TWI696468B - Composition containing short peptide and use of short peptide for inhibiting or alleviating symptoms associated with allergic reactions caused by shrimp - Google Patents

Abstract

The present invention relates to a composition containing a short peptide, which includes a unglycosylated short peptide as an active ingredient, thereby significantly inhibiting alleviating symptoms associated with allergic reactions caused by shrimp. Therefore, the short peptide can be used in preparation of a medicinal composition for inhibiting or alleviating symptoms associated with allergic reactions caused by shrimp.

Description

Compositions containing short peptides and short peptides are used to prepare inhibitory or Use of pharmaceutical composition for slowing shrimp allergic reaction

The present invention relates to a composition for inhibiting or alleviating allergic reactions, and in particular to a composition containing short peptides and the use of short peptides for preparing a pharmaceutical composition for inhibiting or reducing shrimp allergic reactions.

Food allergy is a hypersensitivity state caused by an acute and excessive immune response to foreign, harmless antigens contained in food. In general, foods that are likely to cause allergies include crustacean seafood (shellfish; for example, shrimps), foods containing artificial food additives, pods, stone fruits, caffeinated foods, and some fruits (such as mangoes and strawberries) , Tomatoes, oranges), alcoholic beverages or food, other foods (such as eggs, milk, mushrooms, bamboo shoots, pesticide-residue vegetables), etc.

Shrimps are widely used in various foods, dishes and processed foods. Take the shrimp allergic reaction as an example, in addition to the shrimp itself, but also with the individual Genetics, age, and asthma are related. To avoid allergic reactions to shrimp, in addition to avoiding ingestion of shrimps or foods containing shrimp ingredients in advance, after shrimp allergic reactions, depending on the severity of the symptoms and the site of allergic attack, appropriate drugs (such as antihistamines, steroids) Drugs, sodium cromoglycate, etc.) to inhibit allergic reactions of shrimp in the body.

However, the above drugs have side effects of varying degrees. For example, the common side effects of antihistamines are drowsiness, fatigue, and inattention, the common side effects of steroids are moon face, buffalo back, etc., and the common side effects of sodium cromoglycate are thorns at the beginning of use Painful.

In view of this, it is necessary to develop new pharmaceutical ingredients to effectively inhibit or slow down food allergic reactions and reduce side effects.

Therefore, one aspect of the present invention is to provide a composition containing a short peptide, which contains an unglycosylated short peptide as an active ingredient, so as to inhibit or slow the shrimp allergic reaction of the test subject.

Another aspect of the present invention is the use of a short peptide for preparing a pharmaceutical composition for inhibiting or slowing shrimp allergic reactions, which comprises an unglycosylated short peptide.

According to the above aspect of the present invention, a composition containing a short peptide is proposed, which contains a short peptide as an active ingredient. In this embodiment, the short peptide may be, for example, an unglycosylated short peptide composed of the amino acid sequence shown in SEQ ID NO: 1, so as to inhibit or slow the shrimp allergic reaction of the subject.

In an embodiment of the present invention, the above composition may be, for example, Food composition or pharmaceutical composition.

In an embodiment of the present invention, the test object may be, for example, animal cells or mammals.

According to another aspect of the present invention, a use of a short peptide for preparing a pharmaceutical composition for inhibiting or slowing shrimp allergic reactions is proposed. In this embodiment, the short peptide is an unglycosylated short peptide composed of the amino acid sequence shown in SEQ ID NO:1.

In an embodiment of the present invention, the shrimp allergic reaction includes the expression of histamine, β-hexosaminidase, TNF-α, IL-4, IL-6, IL-13, COX-2 and/or IgE antibodies The amount is improved.

In an embodiment of the present invention, the pharmaceutical composition is administered to a test subject, and the test subject is an animal cell or a mammal.

In an embodiment of the present invention, the effective dose of the above short peptide in the pharmaceutical composition may be, for example, 5.7 μg/g body weight to 9.6 μg/g body weight.

The short peptide-containing composition of the present invention contains an unglycosylated short peptide as an active ingredient, which can significantly inhibit or reduce the symptoms of shrimp allergic reactions, and is further used to prepare a pharmaceutical composition for inhibiting or slowing shrimp allergic reactions. use.

In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious and understandable, the drawings are described in detail as follows:

[FIG. 1A] shows that a pharmaceutical composition containing a short peptide according to an embodiment of the present invention is administered to rat basophilic leukemia (rat basophilic Leukemia (RBL) cell line RBL-2H3 (hereinafter referred to as RBL-2H3 mast cells), a histogram of its cell survival rate.

[FIG. 1B] A bar graph showing the release rate of histamine after administration of a pharmaceutical composition containing short peptides to RBL-2H3 mast cells according to an embodiment of the present invention.

[FIG. 1C] A bar graph showing the β-hexosaminidase activity of a short peptide-containing pharmaceutical composition according to an embodiment of the present invention after administration to RBL-2H3 mast cells.

[FIG. 1D] to [FIG. 1H] show the TNF-α mRNA (FIG. 1D) and IL-4 mRNA of the pharmaceutical composition containing short peptides according to an embodiment of the present invention after administration to RBL-2H3 mast cells (Figure 1E) Histogram of the relative content of IL-6 mRNA (Figure 1F), IL-13 mRNA (Figure 1G), and COX-2 mRNA (Figure 1H).

[Figure 1I] shows the expression of related genes in the message transmission pathway dominated by FcεRI after RBL-2H3 mast cells induced allergic reactions via IgE/DNP and then administered to a pharmaceutical composition containing short peptides according to an embodiment of the present invention Ink dot analysis chart.

[FIG. 2] Western blot showing the expression of genes related to the message transmission pathway dominated by FcεRI after RBL-2H3 mast cells induced allergic reactions by sensitive serum and then administered to a pharmaceutical composition containing short peptides Point analysis chart.

[FIG. 3] A scatter diagram showing the relative content of IgE antibodies in mice after anaphylaxis induced by shrimp extracts and then administered to a pharmaceutical composition containing short peptides according to an embodiment of the present invention.

The singular forms "a", "an" and "said" mentioned in the present invention include plural references unless the context clearly stipulates otherwise. The numerical range (such as 10%~11% A) includes the upper and lower limits (ie 10%≦A≦11%) if there is no specific description; if the numerical range does not define the lower limit (such as B less than 0.2% , Or B below 0.2%), it means that the lower limit may be 0 (that is, 0%≦B≦0.2%). The above terms are used to illustrate and understand the present invention, not to limit the present invention.

The present invention provides a pharmaceutical composition containing a short peptide, which contains an unglycosylated short peptide as an active ingredient to inhibit or slow down the shrimp allergic reaction of the test subject.

The "shrimp allergic reaction" referred to herein is directly related to food allergies. The only known shrimp allergen is tropomyosin. After eating shellfish such as shrimp, allergens in the food (eg shrimp allogen is tropomyosin) will stimulate the immune system and cause various acute allergic symptoms in various parts of the body, such as the digestive system (E.g. abdominal pain, nausea, vomiting or diarrhea, etc.), skin (e.g. red itchy skin, urticaria, eczema, etc.), respiratory system (e.g. runny nose, sneezing, chest tightness, shortness of breath or difficulty, asthma, cough, red eyes) Itching, swelling, oropharyngeal swelling and tingling, etc.), headache, dizziness, hypotension, and even anaphylactic shock.

The current research results indicate that the aforementioned "shrimp allergic reaction" is highly correlated with mast cells. Eating crustaceans such as shrimp After the seafood, the allergen in the food interacts with the antibody IgE and binds to the surface of cells (such as mast cells) with FcεRI, so that the mast cells (degranulation) are degranulated, releasing pro-inflammatory cytokines ( TNF-α, IL-4, IL-6, IL-13), histamine, β-hexosaminidase and other substances, through FcεRI (receptor for IgE antibody) dominate the message transmission path, activate AKT downstream of FcεRI, ERK, JNK, p38, etc. (for example, the expression level of mRNA and/or the amount of phosphorylated protein), start the de novo synthesis of pro-inflammatory cytokines, increase the expression level of antibody IgE, and then aggravate the symptoms of allergic reactions .

The "inhibition or reduction of shrimp allergic reaction" referred to herein refers to a subject who, after administration of the above-mentioned pharmaceutical composition containing short peptides, can directly or indirectly inhibit or slow down any of the following caused by shrimp allergy One case: various symptoms of acute allergy (digestive system, skin, respiratory system, etc.); pro-inflammatory cytokines (TNF-α, IL-4, IL-6, IL-13), histamine, β-hexosaminidase The content of other substances in the body; AKT, ERK, JNK, p38 and other expression levels downstream of FcεRI (such as the expression level of mRNA and/or the amount of protein phosphorylated); the synthesis of de novo proinflammatory cytokines ; And the amount of antibody IgE performance. In addition, when pro-inflammatory cytokines or cytokines are stimulated, cyclooxygenase-2 (COX-2) is also induced. Since the above-mentioned pharmaceutical composition of the present invention contains a short peptide of a specific sequence, the above-mentioned shrimp allergic reaction can be effectively suppressed or alleviated by means of expendable inhibition or competitive inhibition.

The "short peptide" referred to herein refers to the total amino acid residues For short-chain polypeptides not exceeding 40, the total number of amino acid residues is preferably not more than 37. In one embodiment, the short peptide is an unglycosylated short peptide composed of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence shown in SEQ ID NO: 1 is a reference The sequence of Genbank number NP_002843.2 is also listed as a reference in this case. It is explained here that if the total number of amino acid residues of the short peptide exceeds 37, the resulting short peptide has a large molecular weight and it is difficult to increase the effective dose. Secondly, if the short peptide does not completely contain the specific sequence shown in SEQ ID NO: 1, the resulting short peptide contains insufficient effective amino acid regions, which hinders subsequent application to inhibit or slow shrimp allergic reactions Effect.

In one embodiment, the aforementioned unglycosylated short peptide can be made by any conventional method, for example, a synthetic peptide or a recombinant protein obtained by expressing a recombinant gene in an expression system. The manufacturing method of artificially synthesized peptides or recombinant proteins should be well known to anyone with ordinary knowledge in the technical field to which the present invention belongs, and will not be repeated here.

It is explained here that the short peptide of the amino acid sequence shown in SEQ ID NO: 1 must be unglycated. If the short peptide is saccharified in advance, it is equivalent to having biological activity itself, and it is impossible to inhibit or slow down the shrimp allergic reaction by means of expendable inhibition or competitive inhibition.

In application, the above short peptides can be used to prepare compositions that inhibit or slow shrimp allergic reactions, such as food compositions or pharmaceutical compositions. In one embodiment, the dosage form of the suitable pharmaceutical composition is not limited, and the effective dose of the short peptide in the pharmaceutical composition can be flexibly adjusted according to the dosage form and the site of use. Taking an oral pharmaceutical composition as an example, the effective dose of a short peptide in a pharmaceutical composition can be, for example It is 5.7 μg/g body weight to 9.6 μg/g body weight, but preferably 6.4 μg/g body weight to 8.1 μg/g body weight. In pharmaceutical compositions of other dosage forms, such as oral and nasal sprays, the effective dose of short peptides in the pharmaceutical composition can be adjusted according to the dosage form.

In the above embodiment, the above pharmaceutical composition may optionally add a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier referred to herein refers to a carrier, diluent, filler, and/or vehicle used to deliver short peptides as active ingredients to an individual, which are not active ingredients themselves, or Added to the above composition to improve the handling or storage properties of the composition, or to allow or assist the dosage unit of the composition to form an excipient or any ingredient suitable for the pharmaceutical composition and convenient for administration. The aforementioned pharmaceutically acceptable carrier should not destroy the pharmacological activity of the active ingredient, and should be non-toxic when delivering a sufficient therapeutic dose of the active ingredient.

The aforementioned applicable pharmaceutically acceptable carriers may be well known to those who are generally familiar with the general knowledge of manufacturing pharmaceutical compositions, and include but are not limited to buffers, diluents, disintegrants, adhesives, adhesives, wetting agents, Polymers, lubricants, slip agents, ingredients added to mask or counteract bad tastes or odors, dyes, fragrances, and ingredients added to improve the appearance of the composition. Specific examples of the aforementioned pharmaceutically acceptable carriers may include, but are not limited to, citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide , Sodium and calcium salts of phosphoric acid and sulfuric acid, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulose ingredients (Cellulose esters and cellulose alkyl esters such as alkanoic acid), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerin or powders, polymers (such as (Polyvinylpyrrolidone, polyvinyl alcohol and polyethylene glycol) and other pharmaceutically acceptable ingredients.

In one embodiment, the route for administering the short peptide-containing composition is not particularly limited, and it can be administered by oral route or non-oral route (for example, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, respiratory tract inhalation, etc.).

The above-mentioned short peptide-containing composition has been confirmed by animal cell or mammal experiments, and has the effect of inhibiting or slowing the shrimp allergic reaction of the test subject. In an example, the aforementioned efficacy of inhibiting or slowing the shrimp allergic reaction of the subject may include, but is not limited to, inhibiting or slowing down proinflammatory cytokines (TNF-α, IL-4, IL-6, IL-13), tissue Overdose of amine, β-hexosaminidase, antibody IgE and so on. In other examples, the aforementioned efficacy of inhibiting or slowing the shrimp allergic reaction of the subject may include, but is not limited to, inhibiting or slowing AKT, ERK, JNK, p38, etc. downstream of FcεRI (for example, the expression level of mRNA and/or protein is phosphorylated Amount), thereby inhibiting or slowing shrimp allergic reactions.

The following uses several embodiments to illustrate the application of the present invention, but it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch.

Example 1: Preparation of short peptides

In this example, a short peptide RI37 (having the amino acid sequence shown in SEQ ID NO: 1) was used as a short peptide, and the polypeptide pPTX3 (having the amino acid sequence shown in SEQ ID NO: 2) was used ) As a control group.

The above short peptide RI37 can be synthesized by, for example, Kronai International Technology Co., Ltd. (Taipei, Taiwan). The above-mentioned pPTX3 is a recombinant protein expressed and purified by the inventor of the present invention using a conventional prokaryotic expression system (E. coli). The manufacturing method of artificially synthesized peptides or recombinant proteins should be well known to anyone with ordinary knowledge in the technical field to which the present invention belongs, and will not be repeated here.

Example 2: Establish a cell test mode

Cell culture

In this example, rat basophilic leukemia (RBL) cell line RBL-2H3 (ATCC number: CRL-2256; hereinafter referred to as RBL-2H3 mast cell) was selected, and the short peptide of Example 1 was evaluated for mast cell removal. Inhibiting or slowing effect of granulation.

The above RBL-2H3 mast cells can be cultured using its dedicated cell culture and subculture methods. For example, the RBL-2H3 mast cell line contains 10% fetal bovine serum protein (Fetal Bovine Serum; FBS, which has been heat-inactivated) and antibiotics (50 μg/mL to 100 μg/mL streptomycin and 50 U/mL To 100 U/mL of penicillin) Dulbecco's modified Eagle medium (DMEM; Gibco Co.) cell culture medium was cultured at 37°C with a humidity of 5% carbon dioxide concentration. It is any person who has general knowledge in the technical field to which the present invention belongs, so it will not be described in detail.

2. MTT test

MTT is tetrazolium salt, which is decomposed by dehydrogenase in living cells to produce blue formazan crystals from transparent and colorless cells. Cells without the short peptide of Example 1 added The survival rate was taken as 100%, from which the effect of the short peptide of Example 1 on the survival rate of RBL-2H3 cells of Example 2 was judged.

First, the RBL-2H3 mast cells of Example 2 were cultured into a 96-well cell culture plate (cell density of 2×10 ⁴ cells/well), and co-cultured with the short peptide RI37 (300 μg/mL) of Example 1 at 37°C After 24 hours, 48 hours or 72 hours, add 5 mg/mL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bomide] (Sigma) and react at 37℃ for 3 hours . Afterwards, the precipitated MTT blue formazan crystals were eluted with DMSO, and the absorbance of the cells at 570 nm was measured using a commercially available microplate reader (microplate reader; for example, iMark ^TM Microplate Absorbance Reader, Bio-Rad) to evaluate the cell survival rate .

Please refer to FIG. 1A, which is a bar graph showing the cell survival rate of a short peptide-containing pharmaceutical composition administered to RBL-2H3 mast cells according to an embodiment of the present invention, wherein the vertical axis represents the cell absorbance at 570nm , The figure number "+" below the horizontal axis represents the addition of short peptides during cell culture, the figure number "-" represents that no short peptides are added during cell culture, and the figure number "ns" represents no statistically significant difference (no significance; ns ).

It can be seen from the results in FIG. 1A that the short peptide RI37 and RBL-2H3 mast cells in Example 1 of the present invention have no significant effect on cell survival rate after 24 hours, 48 hours or 72 hours of co-cultivation, representing no cells toxicity.

In addition, the short peptide RI37 and RBL-2H3 mast cells in Example 1 of the present invention have no significant effect on the number of cells and FcεRI (receptor of IgE antibody) mRNA (not shown in the figure), indicating no promotion The effect of increasing the number of RBL-2H3 mast cells and FcεRI.

3. Assess the degranulation of cells

When evaluating the degranulation of cells, first culture the RBL-2H3 mast cells of Example 2 to a 24-well cell culture dish (cell density of 2×10 ⁵ cells/well), and 0.2 μg/mL anti-DNP (2, The 4-dinitrophenylhydrazine)-IgE antibody was cultured overnight to perform a cell sensitization reaction. Then, the cells were washed with 1x PBS (1X PBS). Next, cells were pretreated with short peptides (300 ng/mL) at 37°C for 30 minutes. Then, by using the polypeptide pPTX3 (300ng / mL, referred to as of PTX3) or 1μg / mL of DNP-BSA (dissolved in PIPES buffer solution containing 140mM NaCl, 5mM KCl, 0.6mM MgCl 2, 1mM CaCl 2, 5.5mM glucose, 0.1 % BSA and 10 mM PIPES, pH 7.4, as a control group) as antigen (Ag), stimulate cells for 30 minutes.

After that, the cell supernatant was collected, and the content of histamine contained and the activity of β-hexosaminidase were measured as indicators for evaluating the degranulation of the cells.

3.1. Assess the release rate of histamine from cells

The release rate of histamine is calculated according to the following conventional method. First, after the cell supernatant was mixed with 100μL of 20μL of 1M NaOH, then added 25μL of the reaction solution [containing 1% (w / v) o-phthalaldehyde (o -phthalaldehyde)] was dissolved in methanol, at room temperature (Generally 10°C to 40°C) and react for 4 minutes. Then, 10 μL of 3M HCl was added to the above reaction to terminate the reaction. After that, the fluorescence intensity is detected using an excitation wavelength of 355 nm and an emission wavelength of 460 nm, and the results are shown in FIG. 1B.

Please refer to FIG. 1B, which is a bar graph showing the histamine release rate of a short peptide-containing pharmaceutical composition administered to RBL-2H3 mast cells according to an embodiment of the present invention. "+" represents the addition of short peptides or peptides PTX3 during cell culture, figure number "-" represents the absence of short peptides or peptides PTX3 during cell culture, figure number "＊＊＊" represents statistical significance ( p <0.001), and The amount of cell lysis buffer that breaks the cells and completely releases histamine is used as a 100% benchmark.

It can be seen from the results in FIG. 1B that IgE can activate RBL-2H3 mast cells, causing degranulation and releasing histamine. Using antigen (PTX3) to stimulate IgE-sensitized mast cells can add a de-granulation reaction and release more histamine. However, the RBL-2H3 mast cells sensitized by IgE are pretreated with the short peptide RI37 of Example 1 of the present invention before being stimulated and degranulated by antigen (ie PTX3), which can significantly inhibit or slow down the activation of RBL-2H3 by IgE/Ag The degranulation of mast cells reduces the release rate of histamine, and this difference is statistically significant.

3.2. Evaluation of β-hexosaminidase activity of cells

When evaluating the activity of β-hexosaminidase, in principle, it is the same as the method in point 3 above, except that 25 μL of the cell supernatant collected above is taken with an equal volume of 5 mM substrate solution containing 5 mM P-nitrophenyl N-acetyl-β-D-glucosaminide (p-NAG), dissolved in 0.2M sodium citrate buffer solution, pH 4.5] After mixing, immediately Add 25 μL of the reaction solution [containing 1% ( w / v ) phthalaldehyde ( o- phthalaldehyde) in methanol], and react at 37°C for 1.5 hours. Then, 200 μL of a stop solution (containing 0.1 M Na ₂ CO ₃ /NaHCO ₃ , pH 10.0) was added to the above reaction to terminate the reaction. After that, the absorbance of the cells at 405 nm was detected to determine the activity of β-hexosaminidase, and the results are shown in FIG. 1C.

Please refer to FIG. 1C, which shows a histogram of β-hexosaminidase activity of a pharmaceutical composition containing a short peptide administered to RBL-2H3 mast cells according to an embodiment of the present invention, where the vertical axis represents cells The absorbance at 405nm, the graph number "+" below the horizontal axis represents the addition of short peptides or peptides PTX3 during cell culture, the graph number "-" represents the absence of short peptides or peptides PTX3 during cell culture, and the graph number "＊＊" represents the Data with statistical significance ( p <0.01), the drawing number "***" represents statistical significance ( p <0.001).

From the results in Figure 1C, it can be seen that IgE can activate RBL-2H3 mast cells, causing degranulation and releasing β-hexosaminidase. Using antigen (PTX3) to stimulate IgE-sensitized mast cells can add a de-granulation reaction and release more β-hexosaminidase. However, RBL-2H3 mast cells sensitized by IgE are subjected to the present invention before stimulation and degranulation with antigen (ie PTX3). The short peptide RI37 treatment in Example 1 can significantly inhibit or slow the degranulation of IgE/Ag-activated RBL-2H3 mast cells and reduce the activity of β-hexosaminidase, and this difference is statistically significant.

4. Assess the gene expression of proinflammatory cytokines in cells

The total RNA of mast cells sensitized by IgE stimulated with antigen (PTX3) at point 3 above is extracted with a commercially available extraction kit (eg TRIsure RNA extraction reagent, Invitrogen). Next, a commercially available reverse transcriptase (for example, SuperScript III, Invitrogen) is used to perform a reverse transcription reaction on the total RNA to synthesize cDNA. Then, using commercially available fluorescent real-time quantitative qPCR reaction kits (such as KAPA SYBR FAST qPCR Master Mix, Life Technologies Corporation and Kapa Biosystems Inc.) and the primers listed in SEQ ID NOs: 3~14, the commercially available real-time PCR system (For example, CFX connect Real-Time PCR System, BIO-RAD) quantitative PCR (qPCR) of each target gene is performed, and the results are shown in FIGS. 1D to 1H.

Please refer to FIG. 1D to FIG. 1H, which respectively show the TNF-α mRNA (FIG. 1D) and IL-4 mRNA of the pharmaceutical composition containing short peptides according to an embodiment of the present invention after administration to RBL-2H3 mast cells (Fig. 1E), IL-6 mRNA (Fig. 1F), IL-13 mRNA (Fig. 1G), COX-2 mRNA (Fig. 1H) Histogram of relative content changes, in which β-actin (β- actin) mRNA content as an internal control group (not shown). The figure number "+" below the horizontal axis of Figures 1D to 1H represents the addition of short peptides or peptides PTX3 during cell culture, the figure number "-" represents the absence of short peptides or polypeptide PTX3 during cell culture, and the figure number "*" represents Data with statistical significance ( p <0.05), figure number "**" represents data with statistical significance ( p <0.01), figure number "***" represents statistical significance ( p <0.001), and The mRNA content of β-actin in each group serves as an internal control group.

From the results of Figure 1D to Figure 1H, it can be seen that the use of antigen (PTX3) to stimulate IgE-sensitized mast cells can significantly increase the expression of TNF-α , IL-4 , IL-6 mRNA, IL-13 and COX-2 mRNA . However, IBLE-sensitized RBL-2H3 mast cells are pretreated with the short peptide RI37 of Example 1 of the present invention, and then stimulated by IgE-sensitized mast cells with antigen (ie PTX3), can significantly inhibit or slow down TNF-α , IL -4 , IL-6 mRNA, IL-13 and COX-2 mRNA expression levels, and this difference has statistically significant differences.

5. Evaluate the genetic performance of intracellular message transmission pathways (I)

When evaluating the gene expression of the intracellular message transmission path, in principle, the same as the method of point 3 above, the difference is that different concentrations of short peptides (150ng/mL or 300ng/mL) are used to pre-treat the cells at 37℃ for 30 minutes. , Reuse peptide PTX3, DNP-IgE/DNP to stimulate cells. Afterwards, all cell lysates were collected and subjected to Western blot analysis.

Western blot analysis can be performed using conventional methods. First, use modified radioimmunoprecipitation assay (modified RIPA) buffer The solution draws the protein of the above cells. The aforementioned modified formula of RIPA buffer solution contains 50 mM Tris-HCl [pH 7.4], 150 mM sodium chloride, 1 mM ethylenediamine tetra-acetic acid (EDTA), and 1% ethylphenyl polyethylene glycol (octylphenoxypolyethoxyethanol, Nonidet P 40, NP40), 0.25% sodium deoxycholate, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) , 1μg/ml aprotinin (aprotinin) and 1μg/mL leupeptin (leupeptin).

After the cell lysate obtained above was electrophoresed on SDS-PAGE, the cell protein was transfected from SDS-PAGE colloid to PVDF (polyvinylidene difluoride membrane) membrane (Immobilon®-P), and the primary antibody was used at 4°C until overnight. Primary antibodies include anti-p84 antibody (model: GTX70220, GeneTex), anti-phosphorylated AKT (p-AKT) antibody (model: GTX121937, GeneTex), anti-AKT antibody (model: GTX128414, GeneTex), anti-p-ERK antibody (model : 4377, Cell Signaling), anti-ERK antibody (model: 9102, Cell Signaling), anti-p-p38 antibody (model: 9211, Cell Signaling), anti-p38 antibody (model: 9212, Cell Signaling), anti-p-JNK antibody (Model: 4668, Cell Signaling) and anti-JNK antibody (Model: 9252, Cell Signaling). Then, a secondary antibody conjugated to peroxidase was used for 1.5 hours at room temperature. After that, use commercially available color kits (such as ECL kit, PerkinElmer) to detect protein expression, and p84 protein expression as the internal control group.

Regarding the extraction of proteins and Western blot analysis methods are well known to those of ordinary skill in the technical field to which the present invention belongs, and details will not be repeated here.

Please refer to FIG. 1I, which shows related genes of the message transmission pathway dominated by FcεRI after RBL-2H3 mast cells induce allergic reactions via IgE/DNP and then administered to a pharmaceutical composition containing short peptides according to an embodiment of the present invention. The performance of Western blot analysis chart. In Figure 1I, the figure number "+" represents the addition of short peptides or PTX3 during cell culture, the figure number "-" represents the absence of short peptides or PTX3 during cell culture, and the triangle represents the increasing amount of specific short peptides.

From the results of Figure 1I, we can see that the use of antigen (PTX3) to stimulate IgE/DNP-sensitized mast cells can significantly increase the phosphorylation of AKT, ERK1/ERK2, p38 and JNK1/JNK2 and other proteins into p-AKT and p-ERK1/ The amounts of ERK2 and p-p38 and p-JNK1/JNK2 make the allergic reaction aggravation. However, the RBL-2H3 mast cells sensitized by IgE/DNP are pretreated with the short peptide RI37 of Example 1 of the present invention, and then the antigen (ie PTX3) is used to stimulate the mast cells sensitized by IgE/DNP, which can significantly inhibit or slow down Proteins such as AKT, ERK1/ERK2, and p38 and JNK1/JNK2 are phosphorylated to the amount of p-AKT, p-ERK1/ERK2, and p-p38 and p-JNK1/JNK2.

6. Evaluate the genetic performance of intracellular message transmission pathways (II)

When evaluating the genetic performance of intracellular messaging pathways, principles The above method is the same as the above point 3, the difference is that the cells are pre-treated with different concentrations of short peptides (150ng/mL or 300ng/mL) at 37℃ for 30 minutes, and then the mice sensitized with shrimp extract are used. Serum (sera added at 10 μL/mL) serves as antigen-stimulating cells. Afterwards, all cell lysates were collected and subjected to the same Western blot analysis method as point 5, and the protein expression level of p84 was used as the internal control group.

The preparation method of the above shrimp extract is prepared by the following method. First, the shrimps were deheaded and shelled, and the meat part was added to a fixed proportion of double distilled water (ddH ₂ O; also known as secondary water) (shrimp meat: secondary water=1:2) to grind into meat puree. After the meat slurry was centrifuged at a low temperature of 8000 rpm for 30 minutes, the supernatant was collected and freeze-dried to obtain a shrimp extract. The obtained shrimp extract is injected intraperitoneally (ip) with shrimp extract, and the mice are sensitized and challenged to induce shrimp allergic reactions in the mice. Please refer to point 7 for the sensitization and stimulation of mice. After that, the allergic serum of the shrimp allergic mice was taken as an antigen to stimulate RBL-2H3 mast cells.

Please refer to FIG. 2, which shows the expression of genes related to the signal transmission pathway dominated by FcεRI after RBL-2H3 mast cells induce allergic reactions after sensitizing serum and then administer the pharmaceutical composition containing short peptides according to an embodiment of the present invention. Western blot analysis chart. In Fig. 2, the figure number "+" represents the addition of short peptides or PTX3 during cell culture, the figure number "-" represents the addition of short peptides or PTX3 during cell culture, and the triangle represents the increasing amount of specific short peptides added.

It can be seen from the results in Figure 2 that the use of allergic serum to stimulate IgE-sensitized mast cells can significantly increase p-AKT, p-ERK1/ERK2, p-p38, And the protein expression of p-JNK1/JNK2. However, IBLE-sensitized RBL-2H3 mast cells were pretreated with the short peptide RI37 of Example 1 of the present invention, and then stimulated with IgE-sensitized mast cells using allergic serum, which could significantly inhibit or slow down p-AKT, p-ERK1/ ERK2, p-p38 and p-JNK1/JNK2 protein expression levels.

7. Evaluate the content of IgE in the body

In this example, 3 to 4 week old female Balb/c mice (6 mice per group) were injected with antigen (PBS or shrimp extract) through the abdominal cavity (ip) to induce shrimp allergic reactions. The short peptide RI37 was evaluated to inhibit or slow shrimp allergy The effect of the reaction. The body weight of the aforementioned 3 to 4 week old female Balb/c mice is on average 10.4g/mouse to 17.4g/mouse.

The control group (Group 1 and Group 2) was intraperitoneally injected with PBS and aluminum-containing adjuvant [aluminum hydroxide; Al(OH)] on days 1, 4, 7, 10, 13, 16, 19 and 22 ₃ ] (dissolved in PBS), and injected PBS intraperitoneally on the 30th day. The shrimp allergy group (Groups 3 and 4) was intraperitoneally injected with a double dose of shrimp extract and aluminum-containing adjuvant on days 1, 4, 7, 10, 13, 16, 19 and 22 Alumina; Al(OH) ₃ ] (dissolved in PBS) was sensitized, and mice were challenged by intraperitoneal injection of 5 times the dose of shrimp extract on day 30 to cause shrimp allergy reaction. The aforementioned one-fold dose of shrimp extract is 0.5 mg/g body weight.

On the 19th, 22nd and 30th days, the short peptide RI37 treatment group (Group 2 and Group 4) was injected intraperitoneally with the short peptide RI37 (dissolved in PBS, Group 2), or the short peptide RI37 and shrimp Extract (dissolved in PBS, group 4).

Two hours after the stimulation on the 30th day, the whole blood of all the above mice was collected from the mouse sinus (orbital sinus) to evaluate the content of IgE in the mice.

The whole blood obtained above is allowed to stand (that is, unstirred) at room temperature for 30 minutes to coagulate the whole blood. Next, it was centrifuged at 2000 g for 10 minutes in a refrigerated centrifuge. Then, using a commercially available enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's manual, the absorbance of each well at a wavelength of 450 nm was detected, thereby obtaining IgE (1:20 dilution) content.

Please refer to FIG. 3, which is a scatter diagram showing the relative content of IgE antibody in mice after sensitization induced by shrimp extract and administration of a pharmaceutical composition containing short peptides according to an embodiment of the present invention. In Figure 3, the vertical axis represents the absorbance at a wavelength of 450 nm, and the horizontal axis from left to right is Group 1 to Group 4, Group 1 and Group 2 represent the control group, Group 3 and Group 4 represent shrimp. Allergic group, group 2 and group 4 were treated with short peptides. Figure number "+" means that short peptides were added during cell culture, figure number "-" means that short peptides were not added during cell culture, figure number "＊＊" Represents data with statistical significance ( p <0.01), figure number "***" represents statistical significance ( p <0.001), figure number "ns" represents no statistically significant difference (no significance; ns) .

It can be seen from the results of Group 1 and Group 2 in FIG. 3 that whether the short peptide RI37 stimulates mice or not, there is no statistically significant difference in the change in the content of IgE antibodies. From the results of Group 1 and Group 3 in Figure 3, it can be seen that stimulation of mice with shrimp extract can significantly increase the content of IgE antibodies. However, from Figure 3 The results of Group 4 show that shrimp-allergic mice can significantly inhibit or slow down the content of IgE antibody after treatment with short peptide RI37 in Example 1 of the present invention on days 19, 22 and 30, and this difference is statistically significant Difference, to achieve the purpose of inhibiting or slowing shrimp allergic reactions.

It is added that the present invention uses short peptide RI37 as an effective ingredient, which has been confirmed by in vitro and in vivo tests, and indeed can inhibit or slow down shrimp allergic reactions. It has the potential to be used in the preparation of food compositions or pharmaceutical compositions that inhibit or slow shrimp allergic reactions. the use of. In application, the above-mentioned food composition or pharmaceutical composition of the present invention may be administered first before the granulation of mast cells caused by shrimp allergic reactions, or within 30 minutes after taking shrimp itself or food containing shrimp ingredients To prevent, suppress or slow down the shrimp allergic reaction.

In summary, although the present invention takes short peptides of a specific sequence, a specific dosage form, a specific object, a specific administration method, or a specific evaluation method as examples, the short peptide-containing composition of the present invention and its use The purpose of inhibiting or slowing the allergic reaction of shrimp, but anyone with ordinary knowledge in the technical field to which the present invention belongs will know that the present invention is not limited to this, and the present invention may also use other dosage forms without departing from the spirit and scope of the present invention. Other objects, other methods of administration or other methods of evaluation.

It can be seen from the above examples that the short peptide-containing composition of the present invention has the advantage that it contains an unglycosylated short peptide as an active ingredient, which can significantly inhibit or alleviate the symptoms related to shrimp allergic reaction, and is further used to prepare an inhibitory or alleviating Use of pharmaceutical composition of shrimp allergic reaction.

Although the present invention has been disclosed above in several embodiments, it is not intended to limit the present invention, and any person in the technical field to which the present invention belongs generally has Those with knowledge can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be deemed to be defined by the scope of the attached patent application.

<110> National Success University

<120> Compositions containing short peptides and the use of short peptides to prepare pharmaceutical compositions that inhibit or slow shrimp allergic reactions

<130> None

<160> 14

<210> 1

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 200 to 236 amino acid (RI37) recombinant protein

<400> 1

<210> 2

<211> 381

<212> PRT

<213> Artificial sequence

<220>

<223> Human sapiens PTX3 1 to 381 amino acid recombinant protein

<400> 2

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> β-actin gene upstream primer

<400> 3

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> β-actin gene downstream primer

<400> 4

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> TNF-α gene upstream primer

<400> 5

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> TNF-α gene downstream primer

<400> 6

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> IL-4 gene upstream primer

<400> 7

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> IL-4 Gene Downstream Primer

<400> 8

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> IL-6 gene upstream primer

<400> 9

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> IL-6 Gene Downstream Primer

<400> 10

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> IL-13 gene upstream primer

<400> 11

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> IL-13 Gene Downstream Primer

<400> 12

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> COX-2 gene upstream primer

<400> 13

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> COX-2 gene downstream primer

<400> 14

Claims (6)

A short peptide is used for preparing a pharmaceutical composition for inhibiting or slowing shrimp allergic reaction, wherein the short peptide is an unglycosylated short peptide composed of the amino acid sequence shown in SEQ ID NO: 1. The use of the short peptide according to item 1 of the patent application scope for the preparation of a pharmaceutical composition for inhibiting or slowing the allergic reaction of shrimp, wherein one of the dosage forms of the pharmaceutical composition is an oral composition. Use of the short peptide according to item 1 of the patent application scope for preparing a pharmaceutical composition for inhibiting or slowing shrimp allergic reactions, wherein the shrimp allergic reactions include histamine, β-hexosaminidase, TNF-α, IL- 4. The performance of IL-6, IL-13, COX-2 and/or IgE antibodies is improved. Use of the short peptide according to item 1 of the patent application scope for the preparation of a pharmaceutical composition that inhibits or alleviates shrimp allergic reactions, wherein the pharmaceutical composition is administered to a subject, and the subject is an animal cell Or a mammal. Use of the short peptide according to item 4 of the patent application scope for preparing a pharmaceutical composition for inhibiting or slowing shrimp allergic reactions, wherein the effective dose of the short peptide in one of the pharmaceutical compositions is 5.7 μg/g body weight to 9.6 μg /g weight. The use of a short peptide according to item 4 of the patent application scope for the preparation of a pharmaceutical composition for inhibiting or alleviating shrimp allergic reactions, wherein the effective dose of the short peptide in one of the pharmaceutical compositions is 6.4 μg/g body weight to 8.1 μg /g weight.

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