POLYPEPTIDE FRAGMENT B (MP-B) AND USE THEREOF

Abstract

A polypeptide fragment B (MP-B) has an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 10 is Arg, Phe, Glu, Thr, or absent, an amino acid Xaa at position 16 is Asn, Val, Leu, Gly, or absent, and an amino acid Xaa at position 25 is Ser, Glu, Met, Arg, or absent. The MP-B can significantly improve the colonic pathologic morphology and decrease a colonic histopathologic score and an expression level of colonic interferon-γ (IFN-γ) in an inflammatory bowel disease (IBD) mouse model, and shows the ability to interfere with the occurrence of IBD in mice.

Claims

1. A polypeptide fragment B (MP-B), having an amino acid sequence set forth in SEQ ID NO: 1.

2. The MP-B according to claim 1, wherein in the amino acid sequence set forth in SEQ ID NO: 1, an amino acid Xaa at position 10 is Arg, Phe, Glu, Thr, or absent; an amino acid Xaa at position 16 is Asn, Val, Leu, Gly, or absent; and an amino acid Xaa at position 25 is Ser, Glu, Met, Arg, or absent.

3. A method of treating inflammatory bowel disease (IBD), comprising administering the MP-B according to claim 1 in a preparation of an anti-IBD drug to a patient in need thereof.

4. A method of treating inflammatory bowel disease (IBD), comprising administering the MP-B according to claim 1 in a preparation of an anti-IBD food or food additive to a patient in need thereof.

5. A method of treating inflammatory bowel disease (IBD), comprising administering the MP-B according to claim 1 in a preparation of an anti-IBD health product to a patient in need thereof.

6. The method according to claim 3, wherein the anti-IBD drug is used for improving pathologic colon shortening of the IBD.

7. The method according to claim 3, wherein the anti-IBD drug is used for reducing a colonic histopathologic score of the IBD.

8. A pharmaceutical preparation, comprising the MP-B according to claim 1 and a pharmaceutically acceptable carrier, an excipient, or a diluent.

9. The pharmaceutical preparation according to claim 8, wherein a dosage form of the pharmaceutical preparation is an injection, a capsule, a tablet, a granule, a suspension, an enema, an emulsion, or a powder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows body weight change trends of mice in the model group and the blank control group, where compared with the blank control group, .sup.#P<0.05, .sup.##P<0.01, and .sup.###P<0.001; and the independent two-sample t-test is conducted for significance test;

[0022] FIG. 2 is a colon length comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-B experimental group, where compared with the model group, *P<0.05, **P<0.01, and ***P<0.001; compared with the blank control group, .sup.#P<0.01; and one-way analysis of variance (ANOVA) is conducted for significance test; and

[0023] FIG. 3A shows histopathological micrographs of colons of mice in the blank control group, the model group, the MIMP positive control group, and the MP-B experimental group (HE staining 20×microscopy; A. blank control group, B. model group, C. MIMP positive control group, and D. MP-B experimental group); and

[0024] FIG. 3B shows a histopathologic score comparison chart (compared with the model group, *P<0.05, **P<0.01, and ***P<0.001; compared with the blank control group, .sup.###P<0.001; and one-way ANOVA is conducted for significance test).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The technical solutions in the examples of the present disclosure are clearly and completely described below with reference to the accompanying drawings in the examples of the present disclosure. Apparently, the described examples are merely a part rather than all of the examples of the present disclosure. The following description of at least one exemplary example is merely illustrative, and not intended to limit the present disclosure and application or use thereof in any way. All other examples obtained by a person of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.

[0026] The reagents, materials, and devices used in the examples are shown in Table 1:

TABLE-US-00001 TABLE 1 Name Manufacturer Male C57BL6 mice, clean grade Shanghai Slack (Shanghai, China) DSS MP Biomedicals (CA, United States) Phosphate-buffered saline (PBS) Shanghai Boguang Biotechnology Co., Ltd. (Shanghai, China) MIMP Suzhou Qiangyao Biotechnology Co., Ltd. (Suzhou, China) 4% Paraformaldehyde (PFA) Shanghai Boguang Biotechnology Co., Ltd. (Shanghai, China)

EXAMPLE 1

Experiment on an Intervention Effect of MP-B on DSS-Induced IBD in Mice

[0027] The MP-B used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 10 was Glu, an amino acid Xaa at position 16 was Gly, and an amino acid Xaa at position 25 was Met, namely, SPLGSLDGRETMYNLGGVKYLFARMDQLKKQ.

1. Experimental Method

1.1 Establishment of an Acute IBD Mouse Model

[0028] The administration of a DSS solution with a specified concentration to mice can induce an acute IBD model characterized by diarrhea, hematochezia, ulcer, and granulocyte infiltration. Mice were randomly grouped according to body weights of the mice. 40 healthy male C57BL6 mice were divided into four groups each with 10 mice:

[0029] blank control group: the mice were each intragastrically administered with water every day at a volume of 0.4 mL/20 g;

[0030] model group: the mice were each intragastrically administered with a DSS aqueous solution of a mass fraction of 2.5 wt % consecutively for 7 days, where the DSS aqueous solution was freshly prepared and changed every day;

[0031] MIMP positive control group: the mice were each given a pre-administration process for one week, that is, the mice were each intragastrically administered with an MIMP solution of a mass fraction of 50 μg/kg for the first 7 days, and then from day 8, the mice were each intragastrically administered with a DSS aqueous solution of a mass fraction of 2.5 wt % (at a volume of 0.4 mL/20 g) and an MIMP solution of a mass fraction of 50 μg/kg (at a volume of 0.4 mL/20 g) every day; and

[0032] MP-B experimental group: the mice were each given a pre-administration process for one week, that is, the mice were each intragastrically administered with an MP-B solution of a mass fraction of 50 μg/kg for the first 7 days, and from day 8, the mice were each intragastrically administered with a DSS aqueous solution of a mass fraction of 2.5 wt % (at a volume of 0.4 mL/20 g) and an MP-B solution of a mass fraction of 50 μg/kg (at a volume of 0.4 mL/20 g) every day.

[0033] The body weight changes of mice in each group were recorded every day to determine whether the acute IBD mouse model was successfully established.

1.2 Sampling

[0034] Mice were each sacrificed by cervical dislocation and placed on an operating table, the abdominal cavity was exposed, and the intestinal conditions were observed to determine whether there was congestion, ulcer, and adhesion. A mouse colon between an anus end to an ileocecal end was integrally collected, and a length of the colon was measured; and the colon was dissected along a longitudinal axis, feces therein were rinsed off, and then the colon was stored in 4% paramethanol.

1.3 Histopathological Evaluation

[0035] The colon sample stored in 4% paramethanol in step 1.2 was subjected to histopathological section, stained with hematoxylin-eosin (HE), and dehydrated, obtained sections were sealed and examined under an optical microscope, and the histopathological scoring was conducted by two blind examination operators:

[0036] Scoring criteria: 0: no obvious inflammation; 1: moderate inflammatory infiltration in the basal layer; 2: moderate hyperplasia or severe inflammatory infiltration in the mucosa; 3: severe mucosal hyperplasia and absence of goblet cells; and 4: absence of crypt or ulcer.

1.4 Statistical Analysis

[0037] Experimental data in the above experimental method were expressed as (x±SD), the GraphPad Prism (ver. 8.0, GraphPad Software Inc., San Diego, Calif., USA) was used to plot a chart, the SPSS Program (ver. 25.0, SPSS Inc., Chicago, Ill., USA) was used for statistical test, and one-way ANOVA or independent two-sample t-test were used for significance test when the normality and homogeneity of variances were met. It was assumed that α=0.05, and P<0.05 indicates a statistically significant difference.

2. Experimental Results and Analysis

2.1 The MP-B Intervention Significantly Improved the Pathologic Colon Shortening of IBD Mice.

[0038] FIG. 1 shows weight change trends of mice in the model group and the blank control group, and it can be seen that, after one week of DSS induction, a body weight of mice in the model group decreased significantly (compared with the blank control group, .sup.###P<0.001, indicating a significant difference), indicating that the acute IBD mouse model was successfully established.

[0039] FIG. 2 is a colon length comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-B experimental group, and it can be seen that the colon length (5.3±0.6) of mice in the model group was significantly smaller than the colon length (9.2±0.8) of mice in the blank control group (.sup.###P<0.001), and the colon shortening of mice in the MP-B experimental group (colon length: 8.0±0.5) was significantly different from the colon shortening of mice in the model group (colon length: 5.3±0.6) (compared with the model group, ***P<0.001, indicating a significant difference), indicating that the MP-B intervention can significantly reverse this shortening with a comparable effect to the MIMP positive control group (colon length: 7.6±0.5), thereby improving the pathologic colon morphology of IBD mice.

2.2 The MP-B Intervention Significantly Reduced the Colonic Histopathologic Score of IBD Mice.

[0040] FIG. 3A shows histopathological micrographs of colons of mice in the blank control group, the model group, the MIMP positive control group, and the MP-B experimental group (HE staining 20×microscopy; A. blank control group, B. model group, C. MIMP positive control group, and D. MP-B experimental group); and FIG. 3B shows a histopathologic score comparison chart (compared with the model group, *P<0.05, **P<0.01, and ***P<0.001; compared with the blank control group, .sup.###P<0.001; and one-way ANOVA was conducted for significance test).

[0041] The histopathologic score was as follows: the blank control group: 0.0±0.0; the model group: 7.6±0.6; the MIMP positive control group: 3.1±0.2; and the MP-B experimental group: 1.4±0.6.

[0042] It can be seen from the colonic histopathologic score results that the MIMP intervention and the MP-B intervention both can significantly reduce the colonic histopathologic score of IBD mice (***P<0.001, indicating a significant difference). In the MP-B experimental group, the pathological conditions were improved accordingly, the mucosal epithelial structure was relatively complete, the morphology and structure of epithelial cells were normal, and there was no obvious inflammation, proving that the MP-B intervention can improve the large-area ulcer of the colonic mucosa induced by DSS, reduce the infiltration of lymphocytes and neutrophils, and further interfere with the occurrence of IBD.

EXAMPLE 2

[0043] The reagents, materials, devices, and experimental method used in this example were the same as those in Example 1, except that the MP-B used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 10 was Arg, an amino acid Xaa at position 16 was Asn, and an amino acid Xaa at position 25 was Glu, namely, SPLGSLDGRRTMYNLNGVKYLFAREDQLKKQ.

EXAMPLE 3

[0044] The reagents, materials, devices, and experimental method used in this example were the same as those in Example 1, except that the MP-B used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 10 was Thr, an amino acid Xaa at position 16 was Val, and an amino acid Xaa at position 25 was Ser, namely, SPLGSLDGRTTMYNLVGVKYLFARSDQLKKQ.

EXAMPLE 4

[0045] The reagents, materials, devices, and experimental method used in this example were the same as those in Example 1, except that the MP-B used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 10, an amino acid Xaa at position 16, and an amino acid Xaa at position 25 were each absent, namely, SPLGSLDGRTMYNLGVKYLFARDQLKKQ.

[0046] Examples 2 to 4 were tested according to the experimental method of Example 1, and analysis results were not much different from the results of Example 1, indicating that the intervention of the MP-B of the present disclosure can significantly improve the colonic pathologic morphology of the IBD mouse model and decrease the colonic histopathologic score of the IBD mouse model, and shows the ability to improve and interfere with the occurrence of IBD in mice.

[0047] The objectives, technical solutions, and beneficial effects of the present disclosure are further described in detail in the above specific examples. It should be understood that the above are merely specific examples of the present disclosure, but are not intended to limit the present disclosure. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.