KR102328471B1 - Composition for preventing or treating diabetic neuropathy comprising LCN2 inhibitor - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetic nerve damage disease comprising an LCN2 inhibitor as an active ingredient. ), the expression of LCN2 was increased, and it was confirmed that the symptoms of diabetic nerve damage disease were improved when the LCN2 gene was deficient. Therefore, in the present invention, the LCN2 inhibitor is a diabetic nerve damage disease, specifically pain, numbness, numbness, rats, foot ulcers, tachycardia at rest, orthostatic hypotension (dizziness, dizziness, syncope, visual disturbance, weakness, headache) , suggested the possibility that it will be useful as a preventive and therapeutic agent for symptoms such as cardiac denervation syndrome, constipation, diabetic diarrhea, bladder dysfunction, sexual dysfunction, thermoregulation disorder, hypoglycemia anaesthesia, and pupil dysfunction.

Description

Composition for preventing or treating diabetic neuropathy comprising LCN2 inhibitor as an active ingredient

The present invention relates to a composition for preventing or treating diabetic nerve damage disease comprising a lipocalin-2 (Lipocalin-2; LCN2) inhibitor as an active ingredient.

Diabetic neuropathy (DN) is a clinical syndrome characterized by nerve damage caused by persistent hyperglycemia. Several studies have revealed a close link between persistent hyperglycemia and inflammatory pathways, interacting at multiple levels in causing structural and functional nerve damage leading to neurodegenerative diseases. Unfortunately, there is currently no FDA-approved effective treatment for diabetic neuropathy, and all clinical trials to alter the course of diabetic neuropathy have failed. Moreover, the development of targeted therapeutics has been delayed due to a lack of understanding of the pathological mechanisms of diabetic nerve damage disease. From this point of view, understanding the pathogenesis of diabetic nerve damage disease is important in exploring new targets for treating it.

Neutrophil gelatinase-associated lipocalin or LCN2 is a 25-kDa protein and belongs to a member of the lipocalin superfamily. Recently, in several disease models, LCN2 was reported as a novel marker of aseptic neuroinflammation, a modulator of macrophage polarization, and a modulator of peroxisome proliferator-activated receptor-gamma. Meanwhile, in both animals and human subjects, LCN2 has been reported as a novel inflammatory marker that is up-regulated in several metabolic disorders. However, in the peripheral nervous system (PNS) according to diabetes, the effect on LCN2 expression has not been fully studied. Recently, neuroinflammation in diabetic animals has been reported, and it has been reported that the inflammatory microenvironment is associated with a neurodegenerative phenotype in several disease models. In addition, it has already been reported that LCN2 plays an important role in the pathogenesis of demyelinating diseases such as autoimmune encephalomyelitis and optic neuritis. Another study showed that LCN2 contributes to the pathogenesis of hypersensitivity pain in several animal models. LCN2 has been reported to be a modulator of inflammation, pain, myelination and neurodegeneration, but its association with diabetic nerve damage disease has not been known so far.

Korean Patent No. 10-1576606 (Registered on Dec. 4, 2015)

Accordingly, it is an object of the present invention to provide a composition for preventing, improving or treating diabetic nerve damage disease containing an LCN2 expression or activity inhibitor as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating diabetic nerve damage disease containing an LCN2 expression or activity inhibitor as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diabetic nerve damage disease containing an LCN2 expression or activity inhibitor as an active ingredient.

The present invention relates to a composition for preventing or treating diabetic nerve damage disease comprising an LCN2 inhibitor as an active ingredient. ), the expression of LCN2 was increased, and it was confirmed that the symptoms of diabetic nerve damage disease were improved when the LCN2 gene was deficient. Therefore, in the present invention, the LCN2 inhibitor is a diabetic nerve damage disease, specifically pain, numbness, numbness, rats, foot ulcers, tachycardia at rest, orthostatic hypotension (dizziness, dizziness, syncope, visual disturbance, weakness, headache) , suggested the possibility that it will be useful as a preventive and therapeutic agent for symptoms such as cardiac denervation syndrome, constipation, diabetic diarrhea, bladder dysfunction, sexual dysfunction, thermoregulation disorder, hypoglycemia anaesthesia, and pupil dysfunction.

1 shows the expression results of LCN2 in the sciatic nerve and dorsal root ganglion (DRG) of diabetic mice. After 8 weeks of MLDS administration, the expression of LCN2 protein in the sciatic nerve and DRG was measured by Western blot analysis (A and B). After 3 weeks of HDS administration, similar levels of LCN2 protein upregulation were detected in the tissues (C and D). *p < 0.05 versus vehicle-treated control animals; Student's t-test; n=3 for each group; Data are presented as mean ± SEM. MLDS, multiple doses of low-dose STZ; HDS, a single dose of high-dose STZ; LCN2, Lipocalin-2; w, weeks; SEM, standard error of the mean.
Figure 2 shows the results for LCN2 co-localized on the glial cells of the sciatic nerve and DRG after diabetes induction. Results for LCN2 co-localizing with S100 (Schwann cell and satellite glial marker) in sciatic nerve and DRG tissue after 8 weeks of MLDS administration (A and B). Arrows indicate double-labeled cells. Scale bars: 100 μm (sciatic nerve tissue) and 50 μm (DRG tissue). MLDS, multiple doses of low-dose STZ; LCN2, Lipocalin-2; S100, S100 calcium-binding protein B; w, weeks.
3 shows the effect of Lcn2 - deficiency on the inflammatory response of the sciatic nerve and DRG after diabetes induction. The TNF-α protein content in the sciatic nerve and DRG was measured by ELISA and recorded as pg/mg total protein after 8 weeks of MLDS administration (A and C). These are the results of immunofluorescence staining of macrophages infiltrated with Iba-1 (macrophage marker), satellite glial cells activated with GFAP, and nuclei with DAPI (B, D and E). *p < 0.05 versus vehicle-treated control animals; #p < 0.05 between the indicated groups; Student's t-test; n=3 for each group; Data are presented as mean ± SEM. Scale bar, 100 μm. WT, wild type; KO, knockout; MLDS, multiple doses of low-dose STZ; w, weeks; SEM, standard error of the mean.
Figure 4 is Lcn2 for diabetic nerve damage disease shows a deficiency effect. Representative skin sections from control and diabetic mice were stained with PGP9.5 (a nerve fiber marker) and collagen IV (a basement membrane marker that separates the epidermis from the dermis). Multiple free nerve endings passing into the epidermis were visualized (arrows) (A). After 8 weeks of MLDS administration, motor and sensory nerve conduction velocities (MNCV and SNCV) were measured (B). Eight weeks after MLDS administration, H&E staining of the sciatic nerve and DRG was performed (C and E). Eight weeks after diabetes induction, immunofluorescence staining of the sciatic nerve with fluoromyelin was performed (D). *p < 0.05 versus vehicle-treated control animals; #p < 0.05 between the indicated groups; Student's t-test; n=3 for each group; Data are presented as mean ± SEM. Scale bars, 100 μm (PGP 9.5 and collagen IV stained images) and 50 μm (H&E and fluoromyelin stained images). WT, wild type; KO, knockout; MLDS, multiple doses of low-dose STZ; w, weeks; SEM, standard error of the mean.

Accordingly, the present inventors used a streptozotocin (STZ)-induced diabetic mouse model to confirm LCN2 expression in the peripheral nervous system and evaluate its pathological role in the progression of diabetic nerve injury disease. The present inventors have completed the present invention by confirming that LCN2 is a new potential target in the development of a new drug for diabetic nerve damage disease.

The present invention provides a pharmaceutical composition for preventing or treating diabetic nerve damage disease containing an LCN2 expression or activity inhibitor as an active ingredient.

Specifically, the LCN2 expression inhibitor may be an antisense nucleotide complementary to mRNA of the Lcn2 gene, small interfering RNA (siRNA) or short hairpin RNA (shRNA), and the LCN2 activity The inhibitor may be a compound, peptide, peptide mimetics, aptamer, antibody, or natural product that specifically binds to the LCN2 protein, but is not limited thereto.

Preferably, the diabetic nerve damage disease is pain due to diabetes, numbness, numbness, rats, foot ulcers, tachycardia at rest, orthostatic hypotension, cardiac denervation syndrome, constipation, diabetic diarrhea, bladder dysfunction , sexual dysfunction, thermoregulation disorder, hypoglycemia anaphylaxis, or pupil dysfunction, but is not limited thereto.

The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisense, siRNA oligonucleotides, and natural extracts as active ingredients. The pharmaceutical composition or combination formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, and a lubricant. , a solubilizing agent such as a lubricant or flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical formulation of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, sustained-release formulations of the active compound, etc. can The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered as The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the age, weight, general health status, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and drugs used at the same time. Although not limited thereto, for example, in the case of an adult, when administered once to several times a day, the composition of the present invention is administered once to several times a day, in the case of a compound 0.1ng/kg to 10g/kg, polypeptide, In the case of protein or antibody, it can be administered at a dose of 0.1 ng/kg to 10 g/kg, and in the case of antisense nucleotides, siRNA, shRNAi, or miRNA, 0.01 ng/kg to 10 g/kg.

In addition, the present invention provides a health functional food composition for preventing or improving diabetic nerve damage disease containing an LCN2 expression or activity inhibitor as an active ingredient.

Specifically, the LCN2 expression inhibitor may be an antisense nucleotide complementary to mRNA of the Lcn2 gene, small interfering RNA (siRNA) or short hairpin RNA (shRNA), and the LCN2 activity The inhibitor may be a compound, peptide, peptide mimetics, aptamer, antibody, or natural product that specifically binds to the LCN2 protein, but is not limited thereto.

Preferably, the diabetic nerve damage disease is pain due to diabetes, numbness, numbness, rats, foot ulcers, tachycardia at rest, orthostatic hypotension, cardiac denervation syndrome, constipation, diabetic diarrhea, bladder dysfunction , sexual dysfunction, thermoregulation disorder, hypoglycemia anaphylaxis, or pupil dysfunction, but is not limited thereto.

The health functional food composition of the present invention may be provided in the form of powder, granule, tablet, capsule, syrup or beverage, and the health functional food composition is used together with other foods or food additives in addition to the active ingredient, according to a conventional method. can be used appropriately. The mixed amount of the active ingredient may be suitably determined according to the intended use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition may be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for health and hygiene or health control, it should be less than or equal to the above range. It is certain that the active ingredient can be used in an amount greater than the above range because there is no problem in terms of safety.

The type of health food is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and the like.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Mice Breeding and Care

Male C57BL/6 mice (Samtako, Osan, South Korea) and Lcn2 −/- mice on naive C57BL/6 background were treated with a 12 h light/dark cycle (lights on 07:00–19:00) at a constant temperature of 23 ± 2 °C. Under temperature conditions, food and water were provided freely and bred. Lcn2 -/- mouse is Dr. It was provided by Shizuo Akira (Osaka University, Japan). To generate homozygous animals with no background effect on the phenotype, Lcn2 +/+ and Lcn2 −/− mice were backcrossed for 8-10 generations on the C57BL/6 background. All experiments were performed according to the animal care guidelines of the National Institute of Health, and every effort was made to minimize the number of animals used and animal suffering. Male Lcn2 wild-type (WT, Lcn2 +/+) and Lcn2 -knockout (KO, Lcn2 -/-) mice were used at 8-10 weeks of age.

2. Diabetes Induction

Age-matched Lcn2 -knockout (KO, Lcn2 -/-) mice and Lcn2 wild-type (WT, Lcn2 +/+) mice of the same background (C57BL/6) were used for the study of diabetic neuropathy. Two previously reported experimental protocols were slightly modified and used for diabetes induction. First, multiple administrations of low concentrations of STZ (MLDS; 40 mg STZ/kg body weight for 5 consecutive days) induced both pancreatic β-cytotoxic effects and STZ-specific T-cell dependent immune responses. Second, a single administration of a high concentration of STZ (HDS; 150 mg/kg body weight) induced direct toxicity to pancreatic β-cells, which caused necrosis within 48-72 h and induces permanent hyperglycemia. The first day of STZ administration was designated day 0. Three days after administration, blood samples were collected from the tail vein, and ^{glycemia was measured using an SD CodeFree™} glucometer (SD Biosensor Inc., Suwon, Korea). Animals with a fasting blood glucose level of >260 mg/dl were considered diabetic.

3. western blot analysis

sciatic nerve and dorsal root in protein lysis buffer (20 mM HEPES, pH 7.0, 20 mM NaCl, 10% glycerol, 0.5% Triton X-100) containing a mixture of protease and phosphatase inhibitors (Roche, Basel, Switzerland). Dorsal root ganglion (DRG) tissue was homogenized and centrifuged at 13,000 g at 4° C. for 15 minutes. The supernatant was transferred to a new tube and the concentration was measured using a BCA kit (Thermo Scientific, Waltham, MA). Equal amounts of protein were loaded onto polyacrylamide gels (12-15%) for each sample. Proteins separated through SDS-PAGE were electroblotted on a polyvinylidene difluoride membrane (PVDF; Bio-Rad, Hercules, CA) using an electrophoretic transfer system (Bio-Rad Laboratories), and the membranes were separated at 4°C. incubated overnight with the following primary antibodies: goat lipocalin-2 (LCN2, 1:500; R&D Systems, Inc., Minneapolis, Canada) and mouse anti-α-tubulin (α-tubulin, 1:1000; Sigma-Aldrich, Missouri, United States). After washing, the membrane was reacted with horseradish peroxidase-conjugated specific secondary antibody (1:2000; Sigma-Aldrich) for 1 hour. Blots were processed with ECL Western blotting detection reagents (Thermo Scientific) and analyzed with Micro Chemi system (DNR Bio-imaging Systems, Jerusalem, Israel).

4. Enzyme-linked immunosorbent assay (ELISA)

The level of TNF-α protein in the sciatic nerve and DRG was measured using a mouse TNF-α ELISA kit (R&D Systems). Assays were performed according to the manufacturer's protocol in 96-well plates, using either sciatic nerve or DRG tissue. For standardization, mouse recombinant TNF-α was used in a concentration range of 31.3 to 2000 pg/ml, and absorbance was measured at 450 and 540 nm using a microplate reader (Molecular devices). All measurements were obtained through two replicate analyzes.

5. Department of Immunofluorescence Analysis Hematoxylin and Eosin ( haematoxylin and eosin; H&E) Dyeing

Mice were deeply anesthetized, and a solution of 0.9% NaCl and 4% paraformaldehyde dissolved in 0.1 M PBS (pH 7.4) was used for intracardiac perfusion-fixation. The isolated tissue was immersion-fixed with 4% paraformaldehyde for 24 hours. For low-temperature storage, the sciatic nerve and DRG were reacted with 30% sucrose, embedded with an OCT compound (Tissue-Tek; Sakura Finetek USA, Torrance, CA), and staining was performed with a slight modification in the previous report (Glia 65). (9), 2017, 1471-1490). Tissue sections (10-μm thick) were rinsed with phosphate-buffered saline (PBS) and reacted overnight with the following primary antibody: goat lipocalin-2 (LCN2, 1:100; R&D Systems) , Inc., Minneapolis, Canada), rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1, 1:200; Wako, Osaka, Japan), rabbit S100　calcium-binding protein (S100, 1:500; Glostrup, Denmark), rabbit anti-glial fibrillary acidic protein (GFAP, 1:500; Dako, Denmark). After reaction with primary antibody, sections were rinsed and reacted with FITC-conjugated and Cy3-conjugated secondary antibody (1:200; Jackson ImmunoResearch, West Grove, PA). After washing the slides, coverslips were made with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). For fluoromyelin staining, tissue sections were first rehydrated in PBS, and then reacted with fluoromyelin green fluorescent myelin stain (1:300; Invitrogen, California) at room temperature for 20 minutes. Then, the tissues were washed 3 times each for 10 minutes with PBS and mounted with DAPI. Similarly, tissues were stained with Harris' H & E solution. Additionally, immunofluorescent and H&E-stained tissues were visualized by fluorescence microscopy and bright field microscopy, respectively.

6. Density of nerve fibers in the epidermis ( intraepidermal nerve fiber density; IENFD )

The density of nerve fibers in the epidermis was tested on the sole of the hind paw. Briefly, paw pads were collected from the surface of the hind paws prior to animal perfusion, fixed in 4% paraformaldehyde for 24 h, and cryopreserved via sucrose gradient and OCT embedding.

rabbit anti-protein gene product 9.5 antibody (anti-PGP 9.5, 1:1000; Ultraclone, Isle of Wight, UK) and goat collagen IV (collagen IV, 1:200; Southern Biotechnology Associates, Inc., Birmingham, AL) , 10-μm plantar sections were stained. IENF density analysis was performed using a fluorescence microscope (Leica Microsystems, DM2500, Wetzlar, Germany). Individual IENFs were counted after passing through the basement membrane so that the fibers that split after passing through the membrane were counted as one fiber. Data are expressed as the number of fibers per millimeter length.

7. nerve conduction Nerve conduction velocity; NCV ) measurement

NCV was measured in the sciatic nerve. Mice were anesthetized with a combination of ketamine and xylazine, and body temperature was maintained at 37°C using a heating pad. For optimal conductivity, a ring electrode with contact gel was used. The sensing electrode was positioned at the point where the gastrocnemius muscle had the largest diameter, and the control electrode was positioned just below the sensing electrode. For motor nerve conduction velocity (MNCV) measurements, using a bipolar needle electrode at 4 mm proximal distance and 16 mm distal distance from the sensing electrode, the sciatic nerve was subjected to a single supermaximal square wave pulse (at 0.3 Hz and 1 KHz). Stimulation was performed with 5-10 mA and 100 ms duration with low- and high-pass filter settings, with other settings: delay start 0.2 s, maximum repetition rate 1 Hz, repetition 1-∞, pulse width 0.02 ms). Compound motor action potentials (CMAPs) were recorded (AD Instruments, Model FE180, Castle Hill, Australia) via a personal computer using Lab Chart 8 software, amplified, stored, displayed and digitized. MNCV (in m/s) was calculated by dividing the distance between the stimulated electrodes by the difference in mean latency. Similarly, the stimulation site was reversed for sensory nerve conduction velocity (SNCV) and the sciatic nerve was stimulated with square wave pulses of 100 ms duration. Compound muscle action potentials were recorded, and the SNCV was calculated by dividing the distance between the recorded electrodes by the average latency difference. All measurements were repeated at least 3 times from each group.

8. Quantification and Statistical Analysis

To quantify the Western blot band intensity, ImageJ software (National Institutes of Health, Bethesda, MD) was used. Statistical analysis was performed with GraphPad Software (GraphPad Software, La Jolla, CA, USA). All results were expressed as mean ± standard errors of the mean (SEM). Statistical comparisons were performed using Student's t test. Deviations according to p values of less than 0.05 ( p < 0.05) were judged to have statistical significance.

< Example 1> The sciatic nerve and DRG within of LCN2 expression enhancement

In order to confirm the role of LCN2 in peripheral nerve damage disease, the present inventors first confirmed the expression of LCN2 in the sciatic nerve and dorsal root ganglion (DRG) after diabetes induction. At 8 weeks of MLDS ( FIGS. 1A and 1B ) and 3 weeks of HDS ( FIGS. 1C and 1D ) administration, LCN2 protein showed a significant increase in both sciatic nerve and DRG tissues. Next, in order to confirm the cell morphology in the sciatic nerve and DRG expressing LCN2, the present inventors performed immunostaining. As a result of double immunostaining of LCN2 with S100 (Schwann cell marker in sciatic nerve and satellite glia marker in DRG), 8 weeks after MLDS administration, LCN2 was co-localized mainly in Schwann cells of the sciatic nerve and satellite glia of DRG. (FIGS. 2A and 2B). This indicates that the major cell type expressing LCN2 in the peripheral nervous system (PNS) of diabetic animals is glia. No significant immune response to LCN2 was observed in the sciatic nerve or DRG tissue of vehicle-administered control animals ( FIGS. 2A and 2B ). These results support that LCN2 expressed through glial cells in the PNS is involved in the progression of diabetic nerve injury disease.

< Example 2> Lcn2 Sciatic nerve and sciatic nerve after induction of diabetes by deficiency DRG weaken my inflammatory response

In the present invention, the expression of TNF-α protein was significantly increased in both sciatic nerve and DRG tissues by diabetes induction, whereas TNF-α expression was significantly attenuated in Lcn2 KO mice. Induction of inflammatory cytokines was not observed in the vehicle-administered control group ( FIGS. 3A and 3B ). Moreover, it was previously reported that LCN2 protein acts as a chemokine inducer and amplifies neuroinflammation by collecting additional inflammatory immune cells. Accordingly, the present inventors immunostained the sciatic nerve tissue with anti-Iba-1 to label macrophages. As a result of immunofluorescence analysis, macrophage infiltration into the sciatic nerve was confirmed 8 weeks after MLDS administration. Iba-1-positive macrophages in the diabetic sciatic nerve of Lcn2-deficient mice were significantly reduced ( FIG. 3B ). Similarly, the present inventors observed satellite glia activity and macrophage infiltration in DRG tissues of diabetic mice, and confirmed that they were attenuated by Lcn2 deficiency ( FIGS. 3D and 3E ). Taken together, these results support that LCN2 plays an important role in the induction of inflammatory cytokines and inflammatory infiltration, which are necessary conditions for the progression of diabetic nerve injury in the diabetic state.

< Example 3> Lcn2 Sciatic nerve and sciatic nerve in diabetic mice due to deficiency DRG Weakening of the phenotype of my diabetic nerve injury disease

Based on LCN2 expression and inflammatory response in both sciatic nerve and DRG, the present inventors compared the phenotype of neurological injury disease between WT and Lcn2 KO mice after 8 weeks of MLDS administration. To this end, the present inventors first performed immunostaining of the hind paw tissue, and measured the nerve fiber density in the epidermis. We calculated the intra-epidermal nerve fiber density in the plantar skin of the mouse hindpaw by staining with PGP9.5, a whole-neuron marker present in nerve terminals, and collagen IV, a basement membrane marker at the epidermal dermal border (Fig. 4A). As a result of quantification of the nerve fiber density in the epidermis, a significant decrease in nerve fibers was confirmed in WT diabetic mice compared to Lcn2 KO mice after 8 weeks of MLDS administration. Moreover, as a result of measuring the nerve conduction velocity of the sciatic nerve after 8 weeks of MLDS administration, it showed a significant decrease in both motor and sensory nerve conduction velocity compared to the control animals ( FIG. 4B ). On the other hand, after 8 weeks of MLDS administration, Lcn2 deficiency significantly attenuated the decrease in the motor and sensory nerve conduction velocity induced by diabetes ( FIG. 4B ). The above results confirmed peripheral nerve damage disease in the MLDS diabetic mouse model, supporting that deletion of the Lcn2 gene reverses the diabetic nerve damage disease phenotype.

Additionally, the present inventors performed H&E and fluoromyelin staining on the sciatic nerve to confirm histopathological changes in peripheral nerves following diabetes-induction. According to previous reports, diabetic mice exhibited loose, thin, disorganized, myelinated nerve fibers. Several nerve fibers within the sciatic nerve were demyelinated, the lamellar space enlarged, and separated from each other. Axonal atrophy was confirmed as a visible sign after 8 weeks of STZ administration compared to vehicle-administered control animals. On the other hand, the sciatic nerve derived from Lcn2 -deficient animals showed some resistance to diabetes-induced morphological modification ( FIGS. 4C and 4D ). Similarly, vacuolated neurons were prominently observed in the DRGs of diabetic mice, which were attenuated by Lcn2 deficiency ( FIG. 4E ). These results support that LCN2 is involved in structural and functional alterations of diabetic sciatic nerve and DRG, and contributes to the pathogenesis of diabetic nerve injury disease.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Diabetic neuroinflammation containing Lipocalin-2 (LCN2) expression or activity inhibitor as an active ingredient, numbness, numbness, rat or foot ulcer, diabetic diarrhea, constipation, bladder dysfunction, A pharmaceutical composition for preventing or treating sexual dysfunction, thermoregulation disorder or pupil dysfunction. The method of claim 1, wherein the LCN2 expression inhibitor is selected from the group consisting of antisense nucleotides complementary to mRNA of Lcn2 gene, small interfering RNA (siRNA) and short hairpin RNA (shRNA). Prevention or treatment of diabetic neuroinflammation, tingling, numbness, rat or foot ulcer, diabetic diarrhea, constipation, bladder dysfunction, sexual dysfunction, thermoregulation disorder or pupil dysfunction characterized in any one Pharmaceutical composition for use. The diabetic neuroinflammation according to claim 1, wherein the LCN2 activity inhibitor is any one selected from the group consisting of a compound that specifically binds to LCN2 protein, a peptide, a peptide mimetics, an aptamer, an antibody, and a natural product. , Diabetic numbness, numbness, rat or foot ulcer, diabetic diarrhea, constipation, bladder dysfunction, sexual dysfunction, thermoregulation disorder, or a pharmaceutical composition for preventing or treating pupil dysfunction. delete Diabetic neuroinflammation containing LCN2 expression or activity inhibitor as an active ingredient, numbness, numbness due to diabetes, rat or foot ulcer, diabetic diarrhea, constipation, bladder dysfunction, sexual dysfunction, thermoregulation disorder or pupil function A health functional food composition for preventing or improving disorders. The method of claim 5, wherein the LCN2 expression inhibitor is selected from the group consisting of antisense nucleotides complementary to mRNA of Lcn2 gene, small interfering RNA (siRNA) and short hairpin RNA (shRNA). Prevention or improvement of diabetic neuroinflammation, tingling, numbness, rat or foot ulcer, diabetic diarrhea, constipation, bladder dysfunction, sexual dysfunction, thermoregulation disorder or pupil dysfunction characterized in any one For health functional food composition. The diabetic neuroinflammation according to claim 5, wherein the LCN2 activity inhibitor is any one selected from the group consisting of a compound that specifically binds to LCN2 protein, a peptide, a peptide mimetics, an aptamer, an antibody, and a natural product. , A health functional food composition for preventing or improving numbness, numbness due to diabetes, rat or foot ulcer, diabetic diarrhea, constipation, bladder dysfunction, sexual dysfunction, thermoregulation disorder or pupil dysfunction. delete

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