CN108210886B - Application of Metrnl in preventing and treating ulcerative colitis - Google Patents

Abstract

The invention relates to application of Metrnl in preventing and treating ulcerative colitis. The method adopts Metrnl intestinal epithelium specific knockout mice and corresponding control wild type mice to prepare DSS-induced ulcerative colitis models, and the results show that: the severity of ulcerative colitis of the Metrnl intestinal epithelium specific knockout mouse is obviously higher than that of a control wild type mouse, and the intestinal mucosa erosion degree of the Metrnl intestinal epithelium specific knockout mouse is obviously higher than that of the control wild type mouse, so that the Metrnl protein can be used as a new effective medicament for preventing and treating ulcerative colitis. The invention provides a new method for preventing and treating ulcerative colitis, and the Metrnl protein is an endogenous protein of a human body and has small possible side effect on the human body, so that the Metrnl protein has high safety as a potential medicament.

Description

Application of Metrnl in the prevention and treatment of ulcerative colitis

technical field

The invention relates to the fields of molecular biology and biomedicine, in particular to the application of Metrnl in preventing and treating ulcerative colitis.

Background technique

Inflammatory Bowel Disease (IBD), also known as enteritis, is a group of recurrent inflammatory bowel diseases. Inflammatory bowel diseases mainly include Crohn's disease (CD) and ulcerative colitis (UC). In developed countries, the incidence of IBD reaches 1/1000. In China, the total number of cases of inflammatory bowel disease in the past decade is about 350,000. It is worth noting that the number of patients with the disease is increasing year by year. Compared with the previous 10 years, the total number of cases of inflammatory bowel disease has increased by about 24 times, and the number of patients with Crohn's disease has increased by even more than 15 times. Ulcerative colitis lesions are mainly located in the mucosal layer of the colon, and are mainly ulcers, mostly involving the rectum and distal colon, but can expand proximally, so as to spread throughout the colon, and its important symptoms include diarrhea, pus and blood in the stool, abdominal pain and tenesmus. , The disease has a long course of disease, the severity of the disease varies, and it often recurs. It is related to the incidence of colon cancer. It is often clinically manifested as recurrent and difficult to cure. It is listed as one of the modern intractable diseases by the World Health Organization. Drugs for the treatment of ulcerative colitis include broad-spectrum anti-inflammatory drugs such as sulfasalazine and adrenocortical hormones, as well as immunosuppressive and immunomodulatory agents. Currently, there is no specific drug for the treatment of ulcerative colitis.

The Metrnl protein consists of 311 amino acids in total, and its registration number in NCBI is NM_001004431.1, annotated as meteorin-like molecule, a regulator of human glial cell differentiation. Metrnl proteins have been identified in human, mouse, bovine, chicken, Xenopus laevis and zebrafish.

Chinese patent document CN200980137344.4 discloses the therapeutic use of Metrnl for the preparation of medicines for treating diseases, disorders or damages of the nervous system, and points out that Metrnl is a secreted growth factor that can promote nerve migration, regeneration and differentiation, and can be used for treatment Nervous system disorders in general, particularly for the treatment of disorders involving damage to the brain, brainstem or spinal cord and/or peripheral nerves. Chinese patent ZL201310525184.9 discloses the application of Metrnl protein in the preparation of hypoglycemic drugs or health food. Chinese patent ZL201310525181.5 discloses the application of Metrnl protein in the preparation of hypolipidemic drugs or health food.

However, there is no report on the application of Metrnl protein in the prevention and treatment of ulcerative colitis.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a new use of Metrn1 protein or gene in view of the deficiencies in the prior art.

The second object of the present invention is to provide a method for screening potential substances for preventing and treating ulcerative colitis.

For realizing above-mentioned first purpose, the technical scheme that the present invention takes is:

Application of Metrnl protein or gene or their synergist in preparing medicine for preventing and treating ulcerative colitis.

Application of Metrnl protein or gene or their synergist in preparing medicine for treating ulcerative colitis.

Further, the medicine improves the degree of intestinal mucosal erosion in ulcerative colitis.

Further, the synergist is selected from agonists, up-regulators or stabilizers and the like.

Further, the amino acid sequence of the Metrn1 protein is shown in SEQ ID NO.1.

Further, the medicament may also contain conventional pharmaceutical carriers or pharmaceutically acceptable excipients, and be prepared into various dosage forms in the form of pharmaceutical compositions. For example, it can be prepared into conventional solid preparation drugs such as tablets, powders or capsules; when used for injection, it can be prepared into injection solutions. In various formulations, the weight content of the active ingredient ranges from 0.01% to 99.9%.

For realizing the above-mentioned second purpose, the technical scheme that the present invention takes is:

A method of screening potential substances for the prevention and treatment of ulcerative colitis, comprising the steps of:

a) contacting the candidate substance with a Metrnl protein or gene-containing system;

b) Observing the effect of the candidate substance on the expression and activity of Metrn1 protein or gene, wherein, if the candidate substance can promote the expression of Metrn1 gene or increase the activity of Metrn1 protein, the candidate substance is a potential substance for preventing and treating ulcerative colitis.

The advantages of the present invention are:

The present invention adopts Metrnl intestinal epithelium specific knockout mice and corresponding control wild-type mice to prepare DSS-induced ulcerative colitis model, and the results show that: Metrnl intestinal epithelium specific knockout mice have a significant severity of ulcerative colitis Compared with control wild-type mice, Metrnl intestinal epithelium-specific knockout mice had significantly higher intestinal mucosal erosions than control wild-type mice, indicating that Metrnl protein could be used as a new and more effective drug for the prevention and treatment of ulcerative colitis. The present invention provides a new method for preventing and treating ulcerative colitis, and because Metrnl protein itself is an endogenous protein of human body, its possible side effects to human body are small, so it has high safety as a potential drug.

Description of drawings

Figure 1 is a comparison of body weight changes of two groups of animals under DSS induction.

Figure 2 shows the comparison of the comprehensive scores of the overall indicators of ulcerative colitis in the two groups of animals under DSS induction.

Figure 3 is a picture of the intestinal morphology of the two groups of animals without modeling.

Figure 4 is a picture of intestinal morphology of two groups of animals after DSS modeling for 5 days.

Figure 5 is a comparison of the degree of intestinal mucosal erosion in two groups of animals after DSS induction for 5 days.

Figure 6 is an alignment diagram of the full-length amino acid sequence of Metrn1 of human, rat and mouse.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that those skilled in the art can make various changes or modifications to the present invention after reading the contents described in the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Metrnl protein and gene

The full-length alignment of Metrnl amino acid sequences of human, rat and mouse is shown in Figure 6. Reference may be made to: CNS NeurosciTher. 2014 Apr;20(4):344-354.

Herein, the Metrnl protein used may be naturally occurring, eg, it may be isolated and purified from mammals. In addition, the Metrnl protein can also be artificially prepared, for example, prepared according to conventional genetic engineering techniques. Any suitable Metrnl protein may be suitable for use in the present invention. The Metrnl protein includes the full-length Metrnl protein or a biologically active fragment thereof. Preferably, it can be substantially the same as the amino acid sequence shown in SEQ ID NO.1.

Amino acid sequences of Metrnl proteins formed by substitution, deletion or addition of one or more amino acid residues are also included in the present invention. The Metrnl protein or biologically active fragment thereof includes a portion of conserved amino acid substitution sequences that do not affect its activity or retain a portion of its activity. Appropriate substitution of amino acids is a well-known technique in the art, which can be easily implemented and ensure that the biological activity of a known molecule is not altered. These techniques have enabled those in the art to recognize that, in general, changing a single amino acid in a non-essential amino acid region of a polypeptide does not alter biological activity.

Any biologically active fragment of Metrnl protein can be used in the present invention. Here, the meaning of a biologically active fragment of Metrnl protein refers to a polypeptide that still retains all or part of the function of the full-length Metrnl protein. Typically, the biologically active fragment retains at least 50%, 60% to 99% or 100% of the activity of the full-length Metrnl protein.

The present invention can also employ Metrnl proteins that have modified or improved all or part of their amino acids, eg, Metrnl proteins that can be modified or improved to enhance half-life, effectiveness, metabolism, and/or protein potency. The modified or improved Metrnl protein can be a Metrnl protein conjugate, or a substituted or artificial amino acid thereof. The modified or improved Metrnl protein or gene may have certain differences from the natural Metrnl protein or gene, but can also dilate blood vessels without causing other adverse reactions or toxicity. That is, any variant that does not affect the biological activity of the Metrnl protein or the biological function of the gene can be used in the present invention.

Metrnl synergist and its use

The "Metrnl synergist" includes agonists, up-regulators, stabilizers, etc., and refers to any substance that can improve the activity of Metrnl, improve the stability of Metrnl, up-regulate the expression of Metrnl, and increase the effective time of Metrnl. Substances can be used in the present invention. They can be chemical compounds, small chemical molecules, biomolecules, and the like. The biomolecules can be at the nucleic acid level (including DNA, RNA), at the protein level, or a viral product that upregulates the expression of Metrnl.

Example

1. Method

1. Animals: Metrnl intestinal epithelium-specific knockout mice and corresponding control wild-type mice were used, with 9 to 12 mice in each group. The breeding methods of these mice are detailed in the patent application (application number 201610458874.0) and published Literature (ActaPharmacol Sin. 2016 Aug 22. doi: 10.1038/aps. 2016.70. [Epub ahead ofprint]).

2. Administration of dextran sulfate sodium (DSS): DSS (molecular weight 36000-50000) of MP company was used, prepared at a concentration of 3%, and administered by free drinking to prepare a DSS-induced ulcerative colitis model .

3. Overall index detection: The body weight is measured at 10 am every day, and the weight change is scored (the weight gain and change within 1% is 0 points, the change is 1%-5% is 1 point, the change is 5% -10% is 2 points, change is 10%-15% is 3 points, change is more than 15% is 4 points); observe the feces and anus bleeding of mice, and use graded scoring (no diarrhea is 0, loose stools do not account for 2 for anus, 4 for loose stools; 0 for no bleeding, 2 for moderate bleeding, and 4 for severe bleeding).

4. Morphological examination: tissue sections were stained with HE and observed under a microscope. The degree of each lesion was scored according to a three-level system of mild, moderate and severe. Grade III was the most severe, and - was no obvious lesion. The higher the score, the more severe the lesion. Mucosal erosion: necrosis involving the superficial mucosa is grade I, involving the whole layer is grade II, and if there are two or more superficial mucosal necrosis, it is grade III.

2. Results

1. Before DSS was administered, the average weight of mice in both groups was 27.2g. Under DSS induction, the body weight of the mice decreased significantly; compared with the control wild-type mice, the Metrnl intestinal epithelium-specific knockout mice showed a more significant decrease in body weight (Fig. 1, *P<0.05).

2. Under DSS induction, the overall index comprehensive score was performed according to body weight, feces and anal bleeding, and it was observed that the severity of ulcerative colitis in Metrnl intestinal epithelium-specific knockout mice was significantly higher than that in control wild-type mice (Figure 2, *P<0.05).

3. Morphological test showed that in the absence of DSS induction, no obvious abnormality was found in the intestinal morphological test of the two groups of animals (Figure 3). The degree of mucosal erosion was significantly higher than that in control wild-type mice (Fig. 4, Fig. 5, *P<0.05).

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as It is the protection scope of the present invention.

SEQUENCE LISTING

<110> Shanghai Fengjin Biomedical Technology Co., Ltd.

<120> Application of Metrnl in the prevention and treatment of ulcerative colitis

<130>/

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<170> PatentIn version 3.3

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His Arg Gln Lys Ser Arg Val Phe Gln Pro Ala Pro Glu Asp Ser Gly

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Gly His Gly Glu Phe Leu Phe Thr Gly His Val His Phe Gly Glu Ala

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Claims (4)

1. Application of Metrnl protein or gene in preparation of medicines for improving ulcerative colitis intestinal mucosa erosion degree.
2. 2. The use of claim 1, wherein the Metrnl protein has an amino acid sequence shown in SEQ ID No. 1.
3. 3. The use according to claim 1, wherein the medicament further comprises a conventional pharmaceutical carrier.
4. 4. A method for screening potential substances for improving the degree of intestinal mucosal erosion of ulcerative colitis, which comprises the following steps:

   a) contacting a candidate substance with a system comprising a Metrnl protein or gene;

   b) and observing the influence of a candidate substance on Metrnl protein or gene expression and activity, wherein if the candidate substance can promote the Metrnl gene expression or improve the activity of the Metrnl protein, the candidate substance is a potential substance for improving the intestinal mucosal erosion degree of the ulcerative colitis.

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