CN116350667A - 酿酒酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 - Google Patents
酿酒酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 Download PDFInfo
- Publication number
- CN116350667A CN116350667A CN202310295670.XA CN202310295670A CN116350667A CN 116350667 A CN116350667 A CN 116350667A CN 202310295670 A CN202310295670 A CN 202310295670A CN 116350667 A CN116350667 A CN 116350667A
- Authority
- CN
- China
- Prior art keywords
- saccharomyces cerevisiae
- nafld
- group
- treating
- mice
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 42
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 42
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 title claims abstract description 35
- 239000003814 drug Substances 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 229940079593 drug Drugs 0.000 title abstract description 8
- 210000002966 serum Anatomy 0.000 claims abstract description 17
- 238000011282 treatment Methods 0.000 claims abstract description 17
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 claims abstract description 6
- 108010082126 Alanine transaminase Proteins 0.000 claims abstract description 6
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 6
- 108090000695 Cytokines Proteins 0.000 claims abstract description 4
- 102000004127 Cytokines Human genes 0.000 claims abstract description 4
- 208000004232 Enteritis Diseases 0.000 claims abstract description 4
- 239000002775 capsule Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 239000004518 granules dosage form Substances 0.000 claims 1
- 239000006186 oral dosage form Substances 0.000 claims 1
- 239000006041 probiotic Substances 0.000 abstract description 12
- 235000018291 probiotics Nutrition 0.000 abstract description 12
- 230000000529 probiotic effect Effects 0.000 abstract description 9
- 230000006872 improvement Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 230000001225 therapeutic effect Effects 0.000 abstract description 4
- 208000008589 Obesity Diseases 0.000 abstract description 2
- 235000020824 obesity Nutrition 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 208000006454 hepatitis Diseases 0.000 abstract 1
- 231100000283 hepatitis Toxicity 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 33
- 210000004185 liver Anatomy 0.000 description 21
- 210000001072 colon Anatomy 0.000 description 18
- 210000000952 spleen Anatomy 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 238000001914 filtration Methods 0.000 description 6
- 241000233866 Fungi Species 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 210000001165 lymph node Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 208000004930 Fatty Liver Diseases 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000000112 colonic effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 208000010706 fatty liver disease Diseases 0.000 description 2
- 230000004761 fibrosis Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 244000005709 gut microbiome Species 0.000 description 2
- 210000003547 hepatic macrophage Anatomy 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000007358 intestinal barrier function Effects 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000001565 modulated differential scanning calorimetry Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000702462 Akkermansia muciniphila Species 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 241000237903 Hirudo Species 0.000 description 1
- 101100341519 Homo sapiens ITGAX gene Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241000235388 Mucorales Species 0.000 description 1
- 101001065556 Mus musculus Lymphocyte antigen 6G Proteins 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 241000606210 Parabacteroides distasonis Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 235000021068 Western diet Nutrition 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 238000007688 edging Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000011577 humanized mouse model Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000006372 lipid accumulation Effects 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 229960002181 saccharomyces boulardii Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000007218 ym medium Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/064—Saccharomycetales, e.g. baker's yeast
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Alternative & Traditional Medicine (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明公开了酿酒酵母(Saccharomyces cerevisiae Hansen)在制备治疗和/或改善非酒精性脂肪肝药物中的应用。本发明还公开了所述酿酒酵母在制备治疗和/或改善肥胖体征、肝炎、肠炎、降低血清谷丙转氨酶和/或血清炎性细胞因子浓度药物中的应用。本发明首次证实了酿酒酵母对NAFLD有明显的改善作用，后续可应用于NAFLD治疗益生菌制剂的储备，在NAFLD的临床预防和治疗方面具有潜在应用价值。
Description
技术领域
本发明涉及益生真菌在制备治疗和/或改善非酒精性脂肪肝药物中的应用，尤其涉及酿酒酵母(Saccharomyces cerevisiae Hansen)在制备治疗和/或改善非酒精性脂肪肝药物中的应用，属于益生菌治疗领域。
背景技术
非酒精性脂肪肝(NAFLD)影响高达25％的世界人口，是一种非常普遍但在很大程度上被低估的肝脏疾病，患者可能会表现出炎症或纤维化，并出现糖尿病等长期并发症，导致死亡率增加。尽管其是一个紧迫的公共卫生问题，但全世界缺乏全面的应对措施。鉴于NAFLD“多重打击模型”患病机制的复杂性，通常不会选择病因干预，而是选择脂质代谢、炎症免疫、纤维化进展等关键途径的分子靶点进行药物干预，且均处在临床试验中。NAFLD的患病率仍呈现逐年上升趋势，为此，迫切需要寻找新颖且稳定可靠的治疗方法。
肠道微生物群在维持肝脏稳态方面起着至关重要的作用，因为肝脏是通过门静脉排出肠道的第一个器官。因此，肝脏更容易暴露于肠道细菌或相关的消化细菌产物中，这种关系通常被称为“肠-肝轴”或“肝脏-微生物组轴”。越来越多的研究表明，肠道菌群对NAFLD具有不可忽视的影响。一方面，肠道微生物失调被认为与NAFLD的发病机制相关；另一方面，靶向肠道微生物的治疗方法，如益生菌治疗，以其新颖性和创造性而备受关注。
在益生菌治疗方面，目前发现的或许能用于治疗NAFLD的益生细菌较多，而益生真菌较少。益生细菌方面，已报道P.distasonis和B.xylanisolvens对肥胖和NAFLD具有潜在疗效，而Plovier等人也发现Akkermansia muciniphila或其巴氏杀菌形式可以改善肥胖和糖尿病小鼠的新陈代谢，并进一步纯化出了发挥主要作用的膜蛋白，从而为肠屏障功能在代谢方面的重要作用提供了重要佐证。益生真菌方面，有文章报道NAFLD患者的肠道真菌组成发生变化，毛霉菌属物种/酿酒酵母的对数比以及白色念珠菌/酿酒酵母的对数比随NAFLD的严重程度而升高，并通过人源化小鼠模型证实了真菌在西式饮食诱导的脂肪肝炎中的作用，提示靶向肠道真菌的策略或能用于改善NAFLD。常用的益生真菌Saccharomycesboulardii对脂肪性肝病的治疗作用已得到验证。另有研究表明，经果胶酶处理的益生菌香蕉汁可改善NAFLD，这与增加的肠道酿酒酵母有关，但尚未有实验证明酿酒酵母对NAFLD的直接作用。
发明内容
发明目的：本发明所要解决的技术问题是提供酿酒酵母(Saccharomycescerevisiae Hansen)在制备治疗和/或改善非酒精性脂肪肝药物中的应用。
技术方案：为解决上述技术问题，本发明提供了酿酒酵母(Saccharomycescerevisiae Hansen)在制备治疗和/或改善非酒精性脂肪肝药物中的应用。
其中，所述酿酒酵母(Saccharomyces cerevisiae Hansen)的浓度为108cfu/ml。
其中，所述药物的剂型包括片剂、胶囊剂、散剂、颗粒剂或口服剂。
本发明还提供了酿酒酵母(Saccharomyces cerevisiae Hansen)在制备治疗和/或改善肠炎药物中的应用。
本发明还提供了酿酒酵母(Saccharomyces cerevisiae Hansen)在制备降低血清谷丙转氨酶和/或血清炎性细胞因子浓度的药物中的应用。
有益效果：与现有技术相比，本发明具有如下显著优点：1、首次证实了酿酒酵母对NAFLD有明显的改善作用；2、本发明后续可应用于NAFLD治疗益生菌制剂的储备，在NAFLD的临床预防和治疗方面具有潜在应用价值。
附图说明
图1为酿酒酵母小鼠实验设计示意图；
图2为各组小鼠的体重变化情况；
图3A为各组小鼠脾脏的代表性图像，图3B为各组小鼠脾脏重量统计图；
图4A为各组小鼠肝脏的代表性图像，图4B为各组小鼠肝脏重量统计图；
图5A为各组小鼠结肠的代表性图像，图5B为各组小鼠结肠长度统计图；
图6A为各组小鼠肝切片H&E染色的代表图像，图6B为各组小鼠肝脏NAFLD病变程度的NAS评分；
图7A为各组小鼠结肠H&E染色的代表图像，图7B为各组小鼠结肠病理学评分；
图8为各组小鼠血清谷丙转氨酶水平(U/L)；
图9A为各组小鼠血清IL-1β水平(pg/ml)；图9B为各组小鼠血清IL-6水平(pg/ml)；
图10为各组小鼠脾脏巨噬细胞Mφ和肝脏巨噬细胞Mφ水平；
图11为各组小鼠脾脏、淋巴结和结肠中的髓系抑制细胞MDSC水平。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1酿酒酵母的培养和小鼠灌胃储备液的制备
1)本实验的酿酒酵母菌株(Saccharomyces cerevisiae Hansen)购自北纳生物——河南省工业微生物菌种工程技术研究中心(https://www.bncc.com/)，编号为BNCC336054。依据说明书，在30℃摇床中以灭菌的YM培养基(青岛高科技工业园海博生物技术有限公司)好氧培养。
2)24小时后，在4℃以500g离心2min，弃掉培养基，用灭菌好的1×PBS(PH值为7.4)洗涤两遍菌落，离心，重新用PBS重悬后用细胞计数板用细胞计数仪计数后，调整PBS中总的酿酒酵母个数为108cfu/ml。这些操作全程在超净台中进行，最后用无菌EP管进行分装，作为小鼠灌胃储备液。
实施例2酿酒酵母治疗HFD饮食小鼠非酒精性脂肪肝NAFLD疾病的研究
1)小鼠饲养：5-6周龄健康雄性C57BL/6小鼠(来自南京大学模式动物所)，饲养在南京大学医学院的常规和无病原体设施中。所有小鼠都为SPF级，饲养条件为温度23±2℃，湿度为55±5％。采取12小时光照/黑暗交替，保证小鼠正常饮水。小鼠饮食和研究符合ARRIVE指南清单2.0的各个方面要求。购买小鼠后用维持饲料适应性喂养一周后用于实验。实验方法遵循江苏省实验动物管理委员会的相关规定。所有实验步骤都得到了南京大学医学院动物实验伦理标准委员会的批准。
2)分组：小鼠适应性喂养后均分为3组(n＝7)：Con组，又叫untreated组，采用维持饲料喂养，不作其它处理；HFD组，又叫模型组，采用60％脂肪供能的高脂饲料(购自江苏省协同医药生物工程有限责任公司)喂养，平均20g饲料/只/天；如图1所示，第三组(HFD+SC)进行和模型组相同饮食，从第7周开始，每隔一天灌胃一次如上所述的酿酒酵母PBS混悬液(酿酒酵母个数为108cfu/ml)进行治疗，每次每只灌胃100μL，治疗持续5周。每隔一周，称重所有小鼠。
3)组织取样：第12周时，小鼠摘眼球取外周血，置于1.5mL无酶EP管中静置30min，然后2000rpm离心20min，血清50μL一管分装到200μL EP管，存放于-80℃。取血后，小鼠因颈椎脱位而被安乐死。将脾脏、肝脏、结肠、肠系膜淋巴结精确解剖，进行后续的称量、拍照、切分等处理，并储存于-80℃等待进一步的分析。
4)组织细胞分离
对于脾脏，用注射器背部研磨，细胞滤网过滤一遍，再用滤布过滤到离心管中，300g离心5min，弃上清，加ACK裂解红细胞90s，PBS终止，离心弃上清，PBS重悬，即得到分离的细胞，淋巴结研磨后过滤即可。将用于做流式实验的肝脏放到EP管里剪碎，加消化培养基(RPMI 1640+5％FBS+0.2％ CollagenaseⅣ+1mg/ml DNase I)，37℃摇床30min，用细胞滤网边研磨边滤到6孔板里，再用滤布过滤到新的EP管里，30g离心5min(让组织沉淀)，取上清到新的EP管里，800g离心5min，弃上清，用ACK裂解完红细胞即可。将用于做流式实验的结肠切成1cm的小段，放到EP管中，加入HBSS培养基，37℃摇20min，得到的悬液即含有黏膜微生物的悬液，弃掉加入PBS冲洗，弃掉PBS后用剪刀剪成肉糜状，加入消化培养基(RPMI 1640+5％FBS+0.05％ CollagenaseⅧ+5U/ml DNase I)，37℃摇30min后过滤，即得分离的结肠细胞悬液。
5)流式细胞实验
将上述组织的单细胞悬液吸到流式管中，调整细胞密度，洗涤后，将单细胞与荧光偶联的一抗在4℃下在黑暗中预孵育30分钟。然后，在FACS Calibur流式细胞仪(BDBiosciences，USA)上进行了流式细胞实验。结果由FlowJo软件进行分析。用到以下抗体：FITC-conjugated anti-mouse/human CD11b(BioLegend,#101206，USA)，APC-conjugatedanti-mouse F4/80(BioLegend,#157306，USA)，APC anti-mouse Ly-6G/Ly-6C(Gr-1)Antibody(BioLegend,#108412，USA)，APC anti-mouse CD4 Antibody(BioLegend,#100412，USA)，FITC anti-mouse CD3 Antibody(BioLegend,#100203，USA)，APC anti-mouse CD11c Antibody(BioLegend，#117310，USA)，FITC anti-mouse I-A/I-E Antibody(BioLegend，#107605，USA)。
6)H&E染色
将小鼠的一部分结肠和肝脏固定在4％多聚甲醛中，包埋在石蜡中，制成石蜡切片，然后用苏木精和伊红(H&E)染色，在显微镜(Nikon ECLIPSE Ti-U)下观察组织形态并拍照。
7)血清生化指标检测和ELISA
使用丙氨酸氨基转移酶测定试剂盒(Rayto，S03030)，在全自动生化分析仪(Rayto，Chemray 800)上进行检测。接下来，使用IL-1beta Mouse Uncoated ELISA Kit(Thermo Fisher Scientific，88-7013-88)和IL-6Mouse Uncoated ELISA Kit(ThermoFisher Scientific，88-7064-88)，按试剂盒说明书进行操作。
8)实验结果：
8.1)体重曲线：与chow饮食的正常小鼠相比，HFD饮食的小鼠表现出体重上的显著增加，而灌胃酿酒酵母的HFD饮食小鼠则从第七周开始，体重明显得到改善甚至出现回复迹象(p＝0.0053)，体重有统计学差异(p<0.05)(图2)；
8.2)各组脾脏的代表性图像及重量统计图：炎性会引起脾脏肿大，与untreated组相比，HFD组脾脏明显肿大，重量增加；而HFD+SC组显著改善(p＝0.0117)，脾脏重量具有统计学意义(p<0.05)(图3)；
8.3)各组肝脏的代表性图像及重量统计图：NAFLD会引起肝脏脂质积累，导致肝脏重量增加、颜色变浅。与untreated组相比，HFD组肝脏重量增加，颜色变浅；HFD+SC组则明显改善了肝重和脂肪肝的颜色，颜色明显变深，肝脏重量具有统计学意义(p＝0.0003<0.05)(图4)；
8.4)各组结肠的代表性图像及长度统计图：NAFLD往往伴随着肠炎，造成结肠的不正常缩短。与untreated组相比，HFD组结肠长度明显缩短，酿酒酵母治疗则改善了结肠的不正常缩短(p<0.0001)，结肠长度具有统计学意义(p<0.05)(图5)；
8.5)肝切片苏木精和伊红(H&E)染色的代表图像和反映肝脏NAFLD病变程度的NAS评分：与untreated组相比，HFD组肝细胞产生了明显的气球样变和脂肪变，酿酒酵母治疗后发生明显改善；对肝脏NAFLD病变程度进行NAS评分，可以看到HFD组的肝脏病变严重，酿酒酵母治疗后明显改善(p<0.0001)具有统计学意义(p<0.05)(图6)；
8.6)结肠H&E染色的代表图像和结肠组织学评分：与untreated组相比，HFD组肠屏障遭到破坏，结肠组织学形态发生病理改变，治疗后的HFD+SC组发生明显改善(p＝0.0024)，结肠组织学评分具有统计学意义(p<0.05)(图7)；
8.7)血清生化指标：与untreated组相比，HFD组血清谷丙转氨酶(ALT)显著上升，HFD+SC组经治疗后下降(p＝0.0143)，血清ALT水平具有统计学意义(p<0.05)(图8)；
8.8)血清炎性细胞因子：与untreated组相比，HFD组血清IL-1β和IL-6上升，HFD+SC组经治疗后血清IL-1β和IL-6均有下降趋势但无统计学差异(p＞0.05)(图9)；
8.9)小鼠脾脏、肠系膜淋巴结、肝脏、结肠中的免疫细胞变化：与untreated组相比，HFD组小鼠的脾脏巨噬细胞Mφ明显减少，治疗后回升；与之对应的是HFD组肝脏巨噬细胞Mφ明显增加，治疗后下降(图10)；脾脏和淋巴结的髓系抑制细胞MDSC在NAFLD疾病中都明显下降，治疗后回升；与之对应的是结肠中的髓系抑制细胞MDSC明显升高，治疗后下降(图11)。免疫细胞结果均具有统计学意义(p<0.05)。
Claims (5)
1.酿酒酵母(Saccharomyces cerevisiae Hansen)在制备治疗和/或改善非酒精性脂肪肝药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述酿酒酵母(Saccharomycescerevisiae Hansen)的浓度为108cfu/ml以上。
3.根据权利要求1所述的应用，其特征在于，所述药物的剂型包括片剂、胶囊剂、散剂、颗粒剂或口服剂。
4.酿酒酵母(Saccharomyces cerevisiae Hansen)在制备治疗和/或改善肠炎药物中的应用。
5.酿酒酵母(Saccharomyces cerevisiae Hansen)在制备降低血清谷丙转氨酶和/或血清炎性细胞因子浓度的药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310295670.XA CN116350667A (zh) | 2023-03-24 | 2023-03-24 | 酿酒酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310295670.XA CN116350667A (zh) | 2023-03-24 | 2023-03-24 | 酿酒酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116350667A true CN116350667A (zh) | 2023-06-30 |
Family
ID=86935723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310295670.XA Pending CN116350667A (zh) | 2023-03-24 | 2023-03-24 | 酿酒酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116350667A (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806979A (zh) * | 2022-06-29 | 2022-07-29 | 杭州师范大学附属医院(杭州市第二人民医院) | 一种治疗非酒精性脂肪性肝病的益生菌复合物及其应用 |
-
2023
- 2023-03-24 CN CN202310295670.XA patent/CN116350667A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806979A (zh) * | 2022-06-29 | 2022-07-29 | 杭州师范大学附属医院(杭州市第二人民医院) | 一种治疗非酒精性脂肪性肝病的益生菌复合物及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| 刘巧红;赵瑜;胡义扬;: "调节肠道菌群治疗非酒精性脂肪肝的研究进展", 世界中医药, vol. 15, no. 07, pages 1075 - 1079 * |
| 王丽;李显辉;: "益生菌对大鼠非酒精性脂肪肝的治疗作用及机制", 世界华人消化杂志, no. 36, pages 1285 - 1287 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11291694B2 (en) | Composition for treating or preventing metabolic disease, containing, as active ingredient, extracellular vesicles derived from Akkermansia muciniphila bacteria | |
| US11744847B2 (en) | Use of beta-1,3-glucan for modulating immune function and treating intestinal inflammation | |
| Javadi et al. | Pro-and prebiotic effects on oxidative stress and inflammatory markers in non-alcoholic fatty liver disease | |
| KR20160036016A (ko) | 발효식품에서 유래된 세포밖 소포체를 포함하는 조성물 및 이의 용도 | |
| US20050159483A1 (en) | Method of using punicic acid to enhance immune response and prevent metabolic disorders | |
| HUE034628T2 (hu) | Emberi és/vagy állati élelmiszer készítmény, ennek alkalmazásai, élesztõk | |
| CN114344325A (zh) | 脆弱拟杆菌及其两性离子荚膜多糖在制备用于防治生殖泌尿系统肿瘤的药物中的应用 | |
| CN113073126A (zh) | 亚麻籽活性多肽在制备预防、干预/治疗结肠炎产品中应用 | |
| JP2021052753A (ja) | 健康な妊産婦の母乳由来嫌気性ヒト菌株及びそれを用いた代謝性疾患の予防又は治療方法 | |
| Mao et al. | Intestinal immune response to oral administration of Lactobacillus reuteri R2LC, Lactobacillus plantarum DSM 9843, pectin and oatbase on methotrexate-induced enterocolitis in rats | |
| KR101391911B1 (ko) | 증체율 및 면역기능이 향상된 봉독조성물 | |
| Du et al. | Blueberry and blackberry anthocyanins ameliorate metabolic syndrome by modulating gut microbiota and short-chain fatty acids metabolism in high-fat diet-fed C57BL/6J mice | |
| KR100613625B1 (ko) | 면역기능 향상을 위한 약제학적 조성물 및 복령 추출물 | |
| CN116350667A (zh) | 酿酒酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 | |
| JP2018070568A (ja) | 非アルコール性脂肪性肝疾患治療又は予防剤及び非アルコール性脂肪性肝疾患予防用食品 | |
| WO2018084224A1 (ja) | 非アルコール性脂肪性肝疾患治療又は予防剤及び非アルコール性脂肪性肝疾患予防用食品 | |
| WO2022135503A1 (zh) | 阿伦酸在制备治疗肝纤维化药物中的用途 | |
| WO2022089591A1 (zh) | 氨基葡萄糖在制备治疗非酒精性脂肪性药物中的应用 | |
| EP4248964A1 (en) | Pharmaceutical composition for treating sepsis, and use thereof | |
| CN116832070A (zh) | 扣囊复膜孢酵母在制备治疗和/或改善非酒精性脂肪肝药物中的应用 | |
| Fukasawa et al. | Evaluation of Fermented Product, PS-B1, Obtained from Soybean Milk Using Lactic Acid Bacteria in a Stelic Animal Model (STAM?) of Nonalcoholic Steatohepatitis-A Preliminary Study. | |
| CN117797176A (zh) | 双胞梭菌在制备治疗非酒精性脂肪性肝病药物中的应用和一种药物 | |
| KR20230083871A (ko) | 레몬 껍질 발효물을 포함하는 항비만 조성물 | |
| CN116270752A (zh) | Parasutterella菌属在制备治疗肥胖及其相关代谢疾病的产品中的应用 | |
| TWI609691B (zh) | 台灣冬蟲夏草在第一型糖尿病及其併發症上的醫藥用途 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |