KR20110054952A - Zebrafish model for inflammatory disease and screening method of anti-inflammatory agent using the same - Google Patents

Abstract

PURPOSE: A zebra fish model for inflammatory diseases and a method for screening an anti-inflammatory agent using the same are provided to screen effective anti-inflammatory agent without side effects. CONSTITUTION: A method for screening an anti-inflammatory agent using zebra fish model comprises: a step of treating one selected from the group consisting of lipopolysaccharides, oxidized low-density lipoprotein, and saccharification apolipoprotein to the zebra fish; and a step of administering a candidate drug to the zebra fish and evaluating the development and apoptosis of an embryo. The inflammatory diseases are inflammatory dermatitis, glomerulus nephritis, inflammatory bowel disease, inflammatory collagen vascular disease, osteoarthritis, inflammatory eye disease, IgA nephropathy, rheumatoid arthritis, post-vaccinal encephalomyelitis, and diabetes.

Description

Zebrafish model for inflammatory disease and screening method of anti-inflammatory agent using the same

The present invention relates to a zebrafish model for inflammatory diseases and a screening method of an anti-inflammatory agent using the same, which is very useful for developing a drug capable of preventing or treating an inflammatory disease.

Inflammation is a major cause of almost all chronic metabolic diseases and plays an important role in cardiovascular disease. High-density lipoprotein (HDL) is a potent antioxidant and anti-inflammatory molecule in plasma, and HDL-cholesterol levels are inversely correlated with the risk of cardiovascular disease. Normal apoA-I (nA-I) is a major component of HDL that exhibits antioxidant and anti-inflammatory activity through the removal of LDL lipid peroxide in vitro and in vivo when injected into mice and humans. In addition, reconstituted HDL (rHDL) containing apoA-I is known to reduce inflammation by neutralizing the toxicity of endotoxin or lipopolysaccharide (LPS).

In order to mimic acute inflammatory conditions in cells or tissues, LPS treatment has been widely used. Injection of high doses of LPS into the abdominal cavity is well known to be toxic in mammals. However, apoA-I has been proposed to show a protective effect by neutralizing or detoxifying LPS. Expression of human apoA-I is known to reduce LPS-induced inflammation in mouse models.

apoA-I is an anti-arteriosclerosis and anti-inflammatory protein and a major component of HDL. Mature human apoA-I consists of a single polypeptide chain of 243 amino acids, which has 11 and 22 amino acid homology repeats. Twenty-one lysine and sixteen arginine residues are known to be susceptible to glycosylation, and arginine, lysine and trypsin residues are known to be 45-55% modified by methylglyoxal treatment.

However, the beneficial function of apoA-I may be impaired by chemical modifications such as glycosylation, nitrification and chlorination. Glycosylation of apoA-I is an important feature of patients with uncontrolled diabetes and aging. Glycosylated apolipoproteins are frequently found in the plasma of diabetic patients with impaired anti-inflammatory activity and increased susceptibility to infection.

Zebrafish are well-developed innate and acquired immune systems that are very similar to the mammalian immune system. When they occur, zebrafish embryos are externally generated and are optically transparent. These characteristics make zebrafish a useful animal model for a variety of studies.

On the other hand, therapeutic agents for various inflammatory diseases include inflammatory inhibitors by steroids such as prednisolone, nonsteroidal anti-inflammatory drugs (NASAIDs) such as naproxen, and calcium and calmodine dependent protein kinase enzymes such as cyclosporin or FK506. Inhibitors that bind to and inhibit the action of phosphorus calcineurin (calcineurin) have been most used.

However, these steroids and immunosuppressive agents are accompanied by side effects such as renal toxicity, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, nausea, renal dysfunction.

Therefore, there is an urgent need for the development of screening methods for developing anti-inflammatory agents with good efficacy and low side effects.

In order to solve the problems of the prior art, the inventors of the present invention, using a zebrafish embryo showing inflammation induced by LPS and oxLDL, native ApoA-I (nA-I) and glycosylated apolipoprotein in lipid binding state The present invention was completed by comparing and examining the anti-inflammatory activity of (glycated apoA-I, gA-I).

Accordingly, an object of the present invention is a zebra for a new inflammatory disease obtained by treating any one substance selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL) and glycated apolipoprotein (gA-I) in zebrafish To provide a fish model.

Another object of the present invention is to provide a method for screening an anti-inflammatory agent using the zebrafish model.

In order to achieve the above object, the present invention is an inflammatory disease obtained by treating any one substance selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL) and glycated apolipoprotein (gA-I) in zebrafish Provides a zebrafish model for

The inflammatory diseases include, for example, inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, osteoarthritis, inflammatory eye diseases such as conjunctivitis, IgA nephropathy, rheumatoid arthritis, encephalomyelitis, type 1 diabetes, diabetic nephritis And systemic lupus erythematosus can be any one selected from the group consisting of, preferably an acute inflammatory disease, but is not limited thereto.

The present invention also provides a method of treating zebrafish with any one selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL) and glycated apolipoprotein (gA-I); And it provides a method for screening anti-inflammatory drugs using zebrafish comprising the step of evaluating the development and death of the embryo after administration of the candidate drug.

Evaluating the development and death of the embryo includes, but is not limited to, morphological observation through a microscope and various assays.

The present invention also provides a method of treating zebrafish with any one selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL) and glycated apolipoprotein (gA-I); And after the candidate drug is administered, evaluating the glycosylation level of the apolipoprotein.

Evaluating the glycosylation level of the apolipoprotein can be evaluated by fluorescence analysis or electrophoresis, in particular fluorescence analysis can be evaluated by measuring the fluorescence intensity at 370nm (excitation) and 440nm (emission). In addition, it can be evaluated by electrophoresis and protein analysis liquid chromatography (FPLC), glycosylated apolipoprotein (gA-I) is highly susceptible to proteolytic degradation.

Using the zebrafish model according to the present invention, it is possible to effectively screen an effective anti-inflammatory agent with less side effects. In addition, all drug candidates can be tested quickly and easily for in vivo side effects such as toxicity and inflammation.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Purification and Glycation of Apolipoprotein apoA-I

According to the method proposed by Brewer et al. (Brewer HB, Ronan R, Meng M, Bishop C. Isolation and characterization of apolipoprotein AI, A-II, and A-IV.Meth Enzymol 1986; 28: 223-246) ApoA-I was purified from human plasma by ultracentrifugation, column chromatography and organic solvent extraction. The purified apoA-I was stored by lyophilization at -80 ℃ until use.

apoA-I glycosylation was performed by treating excess fructose. That is, purified lipid free apoA-I (10 mg / mL) was added to 5 mM D-fructose [in 200 mM potassium phosphate / 0.02% sodium azide buffer (pH 7.4) for 7 days at 37 ° C. under 5% CO ₂ containing air. Reacted with The degree of final glycosylation of the protein was determined according to the inventor's prior patents (methods of screening glycosylation inhibitors of apolipoprotein AI, Republic of Korea Application No. 10-2009-0078207, filed Aug. 24, 2009). Was determined by analyzing the fluorescence intensity.

Example 2 Reconstituted HDL (rHDL) Synthesis Using nA-I and gA-I

Preparation of Reconstituted HDL (rHDL) was carried out according to the method described in our prior art ( Mol Cells., 27: 291-297, 2009) and in the prior patent (Korean Application No. 10-2008-0043537). And unglycosylated apoA-I (nA-I) and glycosylated apoA-I (gA-) with phospholipid double membranes through sodium cholate dialysis at a molar ratio of 95: 5: 1: 150 with apoA-I and sodium cholate Recombinant HDL comprising I) was synthesized.

Example 3 Examination of Anti-inflammatory Effects Using Zebrafish Embryo

1) Zebrafish Embryo Preparation

Zebrafish and embryos were administered according to standard methods (Nusslein-Volhard, C., Dahm, R. Zebrafish: A Practical Approach. Oxford University Press, 2002). Zebrafish and embryos were bred in system cages and 6-well plates at 28 ° C. under 10:14 hour light cycles.

2) LPS or oxLDL treatment

Magnetic controller into the embryo 4-5 hours after fertilization [MM33; Kantee, Bensenville, IL, USA; LPS or oxLDL were microinjected using a pneumatic picopump (PV820; World Precision Instruments, Sarasota, FL, USA) equipped with a pulled microcapillary pipette-using device (PC-10). In order to minimize the experimental error, microinjection was performed at the same site of egg yolk. At this time, nA-I-rHDL or gA-I-rHDL were injected or not injected into zebrafish embryos.

After injecting the filter sterilization solution of LPS (L4130, Escherichia coli 0111: B4, Sigma, St. Louis, MO, USA) up to 200 ng (final, 50 nL) into the flask of the embryo, live embryos were subjected to stereomicroscopy (Motic SMZ 168). ), And photographed using a Motic cam2300 CCD camera.

In addition, the oxLDL was prepared as follows. That is, LDL (1.019 <d <1.063) was purified from human plasma by ultracentrifugation according to the standard method (J Clin Invest, 34: 1345-53, 1955). At this time, the density of the LDL was adjusted by the addition of NaCl, was centrifuged for 22 hours at 100,000g, 10 ℃ using Himac CP-90α (Hitachi). Oxidized LDL (oxLDL) was prepared by reacting an LDL (1.019 <d <1.063) fraction with CuSO ₄ (final concentration, 10 μM) at 37 ° C. for 4 hours. LDL was purified from the serum of normal persons received from the blood bank of Yeungnam University Medical Center. The oxLDL was then filtered and the degree of oxidation was determined via thiobarbic acid reactive substrate (TBARS) analysis.

The zebrafish embryos were also treated with nA-I-rHDL or gA-I-rHDL and then immersed in water containing LPS (final concentration 100 μg / mL) in 6-well plates at 28 ° C.

3) results

As in FIG. 1A, LPS treatment resulted in the death of zebrafish embryos. Approximately 85% of embryos were killed by 200ng of LPS treatment. However, treatment with LPS (200 ng) and nA-I-rHDL (50 ng) significantly improved the survival rate of zebrafish embryos, resulting in only 45% of deaths for 48 hours, while LPS and gA-I-rHDL were combined. If treated, up to 78% of embryos died.

Figure 1b shows the developmental stage over time, all embryos were healthy up to 6 hours after treatment, except for LPS and gA-I-rHDL simultaneous treatment group. At 50 hours post-treatment, 85% and 78% embryos died in the LPS and LPS and gA-I-rHDL co-treated groups, respectively.

As shown in FIG. 2A, zebrafish embryos in LPS-containing water (100 μg / mL) did not induce significant mortality. nA-I-rHDL injected embryos showed similar survival, while gA-I-rHDL injected embryos had a low survival rate of 20%. As observed by light microscopy at 11 and 28 hours after treatment (FIG. 2B), gA-I-rHDL showed a detrimental effect on the development of zebrafish embryos in the presence of LPS. In addition, embryos treated with gA-I-rHDL developed much slower after 49 hours compared to nA-I-rHDL and buffer injected embryos.

In addition, injection of 50 nl of oxLDL (1 mg / ml protein, 250 nM MDA / mg protein) into the embryo increased mortality as shown in FIG. 3A (30% survival rate at 48 hours after treatment). Injecting normal LDL (60 nM MDA / mg protein) into the embryo shows a survival rate of 50% or higher, indicating that oxLDL is more cytotoxic in embryos.

However, at the same time, the survival rate was increased to approximately 70% when treated with oxLDL and nA-I-rHDL, while the survival rate was 18% when treated with oxLDL and gA-I-rHDL mixtures. Thus, nA-I-rHDL treatment prevented cytotoxicity by oxLDL injection, whereas gA-I-rHDL treatment promoted embryonic development delay and death by oxLDL.

Figure 1a is an electrophoretic pattern before and after glycosylation of apolipoprotein (apoA-I), lane 1 is pure apoA-I (nA-I) before glycosylation, lane 2 is glycosylated apoA-I (gA- I), lane M is the molecular weight standard (BioRad, low-range standard),

1B shows the viability of zebrafish embryos treated with nA-I-rHDL or gA-I-rHDL with LPS at 1 day post fertilization,

FIG. 1C shows time dependent changes of zebrafish embryos treated with nA-I-rHDL or gA-I-rHDL with LPS,

2A shows the viability of zebrafish embryos exposed to water containing LPS after treatment with nA-I-rHDL or gA-I-rHDL,

2B shows the time dependent change of zebrafish embryos exposed to water containing LPS after treatment with nA-I-rHDL or gA-I-rHDL,

Figure 3a shows the viability of zebrafish embryos treated with nA-I-rHDL or gA-I-rHDL with oxLDL,

Figure 3b shows the change in zebrafish embryos 49 hours after treatment with nA-I-rHDL or gA-I-rHDL with oxLDL.

Claims (5)

A zebrafish model for inflammatory diseases obtained by treating zebrafish with any one selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and glycated apolipoprotein (gA-I). The method of claim 1, wherein the inflammatory disease is inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, osteoarthritis, inflammatory eye disease, IgA nephropathy, rheumatoid arthritis, encephalomyelitis, type 1 diabetes, diabetic nephritis and systemic redness Zebrafish model for inflammatory diseases, which is any one disease selected from the group consisting of seminal lupus. Treating zebrafish with any one material selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and glycated apolipoprotein (gA-I); And Evaluating embryo development and death after administration of candidate drug Screening method of anti-inflammatory agent using zebrafish comprising a. Treating zebrafish with any one material selected from the group consisting of lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and glycated apolipoprotein (gA-I); And Assessing glycation levels after administration of the candidate drug Screening method of anti-inflammatory agent using zebrafish comprising a. The method of claim 4, wherein the evaluating the glycosylation level of the apolipoprotein is evaluated by fluorescence analysis or electrophoresis.

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