Composition comprising sestrin 2 for preventing or treating prostate diseases

Abstract

The present invention relates to a composition comprising sestrin 2 for preventing or treating prostate diseases. Specifically, it has been identified that prostate diseases are inhibited by reducing the size, weight, or epithelial thickness of the prostate in an animal model in which prostate diseases are induced, and thus prostate diseases can be treated or prevented and urination disorders caused by prostate diseases can be alleviated.

Description

Composition for preventing or treating prostate disease containing sestrin 2

[0001] The present invention relates to a composition for preventing or treating prostate disease comprising Sestrin2.

[0002] The prostate is a male reproductive organ that produces prostatic fluid, a component of semen, and drains it into the urethra. It surrounds the urethra like a ring, just below the bladder. Prostatic fluid, produced by the prostate, nourishes sperm produced in the testes and transported there. It also maintains a liquid state in ejaculated semen, preventing it from solidifying, thereby facilitating sperm movement.

[0003] Representative diseases related to the prostate include benign prostatic hyperplasia (BPH), prostatitis, and prostate cancer. Benign prostatic hyperplasia (BPH) is caused by abnormal growth of the prostate gland around the urethra, which compresses the urethra, causing various symptoms such as urinary difficulties. The uncontrolled enlargement of the prostate and suppression of the urethra cause lower urinary tract dysfunction, including increased urinary urgency, increased urinary frequency, urgency, weakened urinary flow, and incomplete bladder emptying, which lowers the quality of life and negatively impacts daily life. The cause of BPH is not yet clearly known, but it is known that physical aging and changes in male hormones have an effect. As prostate weight often increases with aging, and benign prostatic hyperplasia (BPH) is commonly observed in older men, making aging the biggest cause. However, the incidence of BPH in younger men is also on the rise, with the average annual increase rate of 12.6% in the number of patients receiving treatment for BPH in their 20s recently showing a high increase. Even in people who are not elderly, excessive testosterone in the blood leads to the synthesis of large amounts of dihydrotestosterone (DHT) by 5-alpha reductase in the prostate tissue. DHT is known to bind to androgen receptors in the nuclei of prostatic epithelial and supportive cells, inducing growth of the epithelium and supportive cytoplasm. In addition, as aging progresses, cellular senescence accumulates in all tissues, including the prostate, and senescent cells become metabolically activated, exhibiting excessive inflammatory secretions that can induce the development of BPH, and increasing cell proliferation, prostatic fibrosis, and epithelial-mesenchymal transition (EMT).In addition, various pathophysiological factors have been reported, such as imbalances in cell proliferation and apoptosis, abnormal ratios of sex hormones and cytokines, inflammation, cellular aging, and oxidative stress. However, the fundamental cause of BPH remains largely unknown. While benign prostatic hyperplasia (BPH) is not a fatal disease in itself, it can diminish the quality of life. If left untreated, it can lead to various kidney stones, persistent bladder residual urine-induced urinary tract infections, and in severe cases, urosepsis, which can even affect life.

[0004] Prostatitis, a disease that causes inflammation of the prostate, is so common that approximately 50% of adult men experience symptoms at least once in their lifetime. Bacterial prostatitis accounts for only 5-10% of prostatitis cases, with chronic nonbacterial prostatitis accounting for the majority. Bacterial prostatitis is most commonly caused by direct bacterial infection through the urethra during a urinary tract infection. It can also be caused by impaired prostatic fluid excretion, urinary reflux into the prostate, or the spread of inflammation such as hemorrhoids or colitis. Chronic nonbacterial prostatitis is thought to be caused by poor lifestyle habits such as excessive drinking, smoking, overwork, and stress, excessive inflammatory responses due to aging, and persistent irritation to the prostate, such as prolonged sitting or cycling.

[0005] Meanwhile, Sestrin2, a highly conserved stress-inducible protein, is known to regulate cellular stress stimuli and maintain cellular homeostasis, protecting organisms from various stimuli such as oxidative stress, hypoxia, starvation, and DNA damage. Several studies have shown that Sestrin2 plays a positive role in various pathological conditions through activation of the AMPK / mTOR signaling pathway. Sestrin2 plays an essential role in improving ischemic stress in the aged heart through AMPK activation (Quan N., et al., 2018), and has been reported to prevent denervated muscle atrophy through AMPK / mTOR by upregulating mitophagy (Yang, X., et al., 2021). Nevertheless, the expression level of Sestrin2 has been found to decrease with age in various tissues, including the heart and skeletal muscle, which may indicate age-related metabolic homeostasis disorders.

[0006] In addition, according to Hao Fu et al. (2018), Sestrin 2 has a low expression in prostate cancer cell lines, and overexpression of Sestrin 2 in human prostate cancer PC3 inhibits tumor proliferation and increases sensitivity to ionizing radiation (IR). However, the exact role of Sestrin 2 in benign prostatic hyperplasia caused by aging or sex hormones is completely unknown.

[0007] The purpose of the present invention is to provide a composition for preventing or treating prostate disease containing Sestrin2.

[0008] In order to solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating prostate disease, which comprises sestrin2 protein or a polynucleotide encoding the same as an active ingredient.

[0009] The above prostate disease may be benign prostatic hyperplasia or prostatitis.

[0010] Sestrin2, also known as Hi95, is a protein encoded by the SESN2 gene in humans. Sestrin2 is known to regulate cell growth and survival and may be involved in cellular responses to various stress conditions. It also constitutes a family of evolutionarily conserved stress-inducible proteins that suppress oxidative stress and modulate adenosine monophosphate-dependent protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling, acting as a key regulator of metabolic homeostasis.

[0011] In order to achieve the purpose of the present invention, various derived and / or formed Sestrin2 proteins (or genes) may be used as long as the effects of the present invention are achieved. This includes a wild-type sequence, a gene in which a portion of the base sequence has been artificially modified to favor characteristics such as expression in a cell or protein stability, a gene in which a portion of the base sequence found naturally has been modified, or fragments of all of these. The modification of the gene base sequence may or may not involve a modification of the corresponding amino acid, and when the modification involves an amino acid, the gene in which such a modification has occurred encodes a protein composed of an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the protein encoded by it, and includes mutants, derivatives, alleles, variants, and homologues. When a mutation of the gene base sequence does not involve a modification of an amino acid in the protein, there is, for example, a degeneracy mutation, and such degeneracy mutants are also included in the gene of the present invention. Modifications of artificial genetic sequences can be produced by methods well known to those skilled in the art, such as site-directed mutagenesis, error-prone PCR, and point mutation.

[0012] The protein used herein can be produced using methods known in the art. In one embodiment, the method for producing the protein utilizes genetic recombination technology. For example, a vector containing a corresponding gene encoding the protein can be delivered to prokaryotic or eukaryotic cells, such as insect cells or mammalian cells, expressed, and then purified for use. The plasmid can be used by cloning the corresponding gene into an expression vector such as pET28b (Novagen), transfecting the cell line, and then purifying the expressed protein, but is not limited thereto. The synthesized protein can be separated and purified by column chromatography, including precipitation, dialysis, ion exchange chromatography, gel-permeation chromatography, HPLC, reverse-phase HPLC, preparative SDS-PAGE, and affinity columns using anti-screening protein antibodies. In the present invention, the Sestrin 2 protein (hereinafter referred to as Sestrin 2) can preferably be composed of the amino acid sequence of SEQ ID NO: 1.

[0013] The term "protein" of the present invention means a compound in which amino acids are linked by peptide bonds, and in this specification, the protein can be used in the same meaning as peptide.

[0014] The amino acid sequence of the present invention can be easily modified by substitution, deletion, insertion, or a combination of one or more amino acids. Therefore, peptides or proteins with high homology to SEQ ID NO: 1, for example, peptides and proteins with a high homology of 70% or more, specifically 80% or more, and preferably 90% or more, should also be construed as being included within the scope of the present invention.

[0015] In the present invention, "polynucleotide" may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture thereof, and refers to a polymer of nucleotides in which nucleotide units are long and connected in a chain shape by covalent bonds. The polynucleotide of the present invention refers to a polynucleotide encoding the Sestrin2 protein of the present invention. In addition, the polynucleotide of the present invention may have various modifications in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, taking into account the codon preferred in the organism to which the protein is to be expressed, and various modifications or modifications may be made in a portion excluding the coding region within a range that does not affect the expression of the gene. That is, the polynucleotide of the present invention may have one or more nucleic acid bases mutated by substitution, deletion, insertion, or a combination thereof, as long as it encodes a protein having an activity equivalent thereto, and these are also included in the scope of the present invention.

[0016] The recombinant vector of the present invention is a means for introducing the protein of the present invention into a cell and expressing the protein of the present invention. A known expression vector such as a plasmid vector, a cosmid vector, a bacteriophage vector, etc. can be used, and the vector can be easily manufactured by a person skilled in the art according to any known method using DNA recombination technology.

[0017] The present invention provides a pharmaceutical composition for preventing or treating benign prostatic hyperplasia, which comprises as an active ingredient a polynucleotide encoding sestrin2, a vector comprising the polynucleotide, a sestrin2-overexpressing cell line transformed with the vector, or a culture medium thereof.

[0018] The term "benign prostatic hyperplasia" of the present invention refers to a symptom in which the size or weight of the prostate increases, and the enlarged prostate causes bladder outlet obstruction, resulting in various lower urinary tract symptoms. Histologically, it can be defined as proliferation of stromal or epithelial tissue cells of the prostate.

[0019] The term "prevention" in the present invention means any act of suppressing or delaying benign prostatic hyperplasia by administering the composition.

[0020] The term "treatment" in the present invention means any action by which the symptoms of prostate disease are improved or beneficially changed by administration of the composition.

[0021] In the present invention, the pharmaceutical composition can be used in a method for preventing or treating prostate disease, and specifically, the method for preventing or treating prostate disease can include a step of administering the composition to a subject other than a human who has developed or is expected to develop a prostate disease.

[0022] The term "administration" in the present invention means introducing the composition to a subject by an appropriate method.

[0023] The term "subject" of the present invention means all animals, including rats, mice, and livestock, including humans, that have developed or may develop prostate disease, and may be a mammal, including humans, as a specific example, but is not limited thereto.

[0024] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. For example, the composition can be administered as an active ingredient at a dosage of 0.01 to 500 mg / kg per day, specifically 5 to 100 mg / kg, and the administration can be administered once a day or in several divided doses. In addition, the pharmaceutical composition of the present invention can contain the Sestrin2 protein of the present invention at a weight percentage of 0.001 to 50% based on the total weight of the composition.

[0025] The composition of the present invention can be administered as an individual treatment or in combination with other treatments, either sequentially or simultaneously with conventional treatments. It can be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily available to those skilled in the art.

[0026] The pharmaceutical composition for preventing or treating prostate disease of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-described effective ingredient. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0027] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions, respectively, according to conventional methods. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules. Such solid preparations can be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. In addition to liquid paraffin for oral administration, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be added to prepare the formulation. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0028] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time, but can be appropriately selected by a person skilled in the art.

[0029] Sestrin2 according to the present invention can reduce the size, weight or epithelial thickness of the prostate.

[0030] The present invention provides a pharmaceutical composition for improving urinary dysfunction caused by prostate disease, comprising Sestrin2 as an active ingredient. In this case, the prostate disease may be benign prostatic hyperplasia or prostatitis.

[0031] In addition, the present invention provides a health functional food for preventing or improving benign prostatic hyperplasia, which contains Sestrin 2 as an active ingredient.

[0032] The present invention relates to a composition for preventing or treating prostate disease, including Sestrin2, and more specifically, it is possible to treat or prevent prostate disease by confirming that the composition reduces the size, weight, or epithelial thickness of the prostate in an animal model in which prostate disease is induced, and suppresses benign prostatic hyperplasia by regulating apoptosis or cell proliferation of prostate tissue.

[0033] Figure 1 shows the results showing the expression level of Sestrin 2 in prostate tissues in an animal model of aging prostate disease. (A) mRNA expression level of Sestrin 2 in prostate tissues of 3-month-old, 13-month-old, and 24-month-old mice. (B) Immunohistochemical staining of Sestrin 2 in prostate tissues of 3-month-old, 13-month-old, and 24-month-old mice (scale bar: 20 μm). (C) Western blot results of Sestrin 2 in prostate tissues of 3-month-old, 13-month-old, and 24-month-old mice. (D) (C) Quantification graph. (* p < 0.05; ** p < 0.01; *** p < 0.001 compared to 3-month-old mice).

[0034] Figure 2 shows the effect of Sestrin2 on prostate size and weight in an animal model of aging prostate disease. (A) Photographs of prostate size measurements in 3-month-old mice, 24-month-old mice, and 24-month-old Sestrin2 protein-administered mice (24 months+Sestrin2). (B) Body weight, prostate weight, and prostate weight normalized to body weight in 3-month-old mice, and 24-month-old Sestrin2 protein-administered mice (24 months+Sestrin2). (C) Graph showing prostate weight normalized to body weight in (B). (*p < 0.05; *p < 0.01; ***p < 0.001 compared to 3 months of age, #p < 0.05; ##p < 0.01; ### p < 0.001 compared to 24 months of age).

[0035] Figure 3 shows the effect of Sestrin2 on prostate epithelial thickness in an animal model of aging prostate disease. (A) H&E staining images of 3-month-old, 24-month-old, and 24-month-old Sestrin2 protein-administered mice (24 months+Sestrin2) (scale bar: 50 μm) (B) (A) Quantification graph. (*p < 0.05; *p < 0.01; ***p < 0.001 compared to 3 months of age, #p < 0.05; ##p < 0.01; ###p < 0.001 compared to 24 months of age).

[0036] Figure 4 shows the results showing the expression level of Sestrin2 in prostate tissue in a testosterone (TP)-induced benign prostatic hyperplasia animal model. (A) mRNA expression level of Sestrin2 in prostate tissue of control and TP group mice. (Compared to the control group, *p < 0.05; *p < 0.01; ***p < 0.001) (B) Immunostaining of Sestrin2 in prostate tissue of control and TP group mice (Scale bar: 400× is 50 μm, 100× is 20 μm).

[0037] Figure 5 shows the effect of Sestrin2 on the size and weight of the prostate in a testosterone (TP)-induced benign prostatic hyperplasia animal model. (A) Photographs of prostate size measurements in mice from the control group (Control), TP administration group (TP), TP and finasteride administration group (TP+Fina), and TP and Sestrin2 administration group (TP+Sestrin2). (B) Body weight, prostate weight, and prostate weight normalized to body weight in mice from the control group (Control), TP administration group (TP), TP and finasteride administration group (TP+Fina), and TP and Sestrin2 administration group (TP+Sestrin2). (C) Graph showing prostate weight normalized to body weight in (B). (*p < 0.05; *p < 0.01; *** p < 0.001 compared to the control group, #p < 0.05; ##p < 0.01; ### p < 0.001 compared to the TP group).

[0038] Figure 6 shows the effect of Sestrin2 on prostate epithelial thickness in a testosterone (TP)-induced benign prostatic hyperplasia (BPH) animal model. (A) H&E staining images of mice in the control group (Control), TP administration group (TP), TP and finasteride administration group (TP+Fina), and TP and Sestrin2 administration group (TP+Sestrin2) (scale bar: 50 μm). (B) (A) Quantification graph. (*p < 0.05; **p < 0.01; *** p < 0.001 compared to the control group, #p < 0.05; ##p < 0.01; ### p < 0.001 compared to the TP group).

[0039] Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The contents introduced herein are provided to sufficiently convey the spirit of the present invention.

[0040] <Example 1. Effect of Sestrin2 in an animal model of aging prostate disease>

[0041] Aging animal models

[0042] Twenty-three-month-old mice were used as a model of prostate disease caused by aging. A total of 24 male C57BL / 6 mice (6 mice aged 8 weeks, 6 mice aged 12 months, and 12 mice aged 23 months) were obtained from the Korea Basic Science Institute Aging Science Animal Facility (Gwangju, Korea) and used in the experiment after an acclimatization period of 7 days at the Chungnam National University Laboratory Animal Center. All animal experiment protocols used in the present invention were approved by the Chungnam National University Animal Ethics Committee (202307A-CNU-136) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were housed under specific pathogen-free conditions at room temperature (20-24℃) and humidity (50-60%).

[0043] Six 23-month-old mice prepared above were intraperitoneally injected with Sestrin2 recombinant protein (0.1 mg / kg, MyBioSource, San Diego, USA) for 14 consecutive days before sacrifice.

[0044]

[0045] prostatectomy

[0046] The day before the end of each animal experiment, all experimental animals were fasted for 24 hours to minimize weight changes due to food intake. After the experiment, mice were anesthetized with CO2 and underwent prostatectomy. The prostate was excised from each mouse using a dissecting microscope, and the fat and foreign substances attached to the excised prostate were removed. The prostate weight was measured. Half of the prostate tissue was immediately fixed in 10% neutral buffered formalin (Sigma Aldrich, St. Louis, USA), and the remaining prostate tissue was frozen at -70°C.

[0047]

[0048] Changes in Sestrin 2 Expression in an Animal Model of Aging Prostate Disease

[0049] To evaluate the correlation between spontaneous BPH and Sestrin2, we examined the expression of Sestrin2 in aged mouse prostates. Quantitative real-time PCR was performed on prostate tissues obtained periodically from 3-, 13-, and 24-month-old mice. Total RNA was isolated from mouse prostate tissues using TRIzol reagent (Takara, Japan). RNA was desynthesized into complementary DNA using Moloney-murine leukemia virus reverse transcriptase (Promega, Madison, USA) and random hexamer primers (BioFact, Korea). Quantitative real-time PCR was performed four times with SYBR green (Smartgene, Daejeon, Korea) using a QuantStudio 1 real-time PCR system (Applied Biosystems, Waltham, USA). Relative mRNA expression was examined using the comparative threshold cycle (Ct) values ​​of each group, which were normalized to β-actin, and the results are shown in Figure 1A. The experiments were performed according to the method of Han, D (2022), and the sequences of the primers used are as follows: mouse sestrin2: forward 5'-TAGCCTGCAGCCTCACCTAT-3', reverse 5'-TATCTGATGCCAAAGACGCA-3'; mouse β-actin: forward 5'- GGCTGTATTCCCCTCCATCG-3', reverse 5'- CCAGTTGGTAACAATGCCATGT-3'.

[0050] As shown in Figure 1A, the mRNA level of Sestrin2 was significantly and gradually downregulated with increasing age.

[0051]

[0052] Immunohistological analysis

[0053] The fixed prostate tissues obtained from the above-described mice were embedded in paraffin and sectioned into 5-μm-thick sections. The paraffin sections were collected and stained with hematoxylin / eosin (H / E). For immunostaining, the paraffin sections were deparaffinized, dehydrated, and subjected to antigen retrieval using citrate buffer (pH 6.0). The sections were then incubated with 3% hydrogen peroxide to inhibit endogenous peroxidase. After blocking with normal goat serum, the sections were incubated overnight at 4°C with primary Sestrin 2 antibody (Proteintech, Illinois, USA). Afterwards, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, and the target antigens were visualized with a diaminobenzidine kit (Vector Lab, Newark, USA), and the sections were counterstained with hematoxylin (Dako, Glostrup, Denmark), and the results are shown in Figure 1B. As shown in Figure 1B, Sestrin2 was clearly expressed (brown) at the apical surface of epithelial cells in prostate progenitors of all ages, and the expression level decreased significantly with increasing age, showing the lowest expression at 24 months.

[0054]

[0055] Western blotting analysis

[0056] The collected prostate tissues were dissolved in radioimmunoprecipitation assay (RIPA; Sigma) buffer and quantified using bicinchoninic acid (BCA; Thermofisher). Western blotting was performed according to the method of Hong, G.-L. et al. (2020), and the primary antibodies used were β-actin (Abcam) and Sestrin2 (Proteintech). After incubation with secondary antibodies, the expression of each protein was visualized using an enhanced chemiluminescence detection kit (LPS Solution, Daejeon, Korea). The signal intensity was quantified using a CS analyzer 4 (ATTO, Tokyo, Japan) and is shown in Figure 1C, which is then quantified and graphically represented in Figure 1D. As shown in Figures 1C and 1D, Western blot analysis showed that the protein level of Sestrin2 gradually decreased with increasing age.

[0057] Results are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey's method was used for all statistical analyses using GraphPad Prism 5. A p < 0.05 value was considered statistically significant. Statistical analysis was applied identically throughout the following experiments.

[0058]

[0059] Changes in prostate size and weight

[0060] We investigated whether administration of Sestrin-2 affects prostate disease in an animal model of aging prostate disease. Figure 2A shows photographs of the excised prostates of mice aged 3 months and 24 months, and mice that were administered Sestrin-2 intraperitoneally at 7 mg / kg / day for 14 days. As shown in Figure 2A, the total anatomical prostate size of 24-month-old mice increased compared to that of 3-month-old mice. However, the prostate size of 24-month-old mice treated with Sestrin-2 decreased compared to that of 24-month-old mice. Figure 2B shows the prostate weights and relative prostate weights normalized to body weight of mice aged 3 months and 24 months, and mice that were administered Sestrin-2 intraperitoneally at 7 mg / kg / day for 14 days, and these are graphically represented in Figure 2C. As shown in Figures 2B and 2C, consistent with the anatomical results, the relative prostate weight was significantly increased in 24-month-old mice compared to 3-month-old mice, but Sestrin2 administration significantly reduced the prostate size in 24-month-old mice.

[0061]

[0062] Measurement of prostate epithelial thickness

[0063] Benign prostatic hyperplasia (BPH) involves increased prostate size and weight, along with increased epithelial thickness. Therefore, we investigated whether administration of Sestrin-2 affects prostate epithelial thickness in an age-induced prostate disease model.

[0064] The prostate tissues of each group obtained above were stained with hematoxylin / eosin (H / E) using the same method as above, and are shown in Fig. 3A, and the epithelial thickness was measured and shown in Fig. 3B. As shown in Fig. 3A and Fig. 3B, the epithelial thickness of the prostates of the aging-induced mouse group increased by approximately 2-fold compared to the control group. The morphology of the prostate cells In addition, the prostates of 3-month-old mice showed a histological pattern of round and regular acinar cells with rectangular to simple columnar epithelium, but the prostates of 24-month-old mice showed irregular outlines of each acinar cell, and a histological pattern of prostatic hyperplasia with compressed, tall columnar epithelium and more folds. However, Sestrin2 administration changed the histological pattern of 24-month-old mice, reducing the growth and folds of acinar cells and changing the epithelial pattern to a simple columnar one.

[0065]

[0066] <Example 2. Effect of Sestrin2 in an animal model of testosterone-induced benign prostatic hyperplasia>

[0067] Testosterone-induced animal model

[0068] Prostate hyperplasia was induced in mice by administering testosterone propionate (TP) for 4 weeks. Twenty-four 7-week-old male C57BL / 6 mice (Orient Bio, Seongnam, Korea) were obtained and used in the experiment after a 7-day acclimatization period at the Chungnam National University Laboratory Animal Center. All animal experiment protocols were approved by the Chungnam National University Animal Ethics Committee (202307A-CNU-136) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were housed under specific pathogen-free conditions at room temperature (20-24°C) and humidity (50-60%).

[0069] The above 24 male C57BL / 6 mice were randomly divided into 4 groups of 6 mice each, and the 3 groups were injected subcutaneously with 7 mg / kg of testosterone to induce benign prostatic hyperplasia. TP was dissolved in corn oil (Sigma Aldrich) and injected subcutaneously once daily for 28 days using a 1 ml syringe with a 27 gauge needle. The control group received an equal volume of corn oil subcutaneously. One group of TP-induced mice received an intraperitoneal injection of 0.1 mg / kg of Sestrin recombinant protein, and one group received an oral administration of 10 mg / kg of finasteride as a positive control. One group of TP-induced mice and the corn oil-administered group received an intraperitoneal injection of phosphate-buffered saline (PBS) (Table 1).

[0070] Group Name Dosage Information Control Group Corn Oil (SC) + PBS (IP) TPTP 7mg / kg (SC) + PBS (IP) TP+FinaTP 7mg / kg (SC) + Finasteride 10mg / kg (Oral) TP+Sestrin2TP (SC) + Sestrin2 Recombinant Protein 0.1mg / kg (IP)

[0071] TP and finasteride administration was performed for 28 days before mouse sacrifice, and Sestrin2 recombinant protein was administered simultaneously for 14 days before mouse sacrifice.

[0072] prostatectomy

[0073] The day before the end of each animal experiment, all experimental animals were fasted for 24 hours to minimize weight changes due to food intake. After the experiment, mice were anesthetized with CO2 and underwent prostatectomy. The prostate was excised from each mouse using a dissecting microscope, and the fat and foreign substances attached to the excised prostate were removed. The prostate weight was measured. Half of the prostate tissue was immediately fixed in 10% neutral buffered formalin (Sigma Aldrich, St. Louis, USA), and the remaining prostate tissue was frozen at -70°C.

[0074]

[0075] Changes in Sestrin2 Expression in a TP-Induced BPH Animal Model

[0076] The expression (mRNA and protein) of Sestrin2 in the prostates of the control and TP groups, which were extracted using the same method as the aging benign prostatic hyperplasia animal model, was confirmed through quantitative real-time PCR, and the results are shown in Figure 4A. As shown in Figure 4A, the expression of Sestrin2 mRNA was significantly reduced in the prostates of mice administered testosterone compared to the control group.

[0077] In addition, the prostate tissues of the control group and the TP group were stained with hematoxylin / eosin (H / E) and immunostained with a Sestrin2 primary antibody (Proteintech, Illinois, USA) using the same method as above, and the results are shown in Figure 4B. As shown in Figure 4B, Sestrin2 is clearly expressed (brown) on the apical surface of epithelial cells in the control prostate gland, while its expression level was significantly reduced in the TP group.

[0078]

[0079] Changes in prostate size and weight

[0080] We examined whether administration of Sestrin2 affects benign prostatic hyperplasia in a TP-induced prostate animal model. Images of the excised prostates of each group of mice obtained above are shown in Figure 5A. As shown in Figure 5A, compared to the control group (Control), the total anatomical prostate size of the TP group mice increased, while the group administered finasteride (TP+Fina) showed a decrease in prostate size. Furthermore, the prostate size of the mice administered Sestrin2 of the present invention (TP+Sestrin2) was significantly reduced.

[0081] Figure 5B shows the prostate weight and relative prostate weight normalized to body weight of each group of mice, and this is graphically represented in Figure 5C. As shown in Figures 5B and 5C, consistent with the anatomical results, the relative prostate weight was significantly increased in the control mice, whereas the prostate size was significantly reduced in the TP-induced BPH mice treated with Sestrin2.

[0082]

[0083] Measurement of prostate epithelial thickness

[0084] Benign prostatic hyperplasia (BPH) is characterized by increased prostate size and weight, along with increased epithelial thickness. Therefore, we investigated whether administration of Sestrin-2 affects prostate epithelial thickness in a TP-induced BPH model.

[0085] The prostate tissues of each group obtained above were stained with hematoxylin / eosin (H / E) in the same manner as above, and are shown in Fig. 6A, and the epithelial thickness was measured and shown in Fig. 6B. As seen in Figs. 6A and 6B, the epithelial thickness of the prostate of the TP-induced mouse group (TP) increased by about 2 times compared to the control group, and the administration of finasteride, the positive control group, significantly inhibited the increase in prostate epithelial thickness induced by TP. In addition, it was confirmed that the administration of Sestrin2 of the present invention also significantly inhibited the increase in prostate epithelial thickness induced by TP, and showed an effect equal to or greater than that of finasteride, the positive control group.

Claims

1. A pharmaceutical composition for preventing or treating prostate disease containing sestrin2 protein as an active ingredient, A pharmaceutical composition for preventing or treating prostate disease, characterized in that the prostate disease is at least one selected from the group consisting of benign prostatic hyperplasia and prostatitis.

2. In paragraph 1, A pharmaceutical composition for preventing or treating prostate disease, characterized in that the above Sestrin 2 reduces the size, weight or epithelial thickness of the prostate.

3. In any one of paragraphs 1 and 2, A pharmaceutical composition for preventing or treating prostate disease, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the effective ingredient.

4. A pharmaceutical composition for preventing or treating prostate disease, comprising a polynucleotide encoding Sestrin 2 as an active ingredient.

5. A pharmaceutical composition for preventing or treating prostate disease, comprising as an active ingredient a polynucleotide encoding Sestrin2, a vector containing the polynucleotide, a Sestrin2 overexpressing cell line transformed with the vector, or a culture medium thereof.

6. A pharmaceutical composition for improving urinary dysfunction caused by prostate disease, comprising Sestrin 2 as an active ingredient.

7. Health functional food for preventing or improving prostate disease containing Sestrin 2 as an active ingredient.

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