KR101472574B1 - A pharmaceutical comprising apamin for treating or preventing acute pancreatitis - Google Patents

Abstract

The present invention relates to a composition for inhibiting the onset of acute pancreatitis in pancreatic tissues and lung tissues by containing afamin as an active ingredient.
The composition according to the present invention reduces inflammation and edema in pancreatic tissue, efficacy to inhibit MPO infiltration in pancreatic tissue, ratio of pancreas weight / body weight (PW / BW), amount of amylase and lipase in serum in pancreatic tissue , The efficacy of proinflammatory cytokine and proinflammatory cytokine mRNA expression in pancreatic tissue, and the activity of MAPKs pathway in pancreatic tissue.
In addition, the composition according to the present invention has an effect of inhibiting inflammation and edema in lung tissue and MPO infiltration in lung tissue.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for preventing, ameliorating or treating acute pancreatitis,

The present invention relates to a composition for inhibiting the onset of acute pancreatitis in pancreatic tissues and lung tissues by containing afamin as an active ingredient.

The composition according to the present invention reduces inflammation and edema in pancreatic tissue, efficacy to inhibit MPO infiltration in pancreatic tissue, ratio of pancreas weight / body weight (PW / BW), amount of amylase and lipase in serum in pancreatic tissue , The efficacy of proinflammatory cytokine and proinflammatory cytokine mRNA expression in pancreatic tissue, and the activity of MAPKs pathway in pancreatic tissue.

In addition, the composition according to the present invention has an effect of inhibiting inflammation and edema in lung tissue and MPO infiltration in lung tissue.

The concentration of the composition according to the present invention is characterized by being 10 to 100 μg / kg once administered intraperitoneally on a mouse basis.

Acute pancreatitis is an inflammatory disease accompanied by swelling or swelling of the pancreas through the process of self-digestion caused by premature activation of the proteolytic enzyme precursor in the pancreas. In many cases, acute pancreatitis is caused by bile duct disease, excessive consumption of alcohol Or hyperlipemia. ≪ / RTI >

In summary, a triggering event converts the digestive proenzymes in the pancreas into active states, resulting in membrane breakdown, edema, intestinal hemorrhage, (intestinal hemorrhage) and necrosis (necrosis).

It also involves an inflammatory process, which causes permeable leukocytes to eventually lead to local pancreatic damage as well as systemic signs. In the inflammatory process, inflammatory cytokines such as TNF-alpha (Tumor necrosis factor-alpha), IL-1 beta (interleukin-1 beta, interleukin-1 beta) and IL- Inflammatory cytokines play a major role. Proinflammatory cytokines are mediators of the inflammatory response, leading to necrosis of linear cells and deepening of local pancreatic injury.

When acute pancreatitis develops, about 25% of the patients reach a critical state, of which 30-50% lead to death. However, since pathologic - physiologic findings of acute pancreatitis have not been elucidated, effective treatment methods are in short supply. Therefore, it is urgent to elucidate the mechanism of acute pancreatitis and to develop therapies and therapeutic substances accordingly.

On the other hand, the present inventors have found out the effect of the bee venom on the prevention of acute pancreatitis of bee cattle ovulation tube, and the effect of bee venom on the bee venom is intensified. As a result, Apamin, a major component of bee venom, has been shown to improve symptoms such as inflammation, edema, tissue damage, and auto-digestion due to digestive enzymes in pancreatic tissue, the main symptom of acute pancreatitis. Accordingly, the present inventors have found a composition for the prophylaxis and treatment of acute pancreatitis containing afamin as an active ingredient.

Hereinafter, the active ingredient of the present invention, afamin, will be briefly described.

In 1964, E. Habermann et al. Named the most toxic neurotoxin peptide from bee venom, known as 'acamin', among the known animal neurotoxins. Apamin contains about 2% of the bee venom content and is composed of 18 amino acids as a kind of polypeptide. The relative molecular mass is 2,035, and it has the effect of relieving neuralgia, pain, immunity and so on.

In the prior art related to the present invention, Korean Patent Laid-Open Publication No. 1994-0005664 discloses a method for producing an acaric acid receptor from a cell material containing a derivatized agarose matrix having a free amino group covalently coupled to a free carboxyl group of an apamin, ≪ / RTI > is disclosed in relation to compositions of matter useful as affinity matrices for the separation of < RTI ID = 0.0 >

This prior art used apamin as a material, but it is not related to the prevention, improvement or treatment of acute pancreatitis.

It is an object of the present invention to provide a composition for inhibiting the onset of acute pancreatitis in pancreatic tissues and lung tissues by containing afamin as an active ingredient.

It is an object of the present invention to provide a method of reducing inflammation and edema in pancreatic tissue, an effect of inhibiting MPO infiltration in pancreatic tissue, a ratio of pancreatic weight / weight ratio (PW / BW), a content of amylase and lipase in serum in pancreatic tissue , The expression of proinflammatory cytokines and proinflammatory cytokine mRNA in pancreatic tissues, and the activity of MAPKs pathway in pancreatic tissues.

It is an object of the present invention to provide a composition for inhibiting inflammation and edema in lung tissue and MPO infiltration in lung tissue.

It is an object of the present invention to provide a composition for preventing, ameliorating or treating acute pancreatitis containing an effective ingredient of afamin in a dose of 10 to 100 μg / kg once administered intraperitoneally on a mouse basis.

In order to accomplish the above object, the present invention aims to solve the technical problem by providing a composition containing afamin as an active ingredient and inhibiting the onset of acute pancreatitis in pancreatic tissue and lung tissue.

The present invention relates to a method for reducing inflammation and swelling in pancreatic tissue, an effect of inhibiting MPO infiltration in pancreatic tissue, a ratio of pancreatic weight / body weight (PW / BW), an effect of reducing the content of amylase and lipase in serum in pancreatic tissue , The expression of proinflammatory cytokines and proinflammatory cytokine mRNA in pancreatic tissues, and the activity of MAPKs pathway in pancreatic tissues.

The present invention aims at solving the technical problem by providing a composition which inhibits inflammation and edema in lung tissue and MPO infiltration in lung tissue.

The present invention aims at solving the technical problem by providing a composition for preventing, ameliorating or treating acute pancreatitis containing an effective ingredient of afamin, which is administered at a dose of 10 to 100 μg / kg once per day in the abdominal cavity on a mouse basis .

The composition for preventing, ameliorating or treating acute pancreatitis according to the present invention has an effect of inhibiting the onset of acute pancreatitis in pancreatic tissues and lung tissues by containing afamin as an active ingredient.

The composition for preventing, ameliorating or treating acute pancreatitis according to the present invention can be used for the treatment of inflammation and swelling in pancreatic tissue, efficacy of inhibiting MPO infiltration in pancreatic tissue, ratio of pancreas weight / weight (PW / BW) The effect of decreasing the content of amylase and lipase in serum, the expression of proinflammatory cytokine and proinflammatory cytokine mRNA in pancreatic tissue, and the activity of inhibiting the activity of MAPKs channel in pancreatic tissues.

The composition for preventing, ameliorating or treating acute pancreatitis according to the present invention has an effect of inhibiting inflammation and swelling in lung tissue, MPO infiltration in lung tissue.

The composition for preventing, ameliorating or treating acute pancreatitis according to the present invention has an efficacy of 10 to 100 μg / kg when administered once per day in the peritoneal cavity on a mouse basis.

The composition for preventing, ameliorating or treating acute pancreatitis according to the present invention has an effect of not only inhibiting the progress of acute pancreatitis but also improving the damaged tissue.

FIG. 1 is a photograph showing the results of comparing the pancreatic tissues of mice treated with cerulane and acute pancreatitis, in which apamin was pretreated, apamin was post-treated, and serine-treated normal groups. The results of staining with siline and eosin were magnified 400 fold.
FIG. 2 is a graph showing swelling and inflammation changes in the group treated with acerin in pancreatic tissues of mice treated with serraine to cause acute pancreatitis.
FIG. 3 is a graph showing swelling and inflammation of the pancreatic tissues of acute pancreatitis-treated mice treated with serurein, followed by treatment with apamin.
FIG. 4 is a photograph showing the results of comparing the lung tissues of mice treated with cerulane to acute pancreatitis by pretreatment with apamin, apamin-treated group, and serine-treated normal group, The results of staining with siline and eosin were magnified 400 fold.
FIG. 5 is a graph showing swelling and inflammation changes in the lung tissue pretreated with acaricin in acute pancreatitis caused by treatment with serulin.
FIG. 6 is a graph showing swelling and inflammation changes in the group treated with aceramin in the lung tissue of mice treated with serraine to cause acute pancreatitis.
7 is a graph showing the results of MPO analysis in pancreatic tissues of mice treated with serraine to cause acute pancreatitis.
8 is a graph showing the results of MPO analysis in lung tissues of mice treated with serraine to cause acute pancreatitis.
9 is a graph showing the results of measurement of pancreatic weight / body weight ratio of mice treated with serraine and induced acute pancreatitis.
10 is a graph showing the results of measuring the content of serum amylase in the pancreas of mice treated with serraine and caused acute pancreatitis.
11 is a graph showing the results of measurement of serum lipase content in pancreas of mice treated with serraine and caused acute pancreatitis.
FIG. 12 is a graph showing the amounts of TNF-.alpha., IL-1.beta. And IL-6 by ELISA after treating pancreas of apamin-pretreated mouse with serraine and inducing acute pancreatitis.
13 is a graph showing the amounts of TNF-a, IL-l [beta] and IL-6 by ELISA after post-treatment of apramine in the pancreas of mice treated with serrene and acute pancreatitis.
FIG. 14 is a graph showing mRNA expression patterns of TNF-.alpha., IL-1 beta and IL-6 after treating acute pancreatitis with serraine in the pancreas of apomethic pretreated mouse.
FIG. 15 is a graph showing mRNA expression patterns of TNF-.alpha., IL-1 beta, and IL-6 after treatment of acute pancreatitis with acute pancreatitis in the pancreas of mice and post-treatment of the pancreas with apamin.
16 is a photograph showing the result of Western blotting performed on the pancreas of mice treated with serraine and induced acute pancreatitis.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments described in the present specification, the reference examples, and the drawings are merely the most preferred examples of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It should be understood that various equivalents and modifications may be present.

Example  One. Apammin  Composition for prevention and treatment of acute pancreatitis containing active ingredient

The composition of the present invention is for the prevention and treatment of acute pancreatitis, and it is intended to contain apamin as an active ingredient.

The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxy, hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as calcium carbonate, sucrose, ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsion-freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, in order to achieve the desired effect, the amount of apamin should be 10 to 100 μg / kg for intraperitoneal injection. In the case of oral administration, the dosage should be adjusted to be at least 10 times the amount injected intraperitoneally. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

In addition, the composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for preventing and ameliorating acute pancreatitis containing an effective ingredient.

The composition of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of acute pancreatitis. Examples of foods to which the composition of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in the form of powders, granules, tablets, have.

The composition of the present invention may be added to food or beverage for the purpose of prevention and improvement of acute pancreatitis. Here, the amount of the composition in the food or beverage is generally from 1 to 5% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition comprises 0.02 to 10 g, 1 g.

The health beverage composition of the present invention does not have any particular limitation on the liquid ingredient other than the ingredients containing the indicated amount of the essential ingredient such as apamin and may contain various flavoring agents or natural carbohydrates as additional ingredients such as ordinary beverages.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention can be applied to various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate etc.), pectic acid and its salts, , Protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Experimental Example  One. Apamin  Inflammation and edema inhibitory effect of pancreatic tissue during treatment

1-1. Experimental preparation and preprocessing

1) Experimental animals

All experiments were performed in compliance with the regulations approved by the Wonkwang University Animal Experiment Committee.

C57BL / 6 Female mice (6- to 8-week-old) were purchased from Orient Bio Co., Ltd. (Sungnam, Kyunggi-do, Korea) at 23 ± 2 ℃ (mean ± SD) The animals were housed for 7 days in an environmentally controlled breeding box of 12 hours (6:00 am to 6:00 pm). During this adaptation period, they were allowed to freely use tap water for animal feed and water purification system.

These animals were free of general symptoms during takeover and acclimatization period and fasted for 18 hours before inducing acute pancreatitis.

2) Experimental reagent

Avidin-peroxidase, 3'3'5'5'-TMB (tetramethylbenzidine), Cerulein, Tris-HCl, NaCl, Triton X-100 , And hexadecyltrimethyl ammonium bromide were purchased from Sigma (St. Louis, Mo., USA). Anti-mouse IL-1β, IL-6, and TNF-α antibodies and recombinant IL-1β, IL-6, and TNF-α were purchased from R & D Systems (Minneapolis, MN, USA).

ERK1 / 2 (extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal protein kinase), and p38 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All MAPK, Iκ-Bα (inhibitory kappa B alpha) and β-actin antibodies were purchased from Santa Cruz (CA, USA). Apamin purchased from Sigma-Aldrich (St. Louis, MO, USA).

3) Acute pancreatitis induction

In order to induce acute pancreatitis, Serulain 50 μg / kg was injected six times per hour for one hour. In the normal group, only six serine injections per hour were injected.

(2) In the pretreatment group of apamin, 10, 50, 100 μg / kg of acampin was injected subcutaneously at 1 hour and 18 hours before the first serurein injection. Acetic acid was subcutaneously injected into the normal group.

③ In the aflatoxin-treated group, 100 μg / kg of acampin was injected subcutaneously at 1 hour, 3 hours, and 5 hours before the first serurein injection.

④ Six hours after the last injection of cerulein, the abdominal cavity of the mouse was opened, blood was collected, and the pancreas and the lung were removed.

⑤ The extracted pancreas and lung were fixed with formalin and stored at -80 ℃.

Here, the serine-induced acute pancreatitis model is highly reproducible and economical in terms of the histopathological findings of acute pancreatitis in humans, and is suitable for studying the cytopathological phenomenon of acute pancreatitis and the self-activation process of digestive enzymes .

The reason for administering serurein 6 times per hour is that the serurein is a cholecystokinin analogue, which is based on the mechanism of inflammation through the pancreas receptor, and also that the half-life of serurein is very short Because.

The reason for injecting the drug through the intraperitoneal injection is that the drug absorbed from the stomach and small intestine goes to the liver through the portal in the case of oral administration and the drug is firstly metabolized (First-Pass Metabolism) It is not.

Considering the bioavailability due to metabolism in the liver, oral dosing should use more dose and delay the onset of action.

Intraperitoneal injection, on the other hand, has a wide absorbed area of the peritoneal membrane, so that when the drug is injected, the absorption is rapid and the action appears quickly. In addition, since the injected amount is absorbed, the pharmacokinetics accuracy is higher than that of oral administration, and the injected drug can freely enter the blood vessel through the pore of endothelial cells. In other words, intraperitoneal injection was chosen to ensure rapidity and dose accuracy of response expression.

Experimental preparation and pre-treatment up to Experimental Example 9 are carried out by the same procedure as described in Experimental Example 1.

1-2. Experimental course

The whole pancreas of the experimental animals was examined, and semi-quantitative analysis was performed on the degree of inflammation, edema, necrosis and vacuolization.

Sections containing at least 100 fields were analyzed for each sample and analyzed for the number of acinar cell necrosis and the presence of 0-3 (0: normal , 3: severe).

The analysis standard also included the degree of ghost, swelling, and structural destruction of the glandular cells.

① Pancreatic tissue was excised and fixed with 10% formalin solution.

Hematoxylin and eosin were stained with a typical tissue specimen preparation method.

③ A photograph was taken after observing with an optical microscope with a camera (Olympus BX 51, Japan).

1-3. Experiment result

The incidence of edema and inflammation of the pancreas may be an indicator for the diagnosis of acute pancreatitis, because edema and inflammation occur in the pancreas at the onset of acute pancreatitis.

In histologic analysis, the degree of edema can be grasped by measuring the interval between tissues, and the degree of inflammation can be determined by infiltration of inflammatory cells between tissues.

FIG. 1 is a photograph showing the results of comparing the pancreatic tissues of mice treated with cerulane and acute pancreatitis, in which apamin was pretreated, apamin was post-treated, and serine-treated normal groups. The results of staining with siline and eosin were magnified 400 fold.

FIG. 2 is a graph showing swelling and inflammation changes in the group treated with acerin in pancreatic tissues of mice treated with serraine to cause acute pancreatitis.

FIG. 3 is a graph showing swelling and inflammation of the pancreatic tissues of acute pancreatitis-treated mice treated with serurein, followed by treatment with apamin.

The results were similarly derived from three additional experiments.

As a result, the inhibition effect on the swelling and inflammation of the pancreatic tissues was observed in the apamin pretreatment group compared with the control group, and the degree of swelling and inflammatory cell aggregation increased as the treatment capacity of the apamin was increased to 10, 50 and 100 μg / kg Respectively.

In the aflatoxin - treated group, edema and inflammatory cell aggregation decreased slightly compared with the control group, but the degree of edema and inflammatory cell aggregation increased with time.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  2. Apamin  Inflammation and edema inhibition efficacy of lung tissue during treatment

2-1. Experimental course

1) Histological observation and analysis

① lung tissue was removed and fixed with 10% formalin solution.

Hematoxylin and eosin were stained with a typical tissue specimen preparation method.

③ A photograph was taken after observing with an optical microscope with a camera (Olympus BX 51, Japan).

2-2. Experiment result

When acute pancreatitis progresses, it attacks other organs such as the lungs, causing complications such as respiratory failure. Therefore, the degree of edema and inflammation of the lung tissue may be an indicator for the diagnosis of acute pancreatitis.

FIG. 4 is a photograph showing the results of comparing the lung tissues of mice treated with cerulane to acute pancreatitis by pretreatment with apamin, apamin-treated group, and serine-treated normal group, The results of staining with siline and eosin were magnified 400 fold.

FIG. 5 is a graph showing swelling and inflammation changes in the lung tissue pretreated with acaricin in acute pancreatitis caused by treatment with serulin.

FIG. 6 is a graph showing swelling and inflammation changes in the group treated with aceramin in the lung tissue of mice treated with serraine to cause acute pancreatitis.

The results were similarly derived from three additional experiments.

Observation results were the same as the results of pancreatic tissue observation. In the control group, apamin suppressed the alveolar edema and inflammation in lung tissues compared to the control group, and the more the treatment capacity of the acamin was increased to 10, 50 and 100 μg / kg, the more the degree of edema and inflammatory cell aggregation Respectively.

In the aflatoxin - treated group, edema and inflammatory cell aggregation decreased slightly compared to the control group, but the degree of edema and inflammatory cell aggregation increased with the passage of time.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  3. Apamin  During processing Pancreatic MPO  Infiltration-inhibiting efficacy

3-1. Experimental course

1) MPO analysis

MPO (myeloperoxidase) activity of pancreatic tissue was measured and neutrophil infiltration was examined in the pancreas.

① Pancreas tissue is dissolved in 20 mmol / L phosphate buffer (pH 7.4), homogenized and centrifuged at 15,000 rpm for 10 minutes.

② Centrifuged pancreatic tissue for 10 minutes was immersed in 50 mmol / L phosphate buffer (pH 6.0) containing 0.5% HTAB (hexadecyl trimethyl ammonium bromide) solution and centrifuged at 15,000 rpm for 5 minutes.

③ Pancreatic tissue was centrifuged for 5 minutes and then suspended in 1.6 mmol / L tetramethylbenzidine, 80 mmol / L sodium phosphate buffer (pH 5.4), and 0.3 mmol / L hydroxan peroxide Injected and reacted again.

(4) Incubate the mixture of tetramethylbenzidine, sodium phosphate buffer, and hydrous peroxidase containing pancreatic tissue at 37 ° C for 110 seconds.

⑤ A mixture of tetramethylbenzidine, sodium phosphate buffer, and hydrous peroxidase containing pancreatic tissue was treated with 2 mol / L sulfuric acid, and the absorbance was measured at 450 nm using a reader.

2) Statistical analysis

The difference between the control and experimental groups was tested by two-way analysis of variance (ANOVA). A P value <0.05 was considered statistically significant. The experimental results were expressed as Mean ± SE.

3-2. Experiment result

Neutrophils account for 50% to 70% of the circulating white blood cells, and the first to arrive at the time of inflammation, the basic function is phagocytosis. This infiltration of neutrophils can be used as an indicator of inflammation and can be measured by measuring the tissue MPO activity.

7 is a graph showing the results of MPO analysis in pancreatic tissues of mice treated with serraine to cause acute pancreatitis.

The results were similarly derived from three additional experiments.

As a result, the inhibitory effect of the apamin treatment group on the MPO infiltration of the pancreatic tissue was higher than that of the control group, and the inhibitory effect was increased as the treatment capacity of the apamin was increased to 10, 50 and 100 μg / kg.

In the aflatoxin-treated group, the MPO infiltration of the pancreatic tissue was slightly inhibited compared to the control group, but the MPO infiltration increased again as the treatment time elapsed.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  4. Apamin  Of lung tissue during treatment MPO  Infiltration-inhibiting efficacy

4-1. Experimental course

MPO (myeloperoxidase) activity of lung tissue was measured to examine neutrophil infiltration in the lung.

① Dissolve lung tissue in 20 mmol / L phosphate buffer (pH 7.4), homogenize and centrifuge at 15,000 rpm for 10 min.

② The lung tissue centrifuged for 10 minutes was immersed in 50 mmol / L phosphate buffer (pH 6.0) containing 0.5% HTAB (hexadecyl trimethyl ammonium bromide) solution and centrifuged at 15,000 rpm for 5 minutes.

(3) Centrifuged lung tissue for 5 minutes was diluted with 1.6 mmol / L tetramethylbenzidine, 80 mmol / L sodium phosphate buffer at pH 5.4, and 0.3 mmol / L hydroxan peroxide Injected and reacted again.

(4) Incubate the mixture of tetramethylbenzidine, sodium phosphate buffer, and hydrous peroxidase containing lung tissue at 37 ° C for 110 seconds.

(5) A mixture of tetramethylbenzidine, sodium phosphate buffer, and hydrous peroxidase containing lung tissue was treated with 2 mol / L sulfuric acid, and the absorbance was measured at 450 nm using a reader.

2) Statistical analysis

The same procedure as described in Experimental Example 3 was carried out.

4-2. Experiment result

8 is a graph showing the results of MPO analysis in lung tissues of mice treated with serraine to cause acute pancreatitis.

The results were similarly derived from three additional experiments.

The measurement results were the same as the result of observation of MPO infiltration of pancreatic tissue. The inhibition of MPO infiltration in lung tissue was more pronounced in the pretreatment group than in the control group, and the inhibitory effect was increased with increasing doses of 10, 50 and 100 μg / kg of apamin.

In the aflatoxin-treated group, MPO infiltration was slightly inhibited compared with the control group. However, MPO infiltration increased again as the treatment time elapsed.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  5. Apamin  Reduction of pancreatic weight / weight ratio during treatment

5-1. Experimental course

1) Measurement of pancreas weight / weight ratio

The pancreas was weighed 11 hours after the first administration of cerulein and then the weight ratio of pancreas to body weight was determined by calculating PW / BW (pancreatic weight / body weight).

2) Statistical analysis

The same procedure as described in Experimental Example 3 was carried out.

5-2. Experiment result

In the case of acute pancreatitis, the pancreas swells due to inflammation, resulting in an increase in the weight of the pancreas and a decrease in body weight. As a result, the ratio of pancreatic weight / body weight (PW / BW) is increased, and the ratio of pancreatic weight to body weight is an important indicator for the diagnosis of acute pancreatitis.

9 is a graph showing the results of measurement of pancreatic weight / body weight ratio of mice treated with serraine and induced acute pancreatitis.

The results were similarly derived from three additional experiments.

As a result, P.W / B.W ratio was significantly († p <0.05) decreased in the apamin pretreated group compared to the control group. The effect of reducing the P.W / B.W ratio was increased as the treatment capacity of apamin was increased to 10, 50 and 100 μg / kg.

In the aflatoxin-treated group, P.W / B.W ratio decreased slightly compared with the control group, but the P.W / B.W ratio increased again with the elapse of the treatment time.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  6. Apamin  Amylase Decreasing Effectiveness in Serum during Treatment

6-1. Experimental course

1) Measurement of Amylase content in serum

The mouse was anesthetized 6 hours after the last injection of Serraine.

② Blood was collected from the heart of mice using a syringe. Blood was centrifuged at 3,000 rpm at 4 ° C for 20 minutes to separate the serum.

③ amylase was quantified in isolated serum. Measurement of serum amylase was performed using a bioassay system (CA, USA).

2) Statistical analysis

The same procedure as described in Experimental Example 3 was carried out.

6-2. Experiment result

Amylase or lipase content in the serum of the pancreas is reported to increase more than three times the normal value when acute pancreatitis occurs. This is a very important indicator for the practical initial diagnosis in the clinic. It can increase the sensitivity and specificity when serum amylase or lipase test is performed simultaneously. In the present invention, the contents of amylase and lipase in serum were all measured.

10 is a graph showing the results of measuring the content of serum amylase in the pancreas of mice treated with serraine and caused acute pancreatitis.

The results were similarly derived from three additional experiments.

As a result, amylase was significantly decreased († p <0.05) in the group treated with apamin compared to the control group. The effect of amylase treatment was increased as the treatment capacity of apamin was increased to 10, 50, and 100 μg / kg.

In the aflatoxin-treated group, the amylase activity was slightly decreased compared to the control group, but the amylase activity increased again with the lapse of time.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  7. Apamin  Serum during treatment Lipase  Reduction efficacy

7-1. Experimental course

1) Determination of lipase content in serum

The mouse was anesthetized 6 hours after the last injection of Serraine.

② The blood was collected from the heart of the mouse using a syringe. The blood was centrifuged at 3,000 rpm at 4 ° C for 20 minutes to separate the serum.

③ Lipase was quantified in the separated serum. Serum lipase was measured by a bioassay system (CA, USA).

2) Statistical analysis

The same procedure as described in Experimental Example 3 was carried out.

7-2. Experiment result

11 is a graph showing the results of measuring the content of lipase in serum in the pancreas of mice treated with serraine and caused acute pancreatitis.

The results were similarly derived from three additional experiments.

As a result, lipase was significantly decreased († p <0.05) in the pretreatment group with apamin compared to the control group. As the treatment capacity of acammin was increased to 10, 50, and 100 μg / kg, the effect of reducing serum lipase was increased.

In the aflatoxin-treated group, lipase was slightly reduced compared with the control group, but the lipase increased again with the elapse of the treatment time.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  8. Apamin  In the pancreas during treatment TNF -α, IL -1β, IL -6 &lt; / RTI &gt;

8-1. Experimental course

① Incubate monoclonal antibodies TNF-α, IL-1β and IL-6 in PBS at pH 7.4 overnight at 4 ° C.

② Incubate 100 μL of incubated anti-mouse TNF-α, IL-1β and IL-6 monoclonal antibody in a 96-well plate.

③ Plates containing anti-mouse TNF-α, IL-1β and IL-6 monoclonal antibodies divided by 100 μl are washed in PBS containing 0.05% Tween-20 and PBS containing 10% FBS for 2 hours.

④ Incubate the additional washed monoclonal antibodies TNF-α, IL-1β and IL-6 at RT for 2 hours and wash the plates.

⑤ Incubate biotinylated anti-mouse TNF-α, IL-1β and IL-6 antibodies in the washed plate for 1 hour at RT.

⑥ Wash plate, add TMB substrate, add Avidin-peroxidase before re-washing and incubate for 30 minutes at RT.

⑦ Measure the color change at 450 nm using an automatic microplate ELISA reader. Standard samples are run on each assay plate by using serial dilutions of recombinant TNF-a, IL-l [beta] and IL-6.

8-2. Experiment result

ELISA (Enzyme-Linked Immunosorbent Assay) is a generic term for enzyme immunoassay, and is a method for measuring the amount of antigen or antibody using an antigen-antibody reaction using an enzyme as a marker. It is used as a diagnostic method for acute pancreatitis.

FIG. 12 is a graph showing the amounts of TNF-.alpha., IL-1.beta. And IL-6 by ELISA after treating pancreas of apamin-pretreated mouse with serraine and inducing acute pancreatitis.

13 is a graph showing the amounts of TNF-a, IL-l [beta] and IL-6 by ELISA after post-treatment of apramine in the pancreas of mice treated with serrene and acute pancreatitis.

The results were similarly derived from three additional experiments.

As a result, the amount of TNF-α, IL-1β and IL-6 was significantly decreased († p <0.05) in the group treated with apamin compared to the control group. The amounts of TNF-α, IL-1β and IL-6 decreased with increasing doses of apamin at 10, 50, and 100 μg / kg.

The amount of TNF-α, IL-1β and IL-6 in the aflatoxin-treated group was comparable to that of the control group.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  9. Apamin  In the pancreas during treatment TNF -α, IL -1β and IL -6 mRNA &Lt; / RTI &gt;

9-1. Experimental course

1) Measurement of mRNA expression of TNF-α, IL-1β and IL-6 in the pancreas

The mRNA transcripts of TNF-α, IL-1β and IL-6 in mouse pancreas were analyzed by RT-PCR. The process is as follows.

Total RNA was isolated from the pancreas using TriZol and then subjected to reverse transcription using SuperScript II RT (Invitrogen, Carlsbad, CA, USA).

② Taqman quantitative RT-PCR was performed using a Light Cycler 2.0 detection system (Roche, Switzerland) according to the manufacturer's instructions.

③ For each sample, the test reaction and the control reaction without the reverse transcriptase were repeated three times to analyze the expression of the gene of interest.

④ The control reaction was normalized with mRNA of 'housekeeping' HPRT (hypoxanthine-guanine phosphoribosyltransferase, hyperoxantin-guanine phosphoribosyltransferase). Any expression unit was calculated by dividing the amount of expression of the gene of interest by the amount of ribosomal protein HPRT mRNA expression.

The forward, reverse and probe primers for multiplex real-time taxan PCR are as follows.

TNF-α:

All directions: 5'-TCT CTT CAA GGG ACA AGG CTG-3 '

Reverse: 5'-ATA GCA AAT CGG CTG ACG GT-3 ';

Probe: 5'-CCC GAC TAC GTG CTC CTC ACC CA-3 '

IL-1?:

Omni direction: 5'-TTG ACG GAC CCC AAA AGA T-3 '

Reverse: 5'-GAA GCT GGA TGC TCT CAT CTG-3 '

Universal Probes, M15131.1-Roche Applied Science,

IL-6:

All directions: 5 '-TTC ATT CTC TTT GCT CTT GAA TTA GA-3'

Reverse: 5 '-GTC TGA CCT TTA GCT TCA AAT CCT-3'

Universal Probes, M20572.1-Roche Applied Science,

2) Statistical analysis

The same procedure as described in Experimental Example 3 was carried out.

9-2. Experiment result

FIG. 14 is a graph showing mRNA expression patterns of TNF-.alpha., IL-1 beta and IL-6 after treating acute pancreatitis with serraine in the pancreas of apomethic pretreated mouse.

FIG. 15 is a graph showing mRNA expression patterns of TNF-.alpha., IL-1 beta, and IL-6 after treatment of acute pancreatitis with acute pancreatitis in the pancreas of mice and post-treatment of the pancreas with apamin.

The results were similarly derived from three additional experiments.

As a result, mRNA expression of TNF-α, IL-1β and IL-6 was significantly decreased (p <0.05) in the pretreatment group compared to the control group. In addition, mRNA expression of TNF-α, IL-1β, and IL-6 decreased with increasing doses of 10, 50, and 100 μg / kg of acamin.

The amount of mRNA expression in the aflatoxin - treated group was comparable to that of the control group.

In other words, the acute pancreatitis inhibitory effect was observed in the treatment group of 10, 50, and 100 μg / kg of acampin, and it can be concluded that the acampine inhibits acute pancreatitis dose-dependently.

Experimental Example  10. Apamin  In the pancreatic tissue at the time of treatment MAPKs  Activity inhibition effect of channel

10-1. Experimental preparation and preprocessing

The preparation of experimental animals and experimental reagents was carried out in the same manner as in Experimental Example 1.

Unlike Experimental Example 1, the process of inducing acute pancreatitis was carried out as follows.

In order to induce acute pancreatitis, Serulain 50 μg / kg was injected six times per hour for one hour. In the normal group, only six serine injections per hour were injected.

In the apamin treatment group, 10, 50, and 100 μg / kg of acampin were injected subcutaneously at 1 hour and 18 hours before the first serulin injection. Acetic acid was subcutaneously injected into the normal group.

③ After 6 hours of the last injection of cerulein, the abdominal cavity of the mouse was opened, blood was collected, and the pancreas and lung were removed.

⑤ The extracted pancreas and lung were fixed with formalin and stored at -80 ℃.

10-2. Experimental course

① After pancreatic tissue was homogenized, it was made with 62.5 mM Tris-HCl, 2% SDS (sodium dodecyl sulfate, sodium dodecyl sulfate), 20% glycerol, 10% 2-mercaptoethanol at pH 6.8 The pancreas tissue was injected into the buffer and boiled.

② Proteins in cell lysates were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane.

After transferring the protein to the nitrocellulose membrane, the membrane was blocked for 2 hours with 5% skim milk in PBST (PBS-Tween-20) at RT and incubated with phosphorylated ERK1 / 2, phosphorylated JNK, Antibodies against p38, Iκ-Bα, all ERK1 / 2, all JNK, all p38, or β-actin were incubated overnight.

④ After washing the incubated antibody three times, each blot was incubated with secondary antibody conjugated with peroxidase for 1 hour. The chemiluminescent detection system was increased to observe the expression of developed antibody-specific proteins.

10-3. Experiment result

The pathway of mitogen-activated protein kinases (MAPKs) acts as an important regulator in both innate and acquired immunity, responds extensively to changes in the cell interior and exterior and interacts with other signaling pathways to regulate intracellular gene expression and cellular function regulation . It also plays a decisive role in regulating the production of proinflammatory cytokines. It is also associated with Ik-Ba (inhibitory kappa-Ba), which functions to inhibit NF-kB, a typical proinflammatory cytokine.

There are three types of MAPKs in mammals: extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38.

The severity of acute pancreatitis is proportional to the degree of acinar cell necrosis and inversely proportional to the degree of apoptosis. Neutropenic infiltration at the onset of acute pancreatitis increases the necrosis of acinar cells and decreases apoptosis, which decreases acute pancreatitis in experimental animals after acute pancreatitis is reduced by neutrophil influences. Therefore, theoretically, if acute pancreatitis develops, it may reduce the severity of acute pancreatitis if it reduces cell death such as necrosis, apoptosis, or reduces necrosis and apoptosis.

Proteins that promote apoptosis include JNK, STAT1 and FOXO3a. Because they are antagonistic, they increase when apoptosis is not performed well.

16 is a photograph showing the result of Western blotting performed on the pancreas of mice treated with serraine and induced acute pancreatitis.

The results were similarly derived from three additional experiments.

As a result of observation, the expression of P-JNK (phosphorylated JNK) is clearly observed in the control group treated with only serurein. In the control group treated with serurein only, the color of the P-JNK band is dark, and it can be seen thick and bold.

No expression was found in the group treated with apamin. In the group treated with apamin, apoptosis was properly performed, indicating that P-JNK and JNK were not expressed.

In other words, it was possible to observe acute pancreatitis inhibiting effect in the acammin treatment group, and it can be concluded that the acamine inhibits acute pancreatitis dose-dependently.

Claims (6)

A composition for the prevention, amelioration or treatment of acute pancreatitis, comprising as an active ingredient afamin.
The method according to claim 1,
A composition for preventing, ameliorating or treating acute pancreatitis, said composition being a complication of acute pancreatitis, which has the effect of inhibiting inflammation, edema and myeloperoxidase (MPO) infiltration in lung tissue.
The method according to claim 1,
The composition is effective for reducing inflammation and edema in pancreatic tissue and for inhibiting MPO infiltration in pancreatic tissue, ratio of pancreatic weight / weight ratio (PW / BW), amount of amylase and lipase in serum in pancreatic tissue , A proinflammatory cytokine and an inflammatory cytokine mRNA expression in the pancreatic tissue, and an activity of inhibiting the activity of the MAPKs channel in the pancreatic tissue, for the prevention, amelioration or treatment of acute pancreatitis.
delete The method according to claim 1,
The composition for preventing, ameliorating or treating acute pancreatitis according to claim 1, wherein the composition contains an apamin having a concentration of 10 to 100 μg / kg once administered on the basis of a subcutaneous injection of a mouse.
A health functional food for preventing or ameliorating acute pancreatitis, comprising a composition according to any one of claims 1 to 3 and 5, with a pharmaceutically acceptable additive.

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