CN112587654A

CN112587654A - Application of mesencephalon astrocyte-derived neurotrophic factor in treatment of ulcerative colitis

Info

Publication number: CN112587654A
Application number: CN202011500878.3A
Authority: CN
Inventors: 沈玉先; 杨琳; 邵伟; 陈滢; 沈玉君
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-04-02
Anticipated expiration: 2040-12-18
Also published as: CN112587654B

Abstract

The invention discloses application of mesencephalon astrocyte-derived neurotrophic factor in treating ulcerative colitis. The research of the invention discovers that the expression of MANF is up-regulated in a human ulcerative colitis patient and a mouse ulcerative colitis model, which indicates that the expression of MANF is increased in ulcerative colitis. Animal experiments show that the MANF can obviously relieve bloody stool caused by ulcerative colitis, increase the weight of a model mouse and improve the diarrhea condition. MANF can reduce inflammatory macrophage infiltration of ulcerative colitis and reduce the expression of inflammatory factors. MANF can be used for treating or ameliorating ulcerative colitis.

Description

Application of mesencephalon astrocyte-derived neurotrophic factor in treatment of ulcerative colitis

Technical Field

The invention relates to application of mesencephalon astrocyte-derived neurotrophic factor in treating ulcerative colitis in the field of biomedicine.

Background

Ulcerative Colitis (UC) is an inflammatory lesion of the intestine characterized by persistent, recurrent inflammation of the intestinal mucosa or lamina propria, which originates in the rectum and invades the proximal colon, with lesions mainly in the mucosa and submucosa, with clinical symptoms of bloody diarrhea, weight loss, mucous stool with tenesmus and abdominal cramps. Ulcerative colitis is considered to be a common disease in economically developed countries and regions, and high-incidence regions mainly include europe, north america and the like, but it is noted that with the development of social economy, the westernization of living habits and dietary habits, and in the last two decades, the incidence rate of ulcerative colitis is rapidly increasing in developing countries and is considered to be an emerging global disease. Because the disease is hidden, easy to recur, expensive in cost, long in time and the like, the disease brings great pain to the mind and body of a patient, and is listed as one of the modern difficult diseases by the world health organization. Ulcerative colitis not only seriously affects the individual quality of life of patients, but also imposes a huge burden on socioeconomic and medical resources.

At present, clinical means for treating ulcerative colitis mainly comprise medicine and operation treatment, and the treatment aims to keep the ulcerative colitis in a long-term static state and avoid occurrence of colon resection and colon cancer. At present, the therapeutic drugs for ulcerative colitis mainly comprise 5-aminosalicylic acid, sulfasalazine, glucocorticoid and immunosuppressant. The anti-TNF-alpha medicines of infliximab and adalimumab have a good curative effect on the treatment of moderate-to-severe ulcerative colitis, and can effectively relieve the symptoms and maintain the rest state of the disease. Colectomies are used in patients with uncontrolled bleeding, perforation and colorectal cancer, and also in patients with refractory severe ulcerative colitis.

The pathogenesis of ulcerative colitis is not completely clear, and at present, genetic susceptibility, external environment stimulation, autoimmune disorder, intestinal flora disorder and other factors are considered to participate in the occurrence and development of ulcerative colitis together. For ulcerative colitis, the existing clinical medicines can only control symptoms but cannot completely cure the ulcerative colitis, and are accompanied by serious side effects, which bring heavy economic and psychological burdens to patients. Furthermore, with prolonged onset of ulcerative colitis, the normal anatomical structure and physiological function of the colon may be compromised, increasing the risk of colitis-associated tumors (CAC) in patients with ulcerative colitis. It is worth noting that no obvious pathological features of inflammatory reaction exist before most colorectal cancers occur, so that it is of great significance to deeply dig the pathophysiological mechanism of the occurrence and development of ulcerative colitis and search for safe and effective new drug treatment targets.

The model of ulcerative colitis induced by Dextran Sodium Sulfate (DSS) is the most common method for establishing mouse ulcerative colitis model by chemical method. In 1985, DSS was first used by japanese researchers to establish animal models of ulcerative colitis. The mechanism of DSS induction of ulcerative colitis may be inhibition of epithelial cell proliferation, destruction of intestinal mucosal barrier, and infiltration of macromolecular substances such as proteins and polysaccharides into intestinal mucosal tissues, which may lead to imbalance of intestinal flora and impaired function of macrophages. In addition, researches prove that the DSS-induced ulcerative colitis model is mediated by Th1/Th2 cell reaction, can obviously cause high expression of TNF-alpha, IFN-gamma and IL-10, and can better simulate the immune response in a human body. The model is simple to operate, only DSS reagent with specific concentration needs to be added into drinking water, the operation is convenient, and the modeling period can be adjusted according to the requirement; the result has good repeatability, can generate symptoms similar to acute and remission stages of the human ulcerative colitis by repeated DSS stimulation, and is an ideal model for simulating the human ulcerative colitis.

Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is a Neurotrophic Factor capable of promoting dopamine neuron survival, also known as ARMET (Arginine Rich, Mutated in Early stage of burners) or ARP (Arginine-Rich Protein), which is isolated from the culture medium of rat Mesencephalic type I astrocytes first cultured in vitro by Petrova equivalent to 2003. The MANF gene is 4.3kb in length, contains 4 exons, and is located on human chromosome 3. The primary transcript of MANF contained 1109bp, encoding a 179 amino acid protein. The N-terminal 21 amino acid signal peptide was used to import newly synthesized MANF into the ER. The secreted form of MANF is a full-length protein without a signal peptide sequence, i.e., 158 amino acids, and has a molecular weight of approximately 18 kDa.

Disclosure of Invention

One technical problem to be solved by the present invention is how to treat and/or prevent and/or alleviate and/or improve ulcerative colitis.

In order to solve the above technical problems, the present invention provides the use of MANF for the preparation of a product (e.g. a medicament, a vaccine, a nutraceutical and/or a food product) for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis.

In the above application, the MANF may be a human MANF. The human MANF may be a recombinant human MANF.

In the above application, the recombinant human MANF may be a protein of R1), R2) or R3):

r1) the amino acid sequence is the protein of SEQ ID No.2,

r2) the amino acid sequence is the protein of SEQ ID No.2 from position 3 to 160,

r3) is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the protein shown by R1) or R2), and the protein has more than 90% of identity with the protein shown by R1) or R2) and has the activity of relieving ulcerative colitis.

In the above applications, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above applications, the 90% or greater identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above application, SEQ ID No.2 consists of 168 amino acid residues, and positions 3-160 of SEQ ID No.2 are human MANF fragments.

For the above uses, said product for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis may comprise said MANF.

In order to solve the above technical problems, the present invention provides the use of biological material related to MANF for the preparation of a product (such as a medicament, a vaccine, a health product and/or a food) for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis; the biomaterial may be any of the following:

B1) a nucleic acid molecule encoding said MANF,

B2) an expression cassette comprising the nucleic acid molecule according to B1),

B3) a recombinant vector containing B1) the nucleic acid molecule or a recombinant vector containing B2) the expression cassette,

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector,

B5) a transgenic animal cell line containing B1) the nucleic acid molecule, a transgenic animal cell line containing B2) the expression cassette or a transgenic animal cell line containing B3) the recombinant vector,

B6) a transgenic plant cell line containing B1) the nucleic acid molecule, a transgenic plant cell line containing B2) the expression cassette or a transgenic plant cell line containing B3) the recombinant vector.

In the above application, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above application, the expression cassette of B2) is a DNA capable of expressing said MANF in a host cell, and said DNA may comprise not only a promoter for initiating transcription of said MANF gene but also a terminator for terminating transcription of said MANF gene. Further, the expression cassette may also include an enhancer sequence.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above application, the microorganism may be bacteria, yeast, algae or fungi.

In the above application, the bacterium may be Escherichia coli, such as BL21(DE 3).

In the above application, the nucleic acid molecule of B1) may be B11) or B12):

B11) the coding sequence is DNA molecule shown in SEQ ID No. 1.

B12) The nucleotide sequence is a DNA molecule shown in the 9 th to 482 th positions of SEQ ID No. 1.

Wherein, SEQ ID No.1 consists of 509 nucleotides, the coding sequence is nucleotides 3 to 509 of SEQ ID No.1, and codes the protein shown in SEQ ID No. 2.

In the above application, the product for treating and/or preventing and/or alleviating and/or improving ulcerative colitis may contain the biological material.

In order to solve the above technical problems, the present invention provides a medicament for treating and/or preventing and/or alleviating and/or ameliorating ulcerative colitis.

The medicine for treating and/or preventing and/or relieving and/or improving ulcerative colitis provided by the invention consists of the MANF and pharmaceutic adjuvant.

In the above medicine, the pharmaceutical adjuvant can be water, ethanol, polyethylene glycol, 1, 3-propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, sodium chloride, glucose or glycerol, cosolvent, buffer or pH regulator.

In the above medicine, the dosage form of the medicine can be injection administration dosage form.

In the above drugs, the injection dosage form may include intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection or intracavity injection.

In order to solve the above technical problems, the present invention provides a method for treating and/or preventing and/or alleviating and/or ameliorating ulcerative colitis.

The method for treating and/or preventing and/or alleviating and/or improving ulcerative colitis provided by the invention comprises the step of administering the MANF or/and the biological material to a receptor animal, and treating and/or preventing and/or alleviating and/or improving ulcerative colitis.

As indicated above, the treatment and/or prevention and/or amelioration of ulcerative colitis may be manifested as a relief of hematochezia caused by ulcerative colitis, a relief of diarrhea caused by ulcerative colitis, a relief of weight loss caused by ulcerative colitis, a relief of colon length shortening caused by ulcerative colitis, a relief of macrophage infiltration caused by ulcerative colitis and/or a reduction in the level of expression of inflammatory factors in colon tissue caused by ulcerative colitis. The inflammatory factor may be IL-6.

As above, the animal may be a mammal, such as a mouse.

As used herein, the MANF may be a native MANF, such as a mammalian-derived MANF, or a non-native MANF, such as a recombinant MANF, provided that the recombinant MANF functions as a native MANF.

The research of the invention discovers that the expression of MANF is up-regulated in a human ulcerative colitis patient and a mouse ulcerative colitis model, which indicates that the expression of MANF is increased in ulcerative colitis. Animal experiments show that the MANF can obviously relieve bloody stool caused by ulcerative colitis, increase the weight of a model mouse and improve the diarrhea condition. MANF can reduce inflammatory macrophage infiltration of ulcerative colitis and reduce the expression of inflammatory factors. MANF can be used for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis.

Drawings

FIG. 1 is a graph of immunohistochemical methods to detect high expression of MANF in patients with ulcerative colitis. In the figure, normal mucosa represents the mucosa of the colon of a healthy person; diseased mucosa refers to the mucosa of the colon of patients with ulcerative colitis; the severe diseased mucosa refers to the mucosa of the colon of patients with severe ulcerative colitis.

FIG. 2 shows that the immunohistochemical method detects the high expression of MANF in mouse ulcerative colitis model. In the figure, H₂O represents the colonic mucosa of the control (ultrapure water drinking) mice; DSS (dextran sulfate sodium salt) represents the colonic mucosa of mice in the ulcerative colitis model (7 days of continuous drinking of 3% DSS in water).

FIG. 3 shows the body weight change index, diarrhea index, rectal bleeding index and disease activity index changes during the modeling of ulcerative colitis model. In the figure, DSS represents the model group, Normal represents the control group; "x" indicates a statistical difference from the control group, P <0.01, "x" indicates a statistical difference from the control group, P < 0.0001.

Fig. 4 is a colon histological section HE staining (a), pathology score (B), colon gross sample (C) and colon length (D) at day 7 of molding of the ulcerative colitis model. In the figure, DSS represents the model group, Normal represents the control group; ". indicates a statistical difference compared to the control group, P < 0.0001.

FIG. 5 shows that MANF treatment ameliorated the symptoms of ulcerative colitis in mice. In the figure, DSS + PBS represents the model control group, Normal + PBS represents the Normal control group, DSS + MANF represents the MANF treatment group; "" indicates a statistical difference compared to the model control group, P < 0.05; ". indicates a statistical difference compared to the model control group, P < 0.01.

FIG. 6 shows that MANF treatment reduced ulcerative colitis impairment. In the figure, DSS + PBS represents the model control group, Normal + PBS represents the Normal control group, DSS + MANF represents the MANF treatment group; "" indicates a statistical difference compared to the model control group, P < 0.05; ". indicates a statistical difference compared to the model control group, P < 0.01.

FIG. 7 shows that MANF treatment reduced inflammatory macrophage cell infiltration. Normal + PBS for Normal control group, DSS + PBS for model control group, DSS + MANF for MANF treatment group; "x" indicates P <0.001, indicating a statistical difference compared to the model control group.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

1. The method of modeling the ulcerative colitis model in the following examples is as follows: SPF-grade female C57BL/6 mice, 6-8 weeks old, weighing 20-23g each, fasted for 8 hours, were randomly divided into two groups: building set (DSS) and control (Normal), 12 each. Each group of C57BL/6 mice was weighed and labeled. Ultrapure water was administered to each mouse in the control group on days 1 to 7, and 3% DSS aqueous solution (liquid obtained by adding DSS to DSS in an amount of 3% by mass) was administered to each mouse in the model group on days 1 to 7. Day 8 the model building block and control group each mouse was dosed with ultrapure water. Histopathological changes were assessed by measuring Disease Activity Index (DAI score), histological change score, and colon length, to demonstrate success of the ulcerative colitis mouse model.

1.1. Disease Activity index score

The Disease Activity Index (DAI) score is a comprehensive evaluation index of clinical ulcerative colitis and reflects the overall conditions of weight change, diarrhea and fecal hemorrhage. The score details are shown in Table 1. The DAI score is the sum of the three indices divided by 3. No mortality occurred in the mice of each group throughout the molding period of ulcerative colitis. But from day 4, the DAI score of the building block showed a rapid increase in comparison to the control. No damage occurred in the control group at day 7, with a DAI score of 0; day 7 building block has a DAI score of 2.3. + -. 0.47 (FIG. 3).

TABLE 1 DAI Scoring rules

Score value	Percentage of body weight loss	Consistency of stool	Occult blood in feces
				0	0	Is normal	Negative of
1	1-5％	Soft stool	Light blue
				2	5-10％	Mucus sample stool	Blue color
3	10-20％	Liquid state stool	Dark blue
				4	＞20％	Dilute liquid stool sample	Bloody stool with naked eyes

Note: the score values are body weight change index, diarrhea index and rectal bleeding index, respectively.

1.1.1 fecal occult blood score

Fecal occult blood is an important clinical indicator of ulcerative colitis. The results showed that the control mice, which had been drunk with ultrapure water, did not develop rectal bleeding. On the 2 nd day of drinking 3% DSS water solution, the mice in the model group have positive fecal occult blood test; strong positive results appeared in the mouse occult blood test starting on day 3; most of the mice of the model group on day 5 showed macroscopic hematochezia; the mice in the model group bleed severe hematochezia overall on day 7. The data were processed using SPSS11.5 statistical software, and the results were expressed as mean. + -. standard deviation using One-way ANOVA test. Rectal bleeding index of mice in the molding group continuously drinking 3% DSS aqueous solution for 7 days was 3.4 ± 0.49, while rectal bleeding index of mice in the control group continuously drinking ultrapure water for 7 days was 0 (fig. 3).

1.1.2 diarrhea index score

Diarrhea is another important clinical indicator of ulcerative colitis. Control mice, which were drinking ultrapure water, did not develop diarrhea. On the 3 rd day when the model-making mice drink the 3% DSS aqueous solution, the excrement of part of the mice is wet and soft; on day 5, all mice developed wet and soft feces; on

days

6 and 7, the mice in the model group developed severe half-loose stools and loose stools. The data were processed using SPSS11.5 statistical software, and the results were expressed as mean. + -. standard deviation using One-way ANOVA test. The diarrhea index of the model group mice continuously drinking 3% DSS aqueous solution for 7 days is 3 plus or minus 1; the control mice, which were continuously drinking ultrapure water for 7 days, did not develop diarrhea with an index score of 0 (fig. 3).

1.1.3 body weight score

Weight loss is also an important clinical indicator of ulcerative colitis. Normal control (Normal) mice, which had been drinking ultrapure water, maintained a steady trend of weight gain and were actively performing throughout the experiment. The weight of the model building (DSS) mouse does not change obviously in the previous 4 days, and the state is active; from day 4 to day 7 of the model, the average body weight percentage of the mice in the model group was significantly decreased and the state was poor as compared with the control group. The data were processed using SPSS11.5 statistical software, and the results were expressed as mean. + -. standard deviation using One-way ANOVA test. On day 8 from the start of the experiment, the rate of weight loss (body weight change index) was 0.78. + -. 0.04 in the model mice, and the rate of weight gain (body weight change index) was 1.1. + -. 0.01 in the control mice (FIG. 3).

1.2 histopathological Scoring

The development of ulcerative colitis results in significant pathological changes in the colon tissue. The present application employs HE staining for colon tissue sections and double-blind methods for pathology scoring. The score criteria for colon section histopathology were 0-4: 0, normal tissue; 1, mild inflammation of the mucosa with some infiltrating monocytes; 2, inflammation in the mucosa worsens, there is more cellular infiltration, crypt glands and epithelium are damaged; 3, extensive infiltration of cells in the mucosa and submucosal regions, epithelial cell destruction. 4, the tissue is heavily infiltrated with cells, and the crypts disappear completely.

The results show that the colon tissue structure of the control mice is represented as: the integrity, no obvious defect of mucosa, regular arrangement of glands, no atrophy, no obvious inflammatory cell infiltration of mucosa lamina propria; the mice in the model group had a large number of inflammatory cells infiltrating the submucosa, and the glands of the lamina propria of the mucosa were atrophic, mostly disappeared, and replaced by a large number of inflammatory cells (a in fig. 4). The histopathological score of the mice in the model group that had been continuously drinking 3% DSS aqueous solution for 7 days was 1.5 ± 0.5, while the histopathological damage of the mice in the control group that had been continuously drinking ultrapure water for 7 days was not observed and the score was 0 (B in fig. 4).

1.3 Colon Length detection

Colon shortening is one of the typical features of mouse models of ulcerative colitis. The colon length of the mice was measured and statistically analyzed. As a result, it was found that: compared with the control group, the colon of the model group mouse is obviously shortened. The colon length of the mice in the model group that had been continuously drunk with 3% DSS aqueous solution for 7 days was 4.4. + -. 0.2cm, and the colon length of the mice in the normal control group that had been continuously drunk with ultrapure water for 7 days was 6.45. + -. 0.31cm (C and D in FIG. 4).

Thus, the C57BL/6 mouse can successfully construct the ulcerative colitis model by continuously drinking the 3% DSS aqueous solution for 7 days, and the ulcerative colitis model mouse is obtained.

EXAMPLE one treatment of ulcerative colitis with recombinant human MANF

Upregulation of MANF expression in colonic tissue of patients with ulcerative colitis

The inventors collected colon samples of 30 ulcerative colitis patients from the first subsidiary hospital of the medical university of Anhui. MANF was found to be highly expressed in colon tissue of patients by immunohistochemical staining. As the degree of inflammatory lesions increased, MANF expression levels increased accordingly (fig. 1). Wherein, the severe patients defecate more than ten times a day, have bloody purulent stool and a large amount of bloody stool; there are significant abdominal pain and tympanites, and severe patients may also have high fever during active period, and some extra-intestinal complications may occur.

Wherein, the immunohistochemical method is used for detecting the expression of the MANF in the colon tissue of the patient as follows: tissue sections are cut, fixed by formalin, embedded by paraffin, and then respectively mixed with an anti-MANF antibody (the anti-MANF antibody is a monoclonal antibody of ARMET, and the monoclonal antibody is prepared according to the method of the following documents, namely the prokaryotic expression of WANGFANCAO, WANGHAPING, LIQI, SQUANYUNYUXIA. ARMET and the preparation of the monoclonal antibody thereof, the university of Anhui medical science, 2009,44(6): 665-. A negative control was set in each experiment using the Cunninghamia lanceolata ABC kit (SP-9000), and was obtained by omitting the primary antibody.

Upregulation of MANF expression in DSS-induced C57 mouse ulcerative colitis model

The expression of MANF in colon tissues of the mouse model for ulcerative colitis is detected by immunohistochemical staining (same as the step 1), and as a result, the MANF is found to be highly expressed in the colon tissues of the mouse for ulcerative colitis (figure 2), which is consistent with the results of patients with ulcerative colitis.

2. Recombinant human MANF for treating mouse ulcerative colitis

2.1 inducible expression and purification of recombinant human MANF protein

(1) The fragment between NcoI and XhoI recognition sites of pET-28a (+) including a small fragment including the NcoI recognition site and the XhoI recognition site was replaced with the human MANF gene shown at positions 1-488 of SEQ ID No.1, and the other sequence of pET-28a (+) was kept unchanged to obtain a recombinant expression vector, which was designated as pET28 a-MANF. pET28a-MANF contains the human recombinant MANF protein-His tag fusion protein hMANF-His coding gene shown in SEQ ID No.1, and the amino acid sequence of the protein hMANF-His coded by the hMANF-His coding gene is shown in SEQ ID No. 2.

(2) The plasmid pET28a-MANF is transformed into BL21 competent cells, kanamycin-resistant screening positive clones are used for screening and culturing, single clones are picked, the plasmid is extracted for sequencing, and the sequencing result shows that the recombinant Escherichia coli containing pET28a-MANF is named as BL21/pET28 a-MANF.

(3) BL21/pET28a-MANF strain was picked up and inoculated into LB medium containing 100. mu.g/ml kanamycin (a medium obtained by adding kanamycin to LB medium to 100. mu.g/ml kanamycin) and shake-cultured at 37 ℃ at 250rpm overnight (13 to 16 hours). The bacterial liquid cultured overnight in the following morning is inoculated into LB culture medium containing 100 mug/ml kanamycin again in a ratio of 1:100, and is placed on a constant temperature shaking table at 37 ℃ and is shaken at 250rpm for about 2-3h of amplification culture. OD of bacterial liquid to be treated₆₀₀When the concentration is 0.6-0.8, IPTG is added to the final concentration of 0.5mmol/L for induction expression for about 2.5 h. Centrifuging the bacterial liquid at 4 ℃, 7000rpm, 5min, removing the supernatant, collecting the thallus, adding a lysis solution for cracking, and carrying out ultrasonic disruption (400w, 3s intermittent, 3s ultrasonic) for 20 min.

(4) Centrifuging the bacteria solution after ultrasonic treatment at 4 ℃, 12000rpm, taking the supernatant, adding the supernatant into a pre-assembled Ni-beads column, sealing the column port, fixing the column port on a silent mixer, and combining for about 2 hours at 4 ℃. Taking off the protein chromatography column, vertically hanging in a refrigerator at 4 deg.C, opening the cover to allow the liquid in the column to flow out vertically, controlling the dripping speed at one drop per 30sec, collecting the protein effluent, and storing for subsequent protein analysis.

(5) 4-5ml of decontamination buffer (solute: 50mmol/L NaH) was added to the column₂PO₄300mmol/L NaCl, 50mmol/L imidazole, water as solvent, pH8.0), closing the column port, fixing the column port again in a silent mixer, mixing for about 1h, allowing the liquid to flow out, and collecting the effluent. This step was repeated twice to wash off proteins non-specifically bound to Ni-beads in the column.

(6) Adding elution buffer (solute: 50mmol/L NaH) into the chromatographic column₂PO₄300mmol/L NaCl, 250mmol/L imidazole, water as solvent, pH8.0)1ml of elution target protein. This procedure was repeated twice and the eluate (about 3ml) containing the protein of interest was collected.

(7) Transferring the purified protein eluent into an ultrafiltration tube for concentration, adding precooled PBS (PBS) with the pH value of 7.4 about 7ml in advance into the ultrafiltration tube, centrifuging (4 ℃, 7500g, 20min), repeating for 3-4 times, and replacing the imidazole buffer solution with the PBS buffer solution with the pH value of 7.4 to obtain the concentrated recombinant human MANF protein solution.

Wherein, the preparation of PBS buffer solution with pH value of 7.4: 8.5g NaCl, 0.2g KCl, 2.9g Na₂HPO₄·12H₂O、0.59g NaH₂PO₄·2H₂O, 1L deionized water.

(8) And (4) subpackaging the concentrated recombinant human MANF protein solution and storing in a refrigerator at the temperature of-80 ℃ for later use. The content of the recombinant human MANF protein in the concentrated solution of the recombinant human MANF protein is 1 mg/mL.

2.2 recombinant human MANF for the treatment of ulcerative colitis in mice

SPF-grade female C57BL/6 mice 6-8 weeks old, each weighing 20-23g, fasted for 8 hours, randomly divided into 3 groups: normal control group (Normal + PBS), model control group (DSS + PBS) and MANF treatment group (DSS + MANF), 12 per group. Each group of C57BL/6 mice was weighed and labeled. The experimental period is 8 days. In the normal control group, ultrapure water was drunk by each mouse on days 1-7, and 200. mu.l of PBS buffer solution with pH value of 7.4 was injected into each mouse from day 4 every day for 3 consecutive days; model control group each mouse was administered 3% DSS aqueous solution (liquid obtained by adding DSS to DSS at 3% by mass content to the above ultrapure water) on days 1 to 7, and each mouse was injected with 200 μ l of PBS buffer having a pH of 7.4 once per day in the tail vein from day 4 for 3 consecutive days. MANF-treated groups mice were each administered a 3% DSS aqueous solution (obtained by adding DSS to 3% DSS in ultrapure water) on days 1-7, and from day 4, each mouse was administered a 200. mu.l daily tail vein injection of a recombinant human MANF protein solution (obtained by diluting a 2.1 recombinant human MANF protein solution with a PBS buffer having a pH of 7.4) at a dose of 1mg/kg body weight of the recombinant human MANF protein for 3 consecutive days. Three groups of mice were each given ultrapure water on day 8 and then sacrificed.

2.2.1 MANF for treating and relieving hematochezia of mice with ulcerative colitis

The fecal blood of the mice was tested according to the fecal occult blood scoring method described above at 1.1.1. The result shows that the normal control group mice drinking ultrapure water do not have hematochezia in the experimental process, and the model control group mice have positive hematochezia experiments on the 2 nd day of drinking 3% DSS aqueous solution; the excrement occult blood test of individual mice shows strong positive on the 3 rd day; most of the mice in the model control group appeared to have hematochezia on day 5; the model control mice developed severe hematochezia throughout the day 7. And the blood stool symptoms of the mice in the MANF treatment group are obviously relieved. The rectal bleeding index of the mice in the model control group, which were continuously drunk with 3% DSS aqueous solution for 7 days, was 3.6 ± 0.47, the rectal bleeding index of the mice in the normal control group, which were continuously drunk with ultrapure water for 7 days, was 0, and the rectal bleeding index of the mice in the MANF treatment group, which was 7 days, was 2.6 ± 0.49 (fig. 5).

2.2.2 MANF treatment to ameliorate diarrhea in mice with ulcerative colitis

And (3) detecting the diarrhea condition of the mice according to the fecal viscosity scoring method of 1.1.2. The results show that the mice in the normal control group drinking ultrapure water have no diarrhea and normal feces in the experimental process. On day 3 of drinking 3% DSS aqueous solution, the individual mice in the model control group developed wet and soft feces, and the individual mice in the MANF treatment group developed wet and soft feces; on the 5 th day when drinking 3% DSS water solution, the mice in the model control group all showed soft and wet feces, and the mice in the MANF treatment group showed soft and wet feces, but had no statistical significance; on

days

6 and 7 of 3% DSS drinking, the model control mice developed severe half-loose stools and loose stools, and the MANF treated mice had reduced diarrhea. The data were processed using SPSS11.5 statistical software, and the results were expressed as mean. + -. standard deviation using One-way ANOVA test. The diarrhea index of the mice in the model control group which continuously drink 3% DSS aqueous solution for 7 days is 3.3 +/-0.94, and the diarrhea index of the mice in the normal control group which continuously drink ultrapure water for 7 days is 0. Mice in the MANF-treated group, who had been drinking 3% DSS in water for 7 days continuously, had diarrhea indices of 2.3 ± 1.37 (fig. 5).

2.2.3 MANF treatment to increase body weight in mice with ulcerative colitis

The change in body weight of the mice was measured according to the 1.1.3 body weight scoring method described above. The results show that the Normal control group (Normal) mice, which had been drinking ultrapure water, maintained a steady trend of weight gain and performed actively throughout the experiment. The body weight of the mice in the model control group (DSS) and the MANF treatment group has no obvious change in the previous 4 days, and the state is active; from day 4 to day 7 of drinking the 3% DSS aqueous solution, the average percentage of body weight of the model control group mice was significantly reduced and the status was poor compared to the normal control group; compared with the model control group, the reduction of the body weight percentage of the MANF treatment group is relieved, and the state is more active. The data were processed using SPSS11.5 statistical software, and the results were expressed as mean. + -. standard deviation using One-way ANOVA test. On day 8 from the start of the experiment, the rate of weight loss (body weight change index) was 0.79. + -. 0.03 in the model control group mice, and the rate of weight gain (body weight change index) was 1.1. + -. 0.03 in the normal group mice. The proportion of body weight loss (body weight change index) was 0.86. + -. 0.02 in the mice of the MANF-treated group (FIG. 5).

2.2.4 MANF treatment increases ulcerative colitis mouse Disease Activity Index (DAI) score

The DAI score was calculated according to the above 1.1 disease activity index scoring method. The results show that no mortality occurred in the groups of mice throughout the experiment. However, from day 4, the DAI scores of the model control group showed a rapid increase in comparison to the normal control group, whereas the DAI scores of the MANF-treated group were significantly reduced. Day 7 the DAI score for the normal control group was 0, day 7 the cohort group was 2.2. + -. 0.75, and day 7 the MANF-treated group was 1.9. + -. 0.3 (FIG. 5).

2.2.5 MANF treatment improves ulcerative colitis mouse Colon histopathology scores

Each group of mice was pathologically scored according to the histological change scoring method described above. The results show that the colon tissue structure of the mice in the normal control group is represented as follows: the integrity, no obvious defect of mucosa, regular arrangement of glands, no atrophy, no obvious inflammatory cell infiltration of mucosa lamina propria; the mouse of the model control group has a large amount of inflammatory cells to infiltrate the submucosa, glands of the mucosa lamina propria are atrophied, most of the glands disappear, and a large amount of inflammatory cells are replaced; pathological scores were reduced in the MANF treated group. MANF treatment significantly improved the inflammation of the colonic tissue as evidenced by a more well-arranged glands in the lamina propria of the mucosa and a significant reduction in the number of inflammatory cells (a in fig. 6). The histopathological score of the mice of the model control group that were continuously fed with the 3% DSS aqueous solution for 7 days was 1.6 ± 0.75, the histopathological score of the mice of the MANF-treated group that were continuously fed with the 3% DSS aqueous solution for 7 days was 1.2 ± 0.39, and the histopathological score of the mice of the normal control group that were continuously fed with ultrapure water for 7 days was 0 (B in fig. 6).

2.2.6 MANF treatment partially restored Colon Length in ulcerative colitis mice

The colon length of each group of mice was measured and statistically analyzed. The results show that: compared with a normal control group, the colon of the mouse of the model control group is obviously shortened; colon recovery was evident after MANF treatment compared to the model control group (C in fig. 6). The colon length of the normal control group mice continuously drinking ultrapure water for 7 days is 6.45 +/-0.31 cm; the colon length of the mice in the model control group, which had been continuously fed with the 3% DSS aqueous solution for 7 days, was 4.37. + -. 0.32cm, and the colon length of the mice in the model control group, which had been continuously fed with the 3% DSS aqueous solution for 7 days, was 5.1. + -. 0.29cm after the MANF treatment (D in FIG. 6).

2.2.7 MANF Mitigation of macrophage infiltration in ulcerative colitis

Inflammatory cell infiltration is a prominent feature of ulcerative colitis. Among them, infiltration and activation of macrophages in colon tissue play an important role in the pathogenesis of ulcerative colitis. To examine the effect of MANF on macrophage infiltration in ulcerative colitis, the macrophage-specific marker molecule CD68 was examined using an anti-CD 68 antibody (Abcam, abbo, ab125212) using immunohistochemical staining. The results show that the expression of CD68 in the colon tissue of the mouse of the model control group is increased compared with that of the normal control group, and the macrophage infiltration in the colon tissue of the mouse of the model control group is serious. Macrophage infiltration was reduced in the MANF treated group compared to the model control group (a in fig. 7).

2.2.8 MANF treatment decreases inflammatory factor expression in colonic tissue of mice with ulcerative colitis

To observe the effect of MANF treatment on the expression of inflammatory cytokines in the intestine of mice with ulcerative colitis, real-time fluorescent quantitative PCR was used to detect the mRNA level of the inflammatory factor IL-6 in colon tissues. The results showed that the IL-6 content of the model control group was significantly increased compared to the normal control group. MANF treatment significantly reduced IL-6 transcript levels (B in FIG. 7). The results suggest that MANF can significantly inhibit IL-6 expression in ulcerative colitis.

In the real-time fluorescent quantitative PCR method, a PCR system is prepared according to the instructions of TOYOBO SYBR reaction enzyme, and a real-time quantitative PCR instrument is adopted to detect the reaction system. mRNA level detection of IL-6 specific primer sequences:

F：5’-ACACATGTTCTCTGGGAAATCGT-3’；

R：5’-AAGTGCATCATCGTTGTTCATACA 3’。

the present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of medical in Anhui

<120> application of mesencephalon astrocyte-derived neurotrophic factor in treatment of ulcerative colitis

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<170> PatentIn version 3.5

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Leu Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr

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Cys Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu

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Claims

Use of MANF for the preparation of a product for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis.
2. Use according to claim 1, characterized in that: the MANF is a recombinant human MANF.
3. Use according to claim 2, characterized in that: the recombinant human MANF is R1), R2) or R3):

r1) the amino acid sequence is the protein of SEQ ID No.2,

r2) the amino acid sequence is the protein of SEQ ID No.2 from position 3 to 160,

r3) is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the protein shown by R1) or R2), and the protein has more than 90% of identity with the protein shown by R1) or R2) and has the activity of relieving ulcerative colitis.
4. Use of biological material related to MANF for the preparation of a product for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis; the biological material is any one of the following materials:

B1) a nucleic acid molecule encoding a MANF according to any one of claims 1-3,

B2) an expression cassette comprising the nucleic acid molecule according to B1),

B3) a recombinant vector containing B1) the nucleic acid molecule or a recombinant vector containing B2) the expression cassette,

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector,

B5) a transgenic animal cell line containing B1) the nucleic acid molecule, a transgenic animal cell line containing B2) the expression cassette or a transgenic animal cell line containing B3) the recombinant vector,

B6) a transgenic plant cell line containing B1) the nucleic acid molecule, a transgenic plant cell line containing B2) the expression cassette or a transgenic plant cell line containing B3) the recombinant vector.
5. Use according to claim 4, characterized in that: B1) the nucleic acid molecule is B11) or B12):

B11) the coding sequence is DNA molecule shown in SEQ ID No.1,

B12) the nucleotide sequence is a DNA molecule shown in the 9 th to 482 th positions of SEQ ID No. 1.
6. A medicament for the treatment and/or prevention and/or alleviation and/or amelioration of ulcerative colitis, which comprises MANF and a pharmaceutically acceptable adjuvant.
7. Use according to claim 6, characterized in that: the MANF is a recombinant human MANF.
8. Use according to claim 7, characterized in that: the recombinant human MANF is R1), R2) or R3):

r1) the amino acid sequence is the protein of SEQ ID No.2,

r2) the amino acid sequence is the protein of SEQ ID No.2 from position 3 to 160,

r3) is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the protein shown by R1) or R2), and the protein has more than 90% of identity with the protein shown by R1) or R2) and has the activity of relieving ulcerative colitis.
9. Use according to any one of claims 1 to 8, wherein: the product is a medicament.
10. A method of treating and/or preventing and/or ameliorating ulcerative colitis, comprising administering MANF according to any one of claims 1-3 or/and biomaterial according to claim 4 or 5 to a recipient animal, thereby treating and/or preventing and/or ameliorating ulcerative colitis.