KR20240086719A - Uses of Lipocalin-2 for diagnosing and treating traumatic brain injury - Google Patents

Abstract

The present invention relates to the use of lipocalin-2 for diagnosis and treatment of traumatic brain injury. More specifically, traumatic brain injury diagnosis, prognosis, and drug responsiveness determination of lipocalin-2, whose expression or activity increases in traumatic brain injury. It relates to the use as a biomarker for , and the use of substances that inhibit its expression or activity in the treatment of traumatic brain injury.
Lipocalin-2 not only shows a significantly increased level in traumatic brain injury patients compared to the normal group, but it is also easy to measure because it can be measured through body fluid samples such as plasma. In addition, lipocalin-2 can be very useful as a target for the treatment and diagnosis of traumatic brain injury because inhibiting its expression or activity can have a therapeutic effect on traumatic brain injury.

Description

{Uses of Lipocalin-2 for diagnosing and treating traumatic brain injury}

The present invention relates to the use of lipocalin-2 for diagnosis and treatment of traumatic brain injury. More specifically, traumatic brain injury diagnosis, prognosis, and drug responsiveness determination of lipocalin-2, whose expression or activity increases in traumatic brain injury. It relates to the use as a biomarker for , and the use of substances that inhibit its expression or activity in the treatment of traumatic brain injury.

Traumatic brain injury (TBI) is damage caused by an external physical force applied to the head, causing a decrease or change in consciousness, resulting in damage to cognitive or physical functions. It can also cause behavioral problems or emotional damage. These injuries or disabilities may be temporary or permanent, and may cause damage to part of the body, impairment of overall function, or psychosocial problems.

Generally, TBI is classified as mild, moderate, and severe depending on the severity of the injury. People with mild TBI may remain conscious or may experience loss of consciousness for seconds or minutes. You may also experience headaches, confusion, lightheadedness, dizziness, blurred vision, eyestrain, ringing in the ears, behavioral or mood changes, or difficulty remembering, concentrating, paying attention, or thinking. People with moderate or severe TBI may exhibit these same symptoms, including headaches that get worse or do not go away, repeated vomiting or nausea, convulsions or seizures, slurred speech, and weakness or paralysis of the extremities.

Disabilities caused by TBI can vary depending on the severity of the injury, location of the injury, and the patient's age and health. Some common disabilities include cognitive impairment (thinking, memory, and reasoning) and sensory processing impairment (visual, auditory). , touch, taste and smell), communication disorders (expression and understanding), and behavioral or mental health disorders (depression, anxiety, personality transformation, aggression and social misfit). To treat this disability, TBI patients receive rehabilitation treatment such as physical therapy, occupational therapy, and speech therapy, but the rehabilitation treatment has the disadvantage of slow improvement in disability. Therefore, there is a need for technology to treat TBI more effectively.

Accordingly, the present inventors conducted repeated research to explore targets that can accurately diagnose and treat traumatic brain injury, and as a result, the expression of lipocalin-2 was significantly increased in the brains of traumatic brain injury patients, and its expression or activity was suppressed. The present invention was completed after discovering that traumatic brain damage can be improved or treated.

Accordingly, the object of the present invention is to provide a pharmaceutical composition for preventing or treating traumatic brain injury containing an inhibitor of the expression or activity of lipocalin-2 as an active ingredient.

Another object of the present invention is the step of (a) contacting a test substance with a cell expressing lipocalin-2; (b) measuring the mRNA expression level of lipocalin-2 gene, or the expression or activity level of lipocalin-2 protein in cells contacted with the test substance and control cells not contacted with the test substance; and (c) comparing the measured expression or activity level with the expression or activity level measured from a control group.

Another object of the present invention is to provide a biomarker composition containing an agent for measuring lipocalin-2 expression level.

Another object of the present invention is to provide a method for qualitative or quantitative analysis of lipocalin-2 expression level from a sample collected from a subject in order to provide information necessary for diagnosing traumatic brain injury.

In order to achieve the above-described object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating traumatic brain injury containing an expression or activity inhibitor of lipocalin-2 as an active ingredient.

In order to achieve another object of the present invention, the present invention includes the steps of (a) contacting a test substance with a cell expressing lipocalin-2; (b) measuring the mRNA expression level of lipocalin-2 gene, or the expression or activity level of lipocalin-2 protein in cells contacted with the test substance and control cells not contacted with the test substance; and (c) comparing the measured expression or activity level with the expression or activity level measured from a control group.

In order to achieve another object of the present invention, the present invention provides a biomarker composition comprising an agent for measuring lipocalin-2 expression level.

In order to achieve another object of the present invention, the present invention provides a method for qualitative or quantitative analysis of lipocalin-2 expression level from a sample collected from a subject in order to provide information necessary for diagnosing traumatic brain injury.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating traumatic brain injury containing an expression or activity inhibitor of lipocalin-2 as an active ingredient.

As used herein, 'treatment' means suppressing the occurrence or recurrence of a disease, alleviating symptoms, reducing direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving, alleviating, or improving the prognosis of the disease state. do. The term 'prevention' used in the present invention refers to all actions that suppress the onset or delay the progression of a disease.

In this specification, 'protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to, for example, a polymer of amino acid residues as commonly found in proteins in their natural state.

In this specification, 'polynucleotide' or 'nucleic acid' refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of a single or double strand. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included. In general, DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), while RNA uses uracil (U) instead of thymine. has. In a nucleic acid double strand, A forms a hydrogen bond with T or U and C forms a hydrogen bond with G base, and this relationship between bases is called 'complementary'.

Meanwhile, 'mRNA (messenger RNA or messenger RNA)' is RNA that serves as a blueprint for polypeptide synthesis (protein translation) by transferring the genetic information of the base sequence of a specific gene to the ribosome during the protein synthesis process. Using a gene as a template, single-stranded mRNA is synthesized through the transcription process.

In the present invention, the specific sequence of the lipocalin-2 protein is not particularly limited as long as it is known to be of human origin, but for example, one known as GenBank accession number: NP_005555.1 (SEQ ID NO: 1) can be used. Preferably, the lipocalin-2 protein of the present invention may be a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1, and more preferably, it may be a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1. The lipocalin-2 protein is known to be encoded by a polynucleotide sequence, for example, GenBank accession number: NM_005564 (mRNA, SEQ ID NO: 2).

[SEQ ID NO: 1]

mplgllwlgl allgalhaqa qdstsdlipa pplskvplqq nfqdnqfqgk wyvvglagna

ilredkdpqk myatiyelke dksynvtsvl frkkkcdywi rtfvpgcqpg eftlgniksy

pgltsylvrv vstnynqham vffkkvsqnr eyfkitlygr tkeltselke nfirfskylg

lpenhivfpv pidqcidg

[SEQ ID NO: 2]

actcgccacc tcctcttcca cccctgccag gcccagcagc caccacagcg cctgcttcct

cggccctgaa atcatgcccc taggtctcct gtggctgggc ctagccctgt tgggggctct

gcatgcccag gcccaggact ccacctcaga cctgatccca gccccacctc tgagcaaggt

ccctctgcag cagaacttcc aggacaacca attccagggg aagtggtatg tggtaggcct

ggcagggaat gcaattctca gagaagacaa agacccgcaa aagatgtatg ccaccatcta

tgagctgaaa gaagacaaga gctacaatgt cacctccgtc ctgtttagga aaaagaagtg

tgactactgg atcaggactt ttgttccagg ttgccagccc ggcgagttca cgctgggcaa

cattaagagt taccctggat taacgagtta cctcgtccga gtggtgagca ccaactacaa

ccagcatgct atggtgttct tcaagaaagt ttctcaaaac agggagtact tcaagatcac

cctctacggg agaaccaagg agctgacttc ggaactaaag gagaacttca tccgcttctc

caaatctctg ggcctccctg aaaaccacat cgtcttccct gtcccaatcg accagtgtat

cgacggctga gtgcacaggt gccgccagct gccgcaccag cccgaacacc attgagggag

ctgggagacc ctccccacag tgccacccat gcagctgctc cccaggccac cccgctgatg

gagccccacc ttgtctgcta aataaacatg tgccctcagg

In the present invention, the expression or activity inhibitor of lipocalin-2 includes an expression inhibitor of the lipocalin-2 gene, or an expression or activity inhibitor of the lipocalin-2 protein.

In the present invention, the 'lipocalin-2 gene expression inhibitor' refers to a substance that reduces the expression or activity of the lipocalin-2 gene in cells, and is a nucleic acid, polypeptide, or protein that specifically binds to the lipocalin-2 gene. , or a combination thereof, and specifically acts directly on the lipocalin-2 gene or indirectly acts on the upstream regulator of the lipocalin-2 gene to reduce the expression of the lipocalin-2 gene at the transcription level. It refers to a substance that reduces the expression level or activity of the lipocalin-2 gene by increasing the degradation of the expressed lipocalin-2 gene or interfering with its activity.

This includes, but is not limited to, biological molecules such as nucleic acids or polypeptides, compounds, extracts isolated from bacteria, plants or animals, etc., which can be processed into cells through standard techniques known in the art to inhibit the genes. Antisense oligonucleotides, short interfering RNA (siRNA), short hairpin RNA (shRNA), aptamers, ribozymes, and small molecule compounds that bind complementary to the mRNA of the lipocalin-2 gene. , gene scissors (CRISPR/Cas-9), DNAzyme, PNA (protein nucleic acid), or a combination thereof. For example, the expression inhibitor may be an antisense oligonucleotide, siRNA, aptamer, or a combination thereof that binds to the lipocalin-2 gene.

In the present invention, the 'antisense oligonucleotide' refers to an oligomer or polymer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar linkages. The term also includes modified or substituted oligomers comprising similarly functioning non-naturally occurring monomers or portions thereof. Incorporation of substituted oligomers is based on factors including increased cellular uptake or increased nuclease resistance and can be selected as known in the art. It may contain an oligomer in which the entire oligonucleotide or a portion thereof is substituted. In addition, the antisense oligonucleotides of the present invention are oligomeric mimetics, for example, modified by methods known in the art to increase affinity for their targets and provide resistance to mismatches in the target sequence. It may include peptide nucleic acids (PNA) and locked nucleic acids (LNA). In addition, the antisense oligonucleotides include natural oligonucleotides, phosphorothioate-type oligodeoxyribonucleotides, phosphorodithioate-type oligodeoxyribonucleotides, methylphosphonate-type oligodeoxyribonucleotides, and phosphoramidate. Type oligodeoxyribonucleotide, H-phosphonate type oligodeoxyribonucleotide, triester type oligodeoxyribonucleotide, alpha-anomeric type oligodeoxyribonucleotide, peptide nucleic acid, other artificial nucleic acids and nucleic acid-modified compounds. It may be in the form of a modified oligonucleotide, but is not limited thereto. Antisense oligonucleotide molecules that bind complementary to the lipocalin-2 base sequence can be isolated or prepared using standard molecular biology techniques, for example, chemical synthesis methods or recombinant methods, or commercially available ones can be used.

In the present invention, the 'short interfering RNA (siRNA)' acts specifically on the lipocalin-2 gene and cleaves lipocalin-2 RNA, thereby inducing RNA interference (RNAi: RNA interference). refers to double-stranded RNA that can Since siRNA can suppress the expression of target genes, it can be provided as an efficient gene knockdown method or as a gene therapy method. siRNA was first discovered in plants, worms, fruit flies, and parasites, but the development/use of siRNA has recently been applied to mammalian cell research. siRNA for the lipocalin-2 gene is composed of a sense RNA strand containing a sequence homologous to part or all of the lipocalin-2 gene nucleic acid sequence and an antisense RNA strand containing a complementary sequence to the lipocalin-2 gene nucleic acid sequence. 2 May contain a nucleotide sequence capable of hybridizing to RNA. The siRNA is not limited to complete pairing of the double-stranded RNA portion where RNAs are paired, but does not pair due to mismatch, i.e., the corresponding bases are not complementary, or bulge, i.e., the absence of the corresponding base in one chain, etc. Parts may be included. The total length may be 10 to 100 bases, preferably 15 to 80 bases, specifically 20 to 70 bases, more specifically 20 to 30 bases. The siRNA molecule may have a short nucleotide sequence, such as about 5-15 nt, inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is hairpinized by intramolecular hybridization. A structure is formed, and an overall stem-and-loop structure may be formed. This stem-and-loop structure can be processed in vitro or in vivo to generate active siRNA molecules capable of mediating RNAi.

In the present invention, the 'short hairpin RNA (shRNA)' refers to an RNA molecule having an RNA sequence that creates a tight hairpin turn that can be used to silence gene expression through RNA interference. The shRNA hairpin structure is cleaved by cellular mechanisms into siRNA, which can then bind to the RNA-induced silencing complex (RISC). This complex can bind to and cleave the mRNA that matches the siRNA to which it binds. The sequence of the siRNA may correspond to the full-length target gene or a subsequence thereof. A siRNA is “targeted” to a gene, where the nucleotide sequence of the duplex portion of the siRNA may be substantially complementary to the nucleotide sequence of the targeted gene. The siRNA sequence duplex needs to be of sufficient length to target the RNA by bringing the siRNAs together through complementary base-pairing interactions, and can be of various lengths. The length of the siRNA is preferably at least 10 nucleotides, sufficient length to stably interact with the target RNA; Specifically 10-30 nucleotides; More specifically any integer between 10 and 30, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, It can be 28, 29 and 30 nucleotides. The term “sufficient length” means nucleotides of at least 10 nucleotides long enough to provide the desired function under expected conditions. shRNA can be cloned into a vector using recombinant DNA technology.

In the present invention, the 'aptamer' refers to a nucleic acid ligand molecule that binds to the lipocalin-2 gene and has an antagonistic effect on it by its ability to adopt a specific three-dimensional conformation. Typically, aptamers can be small nucleic acids 15-50 bases long that fold into defined secondary and tertiary structures, such as stem-loop structures. It may be desirable for the aptamer to bind the target molecule with a kd of less than 10 6, 10-8, 10-10, or 10-12. Aptamers can bind target molecules with a very high degree of specificity. Additionally, aptamers may be composed of multiple ribonucleotide units, deoxyribonucleotide units, or a mixture of both types of nucleotide residues. The aptamer may further comprise one or more modified base, sugar or phosphate backbone units.

In the present invention, the 'ribozyme' refers to an RNA molecule with catalytic activity. Ribozymes with various activities are known, and ribozymes of the lipocalin-2 gene include known or artificially produced ribozymes, and ribozymes with selective target-specific RNA cleavage activity are known. Can be prepared by standard techniques.

In the present invention, the 'low molecular compound' refers to a chemical substance of natural or non-natural origin other than biopolymers existing in the living body, and may be a target tissue-specific compound or a non-natural compound, but is not limited thereto. .

In the present invention, the 'gene scissors (CRISPR/Cas-9)' includes all elements involved in inducing expression or activity, including the guide RNA sequence and the protein or polypeptide of the CRISPR enzyme, and the CRISPR-Cas The -9 system may be an RNP (ribonucleoprotein) system in which guide RNA and CRISPR enzyme form a complex. At this time, the RNP is a protein-RNA complex, which may refer to the form of a complex formed by the interaction of RNA and RNA-binding protein.

In the present invention, the 'lipocalin-2 protein expression inhibitor' refers to a substance that inhibits the expression of the lipocalin-2 gene, which regulates the expression of lipocalin-2 protein, or a substance that decomposes already expressed protein. .

In the present invention, the 'lipocalin-2 protein activity inhibitor' refers to a substance that reduces the activity or action of lipocalin-2 protein, and includes compounds, peptides, and peptide analogs (mimetics) that specifically bind to lipocalin-2 protein. ), it may be an aptamer, antibody, natural extract, synthetic compound, or a combination thereof, for example, it may be an antibody containing a polyclonal antibody, monoclonal antibody, or recombinant antibody that binds to the lipocalin-2 protein. . Additionally, the peptidomimetic refers to a compound that mimics the structure and desirable characteristics of a specific polypeptide as a therapeutic agent.

Since the sequences of the lipocalin-2 gene and protein can be easily obtained by a person skilled in the art from a known database, GenBank, etc., antisense oligonucleotides, siRNA, aptamers, antibodies, etc. that can suppress the expression of the gene using this are known. Can be prepared by standard techniques.

In the present invention, the 'antibody' refers to a specific protein molecule directed to an antigenic site that can bind to lipocalin-2 protein and inhibit the activity of lipocalin-2. The antibody refers to an antibody that specifically binds to lipocalin-2 protein and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the lipocalin-2 protein has been identified, antibodies against the lipocalin-2 protein can be easily prepared using techniques well known in the art. The polyclonal antibody can be produced by a method well known in the art, which involves injecting the lipocalin-2 protein antigen described above into an animal and collecting blood from the animal to obtain serum containing the antibody. These polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows and dogs. The monoclonal antibody can be produced using the hybridoma method or phage antibody library technology well known in the art. Antibodies prepared by the above method can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. Additionally, the antibodies of the invention may include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function and may include, for example, Fab, F(ab'), F(ab') 2, and Fv.

In one embodiment of the present invention, the activity of the lipocalin-2 protein is pharmacologically inhibited using Fatostatin A, or the expression of the lipocalin-2 gene is inhibited using shRNA, resulting in secretion of inflammatory cytokines due to infection, It has been confirmed that the collapse of the vascular barrier is inhibited and the survival rate of sepsis animal models is significantly improved.

In the present invention, the lipocalin-2 expression or activity inhibitor may be used as such or in the form of a pharmaceutically acceptable salt. In the above, 'pharmaceutically acceptable' refers to a non-toxic composition that is physiologically acceptable and does not usually cause allergic reactions or similar reactions when given to humans, and the salt includes pharmaceutically acceptable salts. Acid addition salts formed with free acids are preferred. The free acid may be an organic acid or an inorganic acid. The organic acids are not limited thereto, but include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Includes glutamic acid and aspartic acid. Additionally, the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid.

The pharmaceutical composition according to the present invention may contain the lipocalin-2 expression or activity inhibitor alone or may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may additionally contain an excipient or diluent. The carrier includes all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, it may contain various drug delivery substances used for oral administration. Additionally, the carrier for parenteral administration may include water, suitable oil, saline solution, aqueous glucose, glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and agents may be referred to as described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. It can be.

The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route described above.

In the case of a formulation for oral administration, the composition of the present invention can be formulated into powder, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc. using methods known in the art. You can. For example, oral preparations can be prepared by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose, gelatin, polyvinylpyrrolidone, etc. may be included. Additionally, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives.

In the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a commonly known text in all pharmaceutical chemistry.

The total effective amount of the composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the total preferred dosage of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per kg of patient body weight per day. However, the effective dose for the patient is determined by considering various factors such as the formulation method, administration route, and number of treatments as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate. Therefore, taking this into account, anyone skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

Furthermore, the pharmaceutical composition of the present invention can be administered in combination with known compounds that have the effect of preventing and treating traumatic brain injury.

The present invention also includes the steps of (a) contacting a test substance with a cell expressing lipocalin-2; (b) measuring the mRNA expression level of lipocalin-2 gene, or the expression or activity level of lipocalin-2 protein in cells contacted with the test substance and control cells not contacted with the test substance; and (c) comparing the measured expression or activity level with the expression or activity level measured from a control group.

(a) contacting the test substance with cells expressing lipocalin-2;

Cells or tissues expressing lipocalin-2 protein of the present invention include, but are not particularly limited to, cells or tissues in which lipocalin-2 protein is endogenously expressed or transiently highly expressed lipocalin-2. . The term 'test substance' used while referring to the screening method of the present invention refers to an unknown substance used in screening to test whether it affects the expression or activity of lipocalin-2. The test substances include small interference RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, DNAzyme, peptide nucleic acids (PNA), peptides, antisense oligonucleotides, antibodies, aptamers, and natural substances. Including, but not limited to, extracts or chemicals.

In addition, the cells used in step (a) may be provided in the form of experimental animals, in which case the screening method of the present invention further includes the step of causing traumatic brain damage in the experimental animal, and the test substance and Contact includes, but is not limited to, parenteral or oral administration and stereotaxic injection, and a person skilled in the art will be able to select an appropriate method for testing the test substance on animals.

Contact with a test substance means adding the test substance to the cell or tissue culture medium and then culturing the cells for a certain period of time. When the cells are provided in the form of experimental animals, contact with the test substance is not limited to parenteral or oral administration, including stereotaxic injection, and a person skilled in the art can select an appropriate method for testing the test substance on animals. will be.

(b) measuring the mRNA expression level of lipocalin-2 gene, or the expression or activity level of lipocalin-2 protein in cells contacted with the test substance and control cells not contacted with the test substance;

To measure the mRNA expression level of the lipocalin-2 gene, conventional expression level confirmation methods in the art can be used without limitation, examples of analysis methods include reverse transcription polymerase chain reaction (RT-PCR), competitive Competitive RT-PCR (RT-PCR), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip. , RNA sequencing, etc., but is not limited to these.

In addition, methods known in the art can be used without limitation to measure the expression or activity level of lipocalin-2 protein, such as western blotting, dot blotting, and enzyme-linked immunosorbent assay. (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry ( FACS) or protein chip methods, but are not limited to these.

(c) comparing the measured expression or activity level with the expression level or activity level measured from a control group;

If the expression or activity level measured in step (b) is lower than the expression or activity level measured in the control group, the test substance can be selected as a preventive or therapeutic agent for traumatic brain injury.

The present invention also provides a biomarker composition comprising an agent for measuring lipocalin-2 expression level.

In the present invention, the biomarker is characterized as being used for one or more purposes selected from the group consisting of diagnosis of traumatic brain injury, prediction of prognosis, and determination of drug responsiveness.

In the present invention, the term 'diagnosis' should not only mean determining the presence or absence of a disease, but should be interpreted to encompass determining the prognosis of the disease, drug responsiveness, and severity of the disease.

When the biomarker composition of the present invention is intended to measure the expression level of lipocalin-2 protein, the agent may be an antibody or aptamer that specifically binds to lipocalin-2 protein.

‘Antibody’ refers to an immunoglobulin that specifically binds to an antigenic site. Lipocalin-2 antibody can be prepared by cloning the lipocalin-2 gene into an expression vector to obtain a protein encoded by the gene, and from the obtained protein according to a conventional method in the art. A lipocalin-2 protein-specific antibody can also be prepared using a fragment of the lipocalin-2 protein containing the lipocalin-2 antigenic region. The form of the antibody of the present invention is not particularly limited and includes polyclonal antibody or monoclonal antibody. In addition, if it has antigen-antibody binding, a portion of the total antibody is also included in the antibody of the present invention, and all types of immunoglobulin antibodies that specifically bind to lipocalin-2 are included. For example, a complete antibody with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule, i.e. Fab, F(ab'), F(ab') with antigen-binding function. 2 and Fv, etc. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies and recombinant antibodies as long as they can specifically bind to the lipocalin-2 protein.

Meanwhile, when the biomarker composition of the present invention is intended to measure the expression level of mRNA encoding lipocalin-2 protein, it may be a probe or primer set that specifically binds to the mRNA.

The mRNA encoding lipocalin-2 may be derived from mammals, including humans, and may preferably include the human lipocalin-2 mRNA base sequence represented by SEQ ID NO: 2. The diagnostic composition of the present invention comprising an mRNA-specific probe or primer set encoding lipocalin-2 may further include agents required for known methods for detecting RNA. Using this composition, known RNA detection methods can be used without limitation to measure the level of mRNA encoding lipocalin-2 in a subject.

In the present invention, the 'primer' is a short single strand oligonucleotide that acts as a starting point for DNA synthesis. The primer binds specifically to the polynucleotide as a template under appropriate buffer and temperature conditions, and DNA polymerase adds a nucleoside triphosphate with a base complementary to the template DNA to the primer and connects it to the DNA. is synthesized. Primers generally consist of 15 to 30 base sequences, and the melting temperature (Tm) at which they bind to the template strand varies depending on base composition and length.

The sequence of the primer does not need to be completely complementary to some of the nucleotide sequences of the template, but it is sufficient to have sufficient complementarity within the range to hybridize with the template and perform the original function of the primer. Therefore, in the present invention, primers for measuring the expression level of mRNA encoding lipocalin-2 do not need to have a perfectly complementary sequence to the lipocalin-2 gene sequence, and the mRNA of lipocalin-2 or It is sufficient as long as it has a length and complementarity suitable for the purpose of measuring the amount of lipocalin-2 mRNA by amplifying a specific section of lipocalin-2 cDNA. The primers for the amplification reaction are a set (pair) that bind complementary to the template (or sense) and the opposite side (antisense) at both ends of a specific section of lipocalin-2 mRNA to be amplified. It consists of Primers can be easily designed by those skilled in the art by referring to the mRNA or cDNA base sequence of lipocalin-2.

‘Probe’ refers to a fragment of polynucleotide such as RNA or DNA with a length of several to hundreds of base pairs that can specifically bind to the mRNA or cDNA (complementary DNA) of a specific gene. Since it is labeled, the presence or absence and expression level of target mRNA or cDNA can be confirmed. For the purpose of the present invention, a probe complementary to lipocalin-2 mRNA can be used to diagnose infectious inflammatory diseases by performing hybridization with a sample of a subject and measuring the expression level of lipocalin-2 mRNA. there is. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. Additionally, primers or probes can be modified in various ways according to methods known in the art to the extent that they do not interfere with hybridization of lipocalin-2 with mRNA. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) ) or charged linkers (e.g. phosphorothioate, phosphorodithioate, etc.), and binding of labeling material using fluorescence or enzymes.

The present invention also provides a method of qualitatively or quantitatively analyzing the expression level of lipocalin-2 from a sample collected from a subject in order to provide information necessary for diagnosing traumatic brain injury.

Specifically, the method includes (a) measuring lipocalin-2 expression level from a sample of a subject; and

(b) comparing the expression level of lipocalin-2 with that of a normal person and determining that the subject with an increased expression level of lipocalin-2 compared to the normal person has suffered from traumatic brain injury.

In the present invention, the term 'analysis' preferably means 'measurement', the qualitative analysis may mean measuring and confirming the presence of the target substance, and the quantitative analysis may mean measuring and confirming the presence of the target substance. It may mean measuring and confirming changes in the level of existence (level of expression) or quantity. In the present invention, analysis or measurement can be performed without limitation, including both qualitative and quantitative methods, and preferably, quantitative measurement is performed.

Hereinafter, the method will be described step by step.

Step (a) is a step of measuring lipocalin-2 expression level in a sample provided from a subject (potential patient).

The sample may be used without limitation as long as it is collected from a subject for whom the occurrence of traumatic brain injury is to be diagnosed. For example, cells or tissues obtained through biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, and various secretions. , urine, feces, etc. Preferably, biological liquid samples (body fluid samples) may be used, for example, blood, plasma, serum, saliva, nasal fluid, sputum, joint sac fluid, amniotic fluid, ascites, cervical or vaginal secretions, urine, and cerebrospinal fluid. It could be. The present invention has great technical significance in that it enables diagnosis of traumatic brain injury using body fluid samples (e.g., plasma) other than the direct brain lesion tissue in which traumatic brain injury occurred, thereby increasing the effectiveness, speed, convenience, and safety of diagnosis. In the present invention, the sample may most preferably be blood, plasma, or serum.

In the present invention, the subject may not only be a subject for whom traumatic brain injury is to be diagnosed, but may also be a patient who has already been diagnosed with traumatic brain injury. In this case, the method of the present invention may be used to determine the disease prognosis or severity of a patient with traumatic brain injury.

Additionally, in the present invention, the subject may be a patient who has already been diagnosed with traumatic brain injury and is receiving medication, or may be a patient who has completed medication administration. In this case, the method of the present invention may be used not only to determine the disease prognosis or severity of a patient with traumatic brain injury, but also to determine drug responsiveness.

The sample may be pretreated before use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc.

Measuring lipocalin-2 expression level in step (a) means measuring the expression level of lipocalin-2 protein or lipocalin-2 coding gene.

The measurement method is not particularly limited as long as the protein expression level is measured by a protein expression measurement method known in the art, for example, Western blot, ELISA (enzyme-linked immunospecific assay), radioimmunoassay, radioimmunodiffusion method, Ou Using any one of the following methods: Cteroni immunodiffusion method, rocket immunoelectrophoresis, immunostaining (including immunohistochemical staining and immunofluorescence staining, etc.), immunoprecipitation analysis, complement fixation analysis, FACS (Fluorescence activated cell sorter), or protein chip method. It could be.

The measurement method is not particularly limited as long as the gene expression level is measured by a gene expression measurement method known in the art, but preferably PCR (polymerase chain reaction), RNase protection assay, northern blotting, or Southern blotting. (southern blotting) and DNA chips may be used.

In step (b), the lipocalin-2 expression level of the subject sample measured in step (a) is compared with that of a normal person, and a subject with an increased lipocalin-2 expression level compared to a normal person is considered to have traumatic brain injury. This is the decision stage.

The lipocalin-2 expression level (including both gene or protein levels) of the subject sample measured by the method in step (a) described above is compared with the lipocalin-2 expression level of the normal sample measured by the same method. At this time, it is preferable that the sample is of the same type or obtained by the same method between the subject and the normal person. A subject with an increased expression level of lipocalin-2 compared to a healthy normal person is determined to have traumatic brain injury.

In the present invention, if the subject is a patient who has already been diagnosed with traumatic brain injury, in step (b), a normal sample measured by the same method or a patient who has already been diagnosed with traumatic brain injury determines the course, prognosis, severity, and drug responsiveness of the disease. By comparing the lipocalin-2 expression level of other patients for whom such information is available, the prognosis, severity, drug responsiveness, etc. of the disease can be predicted.

Lipocalin-2 not only shows a significantly increased level in traumatic brain injury patients compared to the normal group, but it is also easy to measure because it can be measured through body fluid samples such as plasma. In addition, lipocalin-2 can be very useful as a target for the treatment and diagnosis of traumatic brain injury because inhibiting its expression or activity can have a therapeutic effect on traumatic brain injury.

Figure 1. Experimental design for creating a traumatic brain injury model and confirming LCN2 expression time. a Fabrication of a closed brain injury model using weights. b Fabrication of an open brain injury model using controlled cortical shock.
Figure 2. Confirmation of the timing of astrocyte LCN2 expression in closed and open brain injury models. a Increased LCN2 expression in hippocampal astrocytes at day 15 of closed brain injury model (weight drop). b Increased Lcn2 mRNA at 1, 7, and 14 days after brain injury in open brain injury model (CCI). c Increased astrocytic LCN2 protein expression in the hippocampal region at day 7 in open brain injury model (CCI).
Figure 3. Increased LCN2 and reactive astrocytes in postmortem brain tissue from patients with chronic traumatic encephalopathy (CTE) and increased LCN2 protein in plasma of patients with traumatic brain injury (TBI). a Increased LCN2 mRNA expression in CTE patient brain. b Increased astrocyte LCN2 in CTE patients. c Increase in reactive astrocytes. c Increased LCN2 protein in plasma collected within 72 hours from patients with traumatic brain injury.
Figure 4. Confirmation of the timing of expression of neuroinflammatory substances related to secondary brain injury in closed and open brain injury models. a Increased Il6 mRNA expression in obstructive brain injury model (weight drop). b Increased Tnf and Il1b mRNA expression in open brain injury model (CCI).
Figure 5. Brain parenchymal, neurobehavioral, and neuroinflammatory glial activity damaged by open traumatic brain injury (CCI) is alleviated by Lcn2 deficiency. a Experimental design. b Visualization and quantification of loss area using cresyl violet staining to identify brain parenchyma. c Cylindrical pole descending behavior test (pole test) to test motor ability. d Adhesive removal test to test sensory ability. e Passive avoidance test for cognitive ability testing. f Y-shaped maze test for spatial cognitive ability testing (y-maze test). g Visualization and quantification of astrocyte and microglial activation using immunostaining. h Measurement of cytokine production using gene amplification technique and quantification of Tnf and Il1b mRNA.
Figure 6. Brain parenchymal, neurobehavioral, and neuroinflammatory glial activity damaged by open traumatic brain injury (CCI) is alleviated by LCN2 neutralizing antibody treatment. a Experimental design. b Visualization and quantification of loss area using neuron (NeuN) staining to identify brain parenchyma. c Cylindrical pole descending behavior test (pole test) to test motor ability. d Adhesive removal test to test sensory ability. e Passive avoidance test for cognitive ability testing. f Y-shaped maze test for spatial cognitive ability testing (y-maze test). g Visualization and quantification of astrocyte and microglial activation using immunostaining. h Measurement of cytokine production using gene amplification technique and quantification of Tnf and Il1b mRNA.
Figure 7. Inhibition of neuroinflammatory response by astrocyte-derived Lcn2 deficiency and treatment with LCN2 neutralizing antibody (LCN2 Ab) in an in vitro model of traumatic brain injury. a Confirmation of cell ratio using immunostaining after cultured astrocytes, microglia, and co-culture of astrocytes and microglia. b Confirmation of Lcn2 mRNA using gene amplification technique after cell scratch injury. c Production of neuroinflammatory substances induced by cell scratch injury after treatment with LCN2 neutralizing antibody (LCN2 Ab) in co-culture of Lcn2-deficient astrocytes and normal microglia or in co-culture of normal astrocytes and microglia ( Tnf and Il1b mRNA) were confirmed using gene amplification technique.

Hereinafter, the present invention will be explained in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Experiment method

1. Preparation of traumatic brain injury (TBI) animal model (Figure 1)

(1) Closed brain injury animal model (Weight-drop model)

Four-month-old wild-type (C57BL/6) mice were used in this study. Mice were provided with standard laboratory diet and water ad libitum in a controlled environment. Closed diffusion TBI was induced using a weight drop device (weight 100 g, drop height 75 cm, angle 90°). The anatomical trajectory of impact was adjusted from bregma -1 to ±4. All mice were initially anesthetized with an intraperitoneal injection of 2% avertin before undergoing weight-drop induced TBI. Mice were exposed to a total of six shocks (once every three days for 15 days after the initial shock) by dropping a single weight (100 g) from a height of 75 cm. Sham-injured animals received the same anesthesia protocol, but no pendulum shock was used. After TBI induction, mice were placed in a supine position in a clean cage heated with a commercially available heating pad. Mice were then returned to their cages after normal behavior (e.g. grooming, walking, exploration) had recovered.

(2) Controlled cortical impact model

Mice were anesthetized with 2-5% isoflurane and placed in a stereotaxic frame. A 4 mm diameter craniotomy window (craniotomy) was created over the right hemisphere centered 1.5 mm lateral to the midline. The skull was removed to expose the dura, and a CCI injury was delivered to animals in the TBI group using a cylindrical 2.5 mm electromagnetic driven impactor (Impact One Stereotaxic Impactor for CCI, Leica Microsystems, Wetzlar, Germany). Diameter tip and impact parameters were as follows: impact velocity, 2 m/s; Penetration depth, 2.5 mm; Dwell time, 300 ms. Sham animals received the same anesthesia and craniotomy. However, no injuries were caused. After the impact or simulation procedure was completed, the severed skull was placed over the craniotomy window and the scalp was sutured. CCI-induced mRNA expression in the brain was measured by RT-PCR at 24 h and 7–14 days after shock. Immunohistochemistry analysis was performed 7 days after shock. Behavioral testing was performed 6–7 days after CCI injury.

2. RT-PCR

Total RNA was extracted from contralateral and ipsilateral brain tissue or cultured cells using QIAzol reagent (QIAGEN) according to the manufacturer's protocol. For conventional RT-PCR, reverse transcription was performed using Superscript II (Invitrogen) and oligo(dT) primers. PCR amplification using specific primer sets was performed at an annealing temperature of 55–60°C for 20–30 cycles. PCR was performed using a C1000 Touch Thermal Cycler (Bio-Rad, Foster City, CA). For PCR product analysis, 10 μl of each PCR was electrophoresed on a 1% agarose gel and detected under UV light. Quantitative RT-PCR (qPCR) analysis was performed using the one-step SYBR ^® PrimeScript™ RT-PCR kit (Perfect Real Time; Takara Bio Inc., Tokyo, Japan) according to the manufacturer's instructions, followed by ABI Prism ^®. It was detected. 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Relative changes in gene expression determined by qPCR were calculated using the 2- ^ΔΔCT method. Primers used for RT-PCR analysis of mouse Lcn2, Tnf, Il1b, Il6, and Gapdh were as follows: Lcn2, 5'-ATG TCA CCT CCA TCC TGG TC-3' (forward), 5'-CAC ACT CAC CAC. CCA TTC AG-3'(reverse); Tnf, 5'-CAT CTT CTC AAA ATT CGA GTG ACA A-3' (forward), 5'-ACT TGG GCA GAT TGA CCT CAG-3'(reverse); Il1b, 5'-AGT TGC CTT CTT GGG ACT GA-3' (forward), 5'-TCC ACG ATT TCC CAG AGA AC-3'(reverse); Il6, 5'-AGT TGC CTT CTT GGG ACT GA-3' (forward), 5'-TCC ACG ATT TCC CAG AGA AC-3'(reverse); Gapdh, 5'-TGG GCT ACA CTG AGG ACC AG-3' (forward), 5'-GGG AGT TGC TGT TGA AGT CG-3' (reverse).

3. Immunofluorescence staining

Animals were anesthetized using diethyl ether and perfused transcardially first with saline and then with 4% paraformaldehyde diluted in 100mM PBS. Brains were fixed in 4% paraformaldehyde for 3 days and then cryoprotected using 30% sucrose solution for an additional 3 days. The fixed brain was coated with OCT compound (Tissue-Tek, Sakura Finetek, Tokyo, Japan), frozen, and then cut into 20-μm-thick slices using a microtome maker. For double immunofluorescence analysis, tissue sections were incubated with antibodies and observed by fluorescence or confocal microscopy. Fluorescence intensity was quantified using ImageJ software version 1.44 (National Institutes of Health (NIH)). For quantitative analysis, images were obtained from three non-overlapping fields within the ipsilateral brain region.

4. Human brain tissue

Neuropathological processing of normal and CTE human brain samples was performed according to previously established procedures at the CTE Center at Boston University. Institutional review board approval for ethical clearance was obtained from the CTE Center. This study was reviewed by the Boston University School of Medicine Institutional Review Board (Protocol H-28974) and approved as exempt because it included only tissue collected from postmortem individuals who were not classified as human subjects. The next of kin provided informed consent for participation and brain donation. This study was conducted in accordance with institutional regulatory guidelines and the principles for the protection of human subjects within the Declaration of Helsinki.

5. Immunohistochemical analysis of human postmortem brain tissue

LCN2 staining: Tissue sections were rehydrated, blocked with blocking solution (1% H2O2), and incubated with goat polyclonal antibody to LCN2 (1:200 dilution; R&D Systems, catalog number: AF1857) for 24 hours. After three washes, slides were processed with the Vector ABC Kit (Vector Laboratories, Inc., Burlingame, CA). Immunoreactive signals were detected using DAB chromogen (Thermo Fisher Scientific, Meridian, Rockford, IL).

GFAP staining: Endogenous alkaline phosphatase was blocked using 3% H ₂ O ₂ in TBS. Sections were treated with 2.5% normal horse serum (Vector Laboratories) before incubation for 24 h with rabbit polyclonal anti-GFAP antibody (1:200 dilution; Sigma Aldrich, catalog number: AB5804). After washing, sections were incubated with ImmPRESS-AP anti-rabbit alkaline phosphatase (IgG) polymer detection reagent (Vector Laboratories) for 30 min at room temperature. Color was used for detection using the Vector Red Alkaline Phosphatase Substrate Kit (Vector Laboratories). Afterwards, the slides were stained for cell nuclei with hematoxylin (Vector Laboratories), and then treated again with xylene using an ethanol gradient of each concentration [70%, 80%, and 95% (once), 100% (twice)]. Next, the tissue piece was sealed by covering it with a cover glass. Images were analyzed using a light microscope.

6. Image analysis of human tissue sections

Quantitative evaluation of immunohistochemical signals and co-localization patterns was performed using CellSens Imaging Software (Olympus Korea Co.). The colocalized region (region of interest) of the merged image from the two overlapping chromogenic dyes was selected by drawing a horizontal line. The signal intensity of interest for the selected line is automatically calculated by the software module and transferred to a Microsoft Excel file to generate statistics and representative graphs. To measure morphological and structural changes in GFAP-positive astrocytes, Sholl analysis was performed using bright-field images immunostained with GFAP antibody. Images of GFAP immunoreactivity in the cortex were taken for analysis. Briefly, the Sholl assay manually draws serial concentric circles at 5-μm intervals from the center of GFAP-positive astrocytes to the end of the process furthest from each single astrocyte and analyzes the number of intersections of GFAP processes in each circle. Sholl analysis was performed using NIH ImageJ software (version 1.44).

7. RNA sequencing

To determine human LCN2 mRNA levels, RNA sequencing data [European Nucleotide Archive (ENA) database under accession no. ERP015139] was utilized, and normal and postmortem human brains diagnosed with CTE were used for analysis. Relative LCN2 mRNA levels were calculated as fold change in fragments per kilobase of exon per million reads (FPKM).

8. Quantification of brain lesion volume

Cresyl violet staining was used to visualize brain damage. Frozen sections were cut in the coronal plane (thickness 20 μm) and thawed and mounted on Superfrost plus slides. Sections were stored at -20°C. Cresyl violet staining was performed to depict brain damage as follows. Cresyl violet staining images were obtained. The lesion was clearly delineated as a pale area within the damaged brain. Calibration standards were used for each image. The evaluator outlined the damaged area using Image J (NIH, version 1.41) in nine standard coronal planes of each brain. Damage was derived by calculating the average damage area between slices and multiplying by the distance between slices. Edema correction was calculated by dividing the ipsilateral hemisphere volume by the contralateral hemisphere volume.

9. Behavioral experiment

Pole test: Mice were positioned head down near the top of a rough-surfaced wooden pole (10 mm diameter, 50 cm height) and the time it took for the mouse to reach the floor was recorded. The test was repeated three times at 30-second intervals, and behavioral changes were evaluated as the average of the three descent times.

Adhesive removal test: Two adhesive tapes (0.3 × 0.4 cm ² ) were attached to the paws of each animal with equal pressure. The mouse was then placed in a plastic cage, and the time of contact and removal of each adhesive tape was recorded up to 120 seconds.

Y-maze test: Spatial cognition was assessed using a voluntary alternation task in a Y-maze apparatus. The Y-maze is a three-armed horizontal maze (40 cm long, 3 cm wide, 12 cm wall height) with the arms at an angle of 120° to each other. Animals were initially positioned in the center and the order and number of arm entries were recorded manually for each animal over a period of 7 min. Voluntary replacement was defined as entry into all three in succession (sequentially). The maze arms were thoroughly washed with water between and after successive placements of animals to remove residual odor. The total number of eight items was used as an indicator of locomotor activity.

Passive avoidance test: This test began with training where the mouse was placed in a brightly lit chamber. When the mouse crossed into a dark room, it received a mild (0.25 mA/sec) electric shock to its paws. The initial latency to enter the dark (shock) compartment was used as a baseline measure. During the probe test 24 hours after training, the mouse was placed back in the bright compartment, and the latency to return to the dark compartment was measured as a passive fear avoidance index.

Cylinder test: To perform this test, mice were individually placed in a transparent cylinder (11.5 cm diameter) and videotaped for 5 min to record aspects of the animals' forelimb use when exploring the cylinder walls. A mirror was placed at an angle behind the cylinder so that forelimb movements could be recorded along 360°. Videos were analyzed in slow motion and frame-by-frame, scored posteriorly, and the number of wall contacts made independently with the left and right forepaws was calculated for a total of 20 wall contacts per mouse and session.

10. Intracisternal administration of LCN2 neutralizing antibody

After anesthetizing with isoflurane, mice were placed in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). The scalp was incised using an operating microscope. A midline incision in the neck muscles exposed the atlanto-occipital membrane covering the cisterna magna. Using a 30G Hamilton syringe, 5 μg of LCN2 monoclonal antibody (R&D systems, Minneapolis, MN, catalog number: MAB1857) or isotype IgG control antibody (R&D systems, catalog number: MAB006) was injected into the cisterna magna of mice after CCI injury within 30 seconds. was injected into the cisterna magna. The syringe was left in place for 5 minutes and then removed. The atlantooccipital membrane was immediately covered with tissue adhesive to prevent leakage. The skin was sutured using 4-0 absorbable sutures.

11. Scratch injury model

Scratch injury was performed as an in vitro model of TBI. Briefly, confluent cell cultures were manually scraped using a sterile pipette tip (200 μL) to create a straight line of damage across the culture well. A 3 mm grid was created by inducing 4 × 4 scratches in each well of a 6-well culture plate. Unscratched cells were used as controls. Cells were exposed to PBS, recombinant LCN2 protein, or LCN2 antibody for 72 h after scratch injury.

Experiment result

As shown in Figure 2, increased LCN2 expression was confirmed in hippocampal astrocytes at day 15 in the closed brain injury model (weight drop) (2a), and at days 1, 7, and 14 after brain injury in the open brain injury model (CCI). An increase in Lcn2 mRNA was identified at day 7 (2b), and an increase in astrocytic LCN2 protein expression was identified in the hippocampal region at day 7 in a open brain injury model (CCI) (2c).

As shown in Figure 3, LCN2 mRNA expression was confirmed to be increased in the brain of patients with chronic traumatic encephalopathy (CTE) (3a), and an increase in LCN2 was confirmed in astrocytes in CTE patients (3b), and the expression of reactive astrocytes was confirmed to be increased (3b). An increase in LCN2 protein was confirmed in plasma collected within 72 hours of patients with traumatic brain injury (3c).

As shown in Figure 4, an increase in the expression of Il6 was confirmed in the closed brain injury model (weight drop) (4a), and an increase in the mRNA expression of TNF and Il1b was confirmed in the open brain injury model (CCI) (4c).

As shown in Figure 5, as a result of visualization and loss area quantification using cresyl violet staining to identify brain parenchyma, it was confirmed that the volume of the brain lesion area caused by open brain injury (CCI) was significantly reduced in the LCN2 deletion model. (5b), and in various behavioral experiments, it was confirmed that abnormal symptoms caused by open brain damage were recovered due to LCN2 deficiency (5c~5f). In addition, as a result of visualizing and quantifying astrocyte and microglial activation using immunostaining, astrocyte and microglial activation was reduced in the LCN2 deletion model (5g), and Tnf and Il1b mRNA quantification results using gene amplification technique also showed that astrocyte and microglial activation was reduced in the LCN2 deletion model. The same trend was confirmed (5h).

As shown in FIG. 6, when LCN2 neutralizing antibody was administered to an open traumatic brain injury animal model, the same effect as the result in FIG. 5 where LCN2 expression was deleted was confirmed.

As shown in Figure 7, as a result of astrocyte-derived Lcn2 deficiency and treatment with LCN2 neutralizing antibody (LCN2 Ab) in a traumatic brain injury in vitro model, it was confirmed that the neuroinflammatory response was suppressed.

Lipocalin-2 not only shows a significantly increased level in traumatic brain injury patients compared to the normal group, but it is also easy to measure because it can be measured through body fluid samples such as plasma. In addition, lipocalin-2 can be very useful as a target for the treatment and diagnosis of traumatic brain injury because inhibiting its expression or activity can have a therapeutic effect on traumatic brain injury, so its industrial applicability is very high.

Sequence list electronic file attached

Claims (11)

A pharmaceutical composition for preventing or treating traumatic brain injury, comprising an expression or activity inhibitor of lipocalin-2 as an active ingredient.
The method of claim 1, wherein the expression inhibitor is an antisense oligonucleotide, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide that binds complementary to the mRNA of the lipocalin-2 gene. A pharmaceutical composition characterized in that it is a tamer, ribozyme, low molecular weight compound, gene scissors (CRISPR/Cas-9), DNAzyme, PNA (protein nucleic acid), or a combination thereof.
The method of claim 1, wherein the activity inhibitor is a compound, peptide, peptide mimetics, aptamer, antibody, natural extract, synthetic compound, or a combination thereof that specifically binds to lipocalin-2 protein. Pharmaceutical composition.
(a) contacting the test substance with cells expressing lipocalin-2;
(b) measuring the mRNA expression level of lipocalin-2 gene, or the expression or activity level of lipocalin-2 protein in cells contacted with the test substance and control cells not contacted with the test substance; and
(c) Comparing the measured expression or activity level with the expression or activity level measured from a control group; A method of screening for a traumatic brain injury prevention or treatment agent comprising a.
A traumatic brain injury biomarker composition comprising an agent for measuring lipocalin-2 expression level.
The composition according to claim 5, wherein the biomarker is one or more biomarkers selected from the group consisting of diagnosing traumatic brain injury, predicting prognosis, and determining drug responsiveness.
The diagnostic composition according to claim 5, wherein the lipocalin-2 expression level is measured by measuring the expression level of lipocalin-2 protein or lipocalin-2 coding gene.
The method of claim 5, wherein the agent
(a) an antibody or aptamer that specifically binds to lipocalin-2 protein; or
(b) A diagnostic composition characterized in that it is a primer or probe that specifically binds to lipocalin-2 mRNA.
The composition according to claim 8, wherein the lipocalin-2 protein comprises an amino acid sequence represented by SEQ ID NO: 1.
The composition according to claim 8, wherein the lipocalin-2 mRNA includes the base sequence of SEQ ID NO: 2.
A method of qualitatively or quantitatively analyzing lipocalin-2 expression levels from samples collected from subjects in order to provide information necessary for diagnosing traumatic brain injury.