CN117298077B - Trifuhalol A application in improving muscular atrophy - Google Patents
Trifuhalol A application in improving muscular atrophy Download PDFInfo
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- 201000000585 muscular atrophy Diseases 0.000 title claims description 27
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Classifications
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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
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
- A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
-
- 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
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- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
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- Neurology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the field of biotechnology, in particular to the application of Trifuhalol A in improving muscular dystrophy, namely the invention provides the application of a compound shown in a formula (I) in improving muscular dystrophy, wherein the compound can effectively improve dexamethasone-induced muscular dystrophy, and the effect is realized through an AKT/mTOR/FOXO3a signal pathway.Formula (I).
Description
Technical Field
The invention relates to the field of biotechnology, in particular to an application of Trifuhalol A in improving muscular atrophy.
Background
Trifuhalol A is a plant derived from AphyllophoralesAgarum clathratum) The extracted polyphenol compound has a chemical structure shown as a formula (I) and is also called 5-2, 6-dihydroxyl-4- (2, 4, 6-trihydroxyphenyl) benzene-1, 2, 3-benzene triphenol.
Formula (I)
Korean patent publication No. KR20220081768A discloses a pharmaceutical composition comprising trifuhalol a or a physiologically acceptable salt thereof as an active ingredient for preventing or treating allergic diseases and diseases associated with vasodilation.
The Chinese patent application with publication number of CN113261676A discloses a food additive composition containing vanadium binding protein and 5-2, 6-dihydroxyl-4- (2, 4, 6-trihydroxyphenyl) benzene-1, 2, 3-benzene-triphenol and application thereof in preparing antioxidant food and anti-obesity food.
Muscle atrophy refers to a disease with reduced muscle strength caused by attenuation of muscle fibers and reduction of muscle volume due to different causes, and the related causes are various, can be caused by various primary diseases, trauma and other factors, and currently lacks a cure method. Dexamethasone is a potent synthetic glucocorticoid that is widely used because of its powerful anti-inflammatory, anti-shock and immunosuppressive functions. However, long-term or excessive use of dexamethasone also accompanies side effects such as muscular atrophy, and the current treatment of muscular atrophy is still a urgent problem, so there is a need in the art for a new effective drug or composition to ameliorate the problem of muscular atrophy induced by dexamethasone.
Disclosure of Invention
As described above, in view of the problems existing in the prior art, it is an object of the present invention to provide a new and effective drug or composition capable of improving the problem of dexamethasone-induced muscular atrophy.
In one aspect, the invention provides a novel use of a compound of formula (I) in the manufacture of a medicament for ameliorating muscle atrophy.
Formula (I)
The invention has the beneficial effects that the invention provides a novel application of the compound shown in the formula (I) in preparing a medicament for improving muscular dystrophy, wherein the compound shown in the formula (I) can effectively improve dexamethasone-induced muscular dystrophy and has potential as a natural functional food ingredient.
Drawings
Fig. 1 is a drug toxicity evaluation result, wherein Maca indicates positive control Maca concentrate, dexa indicates dexamethasone, TFA indicates Trifuhalol a.
FIG. 2 shows the results of myotube cell proliferation over different differentiation times (2, 4,6 days), wherein Maca indicates the positive control Maca concentrate, dexa indicates dexamethasone, and TFA indicates Trifuhalol A.
FIG. 3 shows the proliferation results of myotube cells over different differentiation times (2, 4,6 days), wherein Maca indicates positive control Maca concentrate, dexa indicates dexamethasone, and TFA indicates Trifuhalol A.
FIG. 4 is a diagram of the immunofluorescence of MyHC in dexamethasone-induced C2C12 myotubes.
Fig. 5 shows the results of a dexamethasone-induced immunofluorescence analysis of MyHC in C2C12 myotubes, where (a) - (d) are statistics of fusion index, myotube coverage, total nuclei and myotube diameter, respectively, maca indicates positive control Maca concentrate, dexa indicates dexamethasone, TFA indicates Trifuhalol a.
FIG. 6 is a Western blot analysis of the expression of genes involved in muscular atrophy regulation, wherein Maca indicates positive control Maca concentrate, dexa indicates dexamethasone, TFA indicates Trifuhalol A, and M10 indicates Maca at 10 μg/mL.
Detailed Description
The following description is illustrative of the invention by way of example only and is not intended to limit the scope of the invention, which is defined by the appended claims. And, it is understood by those skilled in the art that modifications may be made to the technical scheme of the present invention without departing from the spirit and gist of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided to better understand the present invention.
Where a range of values is provided, such as a range of concentrations, a range of percentages, or a range of ratios, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range, and any other stated or intervening value in that stated range, is encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.
In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially/mainly of … …". The expression "comprising," "including," or "consisting essentially of … …" is generally understood to mean an open-ended expression that includes not only the individual elements, components, assemblies, method steps, etc., specifically listed thereafter, but also other elements, components, assemblies, method steps. In addition, the expression "comprising," "including," or "consisting essentially of … …" is also to be understood in some instances as a closed-form expression, meaning that only the elements, components, assemblies, and method steps specifically listed thereafter are included, and no other elements, components, assemblies, and method steps are included. At this time, the expression is equivalent to the expression "consisting of … …".
In one aspect, the invention provides the use of a compound of formula (I) in the manufacture of a medicament for ameliorating muscle atrophy.
Formula (I)
In one embodiment, the therapeutically effective concentration of the compound is from 2.5 μg/mL to 10 μg/mL.
In one embodiment, the therapeutically effective concentration of the compound is from 5 μg/mL to 10 μg/mL.
In one embodiment, the therapeutically effective concentration of the compound is 10 μg/mL.
As used herein, the term "therapeutically effective concentration" refers to a concentration of a drug sufficient to produce a therapeutic effect without causing a toxic response.
In one embodiment, the muscle atrophy is induced by dexamethasone.
In one embodiment, the compound is Aphyllophorum praecoxAgarum clathratum) An extract or a purified form.
In one embodiment, the compound ameliorates muscle atrophy by AKT/mTOR/FOXO3a signaling pathway.
Examples
Embodiments of the present invention will be described in detail below with reference to examples and drawings. Those skilled in the art will appreciate that the following examples are illustrative only and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 preparation of Trifuhalol A
Extracting Aphyllophorum powder with 70% acetone as solvent at 30deg.C for 5.75 hr. Subsequently, fractional extraction with Ethyl Acetate Fraction (EAF) and purification by Sephadex LH-20 column chromatography, using a stepwise gradient of water/methanol (1/1 to 1/3 to 0/1) and methanol/acetone (5/1 to 3/1 to 1/1) solvent system, yielded 6 subfractionates (SF 1-SF 6). By passing through 1 H NMR、 13 C NMR and liquid chromatography-Mass Spectrometry analysis onlyTrifuhalol A was identified from SF 5. Thus, SF5 containing Trifuhalol A (83% purity) was used as the purified brown algae polyphenolic compound in the present invention.
Example 2 evaluation of cytotoxicity of Trifuhalol A
First, whether Trifuhalol A is toxic to cells is determined by measuring cell viability.
The viability of C2C12 myoblasts was determined using cell viability assay reagent cell counting kit-8 (CCK-8; dojindo, kumamoto, japan). The cells were mixed at 1X 10 5 The individual cell/ml concentrations were distributed into 96-well plates with DMEM medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin, and at 37℃and 5% CO 2 Cells were attached by incubation for 24 hours. Cells were treated with positive control Maca concentrate (Maca, 100. Mu.g/mL, available from NOW foods, U.S.A.), dexamethasone (Dexa, 10. Mu.M, available from Sigma), and varying concentrations of Trifuhalol A (TFA, 0.625-40. Mu.g/mL) for 24 hours, respectively. After completion of the treatment, CCK-8 reaction solution was added and after 1 hour of the reaction, the absorbance at 450nm was measured by using an ELISA reader (BioTek Synergy HT, woburn, MA, USA) to calculate the cell viability.
As a result, as shown in FIG. 1, the cell viability of the test group treated with Trifuhalol A at 0.625-40. Mu.g/mL was not significantly different from that of the blank group without any treatment, and it was found that all concentration groups were not toxic.
Example 3 influence of Trifuhalol A on proliferation of myotube cells
To differentiate C2C12 myoblasts into myotube cells, the cells were grown at 1X 10 5 The individual cells/ml were seeded at a density in 96-well plates with DMEM medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin and cultured for 24 hours, followed by culturing in DMEM differentiation medium supplemented with 2% horse serum to differentiate myoblasts into myotube cells. The differentiation medium was changed every 2 days and the cells were treated according to the cell groupings shown in Table 1 below, respectively, using positive control Maca at a concentration of 100. Mu.g/mL, dexamethasone at a concentration of 10. Mu.M, and Trifuhalol A at different concentrationsThe unit is μg/mL. Then, the results of cell proliferation of myotube cells differentiated for 2 days, 4 days, and 6 days were examined.
TABLE 1 grouping of cells
Results as shown in fig. 2, dexamethasone treatment inhibited myotube proliferation, while both positive control and Trifuhalol a concentration test groups increased or maintained myotube proliferation of C2C12 myoblasts after 2,4 and 6 days of differentiation. Of particular note, in the Trifuhalol A test group at concentrations of 2.5-10 μg/mL, trifuhalol A was able to significantly inhibit dexamethasone-induced myoblast atrophy of myoblast cells after 2,4 and 6 days of myoblast differentiation. Thus, to further confirm the inhibition of dexamethasone-induced muscle atrophy by Trifuhalol A, the inventors further selected a concentration of Trifuhalol A of 2.5-10 μg/mL for subsequent experiments.
Cells were individually treated according to the cell groupings shown in Table 2 below, with positive control Maca concentrations of 10. Mu.g/mL, dexamethasone concentrations of 10. Mu.M, and different Trifuhalol A concentrations in. Mu.g/mL. Then, proliferation results of myotube cells differentiated for 2 days, 4 days, and 6 days were examined.
TABLE 2 cell grouping process
The results are shown in fig. 3, in which dexamethasone-induced myotube cell atrophy was significantly inhibited in all of the Trifuhalol a concentration-treated test groups after 2,4 and 6 days of C2C12 myoblast differentiation, and in particular, the 10 μg/mL-treated test group showed excellent cell proliferation effects similar to those of the positive control group.
Example 4 influence of Trifuhalol A on dexamethasone-induced C2C12 myotube cell atrophy
To evaluate the effect of Trifuhalol a in improving muscle atrophy, the expression of MyHC (index of muscle maturation) in C2C12 myotube cells of dexamethasone-induced muscle atrophy was measured by immunocytochemistry.
Cells were treated according to the cell groupings shown in Table 2, respectively, and then myoblasts were induced to differentiate for 6 days by using a differentiation medium containing horse serum. Cells were fixed with cold methanol and then blocked with 1% BSA containing glycine. Cells were incubated with primary anti-myosin heavy chain (MyHC) for 1 hour at room temperature. Then, after 2 hours incubation with a secondary anti-Goat anti-Mouse (gold anti-Mouse IgG) at room temperature, the cells were stained with 4', 6-diamidino-2-phenylindole (DAPI). Stained cells were captured using a Lionheart FX automated fluorescence microscope (Bio Tek Instruments, inc.) and analyzed for fusion index, myotube coverage, total nuclei, myotube diameter, etc. using Myotube Analyzer image analysis software.
The results are shown in fig. 4 to 5, and are observed by immunofluorescence analysis, and a plurality of indexes such as fusion index (fig. 5 (a)), myotube coverage (fig. 5 (b)), total nuclei (fig. 5 (c)), and myotube diameter (fig. 5 (d)) are measured. The results show that the fusion index, myotube coverage, total nuclei and myotube diameter were all significantly reduced for the dexamethasone control group compared to the placebo group, indicating that dexamethasone successfully induced muscle atrophy. Whereas the 10 μg/mL concentration of Trifuhalol a treatment significantly increased the fusion index, myotube coverage, total nuclei and myotube diameter to a similar extent as the positive control group, confirming that Trifuhalol a could restore dexamethasone-induced muscle atrophy.
Further, in order to study the molecular mechanism of the modulation of dexamethasone-induced muscle atrophy by Trifuhalol A at the protein level, western blot analysis was performed to compare the expression of the muscle atrophy-modulating related genes. The inventor analyzes a plurality of proteins including MyHC, myoD, myogenin, mTOR, p-mTOR, AKT, p-AKT, foxO3a, muRF-1 and atrogin-1/MAFbx, wherein MyHC, myoD and Myogenin are transcription factors involved in muscle cell differentiation, when physiological stimulus and pathological injury are received, resting muscle satellite cells are activated and quickly converted into myoblasts with proliferation and differentiation capacity, under the control of the muscle differentiation factors (myogenic differentiation, myoD) and the myogenic factors 5 (myogenic factor 5, myf 5), the mononuclear myoblasts proliferate in muscle tissues to form a certain number of myogenic cell groups, initiate differentiation, and the myoblasts proliferate and fuse to form multi-core myotubes, and further form new myofibers with the expression of factors such as Myogenin (Myogenin), myogenin heavy chains (myosin heavy chain, myHC) and the like, so as to repair damaged myofibers. MyoD is the source of repair after skeletal muscle injury, promoting myoblast proliferation and differentiation; the AKT/mTOR signaling pathway is a classical signaling pathway and plays an important role in the aspects of cell growth, metabolism, proliferation, survival and the like; foxO3a is a downstream influencing factor of AKT/mTOR signaling pathway, and plays a role in regulating and controlling cell survival and proliferation; in animal models of muscular atrophy caused by various reasons, atrophy gene-1/muscular atrophy F-box (atrogin-1/MAFbx) and muscle-specific E3 ubiquitin ligase (MuRF-1) mRNA are taken as key regulatory factors for muscle protein decomposition, the expression level of the mRNA is obviously up-regulated, and the mRNA can be taken as a molecular marker of early muscular atrophy.
The western blot analysis method is as follows: cells were treated and differentiated for 4 days according to the cell groupings shown in Table 2, respectively, and then each group of cells was treated with a lysis buffer to isolate proteins in the cells, followed by quantitative analysis with BCA protein quantification reagents (Bio-Rad, irvine, CA, USA). An equal amount of protein was loaded onto Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel, separated by electrophoresis, and then transferred onto nitrocellulose membrane. The nitrocellulose membranes from which the proteins were transferred were reacted with corresponding primary antibodies (phospho-Akt, AKT from Cell Signaling Technology; atrogin-1/MAFbx, muRF-1, phospho-mTOR, mTOR, foxO3a, myHC, myogenin, myoD, GAPDH and α -tubulin from Santa Cruz Biotechnology) and secondary antibodies (from Santa Cruz Biotechnology) respectively, and then analyzed for changes in expression of the specific proteins using the FUSION SOLO Vilber Lourmat system (Paris, france).
The results of Western blot analysis are shown in FIG. 6, in which muscle-specific E3 ubiquitin ligase (MuRF-1), atrophy gene-1/muscle atrophy F-box (atrogin-1/MAFbx) and protein degradation marker FoxO3a were increased in myotubes after dexamethasone treatment, and MAFbx, muRF-1 and FoxO3a were significantly decreased after treatment with Trifuhalol A at a concentration of 2.5-10. Mu.g/mL. Furthermore, after treatment with Trifuhalol a at concentrations of 5 and 10 μg/mL, the expression of the transcription factors MyHC, myoD and Myogenin involved in myoblast differentiation was significantly increased compared to the dexamethasone control group. Phosphorylation of mTOR and AKT was significantly reduced in C2C12 myotube cells stimulated with dexamethasone compared to the placebo group. In the 2.5 μg/mL and 5 μg/mL concentration test groups of Trifuhalol A, there was no increase in the phosphorylated expression of mTOR compared to the dexamethasone control group, but a significant increase occurred in the 10 μg/mL concentration test group of Trifuhalol A. Furthermore, in the 2.5-10 μg/mL concentration test group of Trifuhalol A, the phosphorylated expression of AKT was significantly increased compared to the dexamethasone control group.
Comprehensive study proves that Trifuhalol A can effectively improve dexamethasone-induced muscle atrophy. This effect is related to the mechanisms of action of increased expression of transcription factors MyHC, myoD, myogenin, p-mTOR and p-AKT, and decreased expression of MAFbx, muRF-1 and FoxO3a, which are involved in myoblast differentiation through the AKT/mTOR/FOXO3a signaling pathway. This suggests that Trifuhalol A has an ameliorating effect on dexamethasone-induced muscular dystrophy and has potential as a natural functional food ingredient.
Claims (5)
1. Use of a compound of formula (I) for the preparation of a medicament for ameliorating muscle atrophy induced by dexamethasone
Formula (I).
2. The use of claim 1, wherein the therapeutically effective concentration of the compound is from 2.5 μg/mL to 10 μg/mL.
3. The use of claim 1, wherein the therapeutically effective concentration of the compound is from 5 μg/mL to 10 μg/mL.
4. The use of claim 1, wherein the therapeutically effective concentration of the compound is 10 μg/mL.
5. The use of any one of claims 1-4, wherein the compound ameliorates muscle atrophy by AKT/mTOR/FOXO3a signaling pathway.
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