Composition comprising phospholipase a2 as active ingredient

Abstract

To provide a composition or the like for inhibiting infiltration of pro-inflammatory macrophages.SOLUTION: The composition comprises phospholipase A2, one of the main components of bee venom, as an active ingredient. In addition, a health functional food composition and a feed composition for inhibiting infiltration of pro-inflammatory macrophages comprising phospholipase A2 as an active ingredient are also provided.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to a composition for reducing obesity, which contains phospholipase A2, one of the main components of bee venom, as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating obesity, which contains phospholipase A2 as an active ingredient; a functional health food composition for preventing or reducing obesity, which contains phospholipase A2 as an active ingredient; a feed composition; a method for preventing or treating obesity, which comprises administering to an individual a composition containing phospholipase A2 as an active ingredient; and the use of a composition containing phospholipase A2 as an active ingredient in the prevention or treatment of obesity. [Background technology]

[0002] Obesity, a condition characterized by excessive accumulation of fat in the body, is classified as one of the major metabolic diseases and is a serious health problem facing countries all over the world. It is predicted that the number of people affected will reach more than 300 million by 2025, and the market for obesity medications is expected to grow significantly not only in Korea but also around the world.

[0003] It is known to cause a combination of diabetes, heart disease, high blood pressure, cancer, and other conditions, and in severe cases, lead to early death. Obesity is also classified as a chronic inflammation, and recent research has revealed that the accumulation of inflammatory macrophages in adipose tissue is closely related to obesity. Furthermore, in the case of obesity, an imbalance between inflammatory and anti-inflammatory macrophages in adipose tissue creates an inflammatory environment, and the infiltration of inflammatory macrophages increases the secretion of inflammatory cytokines such as TNF-α and interleukin-6, which can further worsen the symptoms of obesity.

[0004] Therefore, alleviating the inflammatory environment and balancing macrophages is expected to be important in treating obese patients, but there are currently no obesity treatments that target macrophages. Currently, obesity treatments include dietary therapy, exercise therapy, and drug therapy. However, in the case of drug therapy, problems of stability and side effects arise as the administration period becomes longer, and there has been a demand for the development of new obesity treatment agents.

[0005] Currently, only orlistat and lorcaserine are approved for long-term use in Korea, while various treatments such as liraglutide, phentermine / topiramate, and naltrexone / bupropion have been developed and approved in the U.S. However, since many drugs reduce weight by inhibiting the absorption of fat ingested through food and drink, the development of a fundamental obesity treatment drug is urgently needed.

[0006] Phospholipase A2, a protein enzyme found in 12% of bee venom, is an enzyme that hydrolyzes phospholipids and is also known as lecithinase. The phospholipase A2 component liberates fatty acids bound to BDP in glycerol, a constituent lipid of cell membranes, to form lysophosphatides, which are known to cause tissue destruction, hemolysis, and catalytic activity. Various pharmaceutical compositions can be developed by utilizing the functions of phospholipase A2. Patent Document 1 proposes a composition for preventing and treating viral diseases such as influenza and vesicular stomatitis. Patent Document 2 also proposes a composition for treating prion disease containing phospholipase as an active ingredient, which is useful as a pharmaceutical composition for preventing and treating degenerative brain diseases. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Publication No. 10-2015-0049438 [Patent Document 2] Korean Patent Publication No. 10-2012-0139313 Summary of the Invention

[0008] The present inventors have conducted extensive research to develop a method for improving obesity without causing side effects, and as a result have confirmed that phospholipase A2 derived from bee venom has preventive or therapeutic effects against obesity, thereby completing the present invention.

[0009] One object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which contains phospholipase A2 as an active ingredient. Another object of the present invention is to provide a functional health food composition for preventing or improving obesity, which contains phospholipase A2 as an active ingredient.

[0010] A further object of the present invention is to provide a feed composition for preventing or ameliorating obesity, which contains phospholipase A2 as an active ingredient. It is yet another object of the present invention to provide a method for preventing or treating obesity, which comprises administering to an individual a composition containing phospholipase A2 as an active ingredient.

[0011] A further object of the present invention is to provide use of a composition containing phospholipase A2 as an active ingredient in the prevention or treatment of obesity. [Effects of the Invention]

[0012] The composition of the present invention for reducing inflammatory macrophages and preventing or treating obesity, which contains phospholipase A2, a major component of bee venom, as an active ingredient, is useful as a pharmaceutical composition. [Brief explanation of the drawings]

[0013] [Figure 1A] FIG. 1 is a schematic diagram showing experimental steps. [Figure 1B] Photographs showing the condition of mice in each group. [Figure 1C] 1 is a graph showing changes in body weight of mice in each group. [Figure 1D] 1 is a graph showing the food and drink intake of mice in each group. [Figure 1E]1 shows photographs showing white adipocytes in the epididymis and inguinal region for each group. [Figure 1F] 1 shows photographs showing changes in the weight of white adipocytes in the epididymis and inguinal region for each group. [Figure 2A] Photographs showing the condition of CD206-deficient mice in each group. [Figure 2B] 1 is a graph showing changes in body weight for each group. [Figure 2C] 1 shows photographs showing white adipocytes in the epididymis and inguinal region for each group. [Figure 2D] 1 is a graph showing changes in weight of white adipocytes in the epididymis and inguinal region for each group. [Figure 2E] 10 is a graph showing changes in body weight of CD206-deficient mice in each group treated with additional IgG. [Figure 3A] Photographs showing the livers of mice in each group. [Figure 3B] 1 shows photographs showing the liver weight of mice in each group. [Figure 3C] These are photographs of H&E stained livers of mice from each group. [Figure 3D] 1 is a graph showing lipid droplet accumulation in the liver of mice in each group. [Figure 3E] These are photographs of H&E stained kidneys of mice from each group. [Figure 3F] 1 is a graph showing the rate of kidney hypertrophy in mice of each group. [Figure 3G] 1 is a graph showing serum ALT, AST, BUN, and Crea values ​​for mice in each group. [Figure 3H] 1 is a graph showing the serum TG, GLU, HDL-C, and LDL-C levels of mice in each group. [Figure 3I] 1 is a graph showing serum insulin and leptin levels in mice of each group. [Figure 4A] Photographs showing macrophage infiltration in fat in each group. [Figure 4B]1 is a graph showing macrophage infiltration in fat in each group. [Figure 5A] 1 is a graph showing the levels of TNF-α, IL-1β, and IL-12a in each group by quantitative real-time PCR. [Figure 5B] 1 is a graph showing the levels of IL-4, CD206, and Yml in each group by quantitative real-time PCR. [Figure 5C] 1 is a graph showing the levels of PPARγ, C / EBPα, and UCP-1 in each group by quantitative real-time PCR. [Figure 6] This is a graph showing changes in body weight when PLA2 and melittin are administered to mice. DETAILED DESCRIPTION OF THE INVENTION

[0014] To achieve the above object, one embodiment of the present invention provides a pharmaceutical composition for preventing or treating obesity, which comprises phospholipase A2 as an active ingredient. The term "phospholipase A2 (PLA2)" as used herein refers to a protein enzyme. Phospholipase, which accounts for 12% of bee venom, is an enzyme that hydrolyzes phospholipids and is also known as lecithinase. Phospholipases are classified into A1, A2, B, C, and D depending on the position at which they decompose phospholipids.

[0015] The efficacy of PLA2 in improving obesity was not previously known and was discovered for the first time by the present inventors. More specifically, the PLA2 may be derived from the venom of the Western honeybee (Apis mellifera) (bvPLA2) or may be genetically modified (including genetic modification), but is not limited thereto.

[0016] It has been confirmed that PLA2 of the present invention has superior anti-obesity or therapeutic efficacy compared to melittin, a major component of bee venom. Furthermore, the PLA2 may act on mannose receptors, reducing the accumulation of lipid droplets and alleviating fatty liver or kidney damage, alleviating metabolic dysfunction by reducing aminotransferase, alanine aminotransferase, blood urea nitrogen, creatine, triglycerides (TG), glucose (GLU), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), insulin, and leptin, suppressing the infiltration of pro-inflammatory macrophages, regulating M1 / M2 by suppressing M1 macrophages or increasing M2 macrophages, or inhibiting lipid accumulation and lipogenesis, but is not limited thereto.

[0017] The term "obesity" as used herein refers to a state in which the body has an excess of fat. Specifically, obesity is defined as a body mass index (body weight (kg) divided by the square of height (m)) of 25 or greater. Obesity can be induced by a wide variety of factors, including genetic and environmental factors. Ultimately, obesity is induced by the accumulation of fat due to an energy imbalance caused by excessive intake of nutrients relative to energy expenditure over a long period of time. Obesity increases the likelihood of developing diabetes and hyperlipidemia, and increases the risk of developing sexual dysfunction, arthritis, and cardiovascular disease.

[0018] The term "prevention" as used herein may refer to any action of suppressing or delaying obesity by administering to an individual a composition containing the PLA2 of the present invention as an active ingredient. The term "treatment" as used herein may refer to any action of administering the composition of the present invention to an individual suspected of being obese to improve or benefit the symptoms of obesity.

[0019] In one embodiment of the present invention, PLA2 was administered to mice to confirm its efficacy in reducing adipose tissue and body weight (Figure 1), and in mice lacking mannose receptors, it was confirmed that the anti-obesity effect of bvPLA2 was due to the mannose receptor (Figure 2). H&E staining confirmed that it had the effect of reducing lipid droplet accumulation and alleviating fatty liver and kidney damage. Serum analysis of mice in each group confirmed that it had the effect of alleviating metabolic dysfunction (Figure 3). Histological examination confirmed that it had the effect of inhibiting macrophage infiltration, thereby improving obesity. M1 / M2 macrophage marker analysis confirmed that it had the effect of inhibiting or treating obesity by regulating the M1 / M2 macrophage ratio (Figure 4) and adipogenic factor expression (Figure 5). It was also confirmed that it had a superior anti-obesity effect to melittin, a major component of bee venom (Figure 6), thereby completing the present invention.

[0020] The pharmaceutical composition of the present invention may be used as a single preparation, or may be prepared as a combined preparation by further including a drug known to have an approved obesity treatment effect, and may be prepared in a unit dose form or in a multi-dose container by formulating the composition using a pharmaceutically acceptable carrier or excipient.

[0021] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the compound being injected. The type of carrier that can be used in the present invention is not particularly limited, and any pharmaceutically acceptable carrier commonly used in the art can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in combination. The carrier may also include a non-naturally occurring carrier.

[0022] If necessary, other common additives such as antioxidants, buffers and / or bacteriostatic agents may be added, and the composition may be formulated into injection forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders, lubricants, etc.

[0023] The pharmaceutical compositions of the present invention may also contain a pharmaceutically effective amount of PLA2. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and is generally 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, and more preferably 0.1 to 100 mg / kg, administered once or several times daily. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will vary depending on various factors, including the type and degree of response to be achieved, the specific composition, including whether other formulations are used, the patient's age, weight, general health, sex, and diet, the administration time, administration route, and excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical arts.

[0024] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or multiple times. It is important to administer the minimum amount that will provide the maximum effect without inducing side effects, taking into consideration all of the above factors, and this can be easily determined by those skilled in the art.

[0025] The term "administration" as used herein means introducing the pharmaceutical composition of the present invention into a patient by any suitable method, and the administration route of the composition of the present invention can be various routes, such as oral or parenteral, as long as it can reach the target tissue.

[0026] The administration route of the pharmaceutical composition according to the present invention is not particularly limited and may be any route commonly used in the art. Non-limiting examples of the administration route include oral administration and parenteral administration. The pharmaceutical composition according to the present invention may be prepared in various dosage forms according to the desired administration route.

[0027] The administration frequency of the composition of the present invention is not particularly limited, but it can be administered once a day or in divided doses several times a day. Another embodiment of the present invention provides a functional health food composition for preventing or improving obesity, which comprises phospholipase A2 as an active ingredient.

[0028] Yet another embodiment of the present invention provides a feed composition for preventing or ameliorating obesity, which comprises phospholipase A2 as an active ingredient. In the present invention, the above-mentioned "phospholipase A2", "obesity" and "prevention" are as defined above.

[0029] The term "amelioration" as used herein means any action that at least reduces the parameters associated with the condition being treated by administration of a composition containing PLA2 as an active ingredient, for example, the severity of symptoms.

[0030] The term "functional food" used in the present invention is the same as "food for special 24-8 health use (FoSHU)" and refers to a food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrients. The food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain useful effects in preventing or improving obesity.

[0031] The food composition of the present invention may further comprise a nutrient-acceptable carrier. The types of foods to which the PLA2-containing composition of the present invention can be added are not particularly limited, and include, for example, various beverages, gum, tea, vitamin complexes, dietary supplements, etc. The above food compositions can also contain other ingredients that do not interfere with the obesity prevention or improvement effects, and the types of ingredients are not particularly limited.

[0032] For example, like ordinary foods, various herbal extracts, nutrient-acceptable food supplements, natural carbohydrates, and the like may be contained as additional ingredients. The above-mentioned food supplement additives are added to produce health functional foods in various dosage forms, and can be appropriately selected and used by those skilled in the art. Examples include various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and fillers, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc., but the types are not limited to the above examples.

[0033] In this case, the content of the extract contained in the food is not particularly limited, but may be 0.01 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the food composition.

[0034] When the food is a beverage, it may be contained in a proportion of 1 to 30 g, preferably 3 to 20 g, per 100 ml. The composition may also contain additional ingredients commonly used in food compositions to improve odor, taste, or visual appearance. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu). It may also contain amino acids such as lysine, tryptophan, cysteine, and valine. Other food additives that can be added include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydronitrite, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium D-bitartrate, etc.), strengtheners, emulsifiers, thickeners (thickeners), coating agents, gum bases, antifoaming agents, solvents, and improvers. These additives are selected based on the type of food and used in appropriate amounts.

[0035] The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly used in the art. Moreover, unlike general medicines, it has the advantage of being made from food ingredients, which is free from side effects that can occur with long-term use of medicines, and is highly portable.

[0036] Yet another embodiment of the present invention provides a method for preventing or treating obesity, comprising administering to an individual a composition comprising the phospholipase A2 of the present invention as an active ingredient. The term "individual" as used herein may refer to any animal, including humans, that is or may be obese, including, but not limited to, mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar conditions, as well as humans.

[0037] The above-mentioned preventive or therapeutic method of the present invention may specifically comprise the step of administering a pharmaceutically effective amount of the above-mentioned composition to an individual who has become / is likely to become obese. The term "administration" as used herein means introducing the pharmaceutical composition of the present invention into a patient by any suitable method, and the administration route of the composition of the present invention can be various routes, such as oral or parenteral, as long as it can reach the target tissue.

[0038] Yet another embodiment of the present invention provides use of a composition comprising the phospholipase A2 of the present invention as an active ingredient in the prevention or treatment of obesity. The present invention will be described in more detail with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0039] Experimental Example 1: Increase in anti-inflammatory macrophages by bvPLA2 Bee venom-derived phospholipase A2 (bvPLA2, Apis mellifera), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0040] Experimental Example 2: Alleviation of inflammatory environment by bvPLA2 3T3-L1 preadipocytes were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in DMEM supplemented with 10% bovine calf serum (BCS), with the medium replaced every 2–3 days. The cells were then seeded into 96-well plates, and the medium was replaced with MDI medium (DMEM supplemented with IBMX 111 μg / mL, DEX 2 μM, and insulin 2 μg / mL) to induce adipogenesis. The differentiation medium was replaced every 2 days, and after 4 days of differentiation, fully differentiated cells were analyzed by Oil Red O staining.

[0041] Experimental Example 3: Oil Red O staining Differentiated 3T3-L1 cells were washed twice with PBS and then fixed in 4% paraformaldehyde buffer for 20 minutes. Oil Red O (Sigma-Aldrich St. Louis, MO, USA) was dissolved in 60% isopropanol and stained by treating the cells for 20 minutes at room temperature. After washing twice with distilled water, the OD value was measured at 510 nm.

[0042] Experimental Example 4: Experimental Animals Male C57BL / 6 mice (5 weeks old; body weight 18-20 g) and CD206- / - mice (B6.129P2-MRC1tm1Mnz / J) were purchased from Jackson Laboratory (Bar Harbor, ME). After 1 week of adaptation, they were fed a normal diet (ND; normal diet, 10 kcal%) or a high-fat diet (HFD; high-fat diet, 60 kcal%) for 15 weeks. From week 5 to week 11, mice were then injected intraperitoneally with PBS or bvPLA2 (0.5 mg / kg).

[0043] Experimental Example 5: Treg depletion Anti-mouse CD25 (clone: ​​PC61) was produced from a hybridoma obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and mice were injected intraperitoneally with anti-mouse CD25 or rat IgG (Sigma-Aldrich) every 3 days for 11 weeks.

[0044] Experimental Example 6: Quantitative real-time PCR Total RNA was isolated from white adipose tissue (WAT) samples using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Korea) and analyzed using Cyclescript reverse transcriptase (CSTR). cDNA was synthesized using transcriptase (Bioneer, Korea). The synthesized cDNA was subjected to quantitative real-time PCR (95°C for 15 seconds, 55°C for 10 seconds, and 72°C for 10 seconds) using the SensiFAST SYBR no-Rox kit (Bioline, Korea), and this experiment was repeated three times. The primer sequences used in the experiment are as follows: GAPDH: forward, 5'-CCCAGAAGACTGTGGATGG-3' reverse, 5'-CACATTGGGGGTAGGAACAC-3' TNF-α: forward, 5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3' reverse, 5'-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3' IL-1β: forward, 5'-GGACAGAATATCAACCAAACAAGTGATA-3' reverse, 5'-GTGTGCCGTCTTTCATTACACAG-3' IL-12a: forward, 5'-GCTCTAGACCCTGTGCCTTG-3' reverse, 5’-GAAGGCTTACCTGCATCAGC-3’ IL-4: forward, 5’-ACGAAGAACACCACAGAG-3’ reverse, 5’-TGATGTGGACTTGGACTC-3’ CD206 forward, 5’-AGTGGCAGGTGGCTTATG-3’ reverse, 5’-GGTTCAGGAGTTGTTGTG-3’ Ym1 forward, 5’-CATTCAGTCAGTTATCAGATTCC-3’ reverse, 5’-AGTGAGTAGCAGCCTTGG-3’[[ID=ID=19]] PPAR γ forward, 5’-GAAGGCTGAAGTCACCAAGC-3’[[ID=(23]] reverse, 5’-TCAGCCTTGCCAGAGTTTTT-3’ C / EBP α forward, 5’-TTACAACAGGCCAGGTTTCC-3’ reverse, 5’-CTCTGGGATGGATCGATTGT-3’ UCP-1 forward, 5’-TCTCAGCCGGCTTAATGACT-3’ reverse, 5’-GCTGGGTGTATGTGCCTTTT-3’ Experimental Example 7: H&E staining Liver and kidney tissues were fixed overnight in 4% paraformaldehyde, then dehydrated, embedded in paraffin, and sectioned at 4 μm thickness. The sections were deparaffinized in xylene and rehydrated in 100%, 90%, 80%, and 70% ethanol. The hydrated sections were washed with tap water and stained with hematoxylin solution for 5 minutes. They were then immersed in 1% acid alcohol and restained in eosin solution for 3 minutes. The xylene was then removed by dehydration in 70%, 80%, 90%, and 100% ethanol for mounting.

[0045] Experimental Example 8: Statistical Survey The experimental results were expressed as the mean and standard error, and statistical significance was analyzed through the Newman-Keuls test and one-way ANOVA using prism 5 software.

[0046] Example 1: Adipose tissue and weight loss efficacy of bvPLA2 The obesity-improving efficacy of bvPLA2 was evaluated through changes in adipose tissue and body weight in HFD-induced obese mice.

[0047] Specifically, C57BL / 6 mice were fed either a ND or HFD for 15 weeks. After 4 weeks of ND or HFD feeding, PLA2 or PBS was injected starting from week 5 (Figure 1A). Adipose tissue and body weight were then measured in the presence or absence of bvPLA2 injection.

[0048] As shown in Figure 1, the body weight of the HFD group was heavier than that of the ND group, and the HFD+PLA2 group showed a significant decrease in body weight compared to the HFD group not administered PLA2 (Figure 1B, C). Meanwhile, no changes in food intake were observed in any of the groups (Figure 1D). Furthermore, we confirmed that the weights of epididymal WAT and inguinal WAT significantly increased in the HFD group, whereas they significantly decreased in the HFD+PLA2 group (Figure 1E, F).

[0049] Through this, it was confirmed that administration of bvPLA2 does not affect food intake, and furthermore, significantly reduces body weight and adipose tissue weight, demonstrating its efficacy in improving obesity. Example 2: Effects of bvPLA2 on body weight and adipose tissue weight in CD206-deficient and T regulatory cell-deficient mice To confirm the immunomodulatory effects of bvPLA2, CD206-deficient (CD206- / -) mice lacking the mannose receptor were used.

[0050] More specifically, the body weight and adipose tissue weight of CD206- / - mice were measured using the same method as in the above experimental method (Fig. 1A). As a result, as can be seen in Figure 2, no significant changes in body weight or adipose tissue weight were observed between the HED and HFD+PLA2 groups (Figures 2A to 2D).

[0051] To confirm the relationship between bvPLA2 and T regulatory cells, mice were injected every 3 days with anti-mouse CD25 (clone PC61) to deplete T regulatory cells. Rat IgG was used as a control.

[0052] As a result, as shown in Figure 2, the IgG+PLA2-treated obesity-induced mice group showed a significant decrease in body weight compared to the IgG-only-treated obesity-induced mice group. Furthermore, the PC61+PLA2 group also showed a significant decrease in body weight compared to the PC61-treated group (Figure 2E).

[0053] This indicates that PLA2 exerts an anti-obesity effect through the mediation of the mannose receptor, but there was no difference between the IgG+PLA2 group and the PC61+PLA2 group, confirming that the anti-obesity effect of PLA2 is not linked to T regulatory cells mediated by PC61.

[0054] Example 3: Efficacy of bvPLA2 in reducing hepatotoxicity / nephrotoxicity Example 3-1: Efficacy of bvPLA2 in alleviating fatty liver and kidney damage To determine whether bvPLA2 attenuates HFD-induced hepatic steatosis and renal inflammation, liver and kidney tissues obtained from mice were stained with H&E.

[0055] As shown in Figures 3A-3D, the HFD group showed increased liver weight and size due to lipid droplet accumulation and hepatomegaly compared to the ND group. However, the HFD group treated with bvPLA2 suppressed these increases, while the ND+PLA2 group showed no effect.

[0056] We also examined the diameter of glomeruli, which indicate early symptoms of diabetic nephropathy due to kidney damage. As a result, as seen in Figures 3E-3F, renal hypertrophy due to lipid accumulation was found in the HFD group, but renal hypertrophy (increased glomerular diameter) was not found in the ND, ND+PLA2, and HFD+PLA2 groups (Figures 3E, F).

[0057] Through this, it was confirmed that bvPLA2 reduces lipid droplet accumulation and alleviates fatty liver and kidney damage. Example 3-2. Efficacy of bvPLA2 in alleviating metabolic dysfunction The effects of bvPLA2 on liver and kidney function were confirmed.

[0058] More specifically, serum values ​​of aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatine (Crea) were examined for each group.

[0059] As a result, as shown in Figure 3G, the levels of AST, ALT, and Crea were increased in the HED-treated group compared to the ND group, whereas the increased levels of AST, ALT, and Crea were reduced in the HFD group treated with bvPLA2.

[0060] In addition, to confirm the efficacy of bvPLA2 on metabolic syndrome, the levels of triglycerides (TG), glucose (GLU), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) were analyzed for each group.

[0061] As a result, as shown in Figure 3H, TG, GLU, HDL-C, and LDL-C levels were significantly higher in the HFD group compared to the ND group, whereas the increased levels were significantly lower in the HFD+PLA2 group.

[0062] In addition, plasma insulin and leptin levels were analyzed for each group. As a result, as shown in Figure 3I, plasma insulin and leptin levels were increased in the HFD group compared to the ND group, and the increased levels were significantly lower in the HED+PLA2 group (Figure 3I).

[0063] This confirmed that bvPLA2 alleviates metabolic dysfunction induced by HFD. Example 4: Effect of bvPLA2 on macrophage infiltration into adipose tissue Because obesity is characterized by the formation of crown-like structures (CLS) formed by proinflammatory macrophages surrounded by dead adipocytes in AT28, we performed H&E histological examination to determine whether bvPLA2 inhibits the infiltration of these macrophages into WAT.

[0064] As a result, as shown in Figure 4, almost no proinflammatory macrophage infiltration was found in WAT in the ND group, whereas CLS formation was significantly increased in the HFD group. On the other hand, macrophage infiltration and CLS formation were reduced in AT in the HFD+PLA2 group treated with bvPLA2.

[0065] Through this, it was confirmed that bvPLA2 suppresses the infiltration of pro-inflammatory macrophages, which cause obesity, and has the effect of alleviating obesity. Example 5: Efficacy of bvPLA2 to modulate M1 or M2 phenotype markers and adipogenic factors The phenotypic changes of macrophages were analyzed by quantitative real-time PCR.

[0066] More specifically, the expression of TNF-α, IL-1β, and IL-12a was measured to confirm the efficacy of bvPLA2 against M1-associated markers. As a result, as shown in Figure 5A, the expression of TNF-α, IL-1β, and IL-12a was increased in the HFD group compared to the ND group, but in the HFD+PLA2 group, the increased expression of TNF-α, IL-1β, and IL-12a was suppressed to levels similar to those in ND (Figure 5A).

[0067] In addition, the expression of M2-associated markers IL-4, CD206, and Yml was measured. As a result, as seen in Figure 5B, the expression of IL-4 and CD206 was reduced in the HFD group compared to the ND group, but the decreased expression was significantly increased in the HFD+PLA2 group (Figure 5B).

[0068] In addition, the expression of PPARγ, C / EBPα, and UCP-1, markers associated with lipid accumulation and adipogenesis, was measured. As a result, as shown in Figure 5C, the expression of PPARγ and C / EBPα was increased in the HFD group compared to the ND group, but this increase was suppressed in the HFD+PLA2 group. On the other hand, the expression of UCP-1 was significantly decreased in the HFD group compared to the ND group, but significantly increased in the HFD+PLA2 group.

[0069] Through this, we confirmed that HFD conditions increased M1-related markers, decreased M2-related markers, and also changed the expression of adipogenesis-related markers, and confirmed that bvPLA2 had the effect of suppressing and significantly improving these changes.

[0070] Example 6: Comparison with melatin in bee venom To identify which component of bee venom has superior anti-obesity effects, we compared melittin, the component most abundant in bee venom, with bvPLA2, a PLA2 derived from bee venom.

[0071] More specifically, 0.5 mg / kg each of PBS, melittin, and bvPLA2 were intraperitoneally administered to mice in the ND and HFD groups for 16 weeks, and the changes in body weight were compared daily.

[0072] As a result, as shown in Figure 6, the HFD group administered melittin showed a greater increase in body weight than the control group not administered melittin, while the HFD group administered PLA2 showed a decrease in body weight compared to the control group.

[0073] Through this, it was confirmed that melittin, the main component of bee venom, does not have anti-obesity effects, but that PLA2 components do. From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including within the meaning and scope of the claims below, and all modifications and variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

[Claim 1] The invention described in the specification.

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