KR102154235B1 - Composition for improving milk yield comprising C-C motif chemokine ligand 2(CCL2), and Pharmaceutical composition for preventing or treating mastitis comprising the same - Google Patents

Abstract

The present invention relates to a composition containing CCL2 (CC motif chemokine ligand 2) as an active ingredient, and more particularly, a composition for increasing the amount of acid produced containing CCL2 as an active ingredient, feed additive composition, mastitis containing CCL2 as an active ingredient It relates to a pharmaceutical composition and a food composition for prevention or treatment.
The composition comprising CCL2 according to the present invention as an active ingredient is made from proteins produced in the body, so compared to chemical compounds, toxicity or side effects in the body can be minimized, and the expression of proteins in the PI3K/AKT and MAPK signaling pathways is activated. It has the effect of improving the proliferation of breast epithelial cells, reducing the expression of endoplasmic reticulum stress-inducing proteins, and alleviating the inflammatory response. Compositions for increasing milk production, feed additive compositions, pharmaceuticals and functionalities that can prevent and treat mastitis It can be usefully used in food-related fields.

Description

Composition for improving milk yield comprising CCL2 as an active ingredient, and a pharmaceutical composition for preventing or treating mastitis {Composition for improving milk yield comprising C-C motif chemokine ligand 2 (CCL2), and Pharmaceutical composition for preventing or treating mastitis comprising the same}

The present invention relates to a composition containing CCL2 (CC motif chemokine ligand 2) as an active ingredient, and more particularly, a composition for increasing the amount of acid produced containing CCL2 as an active ingredient, feed additive composition, mastitis containing CCL2 as an active ingredient It relates to a pharmaceutical composition and a food composition for prevention or treatment.

Normal milk production is a problem directly related to human health as well as breeding and growth of offspring of cattle (Nissen A et al ., Jopurnal of Dairy Science, 2013). In addition, breast epithelial cells are the most important cells involved in milk production as a functional unit forming the mammary gland (Arendt LM and Kuperwasser C, Mammary Gland Biology and Neoplasia, 2015). Therefore, maintenance of breast epithelial cell proliferation is essential for improving milk production, and various growth factors and cytokines are known to be involved in the proliferation of breast epithelial cells (Inman JL et al ., Development, 2015).

On the other hand, when an inflammatory reaction occurs in the cow's mammary gland continuously and mastitis occurs, casein and calcium in the produced milk decrease, which is a factor that degrades milk quality. Relatedly, ephrin B2 and its target receptor have been reported to be expressed in mammary epithelial cells and induce malignancy in mammary cells (Vaught D et al ., Molecular Biology of the Cell, 2009).

However, until now, the function of the chemokine CCL2 in relation to the proliferation or inflammatory response of breast epithelial cells is unknown.

Under the above-described technical background, the present inventors have made diligent efforts to develop a substance capable of treating mastitis by improving the proliferation of breast epithelial cells, increasing milk production, and alleviating the inflammatory response. The present invention was completed by activating the pathway to improve the proliferation of breast epithelial cells, reducing the expression of endoplasmic reticulum stress-inducing proteins, and alleviating the inflammatory response.

An object of the present invention is to provide a composition for increasing the amount of acid oil containing CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

Another object of the present invention is to provide a feed additive composition comprising CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating mastitis comprising CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving mastitis comprising CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

Another object of the present invention is to provide a method for improving the proliferation ability of breast epithelial cells by using a composition containing CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

The present invention provides a composition for increasing the amount of acid oil containing CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

The present invention also provides a feed additive composition comprising CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating mastitis comprising CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

The present invention also provides a food composition for preventing or improving mastitis comprising CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

The present invention also provides a method of improving the proliferation ability of breast epithelial cells by using a composition containing CCL2 (C-C motif chemokine ligand 2) as an active ingredient.

The composition comprising CCL2 according to the present invention as an active ingredient is made from proteins produced in the body, so compared to chemical compounds, toxicity or side effects in the body can be minimized, and the expression of proteins in the PI3K/AKT and MAPK signaling pathways is activated. It has the effect of improving the proliferation of breast epithelial cells, reducing the expression of endoplasmic reticulum stress-inducing proteins, and alleviating the inflammatory response. Compositions for increasing milk production, feed additive compositions, pharmaceuticals and functionalities that can prevent and treat mastitis It can be usefully used in food-related fields.

1 shows the results of analyzing the effect of CCL2 on the proliferation of bovine breast epithelial cells.
Figure 2 shows the results of analysis of the cell cycle changes in bovine breast epithelial cells according to CCL2 treatment.
Figure 3 shows the results of measuring PI3K/AKT and MAPK signaling pathway changes in bovine breast epithelial cells according to CCL2 treatment.
FIG. 4 shows the results of analysis of changes in the phosphorylation pattern of signaling proteins and proliferative capacity of breast epithelial cells by a mixture composition of AKT, ERK1/2, and JNK target inhibitors and CCL2.
5 shows the results of analyzing the effect of CCL2 treatment on endoplasmic reticulum stress in bovine mammary epithelial cells.
6 shows the results of analyzing the anti-inflammatory effect of CCL2 treatment in bovine breast epithelial cells.
7 shows the mechanism of action of CCL2 related to proliferation of bovine mammary epithelial cells, relieving endoplasmic reticulum stress and anti-inflammatory effects.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, the mechanism of action of CCL2 related to proliferation of breast epithelial cells, relieving endoplasmic reticulum stress and anti-inflammatory effects was revealed (FIG. 7).

The present invention provides a composition for increasing acid flow, comprising CCL2 as an active ingredient.

The composition includes all of the pharmaceutical composition, food composition, and feed additive composition.

The term "composition" as used in the present invention is considered to include not only products containing specific ingredients, but also any products made directly or indirectly by the combination of specific ingredients.

In the present invention, the mammary epithelial cells may be cells derived from mammals, and the mammals are rodents (eg, mice, rats, hamsters, gerbils and guinea pigs), right titles (eg, Cattle, sheep, pigs, goats, deer, giraffes and antelopes), base titles (e.g. horses, donkeys, rhinos and tapirs), carnivores (e.g. dogs, cats, tigers, wolves, foxes, lions, cheetahs) , Leopards, raccoons, badgers, mountain lions, jaguars and wildcats), order of rabbits (rabbits and crying rabbits), order of carnivores (such as hedgehogs, moles, and solenoidons) and primates (such as chimpanzees, orangutans, gorillas) , Bonobono, Japanese monkey, rhesus monkey).

In the present invention, the pharmaceutical composition may be characterized in that it contains a pharmaceutically acceptable carrier, wherein the carrier is an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, electrolyte , Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and wool paper. have.

In the present invention, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual or topical administration. It can be characterized in that it further contains any one or more auxiliary agents selected from the group consisting of buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity modifiers, preservatives, thickeners and viscosity modifiers, solutions, suspensions, emulsions , It may be characterized by having a formulation selected from the group consisting of gels and powders.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, weight, age, sex, mode of administration and time of administration of the patient, and generally skilled physicians are effective in the desired treatment or prevention. The dosage can be easily determined.

In the present invention, the health functional food refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No. 6722, and contains nutrients for the structure and function of the human body. It refers to food consumed for the purpose of controlling or obtaining beneficial effects for health purposes such as physiological effects.

The health functional food composition of the present invention may be formulated into a formulation of a general health functional food known in the art, and granules, tablets, pills, suspensions, emulsions, syrups, gums, teas, jellies, various beverages, and drinks. , Alcoholic beverages, etc., and there is no particular limitation on the kind of the health functional food.

The health functional food composition of the present invention is in any herbal form suitable for administration to an animal body, including the human body, more specifically any form conventional for oral administration, for example, food or feed, additives and adjuvants for food or feed, It may be in a solid form such as fortified food or feed, tablets, pills, granules, capsules and foam formulations, or in liquid form such as solutions, suspensions, emulsions, beverages, pastes, etc., and nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, It may contain a preservative or the like, and these ingredients may be used independently or in combination.

In the present invention, the feed additive composition may be a feed additive composition for livestock, and the livestock is not limited thereto, but, for example, cattle, horses, sheep, pigs, deer, chickens, ducks, goose, turkey, quail, ostrich , Pheasant, may be other animals prescribed by Ordinance of the Ministry of Food, Agriculture, Forestry and Livestock, and may preferably be a ruminant. The ruminant is also referred to as a ruminant animal, and is not limited thereto, but refers to, for example, a camelid, a baby deer, a deer, a giraffe, and a bovine.

In the present invention, the ruminant may preferably be a bovine animal, more preferably a cow, and even more preferably a conversion cow.

By feeding the feed additive composition for livestock of the present invention to livestock, it is characterized by improving milk production, oil quality, and alleviating inflammation occurring in the mammary gland, but is not limited thereto.

According to the present invention, the CCL2 may be characterized in that it activates PI3K/AKT and MAPK signaling mechanisms.

According to the present invention, the CCL2 may be characterized by reducing the expression of the endoplasmic reticulum stress-inducing protein.

In this case, the endoplasmic reticulum stress inducing protein may be at least one selected from the group consisting of IRE1α, ATF6α, PERK, eIF2α, and GADD153.

In addition, the present invention provides a composition for preventing, treating or improving mastitis comprising CCL2 as an active ingredient.

The composition includes both a pharmaceutical composition or a food composition.

Since the pharmaceutical composition or food composition contains a pharmaceutical preparation or food preparation containing the above-described CCL2 as an active ingredient, the contents overlapping with the composition of the present invention described above are excessive in the present specification due to the description of the overlapped contents. The description is omitted to avoid complexity.

The present invention also provides a method of improving the proliferation ability of breast epithelial cells by using the composition containing the CCL2 as an active ingredient.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Example

<Experiment method>

Experimental animals and cell culture

Bovine mammary epithelial (MAC-T) was provided by Dr. Hong-Koo Lee's laboratory at Konkuk University (Konkuk University, Republic of Korea), and was immortalized by introducing SV40 large T antibody to primary cultured bovine mammary cells. Bovine breast epithelial cells have a gravel shape when cultured in a plastic substrate, and the bovine breast epithelial cells used in the present invention were passaged 25-30 times. For monolayer culture of bovine breast epithelial cells, 10% fetal bovine serum (FBS) was mixed with DMEM medium (Dulbecco's Modified Eagle's medium, HyClone, Logan, UT). For the experiment, bovine breast epithelial cells were incubated in starvation for 24 hours, and then treated with various concentrations of CCL2. All experiments were repeated three times.

Experiment material

Recombinant CCL2 (catalog number: 279-MC/CF) was purchased from R&D Systems and used, and tunicamycin (catalog number: T7765) and LPS (catalog number: L3491) were purchased from sigma. ^{(: 4060 Ser 473, catalog number} ), P70S6K (Thr 421 / Ser 424, catalog number: 9204), S6 (Ser 235/236, catalog number: 2211), phospho-AKT in order to determine the signaling mechanisms by CCL2 ERK1/2 (Thr ²⁰² /Tyr ²⁰⁴ , catalog number: 9101), JNK (Thr ¹⁸³ /Tyr ¹⁸⁵ , catalog number: 4668), P38 (Thr ¹⁸⁰ /Tyr ¹⁸² , catalog number: 4511), cyclin D1 (Thr ²⁸⁶ , catalog number: 3300), eIF2α (Ser ⁵¹ , catalog number: 3398) protein and total-AKT (catalog number: 9272), P70S6K (catalog number: 9202), S6 (catalog number: 2217), ERK1/2 (catalog number : 4695), JNK (catalog number: 9252), P38 (catalog number: 9212), cyclin D1 (catalog number: 2922), eIF2α (catalog number: 5324), IRE1α (catalog number: 3294) antibodies to the cell cells Purchased from Signaling Techonology. phospho-PERK (Thr ⁹⁸¹ , catalog number: sc-32577), total-PERK (catalog number: sc-13073), ATF6α (catalog number: sc-166659), GRP78 (catalog number: sc-13968), GADD153 (catalog number: sc-7351), PCNA (catalog number: sc-56) protein was purchased from Santa Cruz Biotechnology. In addition, in order to investigate the effect of inhibiting the target signaling process, the PI3K/AKT inhibitor wortmannin (catalog number: 9951) was obtained from Cell Signaling Technology, the ERK1/2 inhibitor U0126 (catalog number: EI282) and the JNK inhibitor SP600125 (catalog number: EI305) was purchased and used from Enzo Life Science.

BrdU Cell proliferation ability analysis

To confirm the effect of CCL2 on the proliferation capacity of bovine mammary epithelial cells, 5×10 ³ cells and 100 μl of medium were dispensed into 96 wells, and the cells were cultured under FBS starvation conditions. CCL2 was then treated dose-dependently (0, 1, 5, 10, 25, 50, 100, 150 ng), tunicamycin (0.25 μg/ml), wortmannin (1 μM), U0126 (20 μM), SP600125 ( 20 μM) were treated together and incubated for 48 hours. Then, the experiment was performed according to the manufacturer's manual using the BrdU kit (Cat No: 1167229001, Roche) (https:// www.sigmaaldrich.com/ content/ dam/ sigma-aldrich/ docs/ Roche/Bulletin/ 1/ 11647229001bul .pdf). After 48 hours of incubation, 10 μM BrdU was additionally added to each well and incubated in a 37° C./5% CO ₂ incubator for 2 hours. The MAC-T cells were labeled with BrdU and the cells were fixed. The anti-BrdU-POD solution was incubated at room temperature for 90 minutes, followed by washing three times. Finally, the cells were reacted with 100 μL of 3,3',5,5'-tetramethylbenzidine substrate, and the absorbance within 370 nm and 492 nm was measured using an ELISA reader to analyze the cell proliferation ability. All experiments were repeated three times.

Immunofluorescence

3×10 ⁴ bovine breast epithelial cells were dispensed and cultured in a confocal dish (catalog number: 100350, SPL Life Science, Republic of Korea) with 300 μl of 10% FBS-containing DMEM medium, followed by 24 hours FBS starvation. After further incubation with CCL2 (50 ng/mL) for 24 hours, the cells were fixed with methanol for 10 minutes, and PCNA and cyclin D1 antibodies diluted 1:100 were treated, and mouse IgG or rabbit IgG was used as a control group. Treated and incubated at 4° C. for 16 hours. After that, after washing twice with PBS containing 0.1% BSA (bovine serum albumin), the secondary antibody is goat anti-mouse IgG Alexa 488 (catalog number: A11017, Invitrogen, Carlsbad, CA) or goat anti-rabbit. IgG Alexa 488 (catalog number: A-11008, Invitrogen, Carlsbad, CA, USA) was diluted 1:200 in antibody dilution buffer and incubated for 1 hour at room temperature. After washing bovine mammary epithelial cells with 0.1% BSA-PBS, DAPI staining was additionally performed so that the nuclei as well as target proteins in bovine mammary epithelial cells could be observed simultaneously. After the end of the experiment, the cells were observed and photographed using an LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope. All experiments were repeated three times. Relative fluorescence intensity was quantified by dividing green fluorescence by DAPI fluorescence intensity using MetaMorph software (Molecular Devices, Sunnyvale, CA).

Cell cycle analysis

In order to confirm the cell cycle change pattern of bovine mammary epithelial cells by CCL2, 5×10 ⁵ cells were cultured in 6 wells and further cultured in FBS starvation for 24 hours when the cells were filled in a 70-80% culture dish. Thereafter, CCL2 was dose-dependently treated (0, 5, 10, 25, and 50 ng/mL) and cultured in a 37°C/5% CO ₂ incubator for 48 hours. Thereafter, the cells were removed from the culture dish using trypsin, washed with 0.1% BSA/PBS, and the cells were fixed in 70% Ethanol for 16 hours. Thereafter, washing with 0.1% BSA/PBS and PI staining was performed under RNase A (Sigma) treatment. The stained solution was transferred to the FACS tube and the fluorescence intensity was analyzed using a flow cytometer to analyze the cell cycle.

Protein expression analysis ( Western Blot )

Bovine breast epithelial cells were treated with a mixture of CCL2 or a cell signaling inhibitor, and then total protein was extracted from MAC-T cells, and the protein was quantified by Bradford protein assay (Bio-Rad, Hercules, CA, USA). Thereafter, the extracted protein was denatured at 95°C for 5 minutes, electrophoresis was performed using 10% SDS/PAGE gel, transferred to a nitrocellulose membrane, and the primary antibody and the secondary antibody were incubated sequentially, followed by chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) reagent, and the ChemiDoc EQ system and Quantity One software (Bio-Rad) instrument were used to analyze the expression of the target protein. Secondary antibody is a goat anti-rabbit IgG polyclonal antibody (Seracare, catalog number: 474-1506) or goat anti-mouse IgG polyclonal antibody (Seracare, catalog number: 474-1806) diluted 1:2500 at room temperature. Incubated for hours. Total protein expression was used to normalize the expression of phosphorylated proteins. All experiments were repeated three times.

mRNA Expression analysis ( qPCR )

After treating bovine breast epithelial cells with CCL2 and LPS, total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's manual (https:// www.qiagen.com/ se/ resources/ resourcedetail ?id = 14e7cf6e -521a -4cf7 -8cbc -bf9f6fa33e24 & lang = en). RNA concentration and purity were confirmed by spectrometry and denaturing agarose gel electrophoresis. Then, 1 μg of RNA was synthesized cDNA using AccuPower RT PreMix (Bioneer, Daejeon, Korea). Primer for amplifying IL8 , IL6 and NFKB1 genes was designed using Primer 3 software (ver 4.0.0, Free Software Foundation Inc., Boston, MA) based on the GenBank database. Gene expression was measured using SYBR Green (Sigma) and StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). PCR conditions were amplified 40 times after incubation at 95° C. for 3 minutes, then 95° C. 30 seconds, 60° C. 30 seconds, and 72° C. 3 minutes, and normalized based on GAPDH expression level. Relative gene expression was normalized using the 2 ^{- ΔΔCT} method. All experiments were repeated three times.

Statistical analysis

The mean and standard error were calculated using SAS (statistical analysis system) statistical program, and one-way ANOVA was performed. A significance test was conducted at the P <0.05 level. Protein expression in Western blot was normalized to expression of total protein or α-tubulin. Students t tests were used to compare the mean between the two samples.

<Results and Discussion>

CCL2 Bovine Breast epithelial cell Effect on proliferative power

In order to analyze the change in proliferative power in bovine mammary epithelial cells by CCL2, the BrdU incorporation assay was performed by treating CCL2 by concentration (0, 1, 5, 10, 25, 50, 100, 150 ng/mL). As a result of the measurement, it was confirmed that the cell proliferation capacity was increased in a dose-dependent manner to 50 ng/mL. Compared to the control group, 10, 25, 50, 100, 150 ng/mL of CCL2 treatment, respectively, 121.8% ( P <0.05), 132.6% ( P <0.01), 145.7% ( P <0.01), 146.5% ( P < 0.05), 120.1% ( P <0.001) cell proliferation was increased. Based on this experiment, 50 ng/mL was set as the optimal concentration of CCL2 used in the subsequent experiment. In addition, as a result of confirming the expression of PCNA, a protein essential for cell proliferation, through immunofluorescence staining, it was confirmed that the expression of PCNA in bovine mammary epithelial cells significantly increased (804.6%, P <0.001) when CCL2 (50 ng/mL) was treated. (Fig. 1). From these results, it was confirmed that CCL2 promotes the proliferation of bovine breast epithelial cells.

CCL2 Bovine Breast epithelial cell Effects on the cell cycle

Propidium iodide (PI) staining was performed to analyze the effect of CCL2 on the cell cycle of bovine mammary epithelial cells, and the ratio of G2/M phase increased in a dose-dependent manner (0, 5, 10, 25, 50 ng/mL). Through this, it was confirmed that CCL2 can promote cell cycle progression. Without CCL2 treatment, the ratio of G2/M phase was 3.90%, increased to 5.78% ( P <0.05) in response to 25 ng/mL CCL2, and 7.10% ( P <0.05) when exposed to 50 ng/mL CCL2. 0.01). In addition, the expression of Cyclin D1, a representative protein that progresses the cell cycle, was also significantly increased (590.1%, P <0.001) when treated with CCL2 (50 ng/mL), which was confirmed through immunofluorescence staining (FIG. 2). From these results, it was confirmed that CCL2 promotes cell cycle progression of bovine breast epithelial cells.

CCL2 Cattle according to dose-dependent administration Breast epithelial cells Analysis of changes before my signaling mechanism

After CCL2 was dose-dependently treated (0, 5, 10, 25, 50 ng/mL) into bovine mammary epithelial cells, intracellular proteins were extracted to analyze phosphorylation of PI3K/AKT and MAPK signaling proteins involved in cell proliferation. As a result, it was confirmed that phosphorylation of all proteins such as AKT, P70S6K, S6, ERK1/2, JNK, and P38 increased, and the phosphorylation of Cyclin D1 protein was also increased (FIG. 3 ). In the case of AKT, phosphorylation increased by 3.27 times ( P <0.01), 13.12 times ( P <0.001), and 29.39 times ( P <0.001), respectively, in response to 10, 25, and 50 ng/mL of CCL2. Phosphorylation of P70S6K increased by 4.18 fold ( P <0.01) and 4.40 fold ( P <0.001), respectively, by 25 and 50 ng/mL of CCL2 treatment. In addition, phosphorylation of S6 increased by 5.56-fold ( P <0.001), 8.97-fold ( P <0.001), and 14.00-fold ( P <0.001), respectively, by 10, 25, and 50 ng/mL of CCL2 treatment. In addition, 50 ng/mL of CCL2 increased phosphorylation of ERK1/2 by 12.59 times ( P <0.001), and 10 and 25 ng/mL of CCL2 by 8.40 times ( P <0.001) and 12.15 times ( P < 0.001) increased phosphorylation of ERK1/2. In addition, phosphorylation of JNK increased by 1.94 times ( P <0.01), 2.50 times ( P <0.001), and 2.53 times ( P <0.01), respectively, by 10, 25, and 50 ng/mL of CCL2. Phosphorylation of P38 was increased by 3.98-fold ( P <0.01) and 4.41-fold ( P <0.01), respectively, by 25 and 50 ng/mL of CCL2 treatment. The phosphorylation of CyclinD1 was 2.81 times ( P <0.01), 2.75 times ( P <0.05), 12.40 times ( P <0.001), 18.29 times ( P <0.001) by CCL2 at 5, 10, 25 and 50 ng/mL, respectively. Increased. In addition, by treatment with CCL2 (50 ng/mL) and selective inhibitors for signaling proteins, Wortmannin (AKT inhibitor, 1 μM), U0126 (ERK1/2 inhibitor, 20 μM), and SP600125 (JNK inhibitor, 20 μM). As a result of confirming the cell proliferation ability, it was confirmed that the cell proliferation ability decreased when the treatment was combined with a signaling protein inhibitor. Wormannin, U0126, and SP600125 reduced the proliferative power of bovine mammary epithelial cells increased by 29.7% ( P <0.01), 38.7% ( P <0.01), and 29.9% ( P <0.05) CCL2, respectively. Cell proliferation through a molecular mechanism based on the complex cross-effects of each protein when CCL2 was treated in combination with a signaling protein inhibitor, and the proteins were extracted and the activation of PI3K/AKT and MAPK signaling pathways was analyzed through Western blot analysis. Was found to increase (Fig. 4). Phosphorylation of AKT, P70S6K, and S6 proteins corresponding to the PI3K/AKT signaling pathway was decreased by the combination treatment of Wortmannin and CCL2. In addition, pretreatment with U0126 or SP600125 increased phosphorylation of AKT compared to treatment with CCL2 alone. However, the phosphorylation of P70S6K was not changed by other inhibitors except Wortmannin. U0126 treatment reduced the phosphorylation of S6 increased by CCL2, but SP600125 did not significantly affect the S6 protein. The phosphorylation of ERK1/2 increased by CCL2 was further increased by treatment with Wortmannin or SP600125. In addition, the phosphorylation of JNK increased by CCL2 was further increased by Wortmannin or U0126 treatment. Phosphorylation of P38 was further increased when Wormannin and CCL2 were co-treated compared to CCL2 alone. The phosphorylation of CyclinD1 was significantly reduced when U0126 or SP600125 and CCL2 were co-treated compared to CCL2 alone. Through these results, it was confirmed that the cross-effect of the signaling pathway regulated by CCL2 is involved in the proliferation of bovine mammary epithelial cells.

CCL2 Bovine Breast epithelial cells Analysis of the effect of relieving stress in endoplasmic reticulum

As a result of confirming the change in cell proliferation by co-treating with Tunicamycin (0.25 μg/mL) and CCL2 (50 ng/mL), which induces endoplasmic reticulum stress in bovine mammary epithelial cells, cell proliferation decreased by Tunicamycin was recovered by CCL2 and IRE1α, Expression or activity of endoplasmic reticulum stress-related proteins such as ATF6α, PERK, eIF2α, GRP78, and GADD153 decreased during CCL2 treatment (FIG. 5 ). When Tunicamycin was treated with bovine mammary epithelial cells alone, cell proliferation decreased by about 30.2% ( P <0.01), and when tunicamycin and CCL2 were co-treated, cell proliferation decreased by about 16.9% ( P <0.05). In addition, CCL2 reduced the expression of endoplasmic reticulum stress sensor proteins IRE1α (82.5%, P <0.01), ATF6α (45.9%, P <0.05) and phosphorylation of PERK (93.0%, P <0.001) compared with tunicamycin alone. Made it. In addition, the activation of eIF2α, a subprotein of PERK, decreased by 96.7% ( P <0.001) when combined with CCL2 compared to tunicamycin alone. In addition, the expressions of GRP78 and GADD153 proteins were also decreased by 50.3% ( P <0.01) and 54.6% ( P <0.01), respectively, compared to tunicamycin alone when CCL2 was treated. From these results, it was confirmed that CCL2 reduced endoplasmic reticulum stress in bovine mammary epithelial cells.

CCL2 Bovine Breast epithelial cells Analysis of my anti-inflammatory effects

In order to analyze the effect of CCL2 on regulating the inflammatory response in bovine mammary epithelial cells, mRNA levels of inflammatory cytokines IL8 and IL6 and NFκB, a factor that regulates transcription of inflammatory cytokines, were treated in combination with lipopolysaccharide (LPS) and CCL2. As a result of analysis, the expression of the gene increased by LPS was decreased by CCL2. LPS increased IL8 and IL6 mRNA levels in a dose-dependent manner, and expressions increased by 7.03 fold ( P <0.001) and 3.27 fold ( P <0.001), respectively, by 5 μg/mL of LPS. However, CCL2 significantly decreased the expression of IL8 and IL6 increased by LPS. In addition, the expression of NFκB increased by LPS was significantly decreased by treatment with CCL2. The proliferative power of bovine mammary epithelial cells decreased by 15.7% ( P <0.01), 22.9% ( P <0.05), and 31.1% ( P <0.01), respectively, by LPS treatment with 0.5, 1, and 5 μg/mL LPS. In addition, it was found that the proliferative capacity of bovine mammary epithelial cells decreased by LPS was recovered again when treatment with CCL2 was combined, and this recovery effect did not appear when treatment with the addition of a signaling protein inhibitor (FIG. 6 ). CCL2 treatment at 25 and 50 ng/mL increased cell proliferation by 137.5% ( P <0.05) and 150.8% ( P <0.01), respectively, compared with LPS treatment alone. On the other hand, the additional treatment of Wortmannin (1 μM), U0126 (20 μM), and SP600125 (20 μM) was 37.4% ( P <0.001), 35.9% ( P <0.001), respectively, compared to the combination treatment group of CCL2 and LPS, respectively. It decreased by 44.2% ( P <0.001). These results suggest that the anti-inflammatory effect of CCL2 can inhibit the anti-proliferative effect induced by LPS, which will mediate the PI3K/AKT and MAPK signaling pathways.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

A composition for increasing acid flow containing CCL2 (C-C motif chemokine ligand 2) as an active ingredient. The method of claim 1,
The CCL2 composition for increasing acid flow, characterized in that to activate PI3K/AKT and MAPK signaling mechanisms. The method of claim 1,
The CCL2 composition for increasing acid flow, characterized in that to reduce the expression of endoplasmic reticulum stress-inducing protein. The method of claim 3,
The endoplasmic reticulum stress-inducing protein is at least one selected from the group consisting of IRE1α, ATF6α, PERK, eIF2α, and GADD153. delete delete delete delete

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