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WO2013007003A1 - Nouvelle utilisation d'acide acétylacétique pour réguler la prolifération des cellules des muscles squelettiques - Google Patents

Nouvelle utilisation d'acide acétylacétique pour réguler la prolifération des cellules des muscles squelettiques Download PDF

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Publication number
WO2013007003A1
WO2013007003A1 PCT/CN2011/076986 CN2011076986W WO2013007003A1 WO 2013007003 A1 WO2013007003 A1 WO 2013007003A1 CN 2011076986 W CN2011076986 W CN 2011076986W WO 2013007003 A1 WO2013007003 A1 WO 2013007003A1
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WIPO (PCT)
Prior art keywords
acetoacetate
cells
skeletal muscle
proliferation
skeletal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2011/076986
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English (en)
Chinese (zh)
Inventor
朱大海
李莉
邹小停
张勤
张勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS and PUMC
Original Assignee
Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS and PUMC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Institute of Basic Medical Sciences of AMMS, Institute of Basic Medical Sciences of CAMS and PUMC filed Critical Institute of Basic Medical Sciences of AMMS
Priority to PCT/CN2011/076986 priority Critical patent/WO2013007003A1/fr
Publication of WO2013007003A1 publication Critical patent/WO2013007003A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • the present invention relates to the regulation of skeletal muscle cell cycle regulation and cell proliferation, and more particularly to the novel use of acetoacetate in the regulation of cell cycle-associated proteins and the regulation of skeletal muscle cell proliferation.
  • the invention further relates to the use of acetoacetate in the manufacture of a medicament for the treatment of skeletal muscle damage. Background technique
  • the long-term research on cell proliferation mostly focuses on the regulation of cell growth by growth factors and other cytokines.
  • the uptake of nutrients by cells is also regulated by growth factors, and some growth factor signaling pathways and their transcriptional levels have been discovered. Adjust the network.
  • IGF1 Insulin-like growth factor 1
  • IGF1 is similar in structure and function to insulin, and plays an important role in normal body growth and cell proliferation, differentiation, and metabolism. IGF is produced in the liver and exerts biological effects through circulation to various tissues of the body. Skeletal muscle is an important target organ. IGF1 plays an important role in the development and maintenance of skeletal muscle. Mice deficient in the IGF1 receptor develop severe muscle atrophy and die shortly after birth due to the inability to perform normal lung respiration. In contrast, skeletal muscle volume in mice overexpressing IGF1 is significantly increased
  • IGF1 binds to its receptor and stimulates the Ras-raf-MEK-ERK signaling pathway and PI3K-Akt signaling pathway. These two pathways are essential for cell proliferation and survival. Among them, most of the studies related to IGF1 are the Akt pathway. Studies have shown that the activation of PI3K is essential for skeletal muscle hypertrophy [3]. PI3K then activates a series of proteins such as downstream Akt, mTOR, up-regulates transcriptional regulators involved in protein translation, and promotes protein synthesis by increasing the initiation and elongation of translation, leading to skeletal muscle hypertrophy.
  • MSTN is a member of the TGF- ⁇ superfamily and is highly conserved among different species, mainly expressed in skeletal muscle [4], and also expressed in non-skeletal muscle tissues such as glands and myocardium [5, 6] .
  • the skeletal muscle of MSTN knockout mice is three times that of wild-type mice, indicating that MSTN has an inhibitory effect on skeletal muscle growth.
  • the double-muscle cattle with MSTN gene mutation and abnormal skeletal muscle also provide the same evidence [7]. .
  • the use of in situ hybridization to study the expression of MSTN during mouse embryonic development also suggests that MSTN also plays a crucial role in skeletal muscle development [4].
  • MSTN regulates the molecular mechanism of cell proliferation, and found that MSTN can up-regulate the cell cycle inhibitor p21, thereby inhibiting the activity of CDK2, and inhibiting the transcriptional activity of E2F by Rb protein, ultimately blocking the cell from G1 to S phase [8] ]. In addition, some studies have found that MSTN can also inhibit the conversion of cells from G2 to M phase through p21 [9]. Our laboratory study provides another pathway for the mechanism of action of MSTN, ie, MSTN can down-regulate the expression of cyclinD1 through the PI3K-Akt-GSK3P pathway, thereby arresting the cell cycle in G1 phase, and finding that this effect can interact with IGF1.
  • MSTN may cooperate with IGF1 to regulate the role of cell proliferation [10].
  • IGF1 insulin growth factor 1
  • both in vivo and in vitro studies have shown that MSTN can also inhibit the proliferation of satellite cells by up-regulating the expression of p21, suggesting an important role for MSTN in skeletal muscle regeneration [11].
  • endogenous metabolites and cell proliferation There are not many studies on endogenous metabolites and cell proliferation in the prior art, and no unified theoretical model has been proposed. But more and more studies have shown that metabolites such as cholesterol and certain unsaturated fatty acids can regulate cell proliferation.
  • the concept of endogenous metabolite lactic acid a very important signal molecule in sugar metabolism, has recently been proposed [12]. Recent studies have found that lactic acid plays a role in wound healing, suggesting that it has independent biological functions outside the metabolic process.
  • acetoacetate is a downstream metabolite of beta-hydroxybutyrate ( ⁇ - ⁇ ).
  • ⁇ - ⁇ beta-hydroxybutyrate
  • acetoacetate and ⁇ -hydroxybutyric acid are synthesized in the liver into the circulatory system, and catabolism in the extrahepatic tissue produces sputum, which provides energy to the body.
  • ketone bodies can be used as a substitute for sugar by the tissues of the brain, heart, kidneys and skeletal muscles to maintain the life activities of the body.
  • a first aspect of the invention provides a novel use of acetoacetate in the regulation of cell cycle associated proteins.
  • the present inventors have found that acetoacetate has a regulatory effect on the cyclin Dl.
  • the inventors have found that acetoacetate can upregulate the expression level of cyclinDl at the transcriptional and translational levels. Meanwhile, the inventors have demonstrated that acetoacetate up-regulates the expression of cyclin by activating the ERK pathway.
  • a second aspect of the invention provides a novel acetoacetate for promoting skeletal muscle cell proliferation Use.
  • the skeletal muscle cells are myoblasts.
  • the inventors have demonstrated that acetoacetate promotes myoblast proliferation by increasing the ratio of s in a dose- and time-dependent manner.
  • the inventors demonstrated that the endogenous metabolite acetoacetate upregulates the expression of the cyclin D1 by activating the ERK pathway, thereby promoting the proliferation of myoblasts.
  • the invention provides a pharmaceutical composition for promoting skeletal muscle cell proliferation comprising acetoacetate and insulin-like growth factor 1, and a pharmaceutically acceptable carrier.
  • acetoacetic acid can synergize with insulin-like growth factor (IGF1) or antagonize the regulation of cell cycle by MSTN.
  • the invention further provides a method of treating skeletal muscle damage comprising administering an effective amount of acetoacetate to an individual in need thereof. Furthermore, the present invention provides a method of treating skeletal muscle damage comprising administering to a subject in need thereof a pharmaceutical composition comprising acetoacetate and insulin-like growth factor 1, and a pharmaceutically acceptable carrier.
  • the invention further provides the use of acetoacetate in the manufacture of a medicament for treating skeletal muscle damage.
  • the skeletal muscle damage is induced by cyclic phosphate.
  • Acetoacetic acid can promote the proliferation of C 2 C 12 cells, while the effect of ⁇ - ⁇ is not obvious (**: ⁇ ⁇ 0.01).
  • Acetoacetic acid (AA) promotes C2C12 cell proliferation in a time-dependent manner (**: p ⁇ 0.01, p ⁇ 0.001).
  • Acetoacetic acid (AA) upregulates the expression of cyclinDl.
  • Acetoacetic acid activates the ERK pathway (* * : p ⁇ 0.01, ***: p ⁇ 0.001) o
  • Acetoacetic acid promotes proliferation of C 2 C 12 cells by activating the ERK pathway (*: p ⁇ 0.05, **: p ⁇ 0.01) o
  • Acetoacetic acid synergizes with IGF1 to promote cell proliferation or antagonizes MSTN's ability to inhibit cells (*: p ⁇ 0.05, **: p ⁇ 0.01) o
  • Acetoacetic acid significantly attenuates cyclic phosphate (CTX damage to skeletal muscle tissue.
  • C 2 C 12 cells are mouse myoblast cell lines established by Yaffe and Saxel. The cells exhibit fibroblast morphology under normal growth conditions, under appropriate conditions (eg, high cell density or serum starvation) C 2 C 12 cells can differentiate into contractile myotubes and express specific muscle differentiation marker proteins. Treatment of the cells with bone morphogenic protein 2 (BMP-2) can result in their muscle formation. Transformation of cells into osteoblasts.
  • the culture conditions of C 2 C 12 cells in the proliferation stage are Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal calf serum. The antibiotic content was 100 Units/mL penicillin and 100 g/mL streptomycin, and the growth group was growth medium (GM).
  • the culture environment of the cells was 37 ° C and contained 5% C0 2 .
  • the cells stored in liquid nitrogen were taken out and quickly placed in a 37 ° C water bath to melt. After centrifugation at 1000 rpm for 5 min, discard the supernatant and collect the cells. Add 10% fetal bovine serum to the complete medium, gently pipe the cells, transfer to a 25 cm 2 flask, and add 5 mL of complete medium, 37 ° C, 5% C0. 2 Incubate in the incubator.
  • the cells were collected by trypsinization at the time of cryopreservation.
  • Add appropriate amount of frozen solution (containing 10% dimethyl sulfoxide, 20% fetal bovine serum in complete medium), resuspend the cells, transfer to a cryotube, freeze at -70 ° C overnight and transfer to liquid nitrogen. save.
  • Acetoacetic acid promotes the proliferation of myoblast C 2 C 12
  • Acetoacetic acid promotes cell proliferation by up-regulating cyclinD1 expression
  • Acetoacetic acid can promote the proliferation of C 2 C 12 cells by up-regulating the expression of cyclinD1.
  • ERK can affect the cell cycle by regulating the expression of cyclinDl. Therefore, we first examined the effect of related proteins on the ERK pathway on acetoacetate regulation of C 2 C 12 cell proliferation. The cells at different times were collected for 5 mM acetoacetate for protein level detection. The results of Western blotting showed that the phosphorylated ERK protein level gradually increased with the treatment time (Fig. 5A, 5B).
  • Proliferating C 2 C 12 cells were treated with acetoacetate (5 mM), IGF1 (50 ng/ml), and MSTN (500 ng/ml), respectively or in combination.
  • Flow cytometry was consistent with previous results.
  • Treatment with acetoacetate and IGF1 alone increased the proportion of S phase in cells, while treatment with MSTN alone reduced the proportion of S phase.
  • the combination of acetoacetate and IGF1 increased the S phase of cells treated with IGF1 alone, indicating that acetoacetate can synergize with IGF1 to promote cell proliferation.
  • IGF1 or MSTN can regulate the cell cycle through the ERK signaling pathway.
  • acetoacetate and IGF1 or MSTN work together to regulate cell proliferation through the ERK pathway.
  • Flow cytometry results showed that PD98059 only partially inhibited cell cycle progression compared to the control group (Fig. 7C). This result suggests that acetoacetate and IGF1 or MSTN can work together to regulate C 2 C 12 cell proliferation through the ERK pathway.
  • Acetoacetic acid can significantly attenuate the damage of CTX to skeletal muscle tissue
  • CTX different doses of acetoacetate were injected alone or in combination with 8-week-old C57BL/6 mouse gastrocnemius muscle.
  • Tissue samples were taken for histological and molecular biological tests 4 days after injection.
  • Tissue section staining and Western Blot results showed that CTX alone caused extensive damage to skeletal muscle tissue, and a large number of immune cell infiltrates were observed under the microscope (Fig. 8A).
  • Western Blot results showed that the expression of muscle satellite cell activation Marker increased.
  • the myostatin gene is a downstream target gene of basic helix-loop-helix transcription Factor MyoD. Mol Cell Biol. 22, 7066-7082
  • Ketosis acetoacetate

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  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Neurology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne l'utilisation d'acide acétylacétique dans la préparation de médicaments destinés à favoriser la prolifération de cellules des muscles squelettiques, les cellules des muscles squelettiques étant des myoblastes, l'acide acétylacétique favorisant la prolifération des cellules des muscles squelettiques par activation de la voie ERK de manière à réguler à la hausse l'expression d'une cycline, telle que la cycline D1. La présente invention concerne en outre l'utilisation d'acide acétylacétique dans la préparation de médicaments destinés au traitement de lésions des muscles squelettiques, et d'une composition médicamenteuse destinée à favoriser la prolifération des cellules des muscles squelettiques, contenant de l'acide acétylacétique et le facteur de croissance de type insuline 1.
PCT/CN2011/076986 2011-07-08 2011-07-08 Nouvelle utilisation d'acide acétylacétique pour réguler la prolifération des cellules des muscles squelettiques Ceased WO2013007003A1 (fr)

Priority Applications (1)

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PCT/CN2011/076986 WO2013007003A1 (fr) 2011-07-08 2011-07-08 Nouvelle utilisation d'acide acétylacétique pour réguler la prolifération des cellules des muscles squelettiques

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104224766A (zh) * 2013-06-19 2014-12-24 中国医学科学院基础医学研究所 乙酰乙酸在治疗肌肉疾病中的用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1552315A (zh) * 1997-03-17 2004-12-08 英国技术集团国际有限公司 治疗组合物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1552315A (zh) * 1997-03-17 2004-12-08 英国技术集团国际有限公司 治疗组合物

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ABDELMEGEED, M.A. ET AL.: "Acetoacetate Activation of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase in Primary Cultured Rat Hepatocytes: Role of Oxidative Stress", JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 310, no. 2, 2004, pages 728 - 736 *
HUANG, ZHENGYUAN ET AL.: "Effects of IGF-1 and TNF-alpha on Proliferation and Differentiation of C2C12 Myoblast in vitro", JOURNAL OF FUJIAN MEDICAL UNIVERSITY, vol. 41, no. 6, November 2007 (2007-11-01), pages 518 - 520 *
LI, LI: "Endogenous metabolites X promote C2C12 cell proliferation via cyclinDl raised by activating ERK pathway", BASIC SCIENCES, CHINA MASTER'S THESES FULL-TEXT DATABASE, no. 10, 15 October 2010 (2010-10-15), pages A006 - 9 *
SHUBERT, P.S. ET AL.: "Ketoacids attenuate glucose uptake in human trophoblasts isolated from first-trimester chorionic villi", AM J OBSTET GYNECOL., vol. 175, no. 1, 1996, pages 56 - 62 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104224766A (zh) * 2013-06-19 2014-12-24 中国医学科学院基础医学研究所 乙酰乙酸在治疗肌肉疾病中的用途

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WO2013007003A1 (fr) Nouvelle utilisation d'acide acétylacétique pour réguler la prolifération des cellules des muscles squelettiques

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