)试剂盒(货号:A042-2-1)、总胆固醇(TC)试剂盒(货号:A111-1)、高密度脂蛋白(HDL-c)试剂盒(货号:A112-1)、低密度脂蛋白(LDL-c)试剂盒(货号:A113-1-1)、甘油三酯TG试剂盒(A110-1-1)购自南京建成生物工程研究所;下述实施例中所涉及的Trizol试剂(货号:15596026)购自美国赛默飞公司;下述实施例中所涉及的逆转录试剂盒(货号:RR047A)和实时定量试剂盒(货号:RR820A)购自宝日医生物技术(北京)有限公司;下述实施例中所涉及的培养基成分均购买自上海源叶公司;下述实施例中所涉及的高脂饲料购自美国Research Diets公司(货号:D12492)。下述实施例中所涉及的正常饲料购自武汉斯莱克公司(大小鼠繁殖饲料)。下述实施例中所涉及的引物均订购自上海生工生物工程有限公司。
下述实施例中涉及的吉氏副拟杆菌(Parabacteroides distasonis)NSP007,与生物材料保藏证明上的Parabacteroides distasonis PD26是同一株菌的不同命名方式,本发明正文采用吉氏副拟杆菌(Parabacteroides distasonis)NSP007的命名。
下述实施例中涉及培养基:
活化培养基的制备(g/L):成分包括了碳源:果胶0.047,木聚糖0.047,阿拉伯半乳聚糖,支链淀粉0.04,可溶性淀粉0.392;氮源:细菌蛋白胨24,胰蛋白胨24;无机盐:七水硫酸镁0.5,磷酸二氢钾2.5,氯化钠4.5,二水合氯化钙0.45,七水合硫酸铁0.005;胆盐0.4,厌氧剂半胱氨酸盐酸盐0.2和酸碱缓冲液(MES)19.52。首先将以上成分配制完成,并调节pH至6后进行除氧和灭菌(121℃,15min)。灭菌结束后将培养基转移至厌氧手套箱中,将不耐高温的血红素1μg、维生素K3(VK3)1μg和维生素混合液(Wolfe's Vitamin Solution)0.1mL过0.22μm滤膜添加至1L培养基,在厌氧手套箱中过夜除氧,即得到所述活化液体培养基。
富集培养基的制备:每升富集培养基由350mLA液,150mLB液,500mL C液,1mL D液和0.08mL维生素混合物(Wolfe's Vitamin Solution)混合而成,配方(g/L)包括:A液:细菌蛋白胨68.57,胰蛋白胨68.57,胆盐1.14,厌氧剂半胱氨酸盐酸盐1.43,硫酸镁1.14,磷酸一氢钾5.48,氯化钠12.86,氯化钙0.97,七水合硫酸铁0.014;B液:酸碱缓冲剂(MES)130,C液:铁皮石斛多糖10,D液:血红素10mg、维生素K3(VK3)8mg。首先将可高压灭菌成分(A-C液)配制完成,调节pH至6后进行除氧,而后进行灭菌(121℃,15min)。灭菌结束后将培养基转移至厌氧手套箱中过夜。最后将D液和Wolfe's Vitamin Solution过0.22μm滤膜后按比例加入培养基中,即得到所述富集培养基。
BHI液体培养基(g/L):蛋白胨10.0,牛心浸粉17.5,氯化钠5.0,葡萄糖2.0,磷酸氢二钠2.5,溶解于1L蒸馏水中,并加入半胱氨酸盐酸盐0.5g,混合均匀,然后调整其pH为7.2-7.6,115-121℃灭菌15-20min后,即得到所述BHI液体培养基。
BHI固体培养基的制备:在BHI液体培养基的基础上添加1.5-2%琼脂。混合均匀,然后调整其pH为7.2-7.6,115-121℃灭菌15-20min后,即得到所述BHI固体培养基。
BHI选择性培养基(g/L):在BHI固体培养基的基础上加入滤膜灭菌的血红素5mg/L,维生素K110 mg/L,万古霉素7.5mg/L,卡那霉素100mg/L。即得到所述BHI选择性培养基。
下述实施例中所涉及的检测方法如下:
小鼠附睾脂肪、肝脏组织的重量测定:第5周实验结束后,将小鼠麻醉后处死,分离出小鼠完整的附睾脂肪和肝脏,称重并记录。
附睾指数(%)=(附睾脂肪重量(g)/小鼠体重(g))×100
小鼠肝脏、结肠的组织形态和免疫组化观察:
(1)H&E染色:将组织包埋于石蜡中,染色前将石蜡切片脱蜡,分别用苏木素和伊红染色,脱水后的切片封片后显微观察;
(2)油红O染色:将组织包埋于石蜡中,冰冻后切割成切片,切片入油红染液浸染后用用苏木素复染,使用甘油明胶封片后显微观察。
(3)免疫组化:将组织包埋于石蜡中,过氧化氢阻断,抗原修复,依次加入一抗和二抗,DAB染色,苏木素复染,脱水后的切片封片后显微观察。
(4)肠屏障相关蛋白免疫组化阳性面积(%):通过ImageJ 1.53k软件计算阳性面积的百分比。
实时荧光定量PCR:
首先,采用trizol法提取组织中的RNA,具体操作参考试剂盒说明书,利用NanoDrop及琼脂糖凝胶电泳测定RNA浓度和纯度。进一步逆转录,将1μg组织中总RNA加入2μL5×gDNABuffer、1μL gDNAEraser以RNase-Free ddH2O补足至10μL简短混匀并离心,置于42℃,孵育2min后以去除样本中的基因组DNA,再加入1μLPrimeScript RT Enzyme MixⅠ、1μL RTPrimer Mix、4μL 5×PrimeScript Buffer 2和RNase-Free ddH2O补足至20μL,于37℃反应15min,然后85℃反应5s,即完成逆转录。
取逆转录后产物稀释10倍作为模板进行实时荧光定量PCR。qPCR反应体系(20μL):2μg cDNA模板、10μL SYBR染液、0.8μL引物,剩余体积用ddH2O补足。反应条件:25-50℃,1.6℃/s,50℃保持2min;50-95℃,1.6℃/s,95℃保持30s;扩增(95℃20s,60℃,1min,共40循环)。各基因分别设2个副孔,数据处理采用2-ΔΔCT进行相对定量分析。
表1实时荧光定量PCR所用引物
口服葡萄糖耐量实验(OGTT):动物禁食6h后,测0min基础血糖后灌胃葡萄糖1.5g/kg,自鼠尾静脉采血用罗氏血糖仪测定葡萄糖灌胃后15,30,60,90min血糖,绘制血糖-时间曲线,并根据各个时间点中血糖值计算各组曲线下面积。绘制血糖-时间曲线,并根据各个时间点中血糖值计算各组曲线下面积AUC。
AUC计算方法(下同):AUC(mg*min/dL)=(BG0+BG15)×15/2+(BG15+BG30)×15/2+(BG30+BG60)×30/2+(BG60+BG90)×30/2
BG0,BG15,BG30,BG60,BG90代表给予处理后0min,15min,30min,60min,90min的血糖。
胰岛素耐量实验(ITT):动物禁食6h后,测0min基础血糖后腹腔注射胰岛素0.8U/kg,自鼠尾静脉采血用罗氏血糖仪测定胰岛素注射后15,30,60,90min血糖,绘制血糖-时间曲线,并根据各个时间点中血糖值计算各组曲线下面积AUC(同上)。
血清胰岛素的测定:实验结束后,将小鼠麻醉后处死,获取血清,参照试剂盒说明书测定其血清中胰岛素含量。
空腹血糖的测定:动物禁食6h后,自鼠尾静脉采血用罗氏血糖仪测定血糖。
胰岛素抵抗指数(HOMA-IR)的计算方法:
HOMA-IR=空腹血清胰岛素(μU/mL)×空腹血糖(mmol/L)/22.5
FFA、TG、TC、HDL-c、LDL-c、LPS、TNF-α、IL-1β和空腹胰岛素的测定:参考对应试剂盒说明书。
实施例1:吉氏副拟杆菌NSP007的分离筛选
1、样品采集
采集江西地区的T2DM病人体粪便样本,样本置于保藏管中,加入5倍重量的保护液(保护剂的制备:称取半胱氨酸盐酸盐1g/L,甘油200-300g/L,均匀溶解于PBS(1×)中,115-121℃灭菌15-20min)保存于装有干冰的保温盒中,带回实验室后迅速置于-80℃冰箱待分离筛选。
2、粪菌的富集
将上述粪菌液从-80℃冰箱取出,解冻后低速低温离心(500g,5min,4℃)获得上清,然后过100μm滤膜除去上清中杂质,将上清粪菌液接种至活化培养基中(粪菌液:活化培养基=1:9,(v/v)),37℃下,140rpm培养16h,然后将其接种至富集培养基中,接种比例为10%(v/v),37℃下,140rpm培养24h。获得用铁皮石斛多糖富集后的粪菌液。以上操作在无菌厌氧环境中进行。
3、拟杆菌的分离纯化
(1)粪菌液梯度稀释:在无菌厌氧环境中,取上述富集后的粪菌液,加入到9mL生理盐水,得到第一梯度稀释液,吸取1mL第一梯度稀释液于9mL生理盐水,得到第二梯度稀释液,以此类推,共配制5个梯度稀释液;
(2)涂布培养:分别吸取100μL上述所有梯度稀释液分别置于BHI固定培养基上,涂布后置于37℃厌氧条件下培养48h,得到稀释涂布平板;
(3)纯化培养:挑取固体培养基上边缘整齐,微白,不透明,表面湿润光滑,且形态一致的纯的单菌落接种于5mL液体BHI选择性培养基中,至于37℃厌氧条件下培养24h,得到纯化培养液。
4、菌种保藏与鉴定
将步骤3获得的长势最好的纯化培养液8000rpm离心10min,弃上清得菌体。用细菌16S rDNA PCR特异性引物(见表2)进行PCR,PCR产物经核酸电泳分析确认后,扩增产物送至公司进行测序,其16S rDNA序列如SEQ ID NO.1所示,测序结果与NCBI数据库中序列进行比对分析;结果显示与吉氏副拟杆菌的核酸序列相似度高达99%,将其命名为吉氏副拟杆菌(Parabacteroides distasonis)NSP007。
表2引物名称
实施例2:吉氏副拟杆菌NSP007对高脂饮食的IR小鼠体重、体脂和饮食的影响
具体步骤如下:
1、吉氏副拟杆菌NSP007冻存剂的制备:
(1)培养方法:在无菌厌氧环境中,将吉氏副拟杆菌NSP007菌种在BHI固体培养基上划线,在厌氧条件下培养48h,形成单菌落后,挑取单菌落,并将其接种至BHI液体培养基,在37℃下厌氧培养16-24h,达到稳定期,此时的OD值为:0.8~1.0,制备得到种子液。
(2)保护剂的制备:称取半胱氨酸盐酸盐1g/L,甘油200-300g/L,均匀溶解于蒸馏水中,115-121℃灭菌15-20min。
(3)冷冻剂的制备:将步骤(1)培养至稳定期的吉氏副拟杆菌NSP007种子液离心(8000rpm,10min,4℃)后,用无菌的磷酸盐缓冲液(pH 7.2)清洗1-2次后,用步骤(2)制备的保护剂重悬菌液,即得吉氏副拟杆菌NSP007冻存剂,于-80℃保存备用。
2、吉氏副拟杆菌NSP007菌剂的制备:
(1)活化菌株:将步骤1制备得到的吉氏副拟杆菌NSP007冻存剂在BHI固体培养基上划线,在厌氧条件下培养48h,形成单菌落后,将单菌接种至BHI液体培养基,在37℃下厌氧培养16-24h,达到稳定期(OD值为:0.8~1.0)。
(2)菌剂的制备:取100μL不同稀释倍数的步骤(1)获得的培养液涂布于BHI固体培养基,对BHI固体平板上的菌落数进行计数,计算步骤(1)液体培养基中的活菌数。用无菌磷酸盐缓冲液(pH 7.2)清洗1-2次后,将菌液制备成浓度为1×109CFU/mL的制剂,灌胃体积为0.1mL。
3、实验方法:
干预治疗实验过程:
本发明采用高脂饲料喂养的方法诱导小鼠产生胰岛素抵抗(IR)。6周龄健康雄性C57BL/6J小鼠32只,随机分为四组(每组8只):正常组(为描述方便,以N表示)、高脂饮食胰岛素抵抗组(模型组,以M表示)、吉氏副拟杆菌NSP007活菌组(吉氏副拟杆菌组,以LPD表示)、热灭活的吉氏副拟杆菌NSP007组(吉氏副拟杆菌组,以KPD表示)。经过8周的高脂饮食,测定各高脂饮食组小鼠空腹血糖、空腹血清胰岛素水平并计算口服葡萄糖耐受量曲线下面积和胰岛素抵抗指数,结果显示,上述指标高脂饮食组小鼠均显著高于正常组小鼠,认为高脂诱导的胰岛素抵抗小鼠模型造模成功(每组8只);具体指标如表3所示:
表3高脂诱导的胰岛素抵抗小鼠模型造模成功指标
实验流程如表4所示,经一周适应期后;正常组(N):在造模期饲喂正常饲料并自由饮水,治疗期继续饲喂正常饲料,每天灌胃一次0.1mL无菌磷酸盐缓冲液,自由饮水;
治疗期间,高脂饲料干预IR的同时,用吉氏副拟杆菌NSP007进行治疗。干预治疗实验过程:
模型组(M):干预期间每天灌胃一次0.1mL无菌磷酸盐缓冲液;
吉氏副拟杆菌NSP007组(LPD):干预期间每天灌胃一次0.1mL吉氏副拟杆菌NSP007菌液(菌浓度为1×109CFU/mL);
热灭活吉氏副拟杆菌NSP007组(KPD):干预期间每天灌胃一次0.1mL热灭活吉氏副拟杆菌NSP007菌液(菌浓度为1×109CFU/mL)。
干预5周后,将小鼠麻醉后处死。从小鼠眼眶取血,收集血液,3000rpm离心15min,获得小鼠血清。血清、附睾脂肪和肝脏保存于-80℃用于后续分析。
表4实验流程
4、吉氏副拟杆菌NSP007对高脂饮食的IR小鼠体重、体脂和饮食的影响
具体实验过程同步骤1~3,区别在于,干预期间每4天对各组小鼠称重,实验结束后,处死小鼠采集其肝脏和附睾脂肪进行称重,通过体重和附睾脂肪重量计算附睾指数。实验结果如表5、6、7所示,LPD的干预对小鼠能量摄入无显著影响,但LPD的干预显著抑制了IR小鼠的体重增长、肝脏增重以及附睾脂肪的堆积。
表5吉氏副拟杆菌干预期间对IR小鼠能量摄入(千卡/天/小鼠)的影响
表6吉氏副拟杆菌干预期间对IR小鼠体重(g)的影响
表7吉氏副拟杆菌对IR小鼠脏器重量和指数的影响
实施例3:吉氏副拟杆菌NSP007对高脂诱导的IR小鼠胰岛素抵抗的影响
具体步骤如下:
具体实验方法同实施例2,区别在于,在小鼠处死前3天,对各组小鼠进行口服葡萄糖耐量测试(OGTT)、胰岛素耐量测试(ITT)。实验结束后,将小鼠麻醉后处死,取其血清测定胰岛素并计算其胰岛素抵抗指数(HOMA-IR)。结果如表8-10所示。
结果显示,LPD组的OGTT和ITT的曲线下面积显著低于M组和KPD组,另外LPD组的HOMA-IR也显著低于M组和KPD组,与M组相比,OGTT曲线下面积、ITT的曲线下面积和HOMA-IR分别降低26.6%、24.4%和58.5%。LPD组的禁食胰岛素和胰岛素抵抗指数均恢复至与对照组相当的水平。这表明,本发明吉氏副拟杆菌NSP007能够提高IR小鼠的葡萄糖耐量、胰岛素敏感性,并改善高胰岛素血症和胰岛素抵抗程度。
表8吉氏副拟杆菌对IR小鼠葡萄糖耐量(mg/dL)的影响
表9吉氏副拟杆菌对IR小鼠胰岛素耐量(mg/dL)的影响
表10吉氏副拟杆菌对IR小鼠胰岛素抵抗的影响
实施例4:吉氏副拟杆菌NSP007对高脂诱导的IR小鼠肝损伤和脂代谢的影响
具体步骤如下:
具体实验方法同实施例2,区别在于,在实验结束后收集小鼠血清,测定小鼠血清中游离脂肪酸(FFA)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL-c)、低密度脂蛋白(LDL-c)的水平;对小鼠肝脏进行拍照、染色,观察各组小鼠肝脏的形态学变化。结果如表11和图1所示。
表11结果显示LPD组的FFA、TG、TC和LDL-c水平显著低于M组和KPD组,与M组相比,FFA、TG、TC和LDL-c水平分别降低24.8%、32.6%、23.6%和17.4%;并且LPD组的FFA和TG恢复至与对照组相当的水平。这表明,LPD明显改善了IR小鼠的脂代谢紊乱。
图1结果显示M组的肝脏存在明显的弥漫性脂肪变性(空泡化)和纤维化,经过吉氏副拟杆菌NSP007的治疗,小鼠肝脏形态、大小、纤维化和脂肪堆积与M组和KPD组相比得到明显的改善,并且与对照组的形态相近。这表明,LPD能够减轻高脂饮食诱导的IR小鼠肝脏纤维化和脂质沉积,降低血清中游离脂肪酸和血脂水平。
表11吉氏副拟杆菌对IR小鼠脂代谢的影响
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实施例5:吉氏副拟杆菌NSP007对高脂诱导的IR小鼠系统性炎症和肝脏炎症的影响
具体实验方法同实施例2,区别在于,在实验结束后收集小鼠血清,测定小鼠血清中游离TNF-α、IL-1β和LPS的水平,对小鼠肝脏进行免疫组化染色,观察各组小鼠肝脏中巨噬细胞的聚集行为。结果如表12和图2所示。
表12结果显示LPD组的TNF-α、IL-1β和LPS水平显著低于M组和KPD组,与M组相比,TNF-α、IL-1β和LPS水平分别降低26.8%、42.5%和45.8%;并且LPD组的IL-1β和LPS水平恢复至与对照组相当的水平。这表明,LPD明显改善了IR小鼠的系统性炎症。
图2结果显示M组的肝脏存在明显的巨噬细胞聚集行为。经过LPD干预后,巨噬细胞聚集行为显著减少。这表明LPD明显改善了IR小鼠的肝脏炎症。
表12吉氏副拟杆菌对IR小鼠系统性炎症的影响
实施例6:吉氏副拟杆菌NSP007对高脂诱导的IR小鼠肠屏障功能的影响
具体实验方法同实施例2,区别在于,在实验结束后收集小鼠结肠组织,测定小鼠结肠组织中肠屏障相关基因(Claudin1、Muc2、Occludin、Zo1)的表达量,对小鼠结肠进行免疫组化染色,观察各组小鼠结肠中肠屏障相关蛋白(Claudin-1、Muc2、Occludin、ZO-1)的表达量。结果如表13、14和图3所示。
表13结果显示LPD组的Claudin1、Muc2、Occludin和Zo1 mRNA表达量显著高于M组和KPD组,与M组相比,Claudin1、Muc2、Occludin和Zo1 mRNA水平分别升高70.6%、135.0%、69.0%和122.8%,这表明,LPD能有效改善肠屏障功能。
表14和图3结果显示LPD组的Claudin-1、Muc2、Occludin和ZO-1蛋白表达量显著高于M组和KPD组,与M组相比,Claudin-1、Muc2、Occludin和ZO-1蛋白水平分别升高24.3%、73.1%、44.8%和267.9%;并且LPD组各基因和蛋白的表达水平恢复至与对照组相当的水平。这表明,LPD能有效改善肠屏障功能。
表13吉氏副拟杆菌对IR小鼠肠屏障相关基因表达量的影响
表14吉氏副拟杆菌对IR小鼠肠屏障相关蛋白表达量(阳性面积,%)的影响(与图3相关)
虽然本发明已以较佳实施例公开如上,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,都可做各种的改动与修饰,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 (10)
1.一株吉氏副拟杆菌(Parabacteroides distasonis)NSP007,已于2021年8月25日保藏于广东省科学院微生物研究所,保藏地址为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC No:61888。
2.一种微生物菌剂,其特征在于,含有权利要求1所述的吉氏副拟杆菌。
3.如权利要求2所述的微生物菌剂,其特征在于,所述微生物菌剂中,吉氏副拟杆菌NSP007的活菌数不低于1×109CFU/mL或1×109CFU/g。
4.一种含有权利要求1所述的吉氏副拟杆菌产品,其特征在于,所述产品为微生物菌剂、食品、药品、保健品或饲料添加剂。
5.如权利要求4所述的产品,其特征在于,所述产品中,吉氏副拟杆菌的活菌数不低于1×109CFU/mL或1×109CFU/g。
6.如权利要求4所述的产品,其特征在于,所述食品包括饮料、奶制品。
7.如权利要求4所述的产品,其特征在于,所述药品包含权利要求1所述的吉氏副拟杆菌,还含有药物载体和/或药用辅料。
8.如权利要求4所述的产品,其特征在于,所述药品的剂型包括颗粒剂、胶囊剂、片剂、丸剂或口服液。
9.权利要求1所述的吉氏副拟杆菌,或权利要求2或3所述的微生物菌剂在制备预防和/或治疗胰岛素抵抗的药品中的应用。
10.权利要求1所述的吉氏副拟杆菌,或权利要求2或3所述的微生物菌剂在制备维持血糖健康水平的保健品中的应用。
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