KR101353570B1 - Composition of prevention and treatment for fatty liver comprising stimulator of expression or activity of clusterin - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of fatty liver comprising a clusterrin expression or activity promoter, the clusterrin can prevent and treat fatty liver by reducing the expression of SREBP-1c in the liver.

Description

Composition of prevention and treatment for fatty liver comprising stimulator of expression or activity of clusterin}

The present invention relates to a composition for treating fatty liver, and more particularly, to a composition for preventing and treating fatty liver, including a clusterin expression or activity promoter.

In addition to the increase in obese population due to the westernization of diet, nonalcoholic fatty liver disease (NAFLD) due to metabolic syndrome is increasing rapidly. Non-alcoholic fatty liver disease is a disease that shows similar histological findings as alcoholic fatty liver but does not have a history of alcohol consumption, and may be the most important cause of obesity (Clark JM et al. Gastroenterology 122: 1649-1657, 2002; Angulo P, N Eng J Med 346: 1221-1231, 2002; Marceau P et al. J Clin Endocrinol Metab 84: 1513-1517, 1999).

Clinically, the accumulation of fat in the liver is considered to be a feature of insulin resistance and is associated with diseases belonging to metabolic syndromes such as abdominal obesity, hypertension, type 2 diabetes and hyperlipidemia. Metabolic syndrome is expressed in the form of. Therefore, the successful treatment of metabolic syndrome requires correction of metabolic abnormalities in liver tissues, which are major organs of fat and glucose metabolism (Kim TH et al. Korean J Intern Med 68: 347-349, 2005; Marchesini G et. al. Diabetes 50: 1844-1850, 2001).

In the development of fatty liver disease caused by metabolic syndrome, increased free fatty acid is introduced into the liver, and hyperinsulinemia plays a key role in the biosynthesis of triglycerides (sterol-regulatory element-binding protein (SREBP)-). Activating 1c) is known to cause fat accumulation in liver tissue. SREBP-1c is an important transcriptional activator that regulates fatty acid and cholesterol synthesis in the liver by activating enzymes involved in the biosynthetic pathways of fatty acids and cholesterol. Hyperinsulinemia due to insulin resistance increases the biosynthesis of fatty acids by increasing the expression of SREBP-1c in the liver and consequently leads to the accumulation of triglycerides in liver tissues (Marchesini G et al. Diabetes 50: 1844-1850, 2001; Foretz M et al. Proc Natl Acad Sci USA 96: 12737-12742, 1999).

Clusterrin (Clusterin, Apolipoprotein J) is a glycoprotein expressed in various tissues and is known to be involved in the survival and death of cancer cells, lipid metabolism, immune and reproductive control, but its main physiological functions are not clear. Trugakos IP et al. Int J Biochem Cell Biol. 34 (11): 1430-48, 2002; Gleave M et al. Ann NY Acad Sci. 1002: 95-104, 2003.

The present inventors have tried to find a substance that can effectively inhibit the formation of fatty liver by inhibiting the expression or activity of SREBP-1c, an early factor involved in fatty liver formation.

In the course of studying the function of clusterrin in hepatocytes, the inventors have confirmed that clusterrin inhibits fatty liver by reducing the expression of SREBP-1c in hepatocytes, and has completed the present invention.

The present invention relates to a composition for preventing and treating fatty liver, comprising a clusterin expression or activity promoter.

In the present invention, 'fatty liver' is a disease in which fat, particularly triglyceride, accumulates in hepatocytes and accounts for 5% or more of the weight of the liver. There are alcoholic fatty liver associated with drinking and non-alcoholic fatty liver not associated with drinking. Fatty liver in the present invention preferably means non-alcoholic fatty liver.

In the present invention, 'clusterin (Apolipoprotein J) expression or activity promoter' refers to a substance which directly or indirectly acts on clusterrin to improve, induce, stimulate, and increase clusterrin expression or activity. Such materials include single compounds such as organic or inorganic compounds, complexes of plural compounds and biopolymers such as peptides, proteins, nucleic acids, carbohydrates and lipids. The mechanism by which the substance promotes the expression or activity of clusterin is not particularly limited. For example, a substance may act as a mechanism for enhancing gene expression such as transcription, translation, or converting an inactive form into an active form.

Preferably, the substance which promotes the expression or activity of the clusterrin is a biopolymer compound such as peptides, proteins, nucleic acids, carbohydrates and lipids. For clusterins with known nucleic acid and protein sequences, those of ordinary skill in the art will appreciate that single compound peptides, such as organic or inorganic compounds that act as promoters, complexes of biopolymeric compounds such as proteins, nucleic acids, carbohydrates and lipids, and plural compounds, etc. Techniques can be used to make or screen.

Clusterrin expression or activity promoter of the present invention may be provided in the form of a vector capable of expressing clusterrin in cells for use in gene therapy and the like.

Accordingly, the present invention relates to a composition for preventing and treating fatty liver, comprising a nucleic acid encoding a clusterrin, preferably a recombinant vector containing the nucleic acid, as an active ingredient.

Nucleic acid sequences encoding clusterins may be mutated by substitution, deletion, insertion or combination of one or more nucleic acid bases so long as they encode a protein having equivalent activity. The sequence of such a nucleic acid molecule may be short or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

The vector of the present invention includes, but is not limited to, liposomes, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. A preferred vector in the present invention is a viral vector.

Examples of preferred viral vectors in the present invention include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus, alpha virus). Adenovirus vectors are particularly preferred in the present invention.

The recombinant vector of the present invention may comprise a nucleic acid encoding a clusterrin and regulatory sequences for its transcription or translation. A particularly important regulatory sequence is the regulation of transcription initiation, such as promoters and enhancers. It may also contain regulatory sequences consisting of initiation codon, termination codon, polyadenylation signal, Kozak, signal sequence for enhancer, membrane targeting and secretion, IRES (Internal Ribosome Entry Site), and the like. Such regulatory sequences and nucleic acids encoding clusterins should be operably linked.

As used herein, the term 'operably linked' means that the binding between nucleic acid sequences is functionally related. The case where any nucleic acid sequence is operably linked is when any nucleic acid sequence is positioned to be functionally related to another nucleic acid sequence. In the present invention, when any transcriptional regulatory sequence affects the transcription of a nucleic acid molecule encoding clusterrin, the transcriptional regulatory sequence is said to be operably linked to the nucleic acid molecule.

In the present invention, treatment includes inhibiting or preventing fatty liver, reducing, alleviating, reversing the symptoms associated with fatty liver disease and inhibiting fatty liver progression.

The pharmaceutically acceptable carriers to be included in the composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical compositions of the present invention may be administered via routes commonly used in gene therapy, with parenteral administration being preferred, eg, using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration. Can be administered.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. In one embodiment, the pharmaceutical composition of the present invention may be administered 1-2 times intravenously with 1 × 10 ⁸ to 1 × 10 ¹¹ pfu of recombinant adenovirus.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The pharmaceutical compositions of the present invention can be lyophilized to increase stability at room temperature, reduce the need for costly cold storage, and to extend shelf-life. The lyophilization process may consist of successive stages of freezing, primary drying and secondary drying. The secondary drying process after freezing the composition is to drop the pressure and heat it for sublimation of water vapor. The secondary drying step is to evaporate the residual moisture absorbed from the dry matter.

According to the present invention, clusterrin can prevent and treat fatty liver by inhibiting the expression of SREBP-1c in the liver.

1 shows that expression of SREBP-1c increased by insulin is inhibited by clusterrin (A: mouse liver cell line (AML12), B: rat liver cancer cell line (H4IIE), C: human liver cancer cell line (HepG2)) (Left: Northern blot, right: Western blot in A and B).
FIG. 2 shows that clusterrin inhibits fatty liver formation by high fat diet in mice (A: H & E staining and Oil Red O staining) and reduces expression of SREBP-1c in liver (B: Northern blot).

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1. Materials and Methods

1-1. Method for preparing adenovirus clusterin

Recombinant adenovirus comprising a nucleic acid encoding the clusterrin was prepared as follows. Full-length clustered cDNA (1 Gene ID: 24854) was converted to pAdTrack-cytomegalovirus [CMV; AdEasy system; containing green fluorescent protein (GFP) and CMV promoter]. PAd-clusteirn was then co-transformed into Kanamycin LB plates with pAdEasy-1 (containing the viral backbone) to BJ5183 bacteria. The recombinant adenovirus containing the nucleic acid encoding the clusterin thus obtained (abbreviated as 'adenovirus clusterin') was amplified and purified using a plasmid midi-prep system (Qiagen, Valencia, CA). It was then further amplified in 293 cells and titrated using adeno-X repid titier kit (BD bioscience, Palo Alto, Calif.).

The overexpression experiment of the clusterrin gene proceeded as follows. When H4IIE or AML12 cells have grown to 80-90% in culture vessels. Adenovirus clusterin was treated by concentration, and the control group was treated with the same concentration of GFP virus. Infection time was 2 hours, after which the culture solution was replaced and cultured for another 24 hours, RNA was isolated to search for genes showing differences in expression.

1-2. Cell culture

Human hepatoma cell line and rat H4IIE hepatoma cell line were treated with antibiotics (100 U / mL penicillin and 100 g / mL streptomycin) and 10% FBS in minimal essential medium (MEM) medium. Bovine serum was added and cultured, and the mouse AML12 hepatocyte cell line was diluted to INS (insulin-transferrin-selenium) (a mixture of insulin, transferrin, selenium (100X), 1X in DMEM / F12 medium). And 40 ng / ml dexamethasone, antibiotics (100 U / mL penicillin and 100 g / mL streptomycin) and 10% FBS were incubated. Cells were cultured with a mixture of air (95%) and CO ₂ (5%) in a 37 ° C. thermostat maintaining constant humidity, and passaged every 3-4 days. HepG2 cells were incubated for 24 hours in medium supplemented with 0.5% FBS prior to insulin treatment to allow the cells to enter the resting period. Insulin was treated for 6 hours, and adenovirus clusterin was treated by concentration at 18 hours before insulin treatment, and the expression of SREBP-1c was confirmed.

1-3. Northern blot analysis

The radiolabeled reporter for SREBP-1c used in northern blots was random primer DNA labeling kit system using [α-32P] 2'-deoxycytidine 5'-triphosphate (dCTP), Amersham, Arlington Heights, IL. ) Radiolabeled subjects were purified on NAP-5 columns (Pharmacia, Uppsala, Sweden). Thereafter, 20 μg of RNA was electrophoresed on 1% formaldehyde-agarose gel and then transferred to a nylon membrane. The RNA was immobilized on the nylon membrane by UV cross-linker treatment to the nylon membrane to which the RNA was transferred. The nylon membrane was then hybridized at 65 ° C. for 24 hours in an Express Hyb ™ solution with radiolabeled media and washed several times. After washing, the nylon membrane was exposed to X-ray film at -70 ° C for 24 to 48 hours, and then mRNA expression was analyzed using a densitometer.

1-4. Western blot analysis

AML12 and H4IIE cells were treated with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% P-40, 1 mM PMSF), 1 μg / mL protease inhibitors (Luepetin, Aprotinin). The protein was extracted by treatment. Each sample was mixed with RIPA buffer, boiled for 5 minutes, and cooled on ice. Proteins were separated by size by electrophoresis on SDS (Sodium dodecyl sulfate) polyacrylamide gel and then transferred to Immobilon-P transfermembrane (Milipore, Billerica). After blocking with blocking buffer and reacting with SREBP-1c antibody at 4 ° C. for 16 hours, the reaction was performed at room temperature with a horseradish peroxidase-labeled secondary antibody for 2 hours. Protein expression was confirmed using ECL plus (Amersham Biosciences). Membrane was reacted with the actin antibody again to confirm that a certain amount of protein was used.

1-5. Luciferase Activity Measurement

To investigate the effect of insulin and alpha-lipoic acid on SREBP-1c transcriptional activity, the SREBP-1c promoter construct (Human SREBP-1c promoter (-780 / + 62) was expressed on HepG2 cells using Lipofectamin (Invitrogen, Carlsbad, CA). Tarling E, Salter A, Bennett A: Transcriptional regulation of human SREBP-1c (sterol regulataroy element binding protein 1c): a key regulator of lipogenesis.Biochem Soc Trans 32: 107-109, 2004]) (300 ng / well) and the pCMV-β-gal plasmid (200 ng / well) were transformed together (co-transfection) to correct the efficiency of transduction. After 5 hours of transduction, the cells were replaced with a medium containing 0.5% FBS, followed by further incubation for 24 hours. Cells were then washed twice with PBS and digested with 100 μl reporter lysis buffer (Promega, WI, USA). After centrifugation at 4 ° C., 12000 rpm for 10 minutes, 20 μl of supernatant was measured for transcriptional activity using a luminometer (SIRUS Luminometer; Berthold, Pforzheim, Germany). β-galactosidase activity was measured by measuring the absorbance at 420 nm 20 μl of the supernatant using an ELISA reader and this value was used to correct the luciferase activity.

1-6. Animal experiment

As the experimental animals, C57BL / 6 mice of about 8 weeks of age were purchased, and the control group was divided into three groups: normal control group, high-fat diet group, and high-fat diet cluster overexpression experiment group. The conditions of the feeding room temperature was 22 ℃, photoperiod was adjusted every 12 hours every day. The high-fat diet was carried out for 4 weeks and mice in the clustered overexpression group were injected with adenovirus clusterrin into the tail vein for the last 3 days. Three days later, livers were isolated from mice to confirm expression of SREBP-1c by Northern blot.

1-7. H & E dyeing and Oil red O dyeing

H & E staining was performed to examine the morphology of liver tissues. The tissues were stained with Harry's hematoxylin stain for 5 minutes, washed with running water for 5 minutes, and destained with 1% HCl-alcohol and ammonia. After washing with running water for 10 minutes, counterstain again with eosin dye for 1 minute, dehydrated in the reverse direction of hydrolysis (100% -95% -90% -80% -70% EtOH), and then sealed. It was observed under a microscope.

Oil red O staining for fat accumulation in liver tissues was performed after dissolving oil red O in 60% isopropanol.

Example 2. Results

2-1. Confirmation of the Inhibitory Effect of SREBP-1c Increased by Insulin on Clusterrin

Insulin treatment of mouse liver cell line (AML12) increased the expression of mRNA and protein of SREBP-1c. At this time, overexpression of clusterin using adenovirus increased the concentration of SREBP-1c by insulin. It was confirmed by the Northern blot and Western blot that the expression of FAS (fatty acid synthase), which is increased depending on SREBP-1c and also decreased by clusterrin, can be confirmed (A of FIG. 1).

Insulin treatment of rat liver cancer cell line (H4IIE) increased the expression of mRNA and protein of SREBP-1c. At this time, overexpression of clusterrin with adenovirus increased the concentration of SREBP-1c by insulin. MRNA and protein expression was reduced by Northern blot and Western blot (Fig. 1 B).

Treatment of insulin with human liver cancer cell line (HepG2) significantly increased the activity of the SREBP-1c promoter, whereby clusterin inhibited the increased SREBP-1c transcriptional activity by insulin, as measured by luciferase activity (FIG. 1C). ).

2-2. Confirmation of Fatty Liver Inhibitory Effect in Clustered Mouse

After providing high fat diet (HFD) to mice and injecting clustered adenovirus into the tail vein, it was confirmed that fatty liver increased by high fat diet was reduced by clusterrin (FIG. 2A).

In addition, as shown in FIG. 2A, the generation of fatty liver by high fat diet can be confirmed through H & E staining and Oil Red O staining (HFD + Ad GFP), and it can be confirmed that fatty liver is reduced by clusterrin. .

In addition, it was confirmed that the expression of SREBP-1c was increased in mouse liver due to the high fat diet (HFD + Ad GFP), and the Northern blot showed that the expression of SREBP-1c increased by the high fat diet was reduced by clusterrin. It confirmed (B of FIG. 2).

Claims (7)

A pharmaceutical composition for preventing and treating alcoholic fatty liver, comprising a recombinant adenovirus vector containing a nucleic acid encoding a clusterrin as an active ingredient. delete delete delete delete delete The pharmaceutical composition for preventing and treating alcoholic fatty liver, according to claim 1, wherein the clusterin reduces expression of SREBP-1c.

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