KR102361485B1 - Pharmaceutical composition comprising Hepcidin expression enhancer for immunopotentiation - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing immunity comprising a hepcidin expression or activity enhancer, and more specifically, to an immune response of interferon-α (Interferon-α, IFN-α) through enhancing the expression of hepcidin. The present invention relates to a method for screening an immune enhancer, comprising selecting a composition for treating or assisting in treatment of a pathogenic infection such as bacteria or virus through activation, and a candidate substance having enhanced expression or activity of hepcidin as an immune enhancer.
The composition according to the present invention is expected to exhibit a therapeutic effect on pathogenic infections such as bacteria and viruses by activating the interferon alpha immune response by increasing the expression of antibacterial proteins, among others, hepcidin. The discovery of immune enhancers may be useful in the treatment of viral diseases.

Description

A pharmaceutical composition for enhancing immunity, comprising hepcidin expression or activity enhancer {Pharmaceutical composition comprising Hepcidin expression enhancer for immunopotentiation}

The present invention relates to a pharmaceutical composition for enhancing immunity comprising a hepcidin expression or activity enhancer, and more particularly, interferon-α (IFN-) through hepcidin expression or activity enhancement. It relates to a composition for treating or adjuvant treatment of pathogenic infections such as bacteria and viruses through immune activation of α).

The innate immune response refers to the immune system that responds immediately to an invasion of external factors without remembering specific pathogens. Immunity does not last long and treats pathogens in a comprehensive way, and macrophages, phagocytosis such as mesophilus, dermatophytes, and antibiotics play important roles in innate immunity. The physiological barriers that contribute to the innate immune response consist of temperature, pH, and several solutes and cell-associated molecules. Many species are not susceptible to disease because normal body temperature suppresses the growth of pathogens. Chickens, for example, have innate immunity to anthrax because high body temperature inhibits the growth of bacteria. Similarly, there are very few microorganisms that can survive in an environment of low pH, so gastric acid also acts as a physiological innate immunity.

Various soluble factors, including the soluble proteins lysozyme, interferon, and complement, also constitute innate immunity. Lysozyme, a hydrolytic enzyme found in mucus secretions and tears, can break down the peptidoglycan layer of the bacterial cell wall. In particular, it can be seen that it is easy to prevent the invasion of gram-positive bacteria, in which about 80-90% of the cell wall is peptidoglycan. Interferon, which is an important cytokine that expresses a number of genes necessary for virus removal among pathogens and induces initiation of adaptive immune response as well as innate immunity, is secreted by immune cells in the body and is used for the purpose of treatment It is also administered by injection. Interferon, composed of a group of proteins, is made from virus-infected cells, and among its many functions is its ability to bind to nearby cells and induce an antiviral state.

On the other hand, hepcidin is one of the AMP (antimicrobial peptide) family, and expression is regulated by iron status, hypoxia, oxidative stress, inflammation and infection. Hepcidin is a hepatic peptide hormone encoded by the HAMP gene and acts as a major regulator of iron uptake into the circulation. Abnormally high hepcidin levels result in decreased serum iron levels due to iron entrapment in the liver cells and liver cells. Abnormally low hepcidin levels result in iron overload. In addition, disulfide-rich peptide hormones have antibacterial activity against bacteria and fungi. Recently, hepcidin in the liver has been shown to be highly related to the regulation of hepatitis C virus (HCV) replication and chronic hepatitis C. .

HCV infection inhibits the expression of hepcidin, and treatment with synthetic hepcidin peptides has broad anti-HCV activity, at least for genotypes 1 and 2. Although this effect is related to the activity of the IFN-induced gene through increased Hepcidin expression (Iron regulator 643 Hepcidin exhibits antiviral activity against hepatitis C virus. PLoS One, 7 (10)), the exact mechanism and activation of the interferon immune response are not relevant. have not been reported at all.

As a result of the study on the mechanism of the activation of the interferon immune response, the present inventors first identified that increasing the expression of hepcidin enhances the mRNA expression and intracellular generation of interferon alpha, thereby increasing or decreasing the immune activity. The present invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for enhancing immunity comprising a hepcidin expression or activity enhancing agent.

Also, the present invention

a) treating the cells with a candidate substance in vitro;

b) measuring hepcidin expression or activity in said cell; and

c) provides an immune enhancer screening method comprising the step of selecting a candidate material whose hepcidin expression or activity is enhanced compared to the candidate material untreated group as an immune enhancer.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for enhancing immunity comprising a hepcidin expression or activity enhancer.

In one embodiment of the present invention, the composition may enhance the expression of interferon alpha.

Also, the present invention

a) treating the cells with a candidate substance in vitro;

b) measuring hepcidin expression or activity in said cell; and

c) provides an immune enhancer screening method comprising the step of selecting a candidate material whose hepcidin expression or activity is enhanced compared to the candidate material untreated group as an immune enhancer.

In one embodiment of the present invention, the candidate material may be selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids and peptides.

In another embodiment of the present invention, the nucleic acid is siRNA, shRNA, microRNA, antisense RNA, aptamer, DNA (locked nucleic acid), PNA (peptide nucleic acid) and morpholino (morpholino) group consisting of can be selected from

The present inventors observed that the immune response of interferon alpha was activated through enhancement of hepcidin expression or activity, and confirmed that 4-PBA effectively acts as a hepcidin expression enhancer for this purpose. Therefore, the hepcidin expression or activity enhancer, which may be included as an active ingredient in the composition of the present invention, can activate the immune response caused by interferon alpha by enhancing the expression or activity of hepcidin, thereby treating pathogenic infections such as bacteria and viruses. It can be usefully used in various compositions for In addition, it is expected that the immune enhancer screening method through whether hepcidin expression or activity is enhanced can be used for the development of new therapeutic agents.

1a is a result confirming the increase in the protein expression of hepcidin by 4-PBA.
1b is a result confirming the increase in the mRNA expression of hepcidin by 4-PBA.
1c and 1d are results confirming the increase in hepcidin expression in the 4-PBA-treated tissue compared to the excipient-treated tissue.
1e is a result confirming the increase in acetylation of hepcidin 3 histone protein by 4-PBA.
1f is a result confirming the increase of histone acetylation in H3K27 in the whole cell lysate by 4-PBA.
1g is a result confirming the increase in hepcidin promoter-attached RNA polymerase II by 4-PBA.
Figure 2a is a result confirming the increase in the activity of the Interferon Stimulated Response Element by 4-PBA.
Figure 2b is a result confirming the increase in interferon alpha mRNA expression and intracellular secretion by 4-PBA.
Figures 2c and 2d are IFN regulators ( IRF1, IRF3, IRF7 ) whose expression is induced by interferon alpha, protein kinase R ( PKR ), IFN-inducing protein containing tetratricopeptide repeat1 ( IFIT1 ) and 2'-5 '-Oligoadenylate synthase 1 ( OAS1 ) shows the increase in the expression of the genes.
3a, 3b, 3c and 3d are results confirming the change in interferon alpha expression according to the inhibition of hepcidin expression.
4a and 4b are results confirming the establishment of an HCV replicon-based mouse model.
4c and 4d are results confirming the decrease in NS5A protein level by 4-PBA.
4e and 4f are results confirming that HCV replication is not inhibited even by 4-PBA treatment when hepcidin expression is inhibited.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for enhancing immunity comprising a hepcidin expression or activity enhancing agent.

In the present invention, "hecpcidin" is a protein encoded by the HAMP gene in humans and is a major regulator that regulates iron influx in mammals. Hepcidin exists as prepro-hormones, pro-hormones and hormones, and is a tightly folded polypeptide with 32% beta sheet properties and a hairpin structure stabilized by four disulfide bonds.

As used herein, the term "enhancer" refers to a pharmaceutical composition used to enhance various body abilities of a living organism, and includes enhancing gene expression or protein activity of a target component.

As used herein, the term “immunity” refers to a phenomenon in which the internal environment of a living body defends against an external factor, an antigen, and is divided into innate immunity and acquired immunity. Innate immunity is the natural immunity that foreign substances have before infection, and it responds quickly to infectious agents that invade the skin, mucosal epithelial cells, and mucus, which are the body's physical defense systems. Components involved in innate immunity are either always active or in a state that can be activated immediately, so they occur more rapidly than adaptive immunity.

The "hepcidin expression or activity enhancer" that can be used in the present invention may be used without limitation as long as it has a function to enhance the expression or activity of hepcidin, and in the present invention, 4-phenylbutyric acid (4-PBA) is used as hepcidin It was confirmed that it is a substance that enhances the expression of

The present inventors specifically identified the effect of enhancing immune activity through hepcidin expression or activity enhancement through specific examples.

That is, in one embodiment of the present invention, it was confirmed that the expression of hepcidin is increased by treatment of 4-PBA in liver cancer cell lines and mice in which HCV replication is implemented, so that the efficacy of 4-PBA to enhance the expression or activity of hepcidin was confirmed (see Example 2).

In another embodiment of the present invention, as a result of confirming the immune enhancement mechanism through hepcidin expression or activity regulation, when the expression of hepcidin is enhanced through 4-PBA treatment, the expression of interferon alpha is enhanced, and conversely, the expression of hepcidin is When the expression was suppressed, it was confirmed that the expression of interferon alpha was not increased (see Example 3).

In another embodiment of the present invention, in order to confirm whether a specific therapeutic effect is exhibited by immune enhancement through increase in the expression or activity of hepcidin, 4-PBA in mice transplanted with Huh7.5-Con1 cells in which HCV replication is implemented As a result of treatment, it was directly confirmed that HCV replication was inhibited in vivo (see Example 4).

The results of the above examples suggest that substances capable of enhancing the expression or activity of hepcidin can induce immune activity by enhancing the expression of interferon alpha, and thus are effective in treating pathogenic infections such as bacteria and viruses.

The immune enhancing agent according to the present invention may further include a pharmaceutically acceptable carrier commonly used in the preparation of pharmaceutical compositions. The pharmaceutically acceptable carrier is commonly used in formulation, and includes saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It is not limited, and may further include other conventional additives, such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but may be formulated as injections, inhalants, external preparations for skin, and the like.

The composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or locally applied) according to a desired method, but preferably may be administered orally, and the dosage may be determined by the patient. Although it varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and time of administration, it may be appropriately selected by those skilled in the art.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by the patient's disease type, severity, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination, in general 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight, may be administered daily or every other day, or may be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

In another aspect of the present invention, the present invention provides a method for screening an immune enhancer, comprising the following steps.

a) treating the cells with a candidate substance in vitro;

b) measuring hepcidin expression or activity in the cell; and

c) selecting a candidate substance whose hepcidin expression or activity is enhanced as an immune enhancer compared to the candidate substance untreated group.

The term "in vitro" used in the present invention means testing a part of a tissue and an organism in an artificial condition in vitro, such as in tissue culture. It is very difficult to directly observe or control a reaction or change occurring in a living organism, and it is very difficult to constantly control factors other than the manipulated variable because there are so many variables that can affect the reaction. On the other hand, in vitro experiments are used because it is possible to easily control the variables in the environment inside the test tube, and because it is easy to observe, the experiments can be performed more easily.

In the present invention, the candidate material may be selected from the group consisting of a compound, a microbial culture medium or extract, a natural product extract, a nucleic acid, and a peptide, and the nucleic acid is siRNA, shRNA, microRNA, antisense RNA, aptamer , LNA (locked nucleic acid), PNA (peptide nucleic acid) and may be selected from the group consisting of morpholino (morpholino), but is not limited thereto.

In step (b), the expression level is western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry. (flow cytometry), immunofluorescence (immunofluorescence), ouchterlony (ouchterlony), complement fixation assay (complement fixation assay) and protein chip (protein chip) to be measured through one or more methods selected from the group consisting of However, the measurement method is not limited thereto.

The level of activity in step (b) may be to measure the degree of acetylation of hepcidin histone protein, which can be suitably performed by a person skilled in the art without limitation through a known method.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental preparation and experimental method

1-1. Quantitative Real-Time Reverse Transcription Polymerase Reaction (qRT-PCR)

qRT-PCR was performed using Power SYBR Green PCR Master Mix in an ABI Prism 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The integrity of the amplified DNA was confirmed by determining the melting temperature. Expression levels were normalized to the expression level of β-actin.

1-2. Determination of cell viability

Cell viability was measured by lactate dehydrogenase (LDH)-release assays. LDH-release assays (Promega) were performed under identical conditions according to the manufacturer's instructions. Each value represents the average of a minimum of 9 wells.

1-3. Chromatin Immunoprecipitation Assay

Chromatin immunoprecipitation (ChIP) analysis was performed according to the simpleChIP ^R Enzymatic Chromatin IP kit protocol (cell signaling, MA, USA).

1-4. Luminase analysis

Luminase assays were performed using the Renilla Luciferase Assay System kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

1-5. immunoblotting

Cells were lysed with a lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 0.1% NP-40) containing a protease inhibitor cocktail (Calbiochem) and sodium vanadate (NaVO ₄ ). The whole lysate was loaded onto a 10-14% SDS-PAGE gel for electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 hour, washed and incubated overnight at 4° C. with primary antibody. After incubation with the secondary antibody for 45 minutes, the protein was visualized by improved western blotting luminol reagent (Santa Cruz).

1-6. Immunofluorescence assay

Cells were seeded on coverslips in 24-well plates and treated as indicated after 1 day. After incubation, the cells were fixed with 4% paraformaldehyde for 1 hour at room temperature and permeabilized with 0.1% (v/v) NP-40 for 30 minutes. Samples were washed three times with phosphate buffered saline (PBS) and 0.1% (v/v) Triton X-100, followed by 1% (w/v) BSA and 1% (v/v) Triton X-100 in phosphate buffered saline. incubated together. After blocking, samples were incubated with primary antibody overnight at 4°C. After washing 3 times, cells were incubated with Alexa Fluor-488 or -546-conjugated secondary antibody (Thermo Fisher Scientific) for 1 hour at room temperature while protecting them from light.

1-7. Immunoprecipitation

Treated or co-transfected cells were lysed with lysis buffer and the cell lysates were washed by centrifugation at 14,000 rpm for 15 minutes. Lysates (2 mg) were incubated with anti-hVAP-33 or anti-NS5A antibody (10 μg) for 12 h at 4°C. Protein-A/G-binding agarose beads were added and incubated at 4°C for 5 hours. Beads were washed three times with 1XTBST. Immunopellets were boiled with SDS-PAGE sample buffer and resolved by electrophoresis.

1-8. DNAs Wow siRNAs transduction

Plasmids and siRNAs were transduced using Xtreamgene HP (Roche, Swiss) according to the manufacturer's instructions. Hepcidin-specific siRNA and scrambled siRNA were purchased from Dharmacon (Denver, CO, USA) and transduced using Viromer BLUE (lipocalyx, Germany) according to the manufacturer's instructions.

1-9. cell culture

HCV genotype 1b Huh7.5-Con 1 cloned cells were provided by Apath (New York, NY, USA). Cells were incubated with 2 mM non-essential amino acids, 100 U/mL penicillin, 10% fetal bovine serum and 300 µg/mL geneticin (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere (5% CO ₂ , 37° C.). Incubated in the added Dulbecco's modified Eagle's medium (DMEM).

1-10. Materials

4-PBA and 4',6-diamino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and daclatasvir (DAV) was obtained from MedChem Express (Monmouth Junction, NJ, USA). Antibodies used in this study were mouse anti-NS5A; Santa cruz (sc-65458), mouse anti-NS3; Santa cruz (sc-69938), mouse anti-a-actinin; Santa cruz (sc-17829), rabbit anti-hepcidin; abCAM (ab75883), rabbit anti-H3K9ac; abCAM (ab10812), rabbit anti-H3K27ac; abCAM (ab4729), mouse anti-RNA pol III; abCAM (ab817), rabbit anti-OSBP; proteintech (#11096-1-AP), rabbit anti-VAP-B; NOVUS (NBP1-89112), rabbit anti-hVAP-33; Santa cruz (sc-98890), rabbit anti-CypB; abCAM (ab16045), mouse anti-OCH1E5; mybiosource (MBS370077), rabbit anti-NS5A (For Immunohistochemistry); mybiosource (MBS485095), anti-grp78; Santa cruz (sc-13968), rabbit anti-PERK; Santa cruz (sc-13073), rabbit anti-IRE1a; Santa cruz (sc-20790), mouse anti-actin; including Santa cruz (sc-8432). Secondary antibodies used were goat anti-mouse IgG; Invitrogen (M32607), goat anti-rabbit IgG; invitrogen (31460), Alexa Fluor 488 goat anti-rabbit IgG; Invitrogen (A-11034), Alexa Fluor488 goat anti-mouse IgG; Invitrogen (A-11001), Alexa Fluor 594 goat anti-mouse IgG; Invitrogen (A-11005), Alexa Flour 594 goat anti-rabbit IgG; Invitrogen (A-11012).

1-11. electron microscope

Huh 7.5-Con1 replicon cells were treated in 1 mM 4-PBA or 2 μM DAV for 24 hours and fixed in 2.5% glutaraldehyde in phosphate buffer (pH7.3). After washing once in a buffer, the cells were fixed with 1% osmium tetroxide in the same buffer and batch stained with a saturated aqueous solution of uranyl acetate. After rapid dehydration in progressively high concentrations of ethanol, the samples were infiltrated and embedded with Epon. Ultrathin sections were cut and collected on a copper grid, stained with uranyl acetate and lead citrate, and observed with a Tecnai G ² Sprit Twin electron microscope (FEI, Hillsboro, OR, USA) at 120 kV. Images were digitally captured with a US4000 charge-coupled device camera (Gatan, Pleasanton, CA, USA) using Digital Micrograph software (Gatan).

1-12. animal testing

Animal care and experimental procedures were performed with the consent of the National Cancer Center Clinical Trial Review Committee (Goyang-gu, Seoul, reference no. NCC-15-249). Animal studies are reported in accordance with the ARRIVE guidelines and recommendations of the British Journal of Pharmacy. The HCV model was established as previously described. That is, male immunodeficiency NOD/SCID mice (6 weeks old, Charles River Laboratories, Wilmington, MA, USA) were injected with Huh7.5-Con1 cells containing HCV replication via non-circular injection. Cell transplantation and surgical procedures were performed under anesthesia and cell injection was observed by MRI scanning 3 weeks after cell transplantation. Seven weeks after cell transplantation, dimethyl sulfoxide or 4-PBA was dissolved in polyethylene glycol and administered to mice daily via intraperitoneal injection. Body weights were measured every other day during treatment. Each group consisted of 5 animals and rat liver tissues were collected for analysis 7 days after the last injection. Surgery and injection were performed and animals were treated in a specific pathogen-free space. Rats were fed a regular diet and housed with corn heaps under specific pathogen-free conditions.

1-13. immunohistochemistry

Immunolabelling was performed using the Dako REAL EnVision Detection System (Agilent Technologies, San Diego, CA, USA). For immunohistochemical detection of NS5A and OCH1E5, samples were incubated overnight at 4°C with monoclonal antibodies to NS5A. (1:500) Deparaffinized and rehydrated liver tissue sections were incubated with anti-NS5A and anti-OCH1E5 antibodies for fluorescence immunohistochemistry. Samples were mounted on Fluoromount Aqueous Mounting medium (Sigma-Aldrich) and visualized with a multifocal fluorescence microscope.

1-14. Data and Statistical Analysis

Data and statistical analyzes were in accordance with the recommendations of the British Journal of Pharmacy for experimental design and analysis of pharmacology. Results are expressed as the mean±SEM of five independent experiments. Depending on the experimental design, data were analyzed by t-test, one-way or two-way ANOVA. When statistical significance ( P < 0.05) and no significant variance inhomogeneity were detected and the required level was achieved in the ANOVA, a post hoc multiple comparison analysis (Tukey's test) was applied. Statistical analysis was performed using GraphPad Prism v.8.2.1. Differences at P<0.05 were considered statistically significant. Group sizes are designed to be identical and always represent the number of independent experimental replicates, each performed with a unique biologic.

Example 2. Verification of the effect of increasing hepcidin expression

In order to confirm that 4-phenylbutyric acid (4-PBA) effectively acts as a hepcidin expression enhancer, 1 mM 4-PBA was treated with the hepatitis c virus gene-expressing hepatocellular carcinoma cell line Huh7.5-Con 1, Dean protein and mRNA expression changes were confirmed by immunofluorescence staining and qRT-PCR, respectively.

As a result, as shown in FIGS. 1A and 1B , it was confirmed that hepcidin protein and mRNA expression were increased according to 4-PBA treatment, thereby confirming that 4-PBA could be used as a hepcidin expression enhancer.

In addition, immunohistochemistry was performed using livers of excipient-treated mice and 4-PBA-treated mice. As a result, as shown in FIGS. 1c and 1d , it was confirmed that hepcidin was expressed higher in the liver tissue treated with 4-PBA compared to the tissue treated with the excipient (Excipient: 0.07; 75 mg/kg: 0.21; 150 mg/kg: 0.32).

On the other hand, additionally, in order to determine whether enhancement of hepcidin expression as a mechanism of the above phenomenon is associated with epigenetic alteration at the hepcidin locus, Huh7.5-Con1 treated with 4-PBA for 6, 12 or 24 hours Chromatin immunoprecipitation (CHIP) qPCR analysis was performed on cell-derived genomic DNA. As a result, as shown in FIG. 1e, it was confirmed that the acetylation of lysine residues 9 and 27 of hepcidin 3 histone protein was increased. In addition, as shown in FIG. 1f , it was confirmed that histone acetylation was increased in H3K27 in the whole cell lysate by treatment with 4-PBA in Huh7.5-Con1 cells.

In addition, as shown in FIG. 1g , an increase in RNA polymerase II attached to the hepcidin promoter through 4-PBA treatment was also confirmed.

Example 3. Immune enhancement mechanism confirmation through hepcidin expression or activity regulation

In order to check whether the induction of immune activity is regulated through hepcidin expression or activity regulation, an experiment was performed as follows.

First, Huh7.5-Con1 cells were treated with 1 mM 4-PBA verified as a hepcidin expression enhancer through Example 2, and changes in the interferon response were confirmed.

As a result, as shown in FIG. 2a , it was confirmed using luminescent enzyme analysis that the activity of Interferon Stimulated Response Element (ISRE), which can indirectly confirm the initiation of interferon reaction, was increased by about three times according to 4-PBA treatment. In addition, as shown in FIG. 2b , it was confirmed that the mRNA expression of interferon alpha and the generation and secretion of interferon alpha also increased. Furthermore, as shown in FIGS. 2c and 2d, IFN regulators ( IRF1, IRF3, IRF7 ), protein kinase R ( PKR ), tetratricopeptide repeat1, which are known to be involved in the removal of pathogens by induced expression by IFN-α It was confirmed that the expression of the containing IFN-induced protein ( IFIT1 ) and 2'-5'-oligoadenylate synthase 1 ( OAS1 ) genes were all increased.

Next, in order to reconfirm whether the enhancement of hepcidin expression or activity is involved in increasing the response of interferon, hepcidin gene (HAMP) expression was suppressed using siRNA and then 4-PBA was treated with 1 mM, and the expression of related genes was changed was confirmed.

As a result, as shown in FIGS. 3a, 3b, 3c and 3d, when hepcidin expression was suppressed, it was confirmed that the interferon response did not increase even when 4-PBA was treated.

Example 4. Confirmation of antiviral effect by immune activity in mouse model

As an example for confirming the enhancement of immune activity of 4-PBA, which was confirmed to cause interferon alpha expression through increased hepcidin expression according to Examples 1 and 2, Huh7. 5-Con1 cells were transplanted into immunodeficient NOD/SCID mice to produce an HCV replicon-based mouse model. To confirm that the model was established, the liver morphology was examined by magnetic resonance imaging (MRI) and anatomical observation at 3 and 7 weeks after surgery ( FIGS. 4A and 4B ).

After 7 weeks of grafting of HCV replicon cells, 4-PBA (vehicle + 75 or 150 mg/kg) was injected daily for 7 days. The antiviral effect of 4-PBA was evaluated by immunohistochemistry and immunofluorescence labeling of HCV NS5A, and transplanted Huh7.5-Con1 cells were identified based on the expression of human hepatocyte-specific antigen OCH1E5. Although there was no significant reduction in body weight associated with the potential toxicity of 4-PBA, the mice treated with 4-PBA showed a significant decrease in the NS5A protein level in the OCH1E5-positive region compared to the vehicle-injected mice ( FIGS. 4c and 4d ). The above results provide strong evidence that 4-PBA inhibits HCV replication in vivo.

Conversely, as shown in FIGS. 4e and 4f, when 4-PBA was treated in Huh7.5-Con1 cells in which hepcidin expression was suppressed, it was confirmed that HCV replication was not inhibited.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (5)

delete delete a) treating the cells with a candidate substance in vitro;
b) measuring hepcidin expression or activity in said cell; and
c) A screening method for an immune enhancer comprising the step of selecting a candidate material whose hepcidin expression or activity is enhanced compared to the candidate material untreated group as an immune enhancer. 4. The method of claim 3,
The candidate material is a screening method, characterized in that selected from the group consisting of compounds, microbial culture or extracts, natural product extracts, nucleic acids and peptides. 5. The method of claim 4,
The nucleic acid is siRNA, shRNA, microRNA, antisense RNA, aptamer (aptamer), DNA (locked nucleic acid), PNA (peptide nucleic acid) and morpholino (morpholino) characterized in that selected from the group consisting of, screening, characterized in that Way.

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