CN112587654B

CN112587654B - Application of mesencephalon astrocyte-derived neurotrophic factor in treatment of ulcerative colitis

Info

Publication number: CN112587654B
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: 2022-10-14
Anticipated expiration: 2040-12-18
Also published as: CN112587654A

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 good curative effects on treatment of moderate to severe ulcerative colitis, and can effectively relieve symptoms and maintain the rest state of diseases. 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, various factors such as genetic susceptibility, external environment stimulation, autoimmune disorder, intestinal flora disorder and the like are considered to participate in the occurrence and development of ulcerative colitis. 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, research proves 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 reaction 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 needs; 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 MANF primary transcript consists of 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, with a molecular weight of approximately 18kDa.

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 ameliorate 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 protein of which the amino acid sequence is SEQ ID No.2,

r2) the protein whose amino acid sequence is 3-160 of SEQ ID No.2,

r3) is a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the protein shown by R1) or R2), 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 the 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 the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2),

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3),

b5 A transgenic animal cell line containing the nucleic acid molecule according to B1), a transgenic animal cell line containing the expression cassette according to B2) or a transgenic animal cell line containing the recombinant vector according to B3),

b6 A transgenic plant cell line containing the nucleic acid molecule according to B1), a transgenic plant cell line containing the expression cassette according to B2) or a transgenic plant cell line containing the recombinant vector according to B3).

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 the DNA may include 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 A DNA molecule having the coding sequence shown in SEQ ID No. 1.

B12 ) the nucleotide sequence is the DNA molecule shown in SEQ ID No.1 at positions 9-482.

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 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 medicinal adjuvants 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 drugs, the dosage form of the drug can be an 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 patient with human ulcerative colitis 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 inflammatory factor expression. 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 means the mucosa of the colon in a healthy person; diseased mucosa refers to the mucosa of the colon of patients with ulcerative colitis; the critically ill mucosa represents the mucosa of the colon in 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, H ₂ O 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 (3% DSS in water for 7 days of continuous drinking).

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 histoslice HE staining (a), pathology score (B), colon gross sample (C) and colon length (D) on 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 is a graph of MANF treatment ameliorating ulcerative colitis symptoms 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; "x" indicates a statistical difference compared to the model control group, P <0.01.

Figure 6 is a graph of MANF treatment reducing 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.

Figure 7 is 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, each weighing 20-23g, were fasted for 8 hours and were randomly divided into two groups: building set (DSS) and control (Normal), 12 each. Each group of C57BL/6 mice was weighed and labeled. The control group was administered with ultrapure water on days 1 to 7 per mouse, and the model group was administered with 3% DSS aqueous solution (a liquid obtained by adding DSS to DSS of 3% by mass to the above ultrapure water) on days 1 to 7 per mouse. 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 to 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 and 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. The molding mice developed positive fecal occult blood test on day 2 of drinking 3% of the DSS aqueous solution; strong positive appeared in the mouse occult blood experiment from 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, which had been continuously drunk 3% of the DSS aqueous solution for 7 days, was 3.4. + -. 0.49, while rectal bleeding index of mice in the control group, which had been continuously drunk 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 day 3 of the mice model drinking 3% DSS aqueous solution, some mice developed wet and soft feces; 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 mice in the molding group was 3. + -.1 when the DSS aqueous solution was consumed continuously for 7 days; 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 scoring

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 colon tissue structure of the control mice is represented as follows: the integrity, no obvious defect of mucosa, regular arrangement of glands, no atrophy and 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). Mice of the model group, which had been allowed to drink 3% DSS aqueous solution for 7 days continuously, had a histopathological score of 1.5. + -. 0.5, whereas mice of the control group, which had been allowed to drink ultrapure water continuously for 7 days, had no histopathological damage and had a score of 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, which had been made to drink 3% DSS aqueous solution for 7 days, was 4.4. + -. 0.2cm continuously, and the colon length of the mice in the normal control group, which had been made to drink ultrapure water for 7 days continuously, was 6.45. + -. 0.31cm (C and D in FIG. 4).

Thus, the model of ulcerative colitis was successfully constructed by continuously drinking 3% DSS aqueous solution for 7 days from C57BL/6 mice, and the mice of ulcerative colitis model were obtained.

EXAMPLE one treatment of ulcerative colitis with recombinant human MANF

Expression of MANF is upregulated in colon 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 hematochezia; there are marked abdominal pain and tympanites, and severe patients may also have high fever during active period, etc., and cause some extra-intestinal complications.

Wherein, the immunohistochemical method is used for detecting the expression of the MANF in the colon tissue of the patient as follows: tissue sections were formalin fixed, paraffin embedded, and then separately combined with anti-MANF antibodies (anti-MANF antibodies are monoclonal antibodies to ARMET, prepared according to the methods of Wang Facai, wang Haiping, li Qi, fang Shengyun, shen Yuxian. Prokaryotic expression of ARMET and preparation of monoclonal antibodies therefor, proceedings of the university of Anhui medical, 2009,44 (6): 665-669) overnight at 4 ℃. A negative control was set in each experiment using the Cunninghamia lanceolata ABC kit (SP-9000), and was obtained by omitting the primary antibody.

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

(2) The pET28a-MANF plasmid is transformed into BL21 competent cells, kanamycin resistance is utilized to screen positive clones 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 the pET28a-MANF is named as BL21/pET28a-MANF.

(3) The BL21/pET28a-MANF strain was picked up and inoculated into LB medium containing 100. Mu.g/ml kanamycin (medium obtained by adding kanamycin to LB medium to 100. Mu.g/ml kanamycin), and shake-cultured at 37 ℃ overnight (13-16 hours) at 250 rpm. The morning of the next day1, the overnight-cultured bacterial liquid is again inoculated into LB medium containing 100. Mu.g/ml kanamycin, placed on a constant temperature shaker at 37 ℃ and shaken at 250rpm, and is subjected to amplification culture for about 2-3 h. Bacterial suspension OD ₆₀₀ When the concentration is 0.6-0.8, IPTG is added to the final concentration of 0.5mmol/L to induce expression for about 2.5 h. Centrifuging the bacterial solution at 4 ℃,7000rpm for 5min, removing supernatant, collecting thallus, adding lysis solution for cracking, and performing ultrasonic disruption (400 w, 3s intermittent, 3s ultrasonic) for 20min.

(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 suspending 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 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 column ₂ PO ₄ 300mmol/L NaCl, 50mmol/L imidazole, water as solvent, pH 8.0), 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 column ₂ PO ₄ 300mmol/L NaCl, 250mmol/L imidazole, water as solvent, pH8.0) 1ml of elution target protein. This procedure was repeated twice and the eluate (about 3 ml) 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 into the ultrafiltration tube in advance for about 7ml, centrifuging (4 ℃,7500g and 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 Na ₂ HPO ₄ ·12H ₂ O、0.59g NaH ₂ PO ₄ ·2H ₂ O,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 1mg/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 3% by mass of DSS to the above-mentioned ultrapure water) on days 1 to 7, and each mouse was injected with 200. Mu.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 Each mouse was administered 3% DSS aqueous solution (liquid obtained by adding DSS to 3% by mass of DSS to the above-mentioned ultrapure water) on days 1 to 7, 200. Mu.l of recombinant human MANF protein solution (liquid obtained by diluting 2.1 of recombinant human MANF protein solution with PBS buffer having a pH of 7.4) was administered to each mouse in a dose of 1mg/kg body weight of recombinant human MANF protein per day for 3 consecutive days, starting on day 4. Three groups of mice were each given ultrapure water on day 8 and then sacrificed.

2.2.1 MANF treatment and alleviation of hematochezia of mice with ulcerative colitis

The fecal blood status of the mice was tested according to the fecal occult blood scoring method described above at 1.1.1. The results showed that the normal control mice, which had been drinking ultrapure water, did not develop hematochezia during the experiment, and that the model control mice developed fecal occult blood test positivity for individual mice on day 2 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. While the blood stool symptoms of the mice in the MANF treatment group are obviously relieved. The rectal bleeding index of mice in the model control group, which were continuously drunk with 3-percent DSS aqueous solution for 7 days, was 3.6. + -. 0.47, the rectal bleeding index of mice in the normal control group, which were continuously drunk with ultrapure water for 7 days, was 0, and the rectal bleeding index of mice in the MANF-treated group, on day 7, was 2.6. + -. 0.49 (FIG. 5).

2.2.2 MANF treatment for improving diarrhea condition of ulcerative colitis mice

And (3) detecting the diarrhea condition of the mice according to the method for scoring the fecal viscosity 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 mice in the model control group developed wet and soft stool, and the mice in the MANF treatment group developed wet and soft stool; on day 5 of drinking 3% dss aqueous solution, the mice in the model control group appeared soft and wet in stools overall, while the MANF treated group had individual mice with soft and wet stools, but had no statistical significance; on

days

6 and 7 when 3% DSS aqueous solution was consumed, the model control group mice developed severe half-and loose stools and the MANF treated group 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 model control group mice, which had been consumed 3% of the DSS aqueous solution for 7 days continuously, was 3.3. + -. 0.94, and the diarrhea index of the normal control group mice, which had been consumed ultrapure water continuously for 7 days, was 0. Mice in the MANF-treated group had diarrhea indices of 2.3. + -. 1.37 (FIG. 5) based on 3% DSS aqueous solution consumed continuously for 7 days.

2.2.3 MANF treatment for increasing 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 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 of the start of the experiment, the rate of weight loss (body weight change index) was 0.79. + -. 0.03 in the mice of the model control group, and the rate of weight gain (body weight change index) was 1.1. + -. 0.03 in the mice of the normal group. 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 to increase Disease Activity Index (DAI) score in mice with ulcerative colitis

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 to improve ulcerative colitis mouse Colon histopathology score

Each group of mice was pathologically scored according to the histological change scoring method 1.2 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, which had been consumed 3% of the DSS aqueous solution for 7 days continuously, was 1.6. + -. 0.75, the histopathological score of the mice of the MANF-treated group, which had been consumed 3% of the DSS aqueous solution for 7 days continuously, was 1.2. + -. 0.39, and the histopathological score of the mice of the normal control group, which had been consumed ultrapure water for 7 days continuously, 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 of the model control group which had consumed 3% DSS aqueous solution for 7 days was 4.37. + -. 0.32cm, and the colon length of the mice of the model mice which had consumed 3% DSS aqueous solution for 7 days after MANF treatment was 5.1. + -. 0.29cm (D in FIG. 6).

2.2.7 MANF relieves 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. In order to detect the effect of MANF on macrophage infiltration in ulcerative colitis, an immunohistochemical staining method was used to detect the macrophage specific marker molecule CD68 using an anti-CD 68 antibody (Abcam, abbo, ab 125212). The result shows that the expression of CD68 in the colon tissue of the mouse in the model control group is increased compared with that of the normal control group, which indicates that the macrophage infiltration in the colon tissue of the mouse in 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 to reduce inflammatory factor expression in colon tissue of mice with ulcerative colitis

To observe the effect of MANF treatment on the expression of inflammatory cytokines in the intestinal tract 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 within 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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Arg Phe Tyr Gln Asp Leu Lys Asp Arg Asp Val Thr Phe Ser Pro Ala

20 25 30

Thr Ile Glu Asn Glu Leu Ile Lys Phe Cys Arg Glu Ala Arg Gly Lys

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Glu Asn Arg Leu Cys Tyr Tyr Ile Gly Ala Thr Asp Asp Ala Ala Thr

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Lys Ile Ile Asn Glu Val Ser Lys Pro Leu Ala His His Ile Pro Val

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Glu Lys Ile Cys Glu Lys Leu Lys Lys Lys Asp Ser Gln Ile Cys Glu

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Leu Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys

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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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Leu Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ala Arg Thr Asp Leu

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Leu Glu His His His His His His

165

Claims

Use of MANF for the preparation of a product for the treatment 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 a protein of R1) or R2):

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

r2) the amino acid sequence is the protein at the 3 rd to the 160 th positions of SEQ ID No. 2.
4. Use of biological material related to MANF for the preparation of a product for the treatment 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 containing the nucleic acid molecule according to B1),

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2),

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3),

b5 A transgenic animal cell line containing the nucleic acid molecule according to B1), a transgenic animal cell line containing the expression cassette according to B2) or a transgenic animal cell line containing the recombinant vector according to B3),

b6 A transgenic plant cell line containing the nucleic acid molecule according to B1), a transgenic plant cell line containing the expression cassette according to B2) or a transgenic plant cell line containing the recombinant vector according to B3).
5. Use according to claim 4, characterized in that: b1 ) the nucleic acid molecule is B11) or B12):

b11 A DNA molecule having the coding sequence shown in SEQ ID No.1,

b12 ) the nucleotide sequence is the DNA molecule shown in SEQ ID No.1 at positions 9-482.