KR20200055572A - Composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin - Google Patents

Abstract

The present invention relates to a composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin. More specifically, the present invention relates to a composition for preventing or treating ischemia-reperfusion injury which comprise alpha-1 antitrypsin, exhibiting an effect of inhibiting the expression of inflammatory markers in a tissue damage environment due to ischemia-reperfusion, as an active component. The composition of the present invention inhibits the expression of inflammatory factors in an organ damage environment due to ischemia-reperfusion, and has a very excellent effect of inhibiting collagen accumulation and apoptosis, thereby being able to be very usefully used for developing an agent for preventing or treating organ damage due to ischemia-reperfusion.

Description

Composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin}

The present invention relates to a composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin, and more particularly, alpha-1 showing the effect of inhibiting the expression of inflammatory markers in the tissue damage environment by ischemia-reperfusion. It relates to a composition for preventing or treating ischemia-reperfusion injury comprising antitrypsin as an active ingredient.

During surgical procedures such as organ transplant surgery or surgery to treat cardiovascular disease, blood supply to certain tissues may be restricted. In this case, blood circulation to organs in which continuous blood supply, such as the liver, kidney, heart, and brain, is important As it is blocked, damage due to ischemia occurs. In addition, if the blood flow is suddenly increased by reperfusion in an ischemia state where oxygen is not properly supplied, cells and tissues may be greatly damaged due to various complex causes. In particular, liver or kidney ischemia-reperfusion injury (IRI) is a major complication frequently occurring after liver or kidney transplantation or heart disease surgery. Reperfusion, which is performed after blood supply to the liver or kidney is temporarily interrupted by the blockage of blood flow during surgery, may induce severe acute inflammation response and acute tissue damage. In particular, acute inflammatory reaction or cell apotosis due to breakfast damage is recognized as a very serious risk factor because it is one of the main causes of liver failure or kidney failure. Ischemic reperfusion injury is also caused by a sudden increase in blood flow in the oxygen-deficient state and an increase in the intracellular calcium concentration. Increased intracellular calcium can mediate mitochondrial damage. At this time, mitochondrial damage causes free radicals to react with free substances and ATP, and as the body recognizes it as inflammation, more free radicals attack as white blood cells attack. Can occur and eventually lead to cell damage. Ischemic reperfusion injury is more severe as the rate of blood re-flowing increases, and ischemic reperfusion frequently occurs when blood flow resumes after heart surgery or organ transplant surgery. As such, if ischemic reperfusion injury is predicted, research results have been reported that the damage caused by free radicals is considered as one of the main factors, and it can be prevented by pre-administering a strong antioxidant as a treatment for this. However, powerful antioxidants have also been used limitedly due to limitations in effectiveness and clinical studies have been conducted on some drugs, but drugs that directly prevent or treat ischemic reperfusion injury have not been developed to date. Surgical procedures that can cause ischemic reperfusion tissue damage such as organ transplantation are rapidly increasing worldwide, but after restoring ischemic-reperfusion injury, which is a problem during liver or kidney transplantation, it suppresses inflammatory reactions and cell death and restores organ function. There is an urgent need for medical and social development of new techniques.

On the other hand, ischemic reperfusion injury that occurs in the course of kidney transplant surgery is associated with gradual loss of function and dysfunction of the transplanted kidney later. In addition, it is clinically revealed that the initial ischemic reperfusion injury, acute rejection, and long-term prognosis during transplantation are correlated. Although the exact mechanism causing ischemia reperfusion injury has not been identified, it is known that the formation of oxygen free radicals by ischemia reperfusion is one of the important factors. In addition, ischemia / reperfusion injury during renal transplantation has been reported to cause functional delay during transplantation and is also associated with acute rejection, and acute renal failure due to ischemic injury is inflammatory in addition to factors such as vasoconstriction and tubule occlusion. It is known that the mechanisms of inflammatory and cytotoxic damage accompanied by increased cytokines and neutrophil infiltration into the parenchyma are important.

In this, renal ischemic reperfusion injury (IRI) in renal transplantation surgery is responsible for increasing fatal patient complications and mortality after surgery. In order to prevent this, methods such as ischemic preconditioning, adenosine pretreatment, inhalation anesthetics, and AMPK (AMP-activated protein kinase) activation have been studied. It has been known that preconditioning protects against renal ischemia reperfusion injury through intermediate mediators such as intracellular signaling proteins Akt, ERK, HSP27 (heat shock protein 27), HSP70, and inducible nitric oxide synthase (iNOS). . In addition, studies have been conducted to develop a drug that can treat renal ischemia reperfusion injury in the kidney transplantation procedure, but there are various side effects, and it is difficult to solve problems such as deterioration of kidney function caused by transplantation. There was.

Accordingly, the present inventors have made great efforts to develop a safe new material capable of alleviating tissue damage caused by ischemia-reperfusion, and as a result, alpha-1 antitrypsin (hereinafter referred to as “AAT”) ischemia -It has been discovered that the tissue protective effect can be exhibited by suppressing the expression of inflammatory factors, tissue fibrosis, collagen deposition and apoptosis in the environment of tissue damage due to reperfusion, and the present invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

In order to achieve the object of the present invention described above, the present invention provides a pharmaceutical composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

In the present invention, the alpha-1 antitrypsin (hereinafter referred to as “AAT”) is synthesized in hepatocytes and secreted into the blood. Trypsin, chymotrypsin, and elastase present in plasma, It belongs to the serpin family along with inhibitors for most serine-based proteases such as collagenase, thrombin and plasmin. In addition, AAT is a glycoprotein having a molecular weight of 52 kD (kilodalton), and its physiological function acts as an inhibitor to the elitase of neutral white blood cells. In particular, the elastic fibers present in the alveoli are caused by the elitase of neutral white blood cells. It is known to prevent decomposition.

In the present invention, the AAT is preferably a human-derived AAT, and more preferably, may include the amino acid sequence of SEQ ID NO: 1, most preferably a protein consisting of the amino acid sequence of SEQ ID NO: 1. .

(SEQ ID NO: 1)

mpssvswgil llaglcclvp vslaedpqgd aaqktdtshh dqdhptfnki tpnlaefafs

lyrqlahqsn stniffspvs iatafamlsl gtkadthdei leglnfnlte ipeaqihegf

qellrtlnqp dsqlqlttgn glflseglkl vdkfledvkk lyhseaftvn fgdteeakkq

indyvekgtq gkivdlvkel drdtvfalvn yiffkgkwer pfevkdteee dfhvdqvttv

kvpmmkrlgm fniqhckkls swvllmkylg nataifflpd egklqhlene lthdiitkfl

enedrrsasl hlpklsitgt ydlksvlgql gitkvfsnga dlsgvteeap lklskavhka

vltidekgte aagamfleai pmsippevkf nkpfvflmie qntksplfmg kvvnptqk

The AAT of the present invention includes functional equivalents that exhibit equivalent physiological activity, and includes all kinds of peptides, polypeptides, proteins, peptide replicas, compounds, and biologics. In the above, 'equal physiological activity' refers to inhibiting the death of peritoneal mesenchymal cells. The functional equivalent may be a polypeptide having a sequence homology with at least 70%, preferably 80%, more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 1.

The functional equivalent may be generated as a result of addition, substitution, or deletion of a part of the amino acid sequence of SEQ ID NO: 1. The substitution of amino acids in the above is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). In addition, the functional equivalent includes a variant in which a part of the amino acid is deleted on the amino acid sequence of the AAT protein of the present invention. The deletion or substitution of the amino acid is preferably located in a region not directly related to the physiological activity of the AAT of the invention. In addition, the deletion of amino acids is preferably located in a portion that is not directly involved in the physiological activity of AAT. In addition, a variant in which several amino acids are added to both ends or sequences of the amino acid sequence of the AAT is also included. In addition, the scope of the functional equivalent of the present invention includes derivatives in which some of the chemical structures of AAT are modified while maintaining the basic skeleton of AAT according to the present invention and its physiological activity. For example, structural changes to change the stability, storage, volatile or solubility of the protein of the present invention are included therein.

In addition, the AAT of the present invention is a protein having a native amino acid sequence, as well as amino acid sequence variants thereof are also included within the scope of the present invention. Variant of the protein of the present invention means a protein having different sequences by one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1 by deletion, insertion, non-conservative or conservative substitution, substitution of amino acid analogs, or a combination thereof. Amino acid exchanges that do not alter the activity of the molecule as a whole are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).

In some cases, the protein of the present invention may be modified with phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.

The AAT of the present invention may be derived from nature or may be synthesized using known peptide synthesis methods. The peptides of the present invention can be prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (ChemicalApproaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).

In addition, the AAT of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the protein is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (sold by Biosearch or Applied Biosystems). The produced DNA sequence is a vector that includes one or more expression control sequences (eg, promoters, enhancers, etc.) that are operatively linked to the DNA sequence and regulate the expression of the DNA sequence. , And transformed the host cell with the recombinant expression vector formed therefrom. The resulting transformant is cultured under medium and conditions suitable for allowing the DNA sequence to be expressed, thereby recovering substantially pure protein encoded by the DNA sequence from the culture. The recovery can be performed using methods known in the art (eg, chromatography). In the above, the term "substantially pure protein" means that the protein according to the present invention does not substantially contain any other protein derived from the host. The genetic engineering method for protein synthesis of the present invention can refer to the following documents: (Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.)).

Proteins produced by the genetic engineering method or chemically synthesized proteins include, for example, extraction methods, recrystallization methods, various chromatographs (gel filtration, ion exchange, precipitation, adsorption, reverse phase), electrophoresis, countercurrent distribution, etc. It can be isolated and purified by methods known in the art.

The AAT of the composition according to the invention can be used on its own or in the form of a salt, preferably a pharmaceutically acceptable salt. 'Pharmaceutically acceptable' in the present invention refers to a physiologically acceptable, and when administered to a human, usually does not cause an allergic reaction or a similar reaction, and the salt is a pharmaceutically acceptable free acid. The acid addition salt formed by) is preferred. Organic acids and inorganic acids can be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

In the present invention, the type of the organ in which the ischemia-reperfusion injury appears is not particularly limited, but may be, for example, the liver, heart, kidney, lung, brain, and most preferably kidney.

According to one embodiment of the present invention, the AAT inhibited the expression of markers of renal insufficiency in a renal ischemia-reperfusion injury animal model, and suppressed the expression of inflammatory factors, tissue fibrosis, collagen deposition and apoptosis. It was confirmed that the effect is excellent in alleviating kidney damage due to ischemia-reperfusion.

The pharmaceutical composition of the present invention may be variously formulated according to the route of administration by a method known in the art with a pharmaceutically acceptable carrier for the therapeutic effect of organ damage due to ischemia-reperfusion. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

The route of administration may be administered orally or parenterally. The parenteral administration method is not limited to this, but is not limited to intraperitoneal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. It can be my administration.

When the pharmaceutical composition of the present invention is administered orally, the pharmaceutical composition of the present invention is a powder, granules, tablets, pills, dragees, capsules, liquids, gels according to methods known in the art together with a suitable carrier for oral administration. , Syrups, suspensions, wafers, and the like. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like may be included, including methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropyl methyl cellulose. In addition, if necessary, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In addition, when administered parenterally, the pharmaceutical composition of the present invention may be formulated according to methods known in the art in the form of intraperitoneal, injectable, transdermal and nasal inhalants together with a suitable parenteral carrier. The injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils. Can be. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanol amine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be further included. In addition, the injection may additionally contain isotonic agents such as sugars or sodium chloride in most cases.

In the case of transdermal dosage forms, ointments, creams, lotions, gels, external solutions, pasta agents, linen agents, aerosols, and the like are included. In the above, "dermal administration" means that the pharmaceutical composition is topically administered to the skin to deliver an effective amount of the active ingredient contained in the pharmaceutical composition into the skin. For example, the pharmaceutical composition of the present invention can be administered by a method of preparing a injection-type formulation and lightly pricking the skin with a 30-gauge thin injection needle or directly applying it to the skin. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, a recipe generally known in pharmaceutical chemistry.

In the case of inhalation dosages, the compounds used according to the invention may be used in the form of pressurized packs, using suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated to contain a powder mixture of a compound and a suitable powder base such as lactose or starch.

As other pharmaceutically acceptable carriers, reference may be made to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In addition, pharmaceutical compositions according to the present invention may include one or more buffers (e.g. saline or PBS), carbohydrates (e.g. glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (Eg, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and / or preservatives.

In addition, the pharmaceutical compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In addition, the pharmaceutical composition of the present invention may be administered in combination with a known compound having an effect of preventing or treating against long-term damage due to ischemia-reperfusion.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 1,000 mg per kg of patient body weight per day, and most preferably 0.1 μg to 500 mg per kg of body weight per patient. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, the route of administration and the number of treatments, as well as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, etc. Considering this, one of ordinary skill in the art will be able to determine the appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effect of the present invention.

The present invention also provides a food composition for preventing or treating ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

The food composition of the present invention includes all forms of functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the nogeun extract of the present invention may be prepared in the form of tea, juice, and drink for drinking or granulated, encapsulated, and powdered. In addition, the AAT of the present invention can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have a prophylactic or therapeutic effect on ischemia-reperfusion tissue damage. For example, the food composition of the present invention may further contain trace minerals, vitamins, lipids, sugars, and known ingredients in addition to the AAT component. The minerals may contain nutrients necessary for growth, such as calcium and iron, and vitamins may include vitamin C, vitamin E, vitamin B1, vitamin B2, and vitamin B6. Lipids may include alkoxyglycerol or lecithin, and sugars may include fructooligosaccharides.

In addition, functional foods include beverages (including alcoholic beverages), fruits, and processed foods (such as canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (such as ham and sausage corn beef) Etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein , Retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the AAT of the present invention.

In addition, in order to use the AAT of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate.

The preferred content of AAT in the food composition of the present invention may be contained in a ratio of 0.01 to 90%, preferably 0.1 to 50%, based on the total weight of the food relative to the total weight of the food composition.

The composition of the present invention comprising alpha-1 antitrypsin as an active ingredient has an effect of inhibiting the expression of inflammatory factors in the long-term damage environment due to ischemia-reperfusion, and suppressing fibrosis, collagen accumulation and apoptosis of tissues. It is very excellent and can be very useful in the prevention of long-term damage caused by ischemia-reperfusion or in the development of therapeutic agents.

1 is a view showing an experimental animal group and an experimental schedule in an embodiment of the present invention (NC: untreated control group, Sham: sham group, IR: ischemia-reperfusion experiment group, IR-AAT: ischemia-reperfusion experiment group after administration of AAT)
Figure 2a is the result of analyzing the concentration of BUN (Blood Urea Nitrogen) in the serum of each experimental animal, Figure 2b is the concentration of Cr (Creatinine).
FIG. 3A shows the expression level of NGAL mRNA in the kidneys of each experimental animal, FIG. 3B shows the expression level of KIM-1, and FIG. 3C shows the expression level of L-FABP mRNA through RT-qPCR. The result is quantified by ratio.
4 is a result of observing histological changes by staining kidney sections of each experimental animal with PAS and observing it with an inverted microscope.
Figure 5a is a result of observing the accumulation of collagen in the kidney observed by an inverted microscope after staining the kidney sections of each experimental animal with Trichrome, Figure 5b is a result of quantifying it and graphed.
Figure 6 shows the results observed by an inverted microscope after staining F4 / 80 in kidney sections of each experimental animal.
FIG. 7A is a result obtained by staining TNF-alpha in kidney sections of each experimental animal and observed with an inverted microscope, and FIG. 7B is a graph showing quantitation.
8 is a result of analyzing the expression level of IL-6 protein in the serum of each experimental animal through ELISA.
Figure 9a shows the mRNA expression level of TNF-alpha in the kidney tissue of each animal, Figure 9b shows the mRNA expression level of TGF-beta, Figure 9c shows the mRNA expression level of IL-1beta, Figure 9d shows the mRNA of IL-6 It is a result of analyzing the expression level by RT-qPCR and graphing it.
10 is a result of performing a TUNEL assay to observe apoptosis in the kidney tissue of each animal and observing it under a confocal microscope.
11 shows the results of immunohistochemical staining of the TUNEL assay.

12A is a result of measuring the activity of Caspase-3 in the kidney tissue of each animal using a colorimetric assay kit, and FIG. 12B shows the expression levels of Bcl-2 and Bax proteins in the kidney tissue of each animal by Western blot, , This is quantified and shown in FIGS. 12C and 12D.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited by them.

Experiment method

1. Experimental Animal and Kidney IRI Animal Model

All experiments used 8-week old male C57BL / 6 mice (Samtako, Osan, Korea) weighing 22-25 g. Animals were bred to ingest standard feed and water freely and maintained a 12 hour contrast cycle. Mice were performed according to guidelines approved by the Kyungpook National University Animal Care and Use Committee (KNU-2017-0013). During surgery, animals were anesthetized with tribromoethanol (240 mg / kg, 1.2%). To induce ischemia, the kidney was exposed through a minimal lateral incision of the pedicle and completely closed for 30 minutes using a micro aneurysm clamp. After induction of adequate ischemia for 30 minutes, arterial clamps were removed to allow reperfusion. The sham surgical animal group was subjected to a similar surgical treatment as the experimental group except for clamping the kidney pedicle. During surgery, animals were maintained at 36.5-37 ° C. using a temperature controlled heating device (Harvard Bioscience, Holliston, MA, USA).

2. Experiment group

The mice were randomly divided into four groups (Figure 1). Group 1 was the normal control group ( NC group , n = 5). The second group was the Sham group ( Sham group , n = 6). Group 3 was a group in which renal ischemia was induced, and 0.9% physiological saline was administered for 3 days ( IR group , n = 8). Group 4 was a group in which renal ischemia was induced, and was administered intraperitoneally with 80 mg / kg / day AAT (Aralast®; Baxter Healthcare, Vienna, Austria) for 3 days ( IR-AAT group , n = 8). All mice were sacrificed by heart puncture under anesthesia 24 hours after reperfusion. Blood and kidneys were obtained from animals for analysis.

3. Measurement of kidney function markers

Plasma creatinine (Cr) and blood urea nitrogen (BUN) levels were measured with a Hitachi 7600 automatic biochemical analyzer (Hitachi, Tokyo, Japan). Markers of early kidney damage, NGAL, KIM-1 and L-FABP, were evaluated in homogenized whole kidney tissue using RT-qPCR.

4. Measurement of plasma IL-6

After centrifugation, serum was collected and IL-6 levels were measured using a commercially available ELISA kit (R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Values were calculated using standard curves.

5. Quantitative polymerase chain reaction (qPCR)

Total RNA was extracted from whole kidney tissue homogenized according to manufacturer's instructions using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). 1 μg of total RNA was reverse transcribed into cDNA using PrimeScript cDNA synthesis kit (TaKaRa, Otsu, Japan). qPCR was performed in an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA) using the SYBR Green PCR master mix (Life Technologies). The results were analyzed using a comparative Ct method for the relative quantification of gene expression. All primers used for qPCR were designed using Primer Express V1.5 software (Applied Biosystems) and the sequence was as follows: Neutrophil gelatinase-associated lipocalin (NGAL) (forward 5'- GCC ACT CCA TCT TTC CTG TTG- 3 'and reverse 5'-GGG AGT GCT GGC CAA ATA AG-3'); KIM-1 (forward 5'- CAG GGA AGC CGC AGA AAA A-3 'and reverse 5'-GAT AGC CAC GGT GCT CAC AA-3'); IL-1β (front 5'-AGG CAA TAG GTC TGC CCG AGG AC-3 'and reverse 5'-CCA GTT CGC ACT CCT CCC CCA-3'), IL-1β (front 5'-TCG TGC TGT CGG ACC CAT AT-3 'and reverse 5'-GGT TCTC CTT GTA CAA AGC TCA TG-3'); IL-6 (forward 5'-CCC ACC AAG AAC GAT AGT CAA TT-3 'and reverse 5'-CAC CAG CAT CAG TCC CAA GA-3'); transforming growth factor (TGF) -β (front 5'- GGC TGT GGC CAT CAA GAA TT-3 'and reverse 5'-GCA GAG GGA AGA GTC AAA CAT GT-3'); TNF-α (front 5'- GAC TAG CCA GGA GGG AGA ACA G-3 'and reverse 5'-CAG TGA GTG AAA GGG ACA GAA CCT-3'); And β-actin (front 5'-ACC ACC ATG TAC CCA GGC ATT-3 'and reverse 5'-CCA CAC AGA GTA CTT GCG CTC A-3').

6. Histological analysis and immunohistochemistry research

Kidney tissue was fixed with 4% paraformaldehyde (pH 7.4) and embedded in paraffin. Paraffin fixed tissue was sliced to 2 μm and stained with periodic acid-Schiff (PAS) and Masson's trichrome using standard protocols to determine histological changes and collagen deposition. For immunohistochemical staining, 2 μm kidney sections were deparaffinized and rehydrated, and then endogenous peroxidase was inactivated using 3% hydrogen peroxide. After incubating the samples at 4 ° C. overnight with anti-TNF-α (1: 100, Abcam, Cambridge, UK) and anti-F4 / 80 (1: 200, Serotec, Oxford, UK), EnVision-HRP kit ( Dako, Carpinteria, CA, USA). All sections were counter stained with Mayer's hematoxylin and examined under a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ, USA). Quantification of TNF-α expression was measured as a percentage of total area using Image J software (National Institutes of Health, Bethesda, MD, USA). For quantification of F4 / 80-positive cells, cells in 5 non-overlapping regions at × 400 magnification per slice were counted and normalized per mm ^{2 of} tissue.

7. TUNEL assay

In situ Cell Death Detection Fluorescein Kit (Roche, Mannheim, Germany) using a fluorescence microscope for detection of apoptotic cells and Click-iTTM TUNEL colorimetric IHC detection kit for immunohistochemical staining (Life Technologies, Carlsbad) , CA, USA). Briefly, 2 μm kidney sections were de-paraffinized and rehydrated before treatment with proteinase K. For fluorescent staining of TUNEL assay, incubated with TUNEL reagent at room temperature for 30 minutes and washed three times with phosphate buffered saline. After 5 minutes of treatment, the cells were counterstained with 4 ', 6-diamidino-2-phenyl indole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) for 1 minute to detect nuclei. Finally, sections were mounted with Prolong Gold anti-fading reagent (Invitrogen, Carlsbad, CA, USA) and observed under confocal microscopy (Carl Zeiss, Gottingen, Germany). For immunohistochemical staining of TUNEL assay, TdT (terminal deoxynucleotidyl transferase) enzyme and nucleotide at 37 ° C were mixed for 60 minutes and cultured. Sections were incubated with streptavidin-peroxidase conjugate solution for 30 minutes at room temperature, developed with a 3,3'-diaminobenzidine (DAB) reaction mixture to produce a brown color, and then counterstained with Mayer's hematoxylin.

8. Measurement of caspase-3 activity

Caspase-3 activity was measured by homogenized whole kidney tissue using a colorimetric assay kit (Sigma-Aldrich) according to the manufacturer's protocol. Briefly, the supernatant of kidney homogenate was incubated with fluorescence measuring caspase-3 substrate and Ac-DEVD-pNA in assay buffer for 90 min at 37 ° C. Caspase-3 activity was measured at 405 nm using an ELISA reader. Values are expressed as percentage control.

9. Western Blot

The homogenized kidney tissue was electrophoresed on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, blocked with 10% skim milk for 1 hour, and then Bcl-2 (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), Bax (1: 1000, Cell Signaling Technology) and β-actin (1: 5000, Sigma-Old Rich) were incubated overnight at 4 ° C with primary antibodies. After washing, the membrane was incubated with Horseradish peroxidase-conjugated IgG secondary antibody (1: 2000, Dako, Glostup, Denmark) for 1 hour and detected using ECL Advanced Detection (GE Healthcare, Little Chalfont, UK). Bands were analyzed using Scion Image software (Scion, Frederick, MD, USA) and β-actin was used as the loading control.

10. Statistical Analysis

All data are presented as the standard error of the mean. The experiment was repeated independently at least three times. All statistical analyzes were performed by one-way ANOVA using Turkey's post hoc test using GraphPad prism 5.01 software (GraphPad Software, La Jolla, CA, USA). P <0.05 was considered statistically significant.

Experiment result

1.AAT relieves kidney failure

BUN and Cr are markers of renal function, and a significant increase in BUN and Cr was observed in the IR group compared to the control group (NC, sham), and the IR-AAT group showed a marked decrease in BUN and Cr compared to the IR group (FIG. 2a, 2b). The concentrations of NGAL and KIM-1 in plasma, which are early kidney damage markers, were increased in the IR group and decreased compared to those in the IR-AAT group. L-FABP also showed an increase in the IR group and a decrease in the IR-AAT group, but no statistical difference (Fig. 3a, 3b, 3c).

2. AAT defends against histological kidney damage caused by IRI

PAS and Masson's trichrome staining reflects histological changes and kidney collagen deposition. In the control group (NC, Sham), tubular injury and interstitial fibrosis were hardly observed in PAS and Masson's trichrome staining. However, in the IR group, collagen deposition was significantly increased in tubule damage and renal epilepsy, such as loss of cast formation and brush border, compared to the control group. On the other hand, this histological change was improved in the IR-AAT group (Fig. 4, Fig. 5A, Fig. 5A).

3. AAT relieves increased expression of inflammatory markers according to renal IRI

TNF-α is a cytokine associated with systemic inflammation and F4 / 80 is one of the macrophage markers required for induction of efferent CD8 + regulatory T cells needed for peripheral tolerance. TNF-α and F4 / 80 expression increased in the IR group compared to the control group. And in the IR-AAT group, TNF-α and F4 / 80 expression decreased compared to the IR group (FIG. 6, FIG. 7A, 7B). Serum IL-6 levels showed a significant increase in the IR group compared to the control group, while the IR-AAT group showed a marked decrease compared to the IR group. (Fig. 8)

QRT-PCR was used to measure the expression of mRNA levels of the inflammatory cytokines TNF-α, IL-1β, IL-6 and TGF-ß. The qRT-PCR levels of TNF-α, IL-1β, IL-6 and TGF-ß showed a significant increase in the IR group compared to the control group. Except for the mRNA expression level of TNF-α, this increase was attenuated compared to the IR group in the IR-AAT group (FIGS. 9A-9D).

4. In renal IRI, AAT inhibits apoptosis

The TUNEL assay is a method of detecting apoptotic DNA fragmentation. In the IR group, many TUNEL-positive renal tubular epithelial cells were observed and decreased in the IR-AAT group. It was hardly seen in the control group (Fig. 10, Fig. 11).

Caspase-3 is a protein that causes apoptosis. Caspase-3 activity was increased in the IR group compared to the control group, and this increase was lost in the IR-AAT group.

Bcl-2 and bax also reflect apoptosis. In the IR group, bcl-2 expression decreased and bax expression increased compared to the control group. On the other hand, in the IR-AAT group, bcl-2 expression increased and bax expression decreased compared to the IR group (FIGS. 12A-D).

The composition of the present invention comprising alpha-1 antitrypsin as an active ingredient has an effect of inhibiting the expression of inflammatory factors in the long-term damage environment due to ischemia-reperfusion, and suppressing fibrosis, collagen accumulation and apoptosis of tissues. It is very excellent and can be very useful in preventing long-term damage caused by ischemia-reperfusion or developing a therapeutic agent, and thus has excellent industrial applicability.

<110> Kyungpook National University Industry-Academic Cooperation Foundation          KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL <120> Composition for preventing or treating ischemia-reperfusion          injury comprising alpha-1 antitrypsin <130> NP18-0068 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> human alpha-1 antitrypsin <400> 1 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys   1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala              20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn          35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln      50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser  65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr                  85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro             100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn         115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu     130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu                 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys             180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu         195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val     210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys                 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala             260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu         275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp     290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe                 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys             340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly         355 360 365 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile     370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr                 405 410 415 Gln Lys        

Claims (5)

A pharmaceutical composition for preventing or treating ischemia-reperfusion injury, which includes alpha-1 antitrypsin as an active ingredient.
The pharmaceutical composition of claim 1, wherein the ischemia-reperfusion injury is kidney damage.
The pharmaceutical composition of claim 1, wherein the alpha-1 antitrypsin comprises the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition of claim 1, wherein the alpha-1 antitrypsin inhibits expression of inflammatory markers and apoptosis.
Food composition for preventing or improving ischemia-reperfusion injury comprising alpha-1 antitrypsin as an active ingredient.

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