CN114887046B - Application of SPINK4 in treating inflammatory bowel disease - Google Patents

Application of SPINK4 in treating inflammatory bowel disease Download PDF

Info

Publication number
CN114887046B
CN114887046B CN202210671028.2A CN202210671028A CN114887046B CN 114887046 B CN114887046 B CN 114887046B CN 202210671028 A CN202210671028 A CN 202210671028A CN 114887046 B CN114887046 B CN 114887046B
Authority
CN
China
Prior art keywords
spink4
inflammatory bowel
bowel disease
protein
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210671028.2A
Other languages
Chinese (zh)
Other versions
CN114887046A (en
Inventor
张盛洪
王莹
韩婧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
First Affiliated Hospital of Sun Yat Sen University
Original Assignee
First Affiliated Hospital of Sun Yat Sen University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by First Affiliated Hospital of Sun Yat Sen University filed Critical First Affiliated Hospital of Sun Yat Sen University
Priority to CN202210671028.2A priority Critical patent/CN114887046B/en
Publication of CN114887046A publication Critical patent/CN114887046A/en
Application granted granted Critical
Publication of CN114887046B publication Critical patent/CN114887046B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor or ovomucoid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Rheumatology (AREA)
  • Pain & Pain Management (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses an application of SPINK4 in treating inflammatory bowel disease. According to the invention, through extensive and intensive research, the SPINK4 protein can inhibit the disease activity of inflammatory bowel diseases, improve the intestinal state of inflammatory bowel diseases and inhibit the inflammatory reaction of inflammatory bowel diseases, and provides a new target and thought for the treatment of clinical inflammatory bowel diseases.

Description

Application of SPINK4 in treating inflammatory bowel disease
Technical Field
The invention relates to the technical field of inflammatory bowel disease treatment, in particular to application of SPINK4 in treatment of inflammatory bowel disease.
Background
Inflammatory bowel disease (Inflammatory Bowel Disease, IBD) is a chronic, non-specific inflammatory bowel disease caused mainly by disturbances of the intestinal microbiota and the immune system, including Crohn's Disease (CD) and ulcerative colitis (ulcerative colitis UC). IBD is much more affected worldwide, and is statistically about 100 tens of thousands of people in the united states, about 250 tens of thousands in europe, more worldwide, and the cost of diagnosis and treatment of IBD is relatively high, and IBD treatment remains a bottleneck, bringing a significant psychological and economic burden to the home and society.
Current treatments for IBD rely primarily on: 1) Traditional therapeutic drugs: including 5-aminosalicylic acid, glucocorticoids, immunomodulators, and the like; 2) A biological agent; 3) Other drugs: including probiotics, antibiotics, faecal fungus transplantation, etc. Traditional medicines have poor control curative effects on partial moderately severe patients and have serious side effects; with the rise of various biological agents, good news is brought to part of refractory patients, but part of patients still suffer from disease failure response clinically, and the high cost brings heavy economic pressure to patients and families, besides, other drug treatments are only used as auxiliary schemes of IBD treatment due to the uncertainty of curative effects. Thus, finding a positive and effective therapeutic regimen for IBD is a clinical problem that needs to be addressed urgently, defining the key cause of IBD pathogenesis.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides the application of SPINK4 in treating inflammatory bowel diseases.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides the use of a SPINK4 protein or SPINK4 promoter in the manufacture of a medicament for the treatment of inflammatory bowel disease. Wherein, the gene sequence of the SPINK4 is shown as SEQ ID NO.1, and the amino acid sequence of the SPINK4 is shown as SEQ ID NO. 2.
In one embodiment, the inflammatory bowel disease is Crohn's Disease (CD) or ulcerative colitis (ulcerative colitis UC), and the inflammatory bowel disease treatment medicament has at least one of the following functions:
(1) Inhibiting the disease activity of inflammatory bowel disease;
(2) Improving the intestinal state of inflammatory bowel disease;
(3) Inhibiting inflammatory bowel disease intestinal inflammatory response;
the inflammatory bowel disease therapeutic agent in the above-described scheme, the SPINK4 promoter means a substance that increases the level of SPINK 4.
In particular, the increase in SPINK4 levels may be achieved by various chemical, physical, biological methods. Including but not limited to:
(1) Modulating the SPINK4 pathway to increase SPINK4 levels;
(2) Acting on inflammatory bowel disease cells directly increases the level of SPINK 4.
The method can be used for directly increasing the intracellular SPINK4 level of inflammatory bowel disease by delivering the SPINK4 protein or the SPINK4 mimic to an inflammatory bowel disease patient, or can be used for directly increasing the intracellular SPINK4 level of inflammatory bowel disease by over-expressing the SPINK4, and can be used for improving the SPINK4 activity or promoting the transcription or expression of the SPINK4 by adopting a SPINK4 agonist to regulate the SPINK4 channel, thereby improving the SPINK4 level. Increasing SPINK4 activity refers to increasing SPINK4 activity. Preferably, the SPINK4 activity is increased by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by 70%, most preferably by at least 90% compared to before the increase.
In the above schemes, promotion of SPINK4 transcription or expression means: high expression of SPINK4, or increased transcriptional activity of SPINK4, can be achieved by those skilled in the art using conventional methods for regulating the transcription or expression of SPINK 4.
Preferably, the above regimen increases SPINK4 transcription or expression by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by 70%, and most preferably by at least 90%.
Further, by means of over-expressing SPINK4, the intracellular SPINK4 level of inflammatory bowel disease is directly increased, the disease activity can be inhibited, the intestinal state can be improved, and the inflammatory reaction of the intestinal tract can be inhibited;
the medicine for treating inflammatory bowel disease necessarily comprises a SPINK4 protein or a SPINK4 promoter, and takes the SPINK4 protein or the SPINK4 promoter as the active ingredients of the functions, and in the medicine for treating inflammatory bowel disease, the active ingredients playing the functions can be only the SPINK4 protein or the SPINK4 promoter, and can also comprise other molecules which can play similar roles.
Preferably, the SPINK4 protein or SPINK4 promoter is the only active ingredient or one of the active ingredients of the therapeutic agent for inflammatory bowel disease.
In the above-described embodiment, the therapeutic agent for inflammatory bowel disease may be a single component substance or a multicomponent substance.
In the above-mentioned scheme, the dosage form of the therapeutic agent for inflammatory bowel disease is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, etc.
In the above-mentioned scheme, the subject to which the therapeutic agent for inflammatory bowel disease is mainly directed is mammals, such as rodents, primates, etc.
In a second aspect, the invention provides a medicament for the treatment of inflammatory bowel disease, the medicament comprising a biological or chemical agent that targets SPINK4 and is capable of increasing the amount of SPINK4 expression or promoting the expression of SPINK 4.
In this embodiment, the inflammatory bowel disease treatment comprises an effective dose of a protein SPINK4 or a SPINK4 promoter and a pharmaceutically acceptable carrier.
In this embodiment, i.e., the SPINK4 protein or SPINK4 promoter is the only active ingredient or one of the active ingredients of the inflammatory bowel disease therapeutic drug.
In the above-described embodiment, the therapeutic agent for inflammatory bowel disease may be a single component substance or a multicomponent substance.
In the above-mentioned scheme, the dosage form of the therapeutic agent for inflammatory bowel disease is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, etc.
In the above-mentioned scheme, the subject to which the therapeutic agent for inflammatory bowel disease is mainly directed is mammals, such as rodents, primates, etc.
In this embodiment, the inflammatory bowel disease is Crohn's Disease (CD) or Ulcerative Colitis (UC).
The subject may be a mammal. The mammal is preferably a rodent, artiodactyla, fanciful, lagomorpha, primate, etc., preferably a monkey, ape or human.
The subject may be a patient suffering from inflammatory bowel disease or an individual desiring to prevent or alleviate inflammatory bowel disease. Or may be isolated inflammatory bowel disease cells of a patient suffering from inflammatory bowel disease or of an individual desiring to prevent or ameliorate inflammatory bowel disease.
In such embodiments, the SPINK4 protein or SPINK4 promoter may be administered to the subject before, during, or after receiving the inflammatory bowel disease treatment.
In a third aspect the present invention provides the use of a SPINK4 gene or protein in the manufacture or screening of a medicament for the treatment of inflammatory bowel disease, in which embodiment the SPINK4 gene or protein is the target of action, in which embodiment the use is specifically: screening candidate substances by taking the SPINK4 gene or protein as an action target to find an SPINK4 promoter as an alternative inflammatory bowel disease treatment drug.
In summary, the invention adopts the technical proposal and has the beneficial effects that: according to the invention, through extensive and intensive research, the SPINK4 protein can inhibit the disease activity of inflammatory bowel diseases, improve the intestinal state of inflammatory bowel diseases and inhibit the inflammatory reaction of inflammatory bowel diseases, and provides a new target and thought for the treatment of clinical inflammatory bowel diseases.
Drawings
FIG. 1 is a graph showing the therapeutic effect of recombinant SPINK4 protein on inflammatory bowel disease model, wherein TNBS-treatment is TNBS enema group treated by recombinant SPINK4-Fc protein, TNBS-Fc is TNBS enema group treated by Fc, NC is enema control group; panel a) is weight change, panel B-C) is intestinal change, and panel D-E) is intestinal inflammatory response;
FIG. 2 shows a model of a SPINK4 intestinal epithelium-specific knockout mouse, wherein DSS-KO is a model of a SPINK4 knockout mouse DSS, DSS-WT is a model of a littermate control mouse DSS, KO-H 2 O is the water treatment group fed by SPINK4 knockout mice, and WT-H 2 O is the littermate control mice fed the water treatment group; panels a-B) are post-molding disease activity, panels C-D) are intestinal changes, and panels E-F) are intestinal inflammatory responses;
FIG. 3 is a graph showing the therapeutic effects of recombinant SPINK4 protein on inflammatory bowel disease model with SPINK4 intestinal epithelium-specific knockout, wherein WT-DSS is a littermate control mouse DSS-dosed group, WT-H 2 O is the littermate control mice fed the water treatment group; KO-DSS was used as a DSS administration group for SPINK4 knockout mice, KO-H 2 O is the water treatment group fed by the SPINK4 knockout mice, KO-DSS-rmSPINK4 is the recombinant SPINK4-Fc protein treatment group fed by the SPINK4 knockout mice DSS; panel a) is weight change, panel B) is disease activity, panels C-D) is intestinal change, and panels E-F) is intestinal inflammatory response.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
EXAMPLE 1 establishment of TNBS-induced inflammatory bowel disease model
1.1 Experimental materials
30C 57BL/6J male mice of the barrier environment of experimental animals, 6-8 weeks old, body mass (20+ -5 g), purchased from Jiangsu Jiujiaku Kangyaokang biotechnology Co., ltd., animal production license number: SCXK 2018-0008, SPF-grade feeding.
5% TNBS solution was purchased from sigma company; the absolute ethyl alcohol, the acetone and the olive oil are purchased uniformly by a first hospital affiliated to the university of Zhongshan, and the isoflurane is purchased at an animal center affiliated to the first hospital of the university of Zhongshan; histopathological embedding and slicing are accomplished by entrusted wuhansai wile biotechnology limited; microscope (Olympus corporation, japan); electronic balance (sartorius company, germany); micropipettes (Eppendorf, usa); the small animal anesthetic is provided by a first hospital animal center affiliated with the university of Zhongshan; other conventional reagents and surgical instruments are provided by the department of gastroenterology laboratory of the first hospital affiliated to the university of Zhongshan.
1.2 Experimental methods
Preparation of main reagents: pre-sensitization solution: acetone, olive oil and TNBS are prepared in a ratio of 16:4:5, and TNBS is not added in ethanol control group pre-sensitization; enema liquid: a50% ethanol solution and a TNBS-ethanol mixed solution were prepared (5% TNBS solution and ethanol solution were mixed at a ratio of 1:1 to prepare a mixed solution containing 2.5mg of TNBS per 100. Mu.L of 50% ethanol solution).
Animal grouping and establishment of TNBS colitis model 30C 57BL/6J mice were randomly divided into TNBS and ethanol control groups of 15 mice each. Pre-sensitization of the skin at the back of the neck of a mouse is carried out by taking 4:1 acetone olive oil as a solvent to prepare 1% TNBS (Sigma), fasted for 12-24 hours in advance, preparing 2.5% TNBS solvent containing 50% ethanol on the day of clysis, dipping a small amount of paraffin oil into 4cm of the tail end of a smooth thin catheter with the diameter of 1.2mm as a lubricant, slowly inserting the mouse from anus after isoflurane is inhaled for anesthesia, rapidly injecting 100 mu l of 2.5% TNBS solution to the depth of about 4cm, rapidly drawing a tube, and inverting the mouse for about 1min; the normal control group was given an equal amount of 50% ethanol solution, and then returned to the cage for normal feeding after awakening from anesthesia.
Model evaluation method the general state of mice was closely observed daily, the body mass was weighed, and diarrhea, hematochezia conditions and survival numbers were recorded. The modeling is successful by taking the mouse with loose stool, macroscopic blood stool and fecal occult blood as the model.
Experimental results: after 1d of modeling, TNBS mice begin to have the phenomena of listlessness, anorexia and weight loss accompanied by hypokinesia, and TNBS administration groups have 12 diarrhea with different degrees; the ethanol control group mice have good state and activity, normal fecal character, no diarrhea and hematochezia, and the weight is restored to be before fasted. After 3d of modeling, TNBS mice showed a significant trend of decreasing body mass compared with ethanol control mice, and the quality of the mice in 3-7d of modeling was slowly recovered, while the quality of the mice in ethanol control mice was always in a stable and growing state.
EXAMPLE 2 protective Effect of recombinant SPINK4 protein on inflammatory bowel disease model
On the basis of TNBS molding prepared in example 1, 10 mug of SPINK4-Fc (the SPINK4 recombinant protein purchased from Yizhushen, the product number is 50953-M02H) or Fc recombinant protein is injected intraperitoneally for 4 days from the same day as TNBS enema, the left side and the right side of the abdominal midline of a mouse are selected as injection sites, local alcohol is sterilized, then the mice are Z-shaped, the syringes are pushed in after withdrawal from blood, liquid leakage is prevented by anticlockwise rotation when the syringes are pulled out, the mice are fed normally, weighed and euthanized on the 4 th day after the enema. Colorectal length was recorded and HE staining was performed to observe pathological conditions.
The result shows that: the injection of 10 mug of the recombinant protein SPINK4 is given on the basis of the acute TNBS modeling, so that the intestinal inflammation condition of the mice can be improved, wherein the conditions are shown as lower activity of the diseases (figure 1. A), reduced intestinal shortening (figures 1.B and C) and lighter colon inflammation (figures 1.D and E) after the modeling;
EXAMPLE 3 protection of recombinant SPINK4 protein against SPINK4 knockout inflammatory bowel disease model
A CRISPR/Cas9 technology is adopted to establish a SPINK4 conditional knockout mouse model, and the completion of Jiangsu Jixiaokang biotechnology Co Ltd is entrusted, and two pairs of gRNA sequences are respectively as follows: gps 00001365-spine 4-5S: TCGAATGACATGATCCGATA (SEQ ID NO. 3); gps00001365-spin 4-5S4: AAGTCCTAGCTCTGCCTTAT (SEQ ID NO. 4); gps 00001365-spine 4-3S3: AGAGCCCGGATCAGCCACTG (SEQ ID NO. 5); gps00001365-spin 4-3S4: CAAAGCCTCAGTGGCTGATC (SEQ ID NO. 6). Screening the SPINK 4-/-homozygotes for breeding and constructing an acute DSS colonitis model, and finding that the SPINK4 intestinal epithelium-specific knockout mice are more sensitive to DSS than littermate control mice, and are characterized by higher disease activity after modeling (fig. 2.A and B), shorter intestinal tract (fig. 2.C and D) and more severe colonic inflammation (fig. 2.E and F).
On the basis of modeling of a SPINK4 knockout mouse DSS, a group for treating colonitis by the SPINK4-Fc recombinant protein is established, and concretely comprises a SPINK4 recombinant protein treatment+modeling knockout group, an Fc recombinant protein control+modeling littermate control group, a water knockout group, a water littermate control group and 10 mug of mouse SPINK4-Fc recombinant protein or 10 mug of Fc recombinant protein from the 4 th day of DSS modeling are established, and the mice are killed after 6 days of continuous administration. As a result, the overall disease activity of the SPINK4 recombinant protein treated group was found to be significantly reduced compared with that of the control intraperitoneal injection group, as shown in FIG. 3.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of Zhongshan affiliated first hospital
<120> use of SPINK4 in the treatment of inflammatory bowel disease
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 399
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
gcaggcccca gccagctcag gctacactat cccaggatca gcatggccgt ccgccagtgg 60
gtaatcgccc tggccttggc tgccctcctt gttgtggaca gggaagtgcc agtggcagca 120
ggaaagctcc ctttctcaag aatgcccatc tgtgaacaca tggtagagtc tccaacctgt 180
tcccagatgt ccaacctggt ctgcggcact gatgggctca catatacgaa tgaatgccag 240
ctctgcttgg cccggataaa aaccaaacag gacatccaga tcatgaaaga tggcaaatgc 300
tgatcccaca ggagcacctc aagccatgaa gtgtcagctg gagaacagtg gtgggcatgg 360
agaggatatg acatgaaata aaagatccag cccaactga 399
<210> 2
<211> 86
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 2
Met Ala Val Arg Gln Trp Val Ile Ala Leu Ala Leu Ala Ala Leu Leu
1 5 10 15
Val Val Asp Arg Glu Val Pro Val Ala Ala Gly Lys Leu Pro Phe Ser
20 25 30
Arg Met Pro Ile Cys Glu His Met Val Glu Ser Pro Thr Cys Ser Gln
35 40 45
Met Ser Asn Leu Val Cys Gly Thr Asp Gly Leu Thr Tyr Thr Asn Glu
50 55 60
Cys Gln Leu Cys Leu Ala Arg Ile Lys Thr Lys Gln Asp Ile Gln Ile
65 70 75 80
Met Lys Asp Gly Lys Cys
85
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
tcgaatgaca tgatccgata 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
aagtcctagc tctgccttat 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
agagcccgga tcagccactg 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
caaagcctca gtggctgatc 20

Claims (7)

  1. Use of a SPINK4 protein or a SPINK4 promoter for the manufacture of a medicament for the treatment of inflammatory bowel disease, which is crohn's disease or ulcerative colitis, said SPINK4 promoter being a substance that increases the level of the SPINK4 gene or protein.
  2. 2. The use according to claim 1, wherein the agent modulates the SPINK4 pathway to increase the level of SPINK4 or acts to directly increase the level of SPINK4 in inflammatory bowel disease cells.
  3. 3. The use of claim 2, wherein the SPINK4 agonist is used to promote the transcription or expression of SPINK4, thereby increasing the level of SPINK4 gene or protein.
  4. 4. The use according to claim 1, wherein the medicament comprises a biological or chemical agent that targets SPINK4 and is capable of increasing the amount of SPINK4 expression or promoting the expression of SPINK 4.
  5. 5. The use according to claim 4, wherein the medicament is SPINK4 protein or SPINK4 promoter and is the sole active ingredient or one of the active ingredients of the medicament for the treatment of inflammatory bowel disease.
  6. 6. The use according to any one of claims 1 to 5, wherein the therapeutic agent for inflammatory bowel disease is in the form of a solid, liquid, gel, semifluid or aerosol.
  7. Use of the SPINK4 gene or protein in screening a therapeutic agent for inflammatory bowel disease, characterized in that the SPINK4 gene or protein is used as an action target, and candidate substances are screened to find a SPINK4 promoter, wherein the SPINK4 promoter is a substance for increasing the level of the SPINK4 gene or protein, and the inflammatory bowel disease is crohn's disease or ulcerative colitis, as an alternative therapeutic agent for inflammatory bowel disease.
CN202210671028.2A 2022-06-15 2022-06-15 Application of SPINK4 in treating inflammatory bowel disease Active CN114887046B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210671028.2A CN114887046B (en) 2022-06-15 2022-06-15 Application of SPINK4 in treating inflammatory bowel disease

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210671028.2A CN114887046B (en) 2022-06-15 2022-06-15 Application of SPINK4 in treating inflammatory bowel disease

Publications (2)

Publication Number Publication Date
CN114887046A CN114887046A (en) 2022-08-12
CN114887046B true CN114887046B (en) 2023-05-05

Family

ID=82728333

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210671028.2A Active CN114887046B (en) 2022-06-15 2022-06-15 Application of SPINK4 in treating inflammatory bowel disease

Country Status (1)

Country Link
CN (1) CN114887046B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115094131B (en) * 2022-06-15 2023-05-23 中山大学附属第一医院 Diagnostic marker for inflammatory bowel disease and application thereof
CN117982615B (en) * 2024-01-31 2024-10-08 齐鲁工业大学(山东省科学院) Application of polypeptide LLTRAGL in preparation of medicines for preventing and/or treating inflammatory bowel disease

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090258848A1 (en) * 2005-12-06 2009-10-15 The Johns Hopkins University Biomarkers for inflammatory bowel disease
US20160138113A1 (en) * 2013-06-19 2016-05-19 Memorial Sloan-Kettering Cancer Center Methods and compositions for the diagnosis, prognosis and treatment of brain metastasis
US20210285053A1 (en) * 2018-05-29 2021-09-16 Turun Yliopisto L1td1 as predictive biomarker of colon cancer
CN109613258A (en) * 2018-12-14 2019-04-12 中国农业大学 A kind of application of ulcerative colitis biomarker and therapy target
JP2020191834A (en) * 2019-05-29 2020-12-03 学校法人慶應義塾 Method for determining effectiveness of application of cancer immunotherapy for gastrointestinal cancer patient
CA3180628A1 (en) * 2020-06-03 2021-12-09 Rebecca GONSKY Treatments for a sub-population of inflammatory bowel disease patients

Also Published As

Publication number Publication date
CN114887046A (en) 2022-08-12

Similar Documents

Publication Publication Date Title
CN114887046B (en) Application of SPINK4 in treating inflammatory bowel disease
Caine et al. Effects of mesolimbic dopamine depletion on responding maintained by cocaine and food
Pelleymounter et al. Effects of the obese gene product on body weight regulation in ob/ob mice
McLaughlin et al. The opioid growth factor–opioid growth factor receptor axis: Homeostatic regulator of cell proliferation and its implications for health and disease
Steiner et al. Hypocretin/orexin deficiency decreases cocaine abuse liability
AU2019219743B2 (en) TATk-CDKL5 fusion proteins, compositions, formulations, and use thereof
Bernstein et al. Hypocretin receptor 1 knockdown in the ventral tegmental area attenuates mesolimbic dopamine signaling and reduces motivation for cocaine
Del Bufalo et al. Alpha9Alpha10 nicotinic acetylcholine receptors as target for the treatment of chronic pain
CN111714619B (en) Application of fibroblast growth factor 6 in preparation of medicine for relieving liver injury of non-alcoholic steatohepatitis
CN111888463B (en) Thymosin beta 4 medicinal preparation for treating depression, preparation method and application thereof
Sharma et al. Influence of dynorphin A antibodies on the formation of edema and cell changes in spinal cord trauma
Cameron Effect of Total Parenteral Nutrition on Tumor-Host Responses in Rats¹
Christ et al. The application of gene therapy to the treatment of erectile dysfunction
Holstad et al. A transcriptional inhibitor of TNF-α prevents diabetes induced by multiple low-dose streptozotocin injections in mice
Yokoyama et al. Gene therapy for bladder overactivity and nociception with herpes simplex virus vectors expressing preproenkephalin
CN108114271B (en) Pharmaceutical composition containing insulin-like growth factor-2 and application thereof
WO2008132458A1 (en) Compositions useful in the modulation of immune responses and the treatment or prevention of inflammatory responses and related methods
KR20190034546A (en) TATκ-CDKL5 fusion proteins, compositions, formulations and uses thereof
WO2019232203A2 (en) Methods and compositions to alleviate vascular permeability
Chuang et al. Gene gun particle encoding preproenkephalin cDNA produces analgesia against capsaicin-induced bladder pain in rats
WO2004080476A1 (en) Use of secretin in the treatment of schizophrenia
WO2022229644A1 (en) Combination therapies comprising c/ebp alpha sarna
Liu et al. Epigenetic upregulation of hippocampal CXCL12 contributes to context spatial memory-associated morphine conditioning
KR102237349B1 (en) Pharmaceutical composition for the treatment or prevention of nicotine addiction and withdrawal comprising miRNA
CN103751765B (en) Application of recombined ganoderma lucidum immunoregulation protein (rLZ-8) to preparation of medicine for treating tissue fibrosis

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant