WO2022167001A1 - 一种多肽片段d及其应用 - Google Patents

一种多肽片段d及其应用 Download PDF

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WO2022167001A1
WO2022167001A1 PCT/CN2022/080882 CN2022080882W WO2022167001A1 WO 2022167001 A1 WO2022167001 A1 WO 2022167001A1 CN 2022080882 W CN2022080882 W CN 2022080882W WO 2022167001 A1 WO2022167001 A1 WO 2022167001A1
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amino acid
application
polypeptide fragment
bowel disease
inflammatory bowel
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PCT/CN2022/080882
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English (en)
French (fr)
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张彩华
常英
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上海珑欣生物医学科技有限公司
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Priority to US17/737,064 priority Critical patent/US20220402982A1/en
Publication of WO2022167001A1 publication Critical patent/WO2022167001A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to a polypeptide fragment D and its application.
  • IBD Inflammatory bowel disease
  • the lesions are mainly located in the colorectal segment, and involve the mucosa and muscularis mucosae, and the liver and gallbladder, muscle skin and coagulation are more serious.
  • 20-30% of patients with recurrent disease may be transformed into colorectal cancer, which is a very serious intestinal inflammatory disease, which can be divided into ulcerative colitis (UC) and Crohn's disease. (Crohn's disease, CD) two categories.
  • UC ulcerative colitis
  • Crohn's disease, CD two categories.
  • IBD is an intestinal inflammatory reaction caused by abnormal innate immunity and acquired immunity of the intestinal mucosa under the interaction of multiple factors such as environment, heredity, infection and immunity.
  • IBD treatment drugs such as salicylic acid, steroid hormones and immunosuppressants, mainly reduce inflammation and regulate immune disorders to effectively control the onset of the disease.
  • IBD treatment drugs such as salicylic acid, steroid hormones and immunosuppressants.
  • These traditional methods cannot completely cure them, and are often accompanied by some serious adverse reactions, causing serious harm to the quality of life of patients. Therefore, new IBD treatment methods are urgently needed.
  • MIMP Microintegral membrane protein
  • this fragment can significantly improve the inflammatory state of the intestinal tract and prevent intestinal flora imbalance in IBD mice.
  • MIMP is a biological macromolecule composed of 61 amino acids
  • the larger molecular weight is easy to produce immunogenicity, and it is not easy to make medicine, so its clinical application is limited; in addition, the larger molecular weight is not conducive to the industrial production of drugs. From the perspective of medicinal value and economic benefits, further structural modification and transformation of MIMP are carried out to improve the pharmacological activity or/and druggability of the MIMP fragment, thereby facilitating the clinical application and economic benefit of the active fragment.
  • the object of the present invention is to provide a polypeptide fragment D (referred to as MP-D) and its application,
  • the polypeptide fragment D of the present invention can significantly improve the colon pathological morphology of IBD mice, and reduce the colon histopathological score and colonic interferon- ⁇ expression content of IBD mice.
  • the present invention is to provide a polypeptide fragment D having the amino acid sequence shown in SEQ ID No.1.
  • the 9th amino acid Xaa of the amino acid sequence shown in SEQ ID No.1 is Tyr, Val, Gly, Ser, Gln or absent, and the 20th amino acid Xaa is Ser, Gln, Glu, Tyr, Arg or absent, amino acid Xaa at position 25 is Asn, Thr, Ser, Pro, Leu or absent.
  • the present invention also provides an application of the polypeptide fragment D in the preparation of an anti-inflammatory bowel disease drug.
  • the present invention also provides an application of the polypeptide fragment D in the preparation of anti-inflammatory bowel disease food or food additive.
  • the present invention also provides an application of the polypeptide fragment D in the preparation of anti-inflammatory bowel disease health care products.
  • the polypeptide fragment D is used in the preparation of a medicine for improving pathological colon shortening in inflammatory bowel disease.
  • the polypeptide fragment D is used in the preparation of a medicament for reducing the colonic histopathological score of inflammatory bowel disease.
  • the polypeptide fragment D is used in the preparation of a medicine for down-regulating the expression level of colonic interferon- ⁇ in inflammatory bowel disease.
  • the present invention also provides a pharmaceutical preparation comprising the polypeptide fragment D and a pharmaceutically acceptable carrier, excipient or diluent.
  • the dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, suspension, enema, emulsion or powder.
  • the invention establishes an acute inflammatory bowel disease (IBD) mouse model by chemical induction method of Dextran Sulfate Sodium (DSS), and explores polypeptide fragments by means of colon morphology, histopathology and immune factor expression analysis. Whether D(MP-D) can improve the IBD mouse model.
  • D(MP-D) can improve the IBD mouse model.
  • the results of the study showed that at the same dose as MIMP, the intervention of polypeptide fragment D significantly improved the colon pathological morphology of the IBD mouse model, reduced the colon histopathological score of the IBD mouse model, and could improve and interfere with the inflammation in the mice.
  • MP-D fragment has a smaller molecular weight than MIMP, which is also beneficial to the medicine and application of MP-D fragment, revealing the application potential of polypeptide fragment D in the preparation of active products for prevention, intervention and treatment of inflammatory bowel disease.
  • Figure 1 shows the trend of body weight change between the model group and the blank control group, # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001, compared with the blank control group, two independent samples t-test was performed significantly sex test;
  • Figure 2 is a comparison chart of the colon length of mice in the blank control group, the model group, the MIMP positive control group and the MP-D experimental group, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the model group ; ### P ⁇ 0.001, compared with the blank control group, one-way ANOVA was used for significance test;
  • Figure 3 is the histopathological micrographs of the colons of mice in the blank control group, the model group, the MIMP positive control group and the MP-D experimental group (HE staining 20 ⁇ microscopy; A. blank control group; B. model group; C. MIMP positive control group; D.MP-D experimental group);
  • Figure 4 is a comparison chart of the expression levels of colonic IFN- ⁇ in mice in the blank control group, the model group, the MIMP positive control group and the MP-D experimental group, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, Compared with the model group; # P ⁇ 0.05, compared with the blank control group, one-way ANOVA was used for significance test.
  • Example 1 Experiment on the effect of MP-D intervention on DSS-induced inflammatory bowel disease in mice
  • sequence of the polypeptide fragment D (MP-D) used in this example is the amino acid sequence shown in SEQ ID No.1
  • the 9th amino acid Xaa is Gln
  • the 20th amino acid Xaa is Arg
  • the 25th amino acid Xaa is Asn
  • mice When a certain concentration of DSS solution is administered to mice, an acute inflammatory bowel disease model characterized by diarrhea, blood in the stool, ulcers, and granulocyte infiltration can be induced.
  • the experimental grouping followed the principle of randomization, and the mice were randomly divided into stratified groups according to the weight of the mice. Forty healthy male C57BL6 mice were divided into four groups of 10 mice each:
  • Blank control group gavage with water every day, the gavage volume is 0.4mL/20g;
  • Model group gavage with DSS aqueous solution with a mass fraction of 2.5wt% for 7 consecutive days, and the DSS aqueous solution was freshly prepared and replaced every 1 day;
  • MIMP positive control group The mice were given a pre-administration process for one week, that is, the mice were administrated with MIMP solution with a mass fraction of 50 ⁇ g/kg for the first 7 days, and from the 8th day, 2.5wt% DSS aqueous solution (gavage volume is 0.4mL/20g), and MIMP solution with mass fraction of 50 ⁇ g/kg is gavaged to mice (gavage volume is 0.4mL/20g);
  • MP-D experimental group The mice were given a pre-administration process for one week, that is, the mice were administrated with MP-D solution with a mass fraction of 50 ⁇ g/kg for the first 7 days. wt% DSS aqueous solution (the gavage volume was 0.4mL/20g), and the mice were gavaged with MP-D solution with a mass fraction of 50 ⁇ g/kg (the gavage volume was 0.4mL/20g);
  • mice The body weight changes of each group of mice were recorded daily to determine whether the acute inflammatory bowel disease mouse model was successfully established.
  • mice were killed by cervical dislocation, placed on the operating table, the abdominal cavity was exposed, and the intestinal conditions were observed for the presence of congestion, ulcers and adhesions. At the same time, the mouse colon was completely removed from the anus end to the ileocecal end. After measuring the length of the colon, the intestine was dissected along the longitudinal axis, and the intestinal feces were washed and stored in 4% paramethanol or frozen at -80°C.
  • Histopathological sections were performed on the colon samples preserved in 4% paramethanol in step 1.2. After hematoxylin-eosin staining and dehydration, the sections were sealed and examined under a light microscope. Histopathological scoring was performed by two blind examiners. :
  • Evaluation criteria 0 points, no obvious inflammation; 1 point, moderate inflammatory infiltration in the basal layer; 2 points, moderate mucosal hyperplasia or severe inflammatory infiltration; 3 points, severe mucosal hyperplasia, absence of goblet cells; 4 points, crypts Absent or ulcerated.
  • Fig. 1 is the change trend diagram of the body weight of the mice in the model group and the mice in the blank control group. It can be seen that after one week of induction with DSS, the body weight of the mice in the model group decreased significantly (compared with the blank control group, ### P ⁇ 0.001, a significant difference), indicating that the acute inflammatory bowel disease mouse model was successfully established.
  • Figure 2 is a comparison chart of the colon length of mice in the blank control group, the model group, the MIMP positive control group and the MP-D experimental group.
  • Figure 3 is the histopathological micrographs of the colons of mice in the blank control group, model group, MIMP positive control group and MP-D experimental group (HE staining 20 ⁇ microscopy; A. blank control group; B. model group; C. MIMP positive control group; D. MP-D experimental group); and E. Histopathological score comparison chart ( * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with model group; ### P ⁇ 0.001, compared with the blank control group, one-way ANOVA was used for significance test);
  • Histopathological scores of colon were: blank control group 0.0 ⁇ 0.0, model group 7.4 ⁇ 0.6, MIMP positive control group 2.4 ⁇ 0.8, MP-D experimental group 1.3 ⁇ 0.6.
  • colon cytokine IFN- ⁇ was detected by ELISA, as shown in Figure 4, the comparison of the expression of colonic IFN- ⁇ in the blank control group, the model group, the MIMP positive control group and the MP-D experimental group ( * P ⁇ 0.05, * * P ⁇ 0.01, *** P ⁇ 0.001, compared with the model group; # P ⁇ 0.05, compared with the blank control group, one-way ANOVA was used for significance test).
  • IFN- ⁇ The expression levels of IFN- ⁇ were: blank control group 1145.3 ⁇ 89.6, model group 1385.6 ⁇ 99.9, MIMP positive control group 1188.8 ⁇ 65.4, MP-D experimental group 11048.6 ⁇ 139.9.
  • the reagents, materials, equipment, and experimental methods used in this example are the same as those in Example 1, except that the sequence of the polypeptide fragment D (MP-D) used in this example is the amino acid shown in SEQ ID No.1
  • the 9th amino acid Xaa of the sequence is Tyr, the 20th amino acid Xaa is Glu, and the 25th amino acid Xaa is the sequence when Thr is THTVGSYFYVQNGYVGAFSEALGNTEYAMNS.
  • the reagents, materials, equipment, and experimental methods used in this example are the same as those in Example 1, except that the sequence of the polypeptide fragment D (MP-D) used in this example is the amino acid shown in SEQ ID No.1
  • the 9th amino acid Xaa of the sequence is Gly, the 20th amino acid Xaa is Ser, and the 25th amino acid Xaa is Pro, namely THTVGSYFGVQNGYVGAFSSALGNPEYAMNS.
  • the reagents, materials, equipment, and experimental methods used in this example are the same as those in Example 1, except that the sequence of the polypeptide fragment D (MP-D) used in this example is the amino acid shown in SEQ ID No.1
  • the 9th amino acid Xaa of the sequence is absent, the 20th amino acid Xaa is absent, and the 25th amino acid Xaa is the sequence when it does not exist, namely THTVGSYFVQNGYVGAFSALGNEYAMNS.
  • Example 2 to Example 4 were tested according to the experimental method of Example 1, the analysis results were not much different from the results of Example 1, indicating that the polypeptide fragment D of the present invention can significantly improve the colon pathological morphology of IBD mice, Decrease colon histopathological score and colonic interferon- ⁇ expression content in IBD mice.

Abstract

本发明属于生物医药技术领域，具体涉及一种多肽片段D及其应用，所述多肽片段D具有SEQ ID No.1所示的氨基酸序列，其中，第9位氨基酸Xaa为Tyr、Val、Gly、Ser、Gln或不存在，第20位氨基酸Xaa为Ser、Gln、Glu、Tyr、Arg或不存在，第25位氨基酸Xaa为Asn、Thr、Ser、Pro、Leu或不存在。MP-D显著改善了IBD小鼠模型的结肠病理性形态，降低了结肠组织病理学评分和结肠干扰素-γ表达含量，具有干预小鼠炎症性肠病发生的能力。

Description

一种多肽片段D及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种多肽片段D及其应用。
背景技术
炎症性肠病(inflammatory bowel disease，IBD)是一种特发性慢性肠道炎症性疾病，其病变主要位于结直肠段，并累及黏膜及黏膜肌层，严重的还有肝胆、肌肉皮肤及凝血方面的并发症，病情反复发作者20-30％可能转化为结直肠癌，是一种非常严重的肠道炎症性疾病，可分为溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(Crohn's disease,CD)两大类。目前认为，IBD是在环境、遗传、感染和免疫等多因素相互作用下，肠道黏膜固有免疫及获得免疫出现异常而导致的肠道炎性反应，肠黏膜固有层内炎症反应被认为是IBD发病机制的基石。近几十年来，IBD的发病率呈现逐年上升趋势，而传统的IBD治疗药物，如水杨酸类、类固醇激素类及免疫抑制剂等主要是减轻炎症和调节免疫紊乱，以有效控制疾病发作，但这些传统方法无法使其彻底根治，并常伴随一些严重不良反应的发生，对患者的生存质量造成严重的危害，因此急切需要新的IBD治疗方法。
近年来，微生态制剂正逐渐成为IBD治疗的新思路，经研究发现此类制剂可改善IBD患者肠道中不同程度的菌群失调。乳酸杆菌L.plantarum是一类较为常见的益生菌，研究发现其可在肠道内通过黏 附和定植作用抑制致病菌的损伤，调节免疫缺陷小鼠的肠道通透性，进而干预结肠炎的发展，而微小膜蛋白(micro integral membrane protein,MIMP)则是L.plantarum CGMCC 1258菌株分离出的能与侵袭性致病大肠埃希菌竞争性粘附于肠上皮细胞的活性多肽片段，序列为SEQ ID No.2所示：THTVGSYFSVQNGYVGAFSQALGNSEYAMNSPLGSLDGRTTMYNLLGVKYLFAREDQLKKQ，此片段能显著改善肠道的炎症状态，预防IBD小鼠肠道菌群失调。然而，因MIMP是由61个氨基酸组成的生物大分子，较大的分子量易产生免疫原性，不易成药，使其临床应用受到了限制；另外，较大分子量也不利于药物的工业化生产。从药用价值及经济利益出发，为此对MIMP进行了进一步结构修饰与改造，以提高MIMP片段的药理活性或/和成药性，进而有利于该活性片段的临床应用与经济效益。
发明内容
为了解决现有技术中对炎症性肠病具有改善作用的MIMP存在的易产生免疫原性和不易成药的缺陷，本发明的目的在于提供一种多肽片段D(简称MP-D)及其应用，本发明的多肽片段D能够显著改善IBD小鼠的结肠病理性形态、降低IBD小鼠的结肠组织病理学评分和结肠干扰素-γ表达含量。
本发明是通过如下技术方案实现的：
本发明在于提供一种多肽片段D，具有SEQ ID No.1所示的氨基酸序列。
优选地，所述SEQ ID No.1所示的氨基酸序列的第9位氨基酸 Xaa为Tyr、Val、Gly、Ser、Gln或不存在，第20位氨基酸Xaa为Ser、Gln、Glu、Tyr、Arg或不存在，第25位氨基酸Xaa为Asn、Thr、Ser、Pro、Leu或不存在。
本发明还在于提供一种所述的多肽片段D在制备抗炎症性肠病药物中的应用。
本发明还在于提供一种所述的多肽片段D在制备抗炎症性肠病食品或食品添加剂中的应用。
本发明还在于提供一种所述的多肽片段D在制备抗炎症性肠病保健品中的应用。
优选地，所述多肽片段D在制备改善炎症性肠病的病理性结肠缩短的药物中的应用。
优选地，所述多肽片段D在制备降低炎症性肠病的结肠组织病理学评分的药物中的应用。
优选地，所述多肽片段D在制备下调炎症性肠病结肠干扰素-γ表达含量的药物中的应用。
本发明还在于提供一种药物制剂，包括所述的多肽片段D以及药学上可接受的载体、赋形剂或稀释剂。
优选地，所述药物制剂的剂型为注射剂、胶囊剂、片剂、颗粒剂、混悬剂、灌肠剂、乳剂或散剂。
本发明的有益效果：
本发明通过葡聚糖硫酸钠(Dextran sulfate sodium,DSS)化学诱导法建立急性炎症性肠病(IBD)小鼠模型，以结肠形态学、组 织病理学及免疫因子表达分析的手段，探索多肽片段D(MP-D)是否对IBD小鼠模型有改善作用。研究结果表明，在与MIMP同等剂量下，多肽片段D的干预显著改善了IBD小鼠模型的结肠病理性形态，降低了IBD小鼠模型的结肠组织病理学评分，具有可改善及干预小鼠炎症性肠病发生的能力。且MP-D片段相比于MIMP具有更小的分子量，也有利于MP-D片段的成药与应用，揭示了多肽片段D在制备预防、干预及治疗炎症性肠病活性产品中的应用潜力。
本发明所用缩写具体含义如下：
Thr为苏氨酸；His为组氨酸；Val为缬氨酸；Gly为甘氨酸；Ser为丝氨酸；Phe为苯丙氨酸；Asn为天冬酰胺；Tyr为酪氨酸；Ala为丙氨酸；Leu为亮氨酸；Glu为谷氨酸；Met为甲硫氨酸；Pro为脯氨酸；Asp为天冬氨酸；Arg为精氨酸；Lys为赖氨酸；Gln为谷氨酰胺。
附图说明
图1为模型组小鼠与空白对照组小鼠的体重变化趋势图， #P<0.05， ##P<0.01， ###P<0.001，与空白对照组比较，两独立样本t检验进行显著性检验；
图2为空白对照组、模型组、MIMP阳性对照组和MP-D实验组的小鼠结肠长度对比图， *P<0.05, **P<0.01， ***P<0.001，与模型组比较； ###P<0.001，与空白对照组比较，单因素方差分析进行显著性检验；
图3为空白对照组、模型组、MIMP阳性对照组和MP-D实验组小鼠结肠组织病理学显微图(HE染色20×镜检；A.空白对照组；B.模型组；C.MIMP阳性对照组；D.MP-D实验组)；
以及E.组织病理学评分对比图( *P<0.05， **P<0.01， ***P<0.001，与模型组比较； ###P<0.001，与空白对照组比较，单因素方差分析进行显著性检验)；
图4为空白对照组、模型组、MIMP阳性对照组和MP-D实验组小鼠结肠IFN-γ表达含量的对比图， *P<0.05, **P<0.01， ***P<0.001，与模型组比较； #P<0.05，与空白对照组比较，单因素方差分析进行显著性检验。
具体实施方式
下面将结合本发明实施例中的附图，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。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，决不作为对本发明及其应用或使用的任何限制。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。
实施例中所使用的试剂、材料与设备如表1所示：
表1
名称 制造商
雄性C57BL6小鼠，清洁级 上海斯莱克公司(Shanghai,China)
葡聚糖硫酸钠(DSS) MP Biomedicals(CA,United States)
磷酸缓冲溶液(PBS) 上海博光生物科技有限公司(Shanghai,China)
MIMP 苏州强耀生物公司(Suzhou,China)
4％多聚甲醛 上海博光生物科技有限公司(Shanghai,China)
组织研磨仪 上海净信实业发展有限公司(Shanghai,China)
干扰素-γ酶联免疫反应试剂盒 上海博光生物科技有限公司(Shanghai,China)
实施例1：MP-D的干预对DSS诱导小鼠炎症性肠病作用的实验
本实施例所用多肽片段D(MP-D)的序列为SEQ ID No.1所示的 氨基酸序列的第9位氨基酸Xaa为Gln，第20位氨基酸Xaa为Arg，第25位氨基酸Xaa为Asn时的序列，即THTVGSYFQVQNGYVGAFSRALGNNEYAMNS。
1.实验方法
1.1急性炎症性肠病小鼠模型的建立
当给予小鼠一定浓度的DSS溶液即可诱导以腹泻、便血、溃疡、粒细胞浸润为特征的急性炎症性肠病模型。实验分组遵循随机性原则，依据小鼠的体重进行分层随机分组。将40只健康雄性C57BL6小鼠，按每组10只分成四组：
空白对照组：每天以水灌胃，灌胃体积为0.4mL/20g；
模型组：以质量分数为2.5wt％的DSS水溶液灌胃，连续饮用7天，且DSS水溶液为新鲜配制的，每隔1天更换1次；
MIMP阳性对照组：先给予小鼠一周的预给药进程，即前7天用质量分数为50μg/kg的MIMP溶液灌胃小鼠，第8天开始，每天给小鼠灌胃2.5wt％的DSS水溶液(灌胃体积为0.4mL/20g)，并以质量分数为50μg/kg的MIMP溶液灌胃小鼠(灌胃体积为0.4mL/20g)；
MP-D实验组：先给予小鼠一周的预给药进程，即前7天用质量分数为50μg/kg的MP-D溶液灌胃小鼠，第8天开始，每天给小鼠灌胃2.5wt％的DSS水溶液(灌胃体积为0.4mL/20g)，并以质量分数为50μg/kg的MP-D溶液灌胃小鼠(灌胃体积为0.4mL/20g)；
每日记录各组小鼠体重变化以确定急性炎症性肠病小鼠模型是否成功建立。
1.2样本采集
颈椎脱臼处死小鼠，将其置于手术台上，暴露腹腔，观察肠道情况，有无充血、溃疡及粘连情况出现。同时从肛门端至回盲端完整取出小鼠结肠，测量结肠长度后，将肠道沿纵轴剖开，冲洗肠道粪便，保存于4％多聚甲醇中或-80℃冷冻保存。
1.3组织病理学评价
对步骤1.2中保存于4％多聚甲醇的结肠样本进行组织病理切片，苏木精-伊红染色、脱水后将切片密封并在光学显微镜下检查，由两名盲检人员进行组织病理学评分：
评定标准：0分，无明显炎症；1分，基底层中度炎症浸润；2分，粘膜中度增生或重度炎症浸润；3分，粘膜重度增生，杯状细胞不存在；4分，隐窝不存在或溃疡。
1.4酶联免疫吸附测定(ELISA)实验
选取步骤1.2中-80℃冷冻保存的结肠样本于EP管内，加入PBS和磁珠，放入组织研磨仪进行超声匀浆，后离心取上清。使用商业化的ELISA试剂盒测定结肠样本中促炎细胞因子IFN-γ(即干扰素-γ)的表达水平，根据说明书使用合适的一抗及二抗，OPD显色液显色，终止反应后于490nm波长下酶标仪读数，每个样品三复孔。
1.5统计学分析
上述实验方法中的实验数据以
Figure PCTCN2022080882-appb-000001
表示，使用GraphPad Prism(ver.8.0,GraphPad Software Inc.,San Diego,CA,USA)绘制图表，SPSS Program(ver.25.0,SPSS Inc.,Chicago,IL,USA) 进行统计学检验，符合正态性和方差齐性时采用单因素方差分析或两独立样本t检验进行显著性检验。设α＝0.05，P<0.05为差异有统计学显著性。
2.实验结果分析
2.1 MP-D的干预显著改善炎症性肠病小鼠的病理性结肠缩短
图1为模型组小鼠与空白对照组小鼠的体重变化趋势图，可以看出，在给予DSS诱导一周后，模型组小鼠的体重显著下降(与空白对照组比较， ###P<0.001，有显著性差异)，说明急性炎症性肠病小鼠模型建立成功。图2为空白对照组、模型组、MIMP阳性对照组和MP-D实验组的小鼠结肠长度对比图，可以看出，模型组小鼠的结肠长度(5.3±0.6)与空白对照组(结肠长度9.4±0.6)相比显著缩短(与空白对照组比较， ###P<0.001)，而MP-D实验组(8.0±0.9)小鼠相对于模型组小鼠的结肠缩短有明显差异(与模型组比较， ***P<0.001，有显著性差异)，说明MP-D的干预可以显著逆转这种缩短，与MIMP阳性对照组(7.9±0.5)的效果相当，改善了炎症性肠病小鼠的病理性结肠形态。
2.2 MP-D的干预显著降低炎症性肠病小鼠结肠组织病理学评分
图3为空白对照组、模型组、MIMP阳性对照组和MP-D实验组小鼠结肠组织病理学显微图(HE染色20×镜检；A.空白对照组；B.模型组；C.MIMP阳性对照组；D.MP-D实验组)；以及E.组织病理学评分对比图( *P<0.05， **P<0.01， ***P<0.001，与模型组比较； ###P<0.001，与空白对照组比较，单因素方差分析进行显著性检验)；
结肠组织病理学评分为：空白对照组0.0±0.0、模型组7.4±0.6、MIMP阳性对照组2.4±0.8、MP-D实验组1.3±0.6。
可以看出，在结肠组织病理学评分中，MIMP和MP-D的干预均能使炎症性肠病小鼠的结肠组织学评分显著降低。病理学状况也有相应程度的改善，粘膜层上皮结构较为完整，上皮细胞形态结构正常、未见明显炎症的发生，说明MP-D的干预可改善DSS诱导所致的结肠组织粘膜层的大面积溃疡，一定程度减少淋巴细胞与中性粒细胞的浸润，并进一步干预肠道炎症的发生。
2.3 MP-D的干预显著下调炎症性肠病小鼠结肠干扰素-γ(IFN-γ)的表达
ELISA法检测结肠细胞因子IFN-γ表达，如图4为空白对照组、模型组、MIMP阳性对照组和MP-D实验组小鼠结肠IFN-γ表达含量的对比图( *P<0.05， **P<0.01， ***P<0.001，与模型组比较； #P<0.05，与空白对照组比较，单因素方差分析进行显著性检验)。
IFN-γ表达含量为：空白对照组1145.3±89.6、模型组1385.6±99.9、MIMP阳性对照组1188.8±65.4、MP-D实验组11048.6±139.9。
结果表明，MP-D的干预可显著遏制DSS诱导的炎症性肠病小鼠促炎细胞因子IFN-γ的升高，与MIMP阳性对照组的结果一致，说明所述MP-D具有与MIMP相当的改善炎症性肠病小鼠肠道炎症的作用。
实施例2
本实施例中所使用的试剂、材料与设备，以及实验方法均同实施 例1，区别仅仅在于：本实施例所用多肽片段D(MP-D)的序列为SEQ ID No.1所示的氨基酸序列的第9位氨基酸Xaa为Tyr，第20位氨基酸Xaa为Glu，第25位氨基酸Xaa为Thr时的序列，即THTVGSYFYVQNGYVGAFSEALGNTEYAMNS。
实施例3
本实施例中所使用的试剂、材料与设备，以及实验方法均同实施例1，区别仅仅在于：本实施例所用多肽片段D(MP-D)的序列为SEQ ID No.1所示的氨基酸序列的第9位氨基酸Xaa为Gly，第20位氨基酸Xaa为Ser，第25位氨基酸Xaa为Pro时的序列，即THTVGSYFGVQNGYVGAFSSALGNPEYAMNS。
实施例4
本实施例中所使用的试剂、材料与设备，以及实验方法均同实施例1，区别仅仅在于：本实施例所用多肽片段D(MP-D)的序列为SEQ ID No.1所示的氨基酸序列的第9位氨基酸Xaa为不存在，第20位氨基酸Xaa为不存在，第25位氨基酸Xaa为不存在时的序列，即THTVGSYFVQNGYVGAFSALGNEYAMNS。
将实施例2～实施例4按照实施例1的实验方法测试后，其分析结果与实施例1的结果差异不大，说明本发明的多肽片段D能够显著改善IBD小鼠的结肠病理性形态、降低IBD小鼠的结肠组织病理学评分和结肠干扰素-γ表达含量。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体 实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种多肽片段D，其特征在于，具有SEQ ID No.1所示的氨基酸序列。
  2. 根据权利要求1所述的多肽片段D，其特征在于，所述SEQ ID No.1所示的氨基酸序列的第9位氨基酸Xaa为Tyr、Val、Gly、Ser、Gln或不存在，第20位氨基酸Xaa为Ser、Gln、Glu、Tyr、Arg或不存在，第25位氨基酸Xaa为Asn、Thr、Ser、Pro、Leu或不存在。
  3. 一种权利要求1所述的多肽片段D的应用，其特征在于，在制备抗炎症性肠病药物中的应用。
  4. 一种权利要求1所述的多肽片段D的应用，其特征在于，在制备抗炎症性肠病食品或食品添加剂中的应用。
  5. 一种权利要求1所述的多肽片段D的应用，其特征在于，在制备抗炎症性肠病保健品中的应用。
  6. 根据权利要求3所述的应用，其特征在于，在制备改善炎症性肠病的病理性结肠缩短的药物中的应用。
  7. 根据权利要求3所述的应用，其特征在于，在制备降低炎症性肠病的结肠组织病理学评分的药物中的应用。
  8. 根据权利要求3所述的应用，其特征在于，在制备下调炎症性肠病结肠干扰素-γ表达含量的药物中的应用。
  9. 一种药物制剂，其特征在于，包括权利要求1所述的多肽片段D以及药学上可接受的载体、赋形剂或稀释剂。
  10. 根据权利要求9所述的药物制剂，其特征在于，所述药物制剂的剂型为注射剂、胶囊剂、片剂、颗粒剂、混悬剂、灌肠剂、乳剂 或散剂。
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