POLYPEPTIDE FRAGMENT C (MP-C) AND USE THEREOF

Abstract

A polypeptide fragment C (MP-C) has an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 9 is Tyr, Val, Gly, Ser, or Gln, an amino acid Xaa at position 20 is Ser, Gln, Glu, or Tyr, an amino acid Xaa at position 30 is Asn, Thr, Ser, Pro, or Leu, and an amino acid Xaa at position 42 is Gly, Arg, Met, or absent. The MP-C can significantly improve the colonic pathologic morphology and decrease a disease activity index (DAI) and a colonic histopathologic score 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 C (MP-C) or a pharmaceutically acceptable salt thereof, wherein an amino acid sequence of the MP-C is set forth in SEQ ID NO: 1.

2. The MP-C or the pharmaceutically acceptable salt thereof according to claim 1, wherein in the amino acid sequence set forth in SEQ ID NO: 1, an amino acid Xaa at position 9 is Tyr, Val, Gly, Ser, or Gln; an amino acid Xaa at position 20 is Ser, Gln, Glu, or Tyr; an amino acid Xaa at position 30 is Asn, Thr, Ser, Pro, or Leu; and an amino acid Xaa at position 42 is Gly, Arg, Met, or absent.

3. A method of treating inflammatory bowel disease (IBD), comprising administering the MP-C or the pharmaceutically acceptable salt thereof 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-C or the pharmaceutically acceptable salt thereof 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-C or the pharmaceutically acceptable salt thereof 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 preparation of the anti-IBD drug is used for reducing a disease activity index (DAI) of the IBD.

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

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

9. The method according to claim 3, wherein the preparation of the anti-IBD drug is used for down-regulating an expression level of colonic interferon-γ (IFN-γ) in the IBD.

10. The method according to claim 3, wherein a dosage form of the anti-IBD drug 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 DAI score comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C experimental group (up to the end of the experiment), where compared with the model group, *P<0.05, **P<0.01, and ***P<0.001; compared with the blank control group, .sup.###P<0.001; and one-way analysis of variance (ANOVA) is conducted for significance test;

[0023] FIG. 3 is a colon length comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C 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 ANOVA is conducted for significance test;

[0024] FIG. 4A shows histopathological micrographs of colons of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C experimental group (HE staining 20× microscopy; A. blank control group, B. model group, C. MIMP positive control group, and D. MP-C experimental group);

[0025] FIG. 4B 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<0.05; and one-way ANOVA is conducted for significance test); and

[0026] FIG. 5 is a colonic IFN-γ expression level comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C 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<0.001; and one-way ANOVA is conducted for significance test.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] 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.

[0028] 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) o-tolidine Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) Glacial acetic acid Sinopharm (Beijing, China) 30% Hydrogen peroxide solution Sinopharm (Beijing, China) Tissue grinder Shanghai Jingxin Industrial Development Co., Ltd. (Shanghai, China) IFN-γ enzyme-linked Shanghai Boguang Biotechnology Co., Ltd. (Shanghai, immunosorbent assay (ELISA) kit China)

Example 1: Experiment on an Intervention Effect of MP-C on DSS-induced IBD in Mice

[0029] The MP-C used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 9 was Val, an amino acid Xaa at position 20 was Tyr, an amino acid Xaa at position 30 was Ser, and an amino acid Xaa at position 42 was Arg, namely,

TABLE-US-00002 THTVGSYFVVQNGYVGAFSYALGNSEYAMSSPLGSLDGRTTRYNLL.

1. Experimental Method

1.1 Establishment of an Acute IBD Mouse Model

[0030] 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:

[0031] blank control group: the mice were each intragastrically administered with water every day at a volume of 0.4 mL/20 g;

[0032] 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;

[0033] 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

[0034] MP-C 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-C 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-C solution of a mass fraction of 50 μg/kg (at a volume of 0.4 mL/20 g) every day.

[0035] 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 DAI Scoring and Sampling

[0036] After DSS induction, the body weight changes, activities, and feces viscosity of the mice in each group were recorded every day. A small amount of feces was collected, and a solution of 10 g/L o-tolidine in glacial acetic acid and 3% hydrogen peroxide were sequentially added dropwise, and color development results were observed to determine an occult blood status of mouse feces. After comprehensive evaluation, DAI scoring was conducted according to the scoring criteria shown in Table 2. 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 was rinsed off, and then the colon was stored in 4% paramethanol or frozen at −80° C.

TABLE-US-00003 TABLE 2 Fecal occult DAI score Body weight loss Fecal characteristic blood/hematochezia 0 — Normal − 1 0%-5% + 2  5%-10% Loose ++ 3 11%-15% +++ 4 >15% Watery Hematochezia Notes: The DAI score is an arithmetic mean value of the three scores of body weight, fecal characteristic, and fecal occult blood.

1.3 Histopathological Evaluation

[0037] 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:

[0038] 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 ELISA Experiment

[0039] The colon sample frozen at −80° C. in step 1.2 was placed in an EP tube, PBS and magnetic beads were added, and then the colon sample was subjected to ultrasonic homogenization in a tissue grinder; and a resulting homogenate was centrifuged, and a resulting supernatant was collected. A commercial ELISA kit was used to determine an expression level of the proinflammatory cytokine IFN-γ in the colon sample. Appropriate primary and secondary antibodies were used according to the instructions, an o-phenylenediamine (OPD) chromogenic solution was used for color development, and after the reaction was terminated, reading was conducted on a microplate reader at a wavelength of 490 nm, with three replicate wells for each sample.

1.5 Statistical Analysis

[0040] 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

[0041] 2.1 The MP-C intervention significantly reduced the DAI of IBD mice.

[0042] 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. In the absence of drug intervention, the fecal conditions of the mice in the model group continued to deteriorate; and in the MP-C experimental group and the MIMP positive control group, the one-week intervention of MP-C or MIMP prevented the significant decrease in body weight of the mice, improved the fecal characteristics and occult blood of the mice, and significantly suppressed the increase in DAI score of the mice, thereby improving the symptoms of IBD in the DSS-induced mice. As shown in FIG. 2, the DAI was as follows:

[0043] the blank control group: 0.0±0.0; the model group: 3.7±0.6; the MIMP positive control group: 2.7±0.7; and the MP-C experimental group: 2.0±0.3.

2.2 The MP-C intervention significantly improved the pathologic colon shortening of IBD mice.

[0044] FIG. 3 is a colon length comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C experimental group, and it can be seen that the colon length (6.4±0.5) of mice in the model group was significantly smaller than the colon length (9.5±0.5) of mice in the blank control group (compared with the blank control group, .sup.##P<0.01), and the colon shortening of mice in the MP-C experimental group (colon length: 9.2±0.2) was significantly different from the colon shortening of mice in the model group (compared with the model group, **P<0.01, indicating a significant difference), indicating that the MP-C intervention can significantly reverse this shortening with a comparable effect to the MIMP positive control group (colon length: 9.5±0.3), thereby improving the pathologic colon morphology of IBD mice.

2.3 The MP-C intervention significantly reduced the colonic histopathologic score of IBD mice.

[0045] FIG. 4A shows histopathological micrographs of colons of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C experimental group (HE staining 20× microscopy; A. blank control group, B. model group, C. MIMP positive control group, and D. MP-C experimental group); and FIG. 4B 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).

[0046] The histopathologic score was as follows: the blank control group: 0.0±0.0; the model group: 5.6±0.7; the MIMP positive control group: 1.4±0.7; and the MP-C experimental group: 1.2±0.2.

[0047] It can be seen from the colonic histopathologic score results that the MIMP intervention and the MP-C intervention both can significantly reduce the colonic histopathologic score of IBD mice. In the MP-C 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, revealing that the MP-C intervention can improve the large-area ulcer of the colonic mucosa induced by DSS, reduce the infiltration of lymphocytes and neutrophils to some extent, and further interfere with the occurrence of IBD.

2.4 The MP-C intervention significantly down-regulated the expression of colonic IFN-γ in IBD mice.

[0048] The expression of colonic cytokines was detected by ELISA. FIG. 5 is a colonic IFN-γ expression level comparison chart of mice in the blank control group, the model group, the MIMP positive control group, and the MP-C experimental group (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). The expression level of IFN-γ in each group was as follows: the blank control group: 889.2±74.6; the model group: 1223.1±41.5; the MIMP positive control group: 1011.4±79.5; and the MP-C experimental group: 1068.4±61.2.

[0049] The results show that the MP-C intervention can significantly suppress the increase of the proinflammatory cytokine IFN-γ in DSS-induced IBD mice, which is consistent with the results of the MIMP positive control group, indicating that the MP-C shows a comparable effect of improving intestinal inflammation in IBD mice to MIMP.

Example 2

[0050] The reagents, materials, devices, and experimental method used in this example were the same as those in Example 1, except that the MP-C used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 9 was Tyr, an amino acid Xaa at position 20 was Ser, an amino acid Xaa at position 30 was Thr, and an amino acid Xaa at position 42 was Gly, namely, THTVGSYFYVQNGYVGAFSSALGNSEYAMTSPLGSLDGRTTGYNLL.

Example 3

[0051] The reagents, materials, devices, and experimental method used in this example were the same as those in Example 1, except that the MP-C used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 9 was Gln, an amino acid Xaa at position 20 was Glu, an amino acid Xaa at position 30 was Pro, and an amino acid Xaa at position 42 was Met, namely, THTVGSYFQVQNGYVGAFSEALGNSEYAMPSPLGSLDGRTTMYNLL.

Example 4

[0052] The reagents, materials, devices, and experimental method used in this example were the same as those in Example 1, except that the MP-C used in this example had an amino acid sequence shown in SEQ ID NO: 1, in which an amino acid Xaa at position 9 was Gly, an amino acid Xaa at position 20 was Gln, an amino acid Xaa at position 30 was Leu, and an amino acid Xaa at position 42 was absent, namely,

TABLE-US-00004 THTVGSYFGVQNGYVGAFSQALGNSEYAMLSPLGSLDGRTTYNLL.

[0053] 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 MP-C of the present disclosure can significantly improve the colonic pathologic morphology of the IBD mice and decrease the DAI and colonic histopathologic score of the IBD mice.

[0054] 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.