KR102550603B1 - Supplement for reducing fever comprising tauroursodeoxycholic acid as an active ingredient - Google Patents

Abstract

The present invention relates to an antipyretic supplement comprising taurourusodeoxycholic acid as an active ingredient. Specifically, the tauroursodioxycholic acid of the present invention significantly suppresses the expression of UPR-related genes, which are increased by high heat, and restores the expression of genes related to embryonic differentiation, which were suppressed by high heat, to a normal level, resulting in high heat stress. By inhibiting the abnormal differentiation of embryos by, it can be usefully used as an antipyretic aid for mothers, a feed additive for mammals, or a medium composition for in vitro culture of embryos.

Description

Antipyretic supplement containing tauroursodeoxycholic acid as an active ingredient

The present invention relates to an antipyretic supplement comprising taurourusodeoxycholic acid as an active ingredient.

Hyperthermia or fever refers to a temporary rise in body temperature as a complex physiological response of the whole body that acts as a defense mechanism against cellular stress caused by infection or injury. At this time, cells regulate their transcription and translation mechanisms in response to an increase in body temperature for various reasons. In addition, high heat alters the structure of intracellular enzymes through protein folding or denatures mature proteins, resulting in misfolding or unfolding proteins that extremely disrupt intracellular protein homeostasis. accumulate When suddenly exposed to an extreme environment such as heat shock, cells develop a 'heat stress response', including the heat shock response (HSR) mediated by the transcription factor HSF1 (heat shock factor 1). A self-defense mechanism known as 'response' begins in the cytoplasm. Under normal conditions, the HSF1 protein constitutes a complex that includes the chaperone heat shock protein 90 (HSP90) that keeps HSF1 in an inactive state. However, when exposed to heat, HSF1 is released from the complex as the accumulated unfolded protein competes with HSF1 for binding to the binding site of HSP90. Released HSF1 induces transcription of heat shock genes and is involved in maintaining intracellular homeostasis until heat stress is reduced.

Hyperthermia adversely affects mammals by modulating temperature-dependent mechanisms involved in physiological processes, cell division, proliferation and differentiation, as well as progressive development from the embryonic stage, embryonic, fetal to postnatal stages. Therefore, high fever also affects the formation and development of tissues. In addition, high fever causes extensive structural and functional defects in the body, including the central nervous system, and causes severe damage to cells and tissues. In particular, the developing embryo or fetus has limited thermoregulatory capacity, and exposure to extreme heat stress directly affects cell migration, proliferation, differentiation, and death, potentially leading to abnormal embryonic development, growth retardation, or prenatal death. . Recent studies have reported that hyperthermia causes defects in neural tube and craniofacial development, along with almost all organs including the heart. In addition, when exposed to higher heat or when exposed to high heat for a long time, the above abnormalities occur and the risk of miscarriage increases.

In this regard, Korean Patent Registration No. 10-1417905 relates to a feed additive for reducing high-temperature stress of ruminants by controlling the heat of fermentation in the rumen and a feed mixed therewith, by controlling the heat of fermentation in the rumen of ruminants such as cattle. Disclosed is a feed additive capable of reducing high-temperature stress, reducing heat generation and gas generation in the rumen, and ultimately improving productivity of ruminants.

Republic of Korea Patent Registration No. 10-1417905

An object of the present invention is to provide a use of taurourusodeoxycholic acid.

In order to achieve the above object, the present invention provides an antipyretic supplement comprising tauroursodeoxycholic acid (TUDCA) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a feed additive for reducing high-temperature stress containing TUDCA or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides a medium composition for in vitro culture of embryos comprising TUDCA or a pharmaceutically acceptable salt thereof as an active ingredient.

The taurourusodeoxycholic acid of the present invention significantly inhibits the expression of UPR-related genes, which are increased by high heat, and restores the expression of genes related to the differentiation of embryos suppressed by high heat to normal levels, so that embryos caused by high heat stress By inhibiting the abnormal differentiation of, it can be usefully used as an antipyretic aid for mothers, a feed additive for mammals, or a medium composition for in vitro culture of embryos.

1 is a view showing the results of observing inhibition of differentiation of embryoid bodies exposed to high heat for various times in one embodiment of the present invention with a phase contrast microscope.
Figure 2 is a diagram showing the results of confirming expression changes of UPR-related genes in embryoid bodies exposed to high heat for various times in one embodiment of the present invention.
3 is a diagram showing the results of confirming expression changes of stem marker genes in embryoid bodies exposed to high heat for various times in one embodiment of the present invention (black bar: 37° C., gray bar: 42° C.).
4 is a diagram showing the results of confirming expression changes of differentiation marker genes in embryoid bodies exposed to high heat for various times in one embodiment of the present invention (black bar: 37°C, gray bar: 42°C).
5 to 7 are diagrams showing the results of confirming expression changes of UPR-related genes in embryoid bodies exposed to high heat for various times in one embodiment of the present invention (black bar: 37°C, gray bar: 42°C).
8 is a view showing the result of observing the degree of differentiation of an embryoid body after exposure to high heat in one embodiment of the present invention with a phase contrast microscope.
9 to 12 are heat map results confirming expression changes of genes related to embryoid body differentiation after exposure to high heat in one embodiment of the present invention.
13 is a view showing the result of confirming the expression change of the stem marker gene of the embryoid body after exposure to high heat in one embodiment of the present invention (black bar: 37°C, gray bar: 42°C).
14 to 16 are graphs showing changes in expression of genes related to trigerminal differentiation of embryoid bodies after exposure to high heat in one embodiment of the present invention (black bar: 37°C, gray bar: 42°C).
17 and 18 are diagrams showing the results confirming that the expression of UPR-related genes increased by TUDCA in the embryoid body by exposure to high heat in one embodiment of the present invention (black bar: 37° C., gray bar: 42° C.).
19 to 21 are heat map results confirming that TUDCA decreased the expression of genes related to trigerminal differentiation, which increased in embryoid bodies by exposure to high heat in one embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides an antipyretic supplement comprising tauroursodeoxycholic acid (TUDCA) or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term 'tauroursodeoxycholic acid (TUDCA)' is an amphiphilic bile acid, and refers to a compound in which taurine is conjugated to ursodeoxycholic acid (UDCA). it means. The taurourusodeoxycholic acid may include all those known in the art, and specifically, the taurourusodeoxycholic acid may be a compound represented by Formula 1 below:

[Formula 1]

.

.

The taurourusodeoxycholic acid according to the present invention may include not only pharmaceutically acceptable salts thereof, but also solvates and hydrates that can be prepared therefrom.

The pharmaceutically acceptable salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts are formed with inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It can be obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulphate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide. , iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate , Suberate, Sebacate, Fumarate, Maleate, Butane-1,4-dioate, Hexane-1,6-dioate, Benzoate Chlorobenzoate, Methylbenzoate, Dinitro Benzoate, Hydroxybenzoate , methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxy butyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the taurourusodeoxycholic acid according to the present invention in an excess aqueous acid solution and precipitating the salt in a water-miscible organic solvent. there is. In this case, the organic solvent may be methanol, ethanol, acetone or acetonitrile.

Meanwhile, a pharmaceutically acceptable metal salt of taurourusodeoxycholic acid can be prepared using a base. Alkali metal or alkaline earth metal salts can be obtained by dissolving pirfenidone in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, followed by filtering out the undissolved compound salt and evaporating and drying the filtrate. At this time, it may be appropriate to prepare a sodium, potassium or calcium salt as the metal salt. Silver salts can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt.

The antipyretic adjuvant can suppress the abnormal development of cells that may occur due to high fever by administering together with an antipyretic agent. In one example, abnormal development of the cells may be abnormal development of an embryo. That is, the antipyretic aid may be for mothers. Specifically, the antipyretic supplement may be used to suppress abnormal development of the fetus due to high fever of the mother. In addition, the antipyretic supplement may be used to suppress abnormal development of the fetus due to the high fever of the mother in the early stage of pregnancy.

The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of taurourusodeoxycholic acid, an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include at least one active ingredient exhibiting the same or similar function in addition to the above active ingredient.

The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof commonly used in biological preparations. Any pharmaceutically acceptable carrier may be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is described in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol or mixtures thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatic agents and the like may be added as needed.

When formulating the composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and such a solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or mixtures thereof. In addition, the solid preparation may include a lubricant, examples of which include magnesium stearate and talc. Meanwhile, the liquid formulation may be a suspension, internal solution, emulsion or syrup. In this case, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, and preservatives.

The parenteral formulation may include injections, suppositories, powders for respiratory inhalation, aerosols for sprays, powders and creams, and the like. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like. At this time, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used as non-aqueous solvents or suspending solvents.

The composition of the present invention may be administered orally or parenterally, depending on the desired method. Parenteral administration may include intraperitoneal, intrarectal, subcutaneous, intravenous, intramuscular or intrathoracic injection modes.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the administration time, the route of administration, the treatment period, and the drugs used simultaneously. However, for desirable effects, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg/kg, specifically 0.001 to 500 mg/kg. The administration may be several times a day.

The compositions of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, administration can be sequential or simultaneous.

In addition, the present invention provides a feed additive for reducing high-temperature stress containing TUDCA or a pharmaceutically acceptable salt thereof as an active ingredient.

TUDCA or a pharmaceutically acceptable salt thereof contained in the feed additive according to the present invention may have the characteristics as described above. As an example, the TUDCA may be a compound represented by Formula 1 below:

[Formula 1]

.

.

The feed additive may suppress abnormal development of cells that may occur due to high heat. In one example, abnormal development of the cells may be abnormal development of an embryo. That is, the feed additive may be used to suppress abnormal development of a fetus due to high fever. In addition, the feed additive can be used to suppress abnormal development of the fetus due to high fever in the early pregnancy.

The feed additive may be used for all breeding animals known in the art, and may be specifically used for mammals. As an example, the mammal may include pigs, cows, horses, sheep, dogs, cats, rabbits, and the like.

The feed additive may include auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other live cell type microbial preparations; grains such as ground or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugar and dairy products, such as various powdered milk and whey powder; main components such as lipids, for example, animal fats and vegetable fats optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventives may be further included.

The feed additive may be in the form of a solid or liquid formulation, and may further include an excipient. The excipient may include calcium carbonate, horse powder, zeolite, jade powder, rice bran, and the like.

The feed additive may be administered alone or in combination with other feed additives in an edible carrier, wherein the dosage may be adjusted as is well known in the art. The feed additive may be included in an amount of 0.01 to 50 parts by weight based on 100 parts by weight of the total feed composition, which may be appropriately adjusted by a person skilled in the art.

Furthermore, the present invention provides a medium composition for in vitro culture of embryos comprising TUDCA or a pharmaceutically acceptable salt thereof as an active ingredient.

TUDCA or a pharmaceutically acceptable salt thereof contained in the medium composition for in vitro culture of embryos according to the present invention may have the characteristics described above. As an example, the TUDCA may be a compound represented by Formula 1 below:

[Formula 1]

.

.

As used herein, the term 'in vitro culture' refers to a process of artificially growing living cells in vitro under controlled conditions. In addition, cells may be grown and proliferated by aseptically removing a part of an individual's tissue, decomposing intercellular linking substances with an enzyme, and spreading the liberated suspension on a flat bottom of a culture dish such as a bottle or Petri dish. At this time, a culture medium capable of supporting the growth and survival of cells in vitro is required.

At this time, the medium composition according to the present invention may be used by adding it to a basal medium used for in vitro culture of embryos. The term 'basic medium' varies depending on the species of organism from which the embryo is derived, but generally includes inorganic salts, carbon sources, amino acids, bovine serum albumin, and cofactors as a basis for early embryonic cells. medium for culture. The medium for culturing the early embryonic cells may include all types of media known in the art.

The inorganic salts may include all types of inorganic salts known in the art, and specifically, may be sodium chloride (NaCl), potassium chloride (KCl), and sodium bicarbonate (NaHCO ₃ ). Meanwhile, the amino acid may include all types of amino acids known in the art. Specifically, the amino acids are essential amino acids such as valine, leucine, isoleucine, methionine, threonine, lysine, phenylalanine, tryptophan, and histidine. (histidine) and the like. In addition, the amino acids may include semi-essential amino acids such as glutamine, glycine, cysteine, arginine, tyrosine, and proline. Furthermore, the amino acids may include non-essential amino acids such as alanine, aspartic acid, glutamic acid, and asparagine.

In addition, the cofactor may include all types of cofactors known in the art. For example, the cofactor may include zinc, copper, manganese, seline, cobalt, molybdenum, vanadium, iodine, bromine, fluorine, nickel, silicon, tin, chromium, boron, a buffer, and an antibiotic.

As the basal medium, any type of medium known in the art to be used for mammalian cell culture may be used, which may be appropriately modified and used as necessary.

As used herein, the term 'embryo' generally refers to the period until a zygote in which sperm and eggs of an animal are combined starts cell division to become a 'fetus'. That is, the embryo may be an embryo formed by fertilization of a sperm and an egg. In addition, the embryo may be a stem cell-derived embryoid body.

The term 'stem cell' refers to pluripotency capable of differentiating into cells derived from endoderm, mesoderm and ectoderm of animals, or limited differentiation ability capable of differentiating into cells closely related to tissues or functions ( cells with multipotency). The stem cells may include all types of stem cells known in the art. Specifically, the stem cells may be pluripotent stem cells, and more specifically, the stem cells may be embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

The term 'induced pluripotent stem cell (iPSC)' refers to a cell made in a way to establish undifferentiated stem cells having a differentiation potential similar to that of embryonic stem cells from animal somatic cells using dedifferentiation technology. do. The method for producing induced pluripotent stem cells is well known in the art, and can be prepared by a method appropriately modified by a person skilled in the art if necessary.

In addition, the term 'embryoid body' refers to a spherical-shaped stem cell-derived cell mass generated in a suspension culture state. The embryoid body can potentially differentiate into endoderm, mesoderm and ectoderm.

That is, the medium composition for in vitro culture of embryos according to the present invention can suppress abnormal development of embryos that may occur due to high heat. In addition, the medium composition for in vitro culture of embryos according to the present invention can suppress abnormal development of stem cell-derived embryoid bodies that may occur due to high heat.

Hereinafter, the present invention will be described in detail by the following examples, however, the following examples are only for illustrating the present invention, and the present invention is not limited thereto. Anything that has substantially the same configuration and achieves the same effect as the technical concept described in the claims of the present invention is included in the technical scope of the present invention.

Example 1. Check high heat condition

In an in vitro model, the following experiment was performed to determine the optimal time of exposure to high temperatures.

1-1. embryoid body formation

First, the WA09 hESC (human embryonic stem cell, WiCell Research Institute, USA) cell line was cultured in an undifferentiated state using vitronectin and a TeSr-E8 culture medium (Stem Cell Technologies). Cultivation was performed by dispensing cells on vitronectin-coated culture dishes, and at 37° C. and 5% CO ₂ conditions. After 2 days, the cultured cells were physically removed from the culture dish using a glass pipette, and seeded onto a non-adhesive bacterial culture dish. The seeded cells spontaneously formed embryoid bodies by culturing using a differentiation medium that did not contain basic fibroblast growth factor. Differentiation medium is DMEM/F12 containing 1 mM glutamine, 1% nonessential amino acids, 0.1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol and 20% KnockOut Serum Replacement (Invitrogen). A culture medium (Invitrogen, USA) was used.

1-2. High heat condition check

The formed embryoid body was cultured at 37°C and 5% CO ₂ for 2 days, and exposed to 42°C, a temperature that does not generally show cytotoxicity. The embryoid bodies were exposed to high temperatures for 1, 2, 4, 8, or 16 hours, respectively, and then differentiated using the above differentiation medium containing bFGF (basic fibroblast growth factor) while further culturing at a temperature of 37 ° C. . At this time, embryoid bodies exposed to 37 ° C. were used as a control group. The results of observing the morphology of the differentiated embryoid bodies under a phase contrast microscope are shown in FIG. 1 .

As shown in FIG. 1, the morphology of the embryoid body was well maintained without causing apoptosis even when exposed to high heat.

1-3. UPR related Confirmation of gene expression changes

In Example 1-2, expression changes of UPR (unfolded protein response)-related genes in embryoid bodies exposed to high heat were confirmed by RT-qPCR method.

Specifically, total RNA was extracted from the obtained embryoid body using the easy-BLUE total RNA extraction kit (iNtRon Biotechnology DR, Korea) according to the manufacturer's protocol. The concentration and purity of the extracted RNA was confirmed using a Nanodrop ND-2000 spectrophotometer (ThermoFisher Scientific, USA), and 1 μg of RNA was used as a template in an All-in-One 5 × First Strand cDNA Synthesis Master Mix (All-in -one 5× first strand cDNA Synthesis Master Mix, Cellsafe, Korea) was used to synthesize cDNA in a conventional manner. Thereafter, qPCR was performed in a conventional manner using the synthesized cDNA as a template and rTaq plus 5x PCR Master Mix (Elpisbio). At this time, the primers used for qPCR are shown in Table 1 below. Relative mRNA expression levels were expressed as Ct (comparative threshold cycle) values compared to GAPDH gene expression levels using SYBR Green PCT Master Mix (Applied Biosystems, USA) and CFX96 real-time PCR detection system (Bio-Rad, USA) . As a result, the obtained PCR product was electrophoresed on a 1.2% agarose gel, and the expression level was quantitatively confirmed. The results are shown in FIG. 2 .

primer Sequence (5'→3') sequence number GAPDH forward GGT GTG AAC CAT GAG AAG TAT GA SEQ ID NO: 1 GAPDH reverse GAG TCC TTC CAC GAT ACC AAA G SEQ ID NO: 2 OCT4 forward TAT GGG AGC CCT CAC TTC AC SEQ ID NO: 3 OCT4 reverse AGT TTG TGC CAG GGT TTT TG SEQ ID NO: 4 SOX2 forward GCT AGT CTC CAA GCG ACG AA SEQ ID NO: 5 SOX2 reverse GCA AGA AGC CTC TCC TTG AA SEQ ID NO: 6 NANOG forward CAG TCT GGA CAC TGG CTG AA SEQ ID NO: 7 NANOG reverse CTC GCT GAT TAG GCT CCA AC SEQ ID NO: 8 BMP4 forward GGA GAT GGT AGT AGA GGG ATG T SEQ ID NO: 9 BMP4 reverse CGT GTG TGT GTG GTG TAT GT SEQ ID NO: 10 SOX17 forward CAT GAC TCC GGT GTG AAT CTC SEQ ID NO: 11 SOX17 reverse CAC GTC AGG ATA GTT GCA GTA ATA SEQ ID NO: 12 ATF3 forward CCT CTG CGC TGG AAT CAG TC SEQ ID NO: 13 ATF3 reverse TTC TTT CTC GTC GCC TCT TTT T SEQ ID NO: 14 ATF4 forward ATG ACC GAA ATG AGC TTC CTG SEQ ID NO: 15 ATF4 reverse GCT GGA GAA CCC ATG AGG T SEQ ID NO: 16 ATF6 forward TCC TCG GTC AGT GGA CTC TTA SEQ ID NO: 17 ATF6 reverse CTT GGG CTG AAT TGA AGG TTT TG SEQ ID NO: 18 CHOP forward GGA AAC AGA GTG GTC ATT CCC SEQ ID NO: 19 CHOP reverse CTG CTT GAG CCG TTC ATT CTC SEQ ID NO: 20 BIP forward CAT CAC GCC GTC CTA TGT CG SEQ ID NO: 21 BIP reverse CGT CAA AGA CCG TGT TCT CG SEQ ID NO: 22 GADD34 forward ATG ATG GCA TGT ATG GTG AGC SEQ ID NO: 23 GADD34 reverse AAC CTT GCA GTG TCC TTA TCA G SEQ ID NO: 24 sXBP-1 forward GGT CTG CTG AGT CCG CAG CAG G SEQ ID NO: 25 sXBP-1 reverse GGG CTT GGT ATA TAT GTG G SEQ ID NO: 26 tXBP-1 forward CCC TCC AGA ACA TCT CCC CAT SEQ ID NO: 27 tXBP-1 reverse ACA TGA CTG GGT CCA AGT TGT SEQ ID NO: 28 NEUROD1 forward GAA CGC AGA GGA GGA CTC AC SEQ ID NO: 29 NEUROD1 reverse CTT GGG CTT TTG ATC GTC AT SEQ ID NO: 30 TBX3 forward GAT GAG TCC TCC AGT GAA CAA G SEQ ID NO: 31 TBX3 reverse CTT TGA GGT TCG ATG TCC CTA C SEQ ID NO: 32 Hand1 forward CGC CTA GCC ACC AGC TAC ATC SEQ ID NO: 33 Hand1 reverse CGC CAT CCG CCT TCT TGA GTT SEQ ID NO: 34 SMA forward ACC CAC AAT GTC CCC ATC TA SEQ ID NO: 35 SMA reverse GAA GGA ATA GCC ACG CTC AG SEQ ID NO: 36 GATA6 forward TCC ACT CGT GTC TGC TTT TG SEQ ID NO: 37 GATA6 reverse CCC TTC CCT TCC ATC TTC TC SEQ ID NO: 38 AFP forward AAA TGC GTT TCT CGT TGC TT SEQ ID NO: 39 AFP reverse GCC ACA GGC CAA TAG TTT GT SEQ ID NO: 40 OFLM3 forward ACC CAG TTC AAG GAG GAA ATA AG SEQ ID NO: 41 OFLM3 reverse CTC AGC ACT CTT TGG TGT AGT T SEQ ID NO: 42 NR2F2 forward GAA GAT CTC TCC CTT CAC CTT TC SEQ ID NO: 43 NR2F2 reverse GAA TCT CCT CCT CGG TGT TTA TC SEQ ID NO: 44

As shown in FIG. 2, the stemness marker genes, OCT4 and SOX2 genes, showed no significant change in expression level even when exposed to high heat for up to 16 hours, but the expression level of the NANOG gene slightly decreased after 8 hours. . On the other hand, UPR-related genes GADD34 , CHOP , sXBP -1 and BIP genes showed the highest expression level when exposed to high temperature for 4 hours, and most of the UPR-related genes including the above genes increased expression levels as the exposure time to high temperature increased. returned to normal levels.

Therefore, based on the above results, in subsequent experiments, embryoid bodies exposed to 42° C. for 4 hours were used.

Example 2. Due to high fever embryoid body Confirmation of inhibition of differentiation - (1)

As described above, the embryoid body was exposed to 42° C. for 4 hours, and while spontaneous differentiation was induced, changes in the expression of related genes were confirmed by qRT-PCR for 1 to 24 hours. At this time, embryoid bodies exposed to 37 ° C. were used as a control group. Experiments were performed as described above using embryoid bodies after 0, 0.5, 1, 3, 12 or 24 hours of differentiation, and the results are shown in FIGS. 3 to 7 .

As shown in Figures 3 and 4, there was no significant change in the expression of stem marker genes or differentiation marker genes by high heat exposure. However, as shown in FIGS. 5 to 7 , the expression of the UPR-related genes CHOP, ATF3, sXBP-1, and GADD34 genes continuously increased until 1 hour of differentiation and then decreased thereafter. On the other hand, the expression of ATF4 and ATF6 genes was not significantly changed by high heat stress.

Example 3. by high fever embryoid body Confirmation of inhibition of differentiation - (2)

3-1. microscope observation

As described above, the embryoid body was exposed to 42° C. for 4 hours, and the morphology of the embryoid body was observed under a phase contrast microscope for 12 days while spontaneous differentiation was induced. As a result, a photograph observed under a microscope is shown in FIG. 8 .

As shown in Fig. 8, the morphology of embryoid bodies did not show significant changes by high heat.

3-2. Check gene expression profile

Using the embryoid bodies in which differentiation was induced for 12 days in Example 3-1, expression changes of genes encoding heat shock proteins were analyzed by human heat shock protein & chaperone RT ² -PCR array (Human Heat Shock Proteins & Chaperones RT ^2- PCR Array, Qiagen, Germany).

Specifically, the cDNA synthesized as described above was diluted in nuclease-free distilled water, and a SYBR green master mix was added to prepare a mixture. Then, 25 μl of the mixture was added to each well of the PCR array, and real-time PCR was performed using a StepOne Real-Time PCR system (Applied Biosystems). Fluorescence detection was used with a thermal profile of 40 cycles of initial denaturation at 95 °C for 10 min, followed by denaturation at 95 °C for 15 sec and annealing at 60 °C for 1 min. Then, the differentiation of the embryoid body into three germ layers was evaluated using the TaqMan hPSC Scorecard Panel 2×96w FAST kit (TaqMan hPSC Scorecard Panel 2×96w FAST kit, Applied Biosystems), and the heat map ( heatmap) results are shown in FIGS. 9 to 12.

As shown in FIGS. 9 to 12 , expression of genes related to embryoid body differentiation was significantly suppressed by exposure to high heat.

3-3. Confirmation of gene expression changes

Using the embryoid bodies in which differentiation was induced for 12 days in Example 3-1, expression changes of genes related to differentiation into three germ layers were confirmed by performing qRT-PCR in the same manner as described above. As a result, the expression changes of the identified genes are shown in FIGS. 13 to 16 .

As shown in Figure 13, the expression of OCT4 and NANOG genes was maintained even after 10 days of differentiation by high heat stress. Meanwhile, expression patterns of genes related to trigerminal differentiation were different in embryoid bodies exposed to high heat stress compared to embryoid bodies cultured at normal temperature. Specifically, the expression of Hand1 and SMA genes, which are mesodermal genes, was confirmed after 3 days of differentiation in embryoid bodies exposed to high heat, whereas expression was confirmed after 1 day of differentiation in embryoid bodies cultured at normal temperature. On the other hand, the expression level of the NeuroD1 gene, an ectodermal gene, was very low in embryoid bodies exposed to high heat until day 12 of differentiation. In addition, endoderm ( GATA6 , AFP and SOX17 ) and ectodermal ( NR2F2 ) genes did not show significant expression changes by high fever.

Therefore, it was confirmed from the above that high heat stress had a significant effect on the development of the mesodermal layer of the embryoid body.

Experimental example One. to taurourusodeoxycholic acid Confirmation of gene expression changes by

1-1. UPR Confirm related gene expression changes

After exposure to high heat at 42°C for 4 hours, 200 μM of tauroursodeoxycholic acid (TUDCA) was added to the medium and differentiated for 24 hours, using embryoid bodies obtained to change UPR-related gene expression was confirmed by the qRT-PCR method as described above. As a result, the confirmed gene expression changes are shown in FIGS. 17 and 18 .

As shown in FIGS. 17 and 18, the expression of ATF2 , GADD34 , sXBP1 , and ATF6 genes, which increased within a short period of time by high heat exposure, compared to embryoid body cultured at normal temperature, was significantly reduced by TUDCA treatment.

1-2. tricotyledon Identification of differentiation-related gene profiles

After exposure to high heat at 42 ° C for 4 hours, 200 μM of tauroursodeoxycholic acid (TUDCA) was added to the medium and differentiated for 12 days. Profiles were confirmed by performing the RT ² -PCR array as described above. As a result, the obtained heat maps are shown in FIGS. 19 to 21 .

As shown in FIGS. 19 to 21 , expression of mesoderm genes lowered by high heat exposure was significantly restored by TUDCA treatment. On the other hand, expression of ectodermal and endoderm genes did not show significant changes by TUDCA treatment.

Therefore, it was found from the above results that abnormal development of mesoderm caused by high fever was significantly restored by TUDCA.

<110> SOONCHUNHYANG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> SUPPLEMENT FOR REDUCING FEVER COMPRISING TAUROURSODEOXYCHOLIC ACID AS AN ACTIVE INGREDIENT <130> DP210280 <160> 44 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> artificial sequence <220> <223> GAPDH forward <400> 1 ggtgtgaacc atgagaagta tga 23 <210> 2 <211> 22 <212> DNA <213> artificial sequence <220> <223> GAPDH reverse <400> 2 gagtccttcc acgataccaa ag 22 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> OCT4 forward <400> 3 tatgggagcc ctcacttcac 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> OCT4 reverse <400> 4 agtttgtgcc agggtttttg 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> SOX2 forward <400> 5 gctagtctcc aagcgacgaa 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> SOX2 reverse <400> 6 gcaagaagcc tctccttgaa 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> NANOG forward <400> 7 cagtctggac actggctgaa 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> NANOG reverse <400> 8 ctcgctgatt aggctccaac 20 <210> 9 <211> 22 <212> DNA <213> artificial sequence <220> <223> BMP4 forward <400> 9 ggagatggta gtagagggat gt 22 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> BMP4 reverse <400> 10 cgtgtgtgtg tggtgtatgt 20 <210> 11 <211> 21 <212> DNA <213> artificial sequence <220> <223> SOX17 forward <400> 11 catgactccg gtgtgaatct c 21 <210> 12 <211> 24 <212> DNA <213> artificial sequence <220> <223> SOX17 reverse <400> 12 cacgtcagga tagttgcagt aata 24 <210> 13 <211> 20 <212> DNA <213> artificial sequence <220> <223> ATF3 forward <400> 13 cctctgcgct ggaatcagtc 20 <210> 14 <211> 22 <212> DNA <213> artificial sequence <220> <223> ATF3 reverse <400> 14 ttctttctcg tcgcctcttt tt 22 <210> 15 <211> 21 <212> DNA <213> artificial sequence <220> <223> ATF4 forward <400> 15 atgaccgaaa tgagcttcct g 21 <210> 16 <211> 19 <212> DNA <213> artificial sequence <220> <223> ATF4 reverse <400> 16 gctggagaac ccatgaggt 19 <210> 17 <211> 21 <212> DNA <213> artificial sequence <220> <223> ATF6 forward <400> 17 tcctcggtca gtggactctt a 21 <210> 18 <211> 23 <212> DNA <213> artificial sequence <220> <223> ATF6 reverse <400> 18 cttgggctga attgaaggtt ttg 23 <210> 19 <211> 21 <212> DNA <213> artificial sequence <220> <223> CHOP forward <400> 19 ggaaacagag tggtcattcc c 21 <210> 20 <211> 21 <212> DNA <213> artificial sequence <220> <223> CHOP reverse <400> 20 ctgcttgagc cgttcattct c 21 <210> 21 <211> 20 <212> DNA <213> artificial sequence <220> <223> BIP forward <400> 21 catcacgccg tcctatgtcg 20 <210> 22 <211> 20 <212> DNA <213> artificial sequence <220> <223> BIP reverse <400> 22 cgtcaaagac cgtgttctcg 20 <210> 23 <211> 21 <212> DNA <213> artificial sequence <220> <223> GADD34 forward <400> 23 atgatggcat gtatggtgag c 21 <210> 24 <211> 22 <212> DNA <213> artificial sequence <220> <223> GADD34 reverse <400> 24 aaccttgcag tgtccttatc ag 22 <210> 25 <211> 22 <212> DNA <213> artificial sequence <220> <223> sXBP-1 forward <400> 25 ggtctgctga gtccgcagca gg 22 <210> 26 <211> 19 <212> DNA <213> artificial sequence <220> <223> sXBP-1 reverse <400> 26 gggcttggta tatatgtgg 19 <210> 27 <211> 21 <212> DNA <213> artificial sequence <220> <223> tXBP-1 forward <400> 27 ccctccagaa catctcccca t 21 <210> 28 <211> 21 <212> DNA <213> artificial sequence <220> <223> tXBP-1 reverse <400> 28 acatgactgg gtccaagttg t 21 <210> 29 <211> 20 <212> DNA <213> artificial sequence <220> <223> NEUROD1 forward <400> 29 gaacgcagag gaggactcac 20 <210> 30 <211> 20 <212> DNA <213> artificial sequence <220> <223> NEUROD1 reverse <400> 30 cttgggcttt tgatcgtcat 20 <210> 31 <211> 22 <212> DNA <213> artificial sequence <220> <223> TBX3 forward <400> 31 gatgagtcct ccagtgaaca ag 22 <210> 32 <211> 22 <212> DNA <213> artificial sequence <220> <223> TBX3 reverse <400> 32 ctttgaggtt cgatgtccct ac 22 <210> 33 <211> 21 <212> DNA <213> artificial sequence <220> <223> Hand1 forward <400> 33 cgcctagcca ccagctacat c 21 <210> 34 <211> 21 <212> DNA <213> artificial sequence <220> <223> Hand1 reverse <400> 34 cgccatccgc cttcttgagt t 21 <210> 35 <211> 20 <212> DNA <213> artificial sequence <220> <223> SMA forward <400> 35 acccacaatg tcccccatcta 20 <210> 36 <211> 20 <212> DNA <213> artificial sequence <220> <223> SMA reverse <400> 36 gaaggaatag ccacgctcag 20 <210> 37 <211> 20 <212> DNA <213> artificial sequence <220> <223> GATA6 forward <400> 37 tccactcgtg tctgcttttg 20 <210> 38 <211> 20 <212> DNA <213> artificial sequence <220> <223> GATA6 reverse <400> 38 cccttccctt ccatcttctc 20 <210> 39 <211> 20 <212> DNA <213> artificial sequence <220> <223> AFP forward <400> 39 aaatgcgttt ctcgttgctt 20 <210> 40 <211> 20 <212> DNA <213> artificial sequence <220> <223> AFP reverse <400> 40 gccacaggcc aatagtttgt 20 <210> 41 <211> 23 <212> DNA <213> artificial sequence <220> <223> OFLM3 forward <400> 41 acccagttca aggaggaaat aag 23 <210> 42 <211> 22 <212> DNA <213> artificial sequence <220> <223> OFLM3 reverse <400> 42 ctcagcactc tttggtgtag tt 22 <210> 43 <211> 23 <212> DNA <213> artificial sequence <220> <223> NR2F2 forward <400> 43 gaagatctct cccttcacct ttc 23 <210> 44 <211> 23 <212> DNA <213> artificial sequence <220> <223> NR2F2 reverse <400> 44 gaatctcctc ctcggtgttt atc 23

Claims (9)

An antipyretic supplement for mothers containing tauroursodeoxycholic acid (TUDCA) or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 1, wherein the TUDCA is a compound represented by the following formula (1), antipyretic aid for mothers:
[Formula 1]

.
delete As a feed additive for reducing high-temperature stress containing TUDCA or a pharmaceutically acceptable salt thereof as an active ingredient,
The feed additive is a feed additive for reducing high-temperature stress for pregnant mammals.
The feed additive for reducing high-temperature stress according to claim 4, wherein the TUDCA is a compound represented by Formula 1 below:
[Formula 1]

.
delete A medium composition for in vitro culture of embryos comprising TUDCA or a pharmaceutically acceptable salt thereof as an active ingredient,
The medium composition is a medium composition for in vitro culture of embryos that inhibits the formation of teratoma of embryos due to high temperature.
The medium composition for in vitro culture of embryos according to claim 7, wherein the TUDCA is a compound represented by Formula 1 below:
[Formula 1]

.
delete

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