CN115957227A - Application of morroniside in preparation of health care products and/or medicines for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury - Google Patents
Application of morroniside in preparation of health care products and/or medicines for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury Download PDFInfo
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- CN115957227A CN115957227A CN202211566940.8A CN202211566940A CN115957227A CN 115957227 A CN115957227 A CN 115957227A CN 202211566940 A CN202211566940 A CN 202211566940A CN 115957227 A CN115957227 A CN 115957227A
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- muscle
- morroniside
- skeletal muscle
- inhibiting
- preventing
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides application of morroniside in preparing health products and/or medicaments for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury. According to the research, the morroniside can promote survival, proliferation and differentiation of skeletal muscle stem cells, promote differentiation and skeletal muscle regeneration of skeletal muscle stem cells after muscle injury, maintain type diversity and plump state of skeletal muscle stem cells and mature muscle fibers, inhibit classical and non-classical NF-kB signal transduction, inflammatory mediators (IL 6, IL-1B, CRP, NIRP3, PTGS2 and TNF alpha), zinc ion aggregation signals (ZIP 14), protein degradation signals (Follistatin, myostatin, bmp11, ALK4/5/7 and Smad 7/3), ubiquitin-proteasome molecules (FoxO 3, atrogin-1 and MuRF 1) autophago-lysosomal molecules (Bnip 3, LC3A, LC B) and promote protein synthesis signals (IGF-1/IGF-1R/IRS-1/BMPI 3K/Smat, BMP 14/PR 2/3/6/ALK) and realize obvious effects of preventing and treating skeletal muscle atrophy and preventing and treating liver and kidney injury, and achieving good effects in vivo and mice or in vivo.
Description
Technical Field
The invention relates to the technical field of health care and medicine, in particular to application of morroniside in preparing health care products and/or medicines for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury.
Background
The morroniside is an iridoid glycoside compound with chemical formula of C 17 H 26 O 11 Is the main active ingredient in traditional Chinese herbal medicine plants and a health food material, namely, cornus officinalis (Cornus officinalis, C. Officinalis, sieb. Et Zucc.). Morroniside has been reported to have various biological effects such as neuroprotective, analgesic, cardioprotective, skeletoprotective and diabetes-related hepatoprotective and kidney protective effects as well as anti-inflammatory, antioxidant, anti-apoptotic, etc.
Skeletal muscle makes up about 40% of the body weight. The muscle increase can make male more positive and strong, and make female more beautiful, more upright and sexy. However, health care products and/or medicines which directly act on skeletal muscle to increase muscle are still lacking at present. Although people currently achieve muscle building primarily through fitness and supplementation with high protein, high carbohydrate, high calorie dietary supplements, the results are limited and difficult to persist.
Skeletal muscle atrophy (skeletal muscle atrophy) is characterized by a decline in muscle mass and contractile function, increasing the risk of a range of adverse consequences, including weakness, fall-related fractures and trauma, limb disability, hospitalization, and significant health care costs to society. Currently, no drug is clinically approved for use against skeletal muscle atrophy. Resistance training and nutritional supplementation are currently the two main clinical interventions, but are not applicable to all patients with muscle wasting, such as those who are already infirm and require rest or are paralyzed, and have limited therapeutic efficacy and poor compliance.
Muscular atrophy is caused by various pathological conditions such as aging, rheumatoid arthritis, cachexia, cancer, disuse, denervation, duchenne muscular dystrophy, crohn's disease, diabetes, and the like. Despite the tremendous efforts of pharmaceutical companies to identify effective drug targets and compounds to counteract muscle loss, there is no clinically successful drug treatment for muscle atrophy. In clinical studies, therapeutic interventions including etanercept (TNF α inhibitor) and the neutralizing antibody to block TNF α infliximab have failed to successfully treat cachexia-induced muscle atrophy, perhaps because cachexia is a multifactorial syndrome, and any monotherapy is insufficient to prevent or prevent muscle loss.
Muscle damage accounts for the highest proportion of motor-related contusions. The treatment of muscle damage is related to its extent of damage. The RICE principle (rest, ice compress, pressure, elevation) is generally used clinically with emphasis on physical conservative treatment. In the case of tissue tears, local injections of anesthetics, anti-inflammatory agents, and even surgical repair. Although the use of non-steroidal anti-inflammatory drugs and antithrombotic prophylaxis is reasonable, side effects limit their use. Although some blood-activating and stasis-resolving traditional Chinese medicines are also used for treating muscle strain, the treatment mechanism is mainly related to improving local blood circulation. In addition to the above therapies, there is still a clinical shortage of drugs that directly promote skeletal muscle regeneration.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide the application of the morroniside in preparing health care products and/or medicines for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury; the research finds that the morroniside can realize good effects of muscle proliferation, skeletal muscle atrophy prevention and/or muscle injury prevention and treatment through various unknown action mechanisms.
The invention provides application of morroniside in preparing health care products and/or medicines for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury.
Further, morroniside increases muscle, prevents skeletal muscle atrophy and/or prevents muscle injury by promoting survival proliferation and/or differentiation of skeletal muscle stem cells.
Further, the morroniside increases muscle mass, prevents skeletal muscle atrophy and/or prevents muscle injury by at least one of promoting skeletal muscle stem cell differentiation and promoting skeletal muscle regeneration; preferably, the skeletal muscle stem cell differentiation comprises differentiation of skeletal muscle stem cells into muscle fibers.
Further, the morroniside increases muscle mass, prevents skeletal muscle atrophy, and/or prevents muscle injury by at least one of maintaining skeletal muscle stem cell self-renewal, maintaining type diversity of mature muscle fibers, and maintaining the satiation status of mature muscle fibers.
Further, morroniside prevents skeletal muscle atrophy and/or prevents muscle damage by inhibiting classical and/or non-classical NF- κ B signaling; preferably, inhibiting classical NF-. Kappa.B signaling comprises inhibiting NF-. Kappa. B p65/RelA signaling and/or inhibiting NF-. Kappa. B p50 signaling; inhibiting non-classical NF-. Kappa.B signaling includes inhibiting NF-. Kappa.B RelB signaling and/or inhibiting NF-. Kappa. B p52 signaling.
Further, morroniside prevents skeletal muscle atrophy and/or prevents muscle injury by inhibiting the expression of inflammatory factors downstream of classical and/or non-classical NF-kB signaling; preferably, the inflammatory factor comprises at least one of IL6, IL-1b, CRP, NIRP3, PTGS2 and TNF α.
Further, morroniside augments muscle by activating one of IGF-1/IGF-1R/PI3K/Akt, activating BMP14/BMPR2/ALK2/3/Smad6/5/9 signaling pathway, and promoting myotube protein synthesis.
Further, the morroniside can prevent and treat skeletal muscle atrophy and/or muscle injury by activating one of IGF-1/IGF-1R/IRS-1/PI3K/Akt, activating BMP14/BMPR2/ALK2/3/Smad5/9 signaling pathway and promoting myotube protein synthesis.
Further, the morroniside can increase muscle, prevent skeletal muscle atrophy and/or prevent muscle injury by at least one of inhibiting a Myostatin/activin protein degradation pathway, inhibiting activation of downstream genes/proteins associated with the ubiquitin-proteasome system, and inhibiting activation of downstream genes/proteins associated with autophagy-lysosomal system; preferably, inhibiting the pathway of Myostatin/activin protein degradation comprises at least one of upregulating Follistatin/Smad7 and downregulating Myostatin/Bmp11/Alk4/5/7/Smad 2/3; inhibiting ubiquitin-proteasome system-related downstream gene/protein activation comprises inhibiting at least one of FoxO3, atrogin-1, and MuRF1 expression; inhibiting activation of a downstream gene/protein associated with the autophagy-lysosomal system includes inhibiting at least one of Bnip3, LC3A, and LC3B expression.
Further, morroniside increases muscle mass, prevents skeletal muscle atrophy and/or prevents muscle injury by inhibiting zinc ion concentration signals (ZIP 14).
In the application, the purity of the morroniside is more than or equal to 98 percent; the mouse in vivo effective dose of the morroniside is 10-40 mug/kg/d, preferably 10-20 mug/kg/d; the effective dose of the morroniside for increasing muscle, preventing skeletal muscle atrophy and/or preventing muscle injury in human body is 0.8-4.7 μ g/kg/d, preferably 0.8-2.4 μ g/kg/d.
In the present invention, augmenting muscle includes, but is not limited to, preventing, ameliorating, treating skeletal muscle growth, development, quality, strength, function, and/or the like-associated phenotype and/or disease; preventing skeletal muscle atrophy and/or muscle damage including, but not limited to, preventing, ameliorating, treating diseases associated with skeletal muscle atrophy and/or muscle damage; diseases associated with skeletal muscle atrophy and/or muscle injury include, but are not limited to, aging, rheumatoid arthritis, cachexia, cancer, disuse, loss of nerve, various muscular dystrophies including duchenne, muscle weakness, myositis, myopathy, crohn's disease, diabetes and other pathological conditions resulting in muscle atrophy and/or muscle injury resulting from exercise, trauma, contusion, strain, laceration, toxins, degenerative diseases (e.g., muscular dystrophy) and strain.
The research of the invention finds that: the morroniside can promote survival proliferation and/or differentiation of skeletal muscle stem cells (and high concentration morroniside (640 mu g/mL) has no obvious toxic and side effects on skeletal muscle stem cells), promote skeletal muscle stem cell differentiation and skeletal muscle regeneration after muscle injury, maintain type diversity and plumpness of skeletal muscle stem cells and mature muscle fibers, inhibit classical and non-classical NF-kappa B signaling, inflammatory mediators (IL 6, IL-1B, CRP, NIRP3, PTGS2, TNF alpha), zinc ion aggregation signals (Zip 14), protein degradation signals (Follistatin, myostatin, bmp11, ALK4/5/7, smad 7/3), ubiquitin-proteasome molecules (FoxO 3, atrogin-1, muRF 1) autophagy-molecule (Bnip 3, LC3A, LC B), simultaneously promote protein synthesis signals (IGF-1/PR-1R/IRS-1/PI 3K/14K, akRF 1) and prevent and treat renal atrophy of mouse muscle tissue including any renal muscle atrophy, and further improve functions of mouse liver and kidney, thereby preventing and treating and liver diseases including good mouse liver diseases.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below.
FIG. 1 shows that morroniside directly promotes the survival, proliferation and differentiation of skeletal muscle stem cells, and that high concentrations of morroniside (640. Mu.g/mL) had no significant toxic side effects on skeletal muscle stem cells (p < 0.05;. P < 0.01;. P < 0.001);
figure 2 is the results of morroniside directly promoting skeletal muscle stem cell differentiation and promoting skeletal muscle regeneration after muscle injury (/ p < 0.05);
FIG. 3 shows the results of the protein synthesis signaling pathway that mogrosides directly promote C2C12 myotubes (p < 0.05;. P < 0.01;. P < 0.001;. P < 0.0001);
FIG. 4 shows the results of the direct inhibition of proteasome and autophagy degradation signaling pathways and zinc ion aggregation signaling in C2C12 myotubes by morroniside (p < 0.05;. P < 0.01;. P < 0.001);
figure 5 shows TNF α increase in atrophic skeletal muscle caused by denervation and induction of myotube atrophy in vitro ([ p <0.05, [ p <0.01, [ p < 0.001) ];
figure 6 is the results of morroniside correction of TNF α -induced myotube atrophy in vitro (/ p <0.05,/p < 0.01);
figure 7 shows the results of morroniside blocking skeletal muscle atrophy induced by denervation in vivo (/ p <0.05,/p <0.01,/p <0.001,/p < 0.0001);
figure 8 is the results of morroniside inhibition of classical and non-classical NF- κ B signaling in TNF α -treated C2C12 myotubes and denervated mouse muscles (/ p <0.05,/p <0.01,/p <0.001,/p < 0.0001);
figure 9 shows the results of the morroniside reduction of inflammatory mediators and zinc ion accumulation signal ZIP14 in C2C12 myotubes and denervated mouse muscle treated with TNF α (p <0.05, p <0.01, p <0.001, p < 0.0001);
figure 10 shows the results of morroniside improvement of protein synthesis signaling pathways in TNF α -treated myotubes (/ p <0.05,/p <0.01,/p < 0.001);
figure 11 is the results of morroniside inhibition of proteasome and autophagy degradation signaling pathways in C2C12 myotubes and/or denervated mouse muscle treated with TNF α ([ p ] 0.05, [ p ] 0.01, [ p ] 0.001, [ p ] 0.0001);
FIG. 12 shows that morroniside has no significant side effects on the liver and kidney of denervation-induced mice;
figure 13 schematically illustrates the protective effects, mechanisms and safety of morroniside in promoting skeletal muscle growth and skeletal muscle atrophy, injury.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Experimental Material
1. Morroniside
Purity 99.57% and was purchased from Dowmastt Biotech, inc., CAS No.25406-64-8, cat # A0349.
2. C2C12 cell line
Derived from mouse myoblasts (skeletal muscle stem cells) purchased from
CRL-1772TM。
3. Reagent
C2C12 complete basal medium: 10% P/S + DMEM;
C2C12 differentiation medium: 2% HS +1% P/S + DMEM;
C2C12 frozen stock solution: 10% DMSO + FBS.
4. Animal(s) production
2-month-old C57BL/6 male mice were subjected to skeletal muscle injury induced by skeletal muscle injection of 1.2% barium chloride to investigate the effect of muscle regeneration. The experimental muscle injury model operating method comprises the following steps: mice were first anesthetized by intraperitoneal injection of 250-500mg/kg tribromoethanol (Avertin). Then 50. Mu.l of 1.2% barium chloride (BaCl) was injected Intramuscularly (IM) using a 30G syringe 2 ) The solution was added to the tibialis anterior and then randomized into vehicle saline control and morroniside drug treated groups based on body weight. Starting on day 2, the carrier saline or morroniside was administered at 20. Mu.g/kg/day, and 10 days after administration, the hind limb tibialis anterior muscle of the mouse was taken, embedded in O.C.T. (Sakura Finetek, cat # 4583), cryosectioned, and analyzed for cross-sectional area (CSA) of the nascent muscle fiber (CNF) by Laminin (green) and DAPI (blue) immunofluorescence staining. The muscle fiber with the nucleus in the center is new muscle fiber, and the capability of the skeletal muscle stem cell to differentiate to form the muscle fiber and the capability of the muscle to regenerate after the skeletal muscle injury are observed by counting the cross section area of the new muscle fiber.
The experimental muscle atrophy model was operated as follows: male C57BL/6 mice, 2 months old, were randomly grouped according to body weight and anesthetized by intraperitoneal injection of 250-500mg/kg tribromoethanol (Avertin). Hair on the hind limbs was shaved using an electric shaver. A small incision was made in the hind limb skin, the sciatic nerves of both legs were separated, approximately 5mm of the nerves were cut with surgical scissors, and the incision was closed with sutures. Mice received 5mg/kg of meloxicam subcutaneous injections once a day for three days to control pain. In the sham group, mice received similar surgery, but did not undergo sciatic nerve transection. To study the effect of morroniside, mice in sham and denervation groups were intraperitoneally injected daily with vehicle saline, starting on the first day after surgery; the nervus-loss low-dose treatment group is injected with 10 mu g/kg of morroniside per day in the abdominal cavity, and the nervus-loss high-dose treatment group is injected with 20 mu g/kg of morroniside per day for 12 days. Thereafter, the mice were sacrificed by euthanasia and blood, various skeletal muscle tissues, liver and kidney tissues were collected for further experiments. Tibialis Anterior (TA) muscle tissue was dehydrated in 30% sucrose PBS solution for 24 hours, then embedded in o.c.t (Sakura Finetek, cat # 4583), frozen in dry ice cooled isopentane, and stored at-80 ℃ for subsequent sectioning and staining.
2. Experimental methods
1. Cell survival proliferation and induction of myotube formation
Survival and proliferation: C2C12 myoblasts were given a morroniside treatment of 0-640. Mu.g/mL for 48 hours, and the survival and proliferation of the cells were examined with the CCK8 kit ((Vazyme, cat. No. A311-02).
Induction of myotubes: separating myogenic cells (CD 45) - ；CD31 - ；CD11b - ；Sca1 - ) Or C2C12 cells were seeded in growth medium (DMEM medium containing 10% FBS, 1% Pen/Strep, 1% Glu) until cell fusion reached 70%. The differentiation medium (DMEM medium containing 2% horse serum, 1% Pen/Strep, 1% Glu) with or without low-dose morroniside (Mor-L, 80. Mu.g/mL) or high-dose morroniside (Mor-H, 160. Mu.g/mL) containing 5ng/mL, 10ng/mL or 50ng/mL TNF α was incubated for 8-72 hours, with medium replacement every 12-48 hours.
2. Real-time quantitative PCR
Gastrocnemius muscles were homogenized in tissue lyser II (Qiagen, USA) in TRIzol reagent (Invitrogen, thermo Fisher Scientific). Total RNA was extracted from muscle homogenates or C2C12 myotubes using TRIzol reagent. The cDNA was synthesized using HiScript II cDNA Synthesis kit (Vazyme, cat. No. R222-01). Quantitative RT-PCR amplification was performed in Jena Qtower384G machine using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, cat # Q711-02). Each sample was triplicated, and each experiment was repeated at least 3 times. Gene expression fold change was calculated using the 2^ - (Δ Δ Ct) method, normalized to Gapdh expression.
3. Western blot analysis and co-immunoprecipitation
Gastrocnemius muscle was placed in protein lysate (1 x RIPA lysis buffer (Millipore, cat # 20-188) supplemented with 5mm nem,1mm dtt,1mm PMSF and protease inhibitor (Roche, cat # 11836170001)) and muscle Tissue was lysed using Tissue Lyser II instrument (Qiagen, USA) at 30 Hz. For in vitro experiments, C2C12 cells were resuspended in protein lysis buffer. The muscle homogenate and C2C12 cell lysis buffer were shaken on ice for 40 minutes and centrifuged at 13,300r.p.m. for 15 minutes at 4 ℃. Protein concentrations were quantified using the BCA protein assay kit (thermolfisher, cat # 23225). Whole cell lysates (10-30. Mu.g protein/lane) were loaded on 4% -15% SDS-PAGE gels and transferred to PVDF membrane. Immunoblotting was performed using TNF α antibodies (1, 1000, cell Signaling, cat No. 11948T), relA (1. For the ubiquitination assay, 500 μ g of protein lysate containing 103nM ubiquitin-aldehyde (South Bay Bio, CA, USA, cat # SBB-PS 0031) was incubated with MyHC antibody (1, R &D, cat # MAB 4470), and the precipitated protein was immunoblotted with Ub antibody (santa cruz, cat # sc-8017). The bands were visualized using ECL chemiluminescence (Thermo Fisher, cat No. 34577).
4. Immunofluorescence staining
Frozen sections (10 μm thick) were fixed with 4% PFA for 10 min, washed and blocked with 0.2% Triton-100 and 10% normal goat serum in PBST for 30 min at room temperature and incubated with 3% affinity pure Fab fragments anti-mouse IgG (H + L) and anti-mouse IgM (Jackson Immuno Research, west Grove, pa., USA, cat # 115-007-003 and 115-006-020) for 1H at room temperature. Sections were incubated with primary anti-MyHC-IIA (1, DSHB, iowa City, IA, USA, cat # SC-71), myHC-IIB (1. The next day, sections were incubated with Alexa Fluor 568/488-conjugated goat anti-mouse IgG1/IgM secondary antibody (1. MyHC primary antibody (1, R &D systems, minneapolis, MN, USA, cat # MAB 4470) was incubated overnight at 4 ℃ for myotube staining of primary myoblasts and C2C12 cell differentiation. The following day, alexa Fluor 488-conjugated goat anti-mouse IgG (1, 200, invitrogen, cat # A11001) was incubated at room temperature for 1 hour. Stained slides were mounted with VECTASHIELD Antifade Mounting Medium slides with and without DAPI (Vector Laboratories, burlingame, CA, USA, catalog H-1200-10 or H-1000-10) followed by imaging using Olympus (Shinjuku, japan) FV3000 inverted confocal laser scanning microscope. The stained plates were imaged using an Olympus (Shinjuku, japan) IX83 inverted electric fluorescence microscope.
5. Serum alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), urea nitrogen and creatinine quantification
Mice were sacrificed by euthanasia and blood was drawn from the heart with a 1mL syringe. Serum was isolated according to manufacturer's instructions (national institute for bioengineering, nanjing, china) for measurement of serum ALT (catalog # C009-2-1), serum AST (catalog # C010-2-1), serum urea nitrogen (catalog # C013-2-1) and serum creatinine (catalog # C011-2-1).
6. Hematoxylin and Eosin (HE) staining
The liver was cut into 0.5c wide pieces, the kidney was cut into Cheng Liangkuai identical pieces along the coronal axis and fixed in 10% buffered formalin at 4 ℃ for 1-2 days. The samples were dehydrated in a series of graded ethanol and xylene solutions and embedded in paraffin. Sections 4 μm thick were line HE stained.
7. Statistical analysis
In the experimental study, all results are given as mean ± standard deviation. Statistical analysis was performed using GraphPad prism8.0.1 Software (GraphPad Software inc., san Diego, CA, USA). The unpaired two-tailed Student's t test was used for comparison between the two groups. One-way anova and Turkey's multiple comparison test were used for comparisons between 3 or more groups. Two-factor analysis of variance and Tukey's multiple comparison test was used for multiple sets of comparisons between two factors. A p-value of less than 0.05 is considered statistically significant.
3. Results of the experiment
1. The morroniside directly promotes the survival, proliferation and differentiation of skeletal muscle stem cells, and the high-concentration morroniside (640 mu g/mL) has no obvious toxic and side effects on the skeletal muscle stem cells
In order to detect the proliferation and differentiation effects of morroniside on skeletal muscle stem cells directly, C2C12 myoblasts are firstly treated by 0-640 mu g/mL of morroniside in vitro for 48 hours, and the results show that the cell activity increasing capability is obviously increased along with the increase of the administration dose of the morroniside, the survival and proliferation of the C2C12 myoblasts can be effectively promoted when the concentration of the morroniside is 10-160 mu g/mL, and the concentration of the morroniside reaches 640 mu g/mL without any obvious toxic and side effects on the cells (figure 1A). Then C2C12 cells are treated by morroniside while induction of myotube formation, and an immunofluorescence staining result shows that the morroniside can remarkably promote MyHC positive mature myotube differentiation with more than three nuclei and has dose dependence (figure 1B); qPCR results showed that morroniside significantly promoted the expression of myotube differentiation indicators MyoD, myoG and was dose dependent (fig. 1C).
2. Morroniside directly promotes differentiation of skeletal muscle stem cells after muscle injury and promotes skeletal muscle regeneration
2-month-old C57BL/6 male mice were subjected to skeletal muscle injury induced by skeletal muscle injection of 1.2% barium chloride to investigate the effect of muscle regeneration. Starting on day 2 of muscle injury, morroniside was administered at 20. Mu.g/kg/day, and after 10 days of administration, the posterior tibialis anterior muscles of mice were harvested, embedded in O.C.T., frozen sections were taken, and the cross-sectional area (CSA) of the myofibers (CNF) of the newly born nucleus in the center was analyzed by Lamin (green) and DAPI (blue) immunofluorescence staining (FIG. 2A) (FIG. 2B). The muscle fiber with the nucleus in the center is new muscle fiber, and the capability of the skeletal muscle stem cell to be differentiated into the muscle fiber and the capability of the skeletal muscle to be regenerated after the skeletal muscle injury are observed by counting the cross sectional area of the new muscle fiber; the results show that the cross-sectional area of the core-centered neomyofibers was significantly larger than the carrier saline group (Veh) after morroniside treatment (fig. 2A, fig. 2B). Therefore, the morroniside can effectively promote the skeletal muscle stem cells to differentiate and form new muscle fibers, thereby achieving the therapeutic effect of promoting the skeletal muscle regeneration after the injury.
3. Monoroside directly promotes protein synthesis signal path of C2C12 myotube
Protein synthesis is critical to myotube formation, muscle growth, development, function, and the IGF-1/IGF-1R/Akt and BMP pathways are two key pathways for skeletal muscle protein synthesis. The results of the direct effect of morroniside on the C2C12 myotube IGF-1/IGF-1R/Akt protein synthesis pathway indicate that: morroniside up-regulated the expression of IGF-1, IGF-1R, PI K kinase catalytic subunit PIK3CA, akt2 mRNA (fig. 3A-fig. 3D).
Furthermore, the results of studies on the direct effect of morroniside on the BMP protein synthesis pathway indicate that: morroniside up-regulates BMP ligand BMP14 (fig. 3E), mRNA expression of the BMP type II receptor BMPR2 (fig. 3F), and the level of recruitment of the type 1 receptor ALK2/3 (fig. 3G-fig. 3H). At the same time, morroniside attenuated the expression of the BMP pathway down-regulator Smad6 (fig. 3I) and up-regulated Smad5/9mRNA levels (fig. 3K-fig. 3L).
The above results show that: the morroniside directly promotes IGF-1/IGF-1R/PI3K/Akt and BMP14/BMPR2/ALK2/3/Smad6/5/9 signaling pathways in the differentiation and formation process of C2C12 myotubes, and promotes protein synthesis and expression.
4. Morroniside directly inhibits proteasome and autophagy degradation signal pathway and zinc ion aggregation signal of C2C12 myotube
The ubiquitin-protease system and autophagy-lysosome system induce muscle protein degradation. Myostatin/activin and GDF11 (BMP 11) bind to type II receptors (e.g., actRIIB/IIA) and recruit type I receptors ALK4/5/7, which activate the transcription factor Smad2/3 via Smad4, promoting ubiquitination-related protein degradation. Myostatin/activin ligands are inhibited extracellularly by the cytokine Follistatin. Smad7 down regulates the Myostatin/activin signaling pathway in the cytoplasm. Transcription factor forkhead box O3 (FoxO 3) can not only up-regulate ubiquitin-proteasome systems (Atrogin 1 and MuRF 1), but also activate autophagy-lysosome systems (BNIP 3 and LC 3). In addition, ZIP-14 dependent zinc ion accumulation in cachexia leads to loss of MyHC (Wang et al, 2018).
The involvement of protein degradation signals was studied in the muscle-increasing effect of morroniside, the protein degradation pathway of myostatin is shown in fig. 4A-4E, and morroniside directly significantly increased mRNA levels of myostatin pathway inhibitory cytokine Follistatin and negative regulator Smad7 in C2C12 myotubes, and decreased ligand Bmp11 and effector Smad3mRNA expression (fig. 4A-4E).
To investigate whether morroniside inhibits the expression of FoxO3, atrogin1 and MuRF1, qPCR was performed and as a result: morroniside was able to directly reduce FoxO3 as well as Atrogin1 and MuRF 1mRNA levels in C2C12 myotubes (fig. 4F-fig. 4H).
To investigate another protein degradation pathway (autophagy-lysosomal system); the results show that: mRNA levels of autophagy-related proteins Bnip3, LC3a in C2C12 myotubes were significantly reduced after morroniside treatment (fig. 4I-fig. 4J).
In addition, the zinc ion aggregation signal is studied, and the result shows that: morroniside significantly reduced Zip14 mRNA expression in C2C12 myotubes (fig. 4L).
The above results show that: in the C2C12 myotube differentiation process, morroniside attenuated the activation of Myostatin/activin pathway (Follistatin/Smad 7 up-regulated, bmp11/Smad3 down-regulated), foxO3 and genes related to ubiquitin-proteasome system (Atrogin 1, muRF 1), autophagy-lysosome system (Bnip 3, LC3 a) and zinc ion accumulation signal (ZIP 14) (fig. 4A-4L), thereby inhibiting the degradation of myosin heavy chain MyHC etc., consistent with the results of fig. 1B.
5. TNF alpha is increased in atrophic skeletal muscle caused by denervation and induces myotube atrophy in vitro
To examine the role of TNF α in denervation-induced muscle atrophy, a portion of the sciatic nerve of adult wild-type (WT) mice was removed and TNF α levels in gastrocnemius muscles of sham-operated and denervated mice were compared; the results show that: mean mRNA and protein levels of TNF α in skeletal muscle samples of denervated mice were higher than those of sham operated mice (fig. 5A and 5B). Skeletal muscle phenotype of sham-operated and denervated mice was compared; the results show that: weight (BW), tibialis Anterior (TA), extensor Digitorum Longus (EDL), soleus muscle (Soleus) and gastrocnemius muscle (Gas) of denervated mice were reduced (fig. 5C). Treating a murine myoblast cell line C2C12 with TNF α and inducing cell differentiation into myotubes; the statistics of the number of mature myotubes with more than three nuclei positive by MyHC shows that: TNF α significantly inhibited C2C12 cell myotube formation in a dose-dependent manner (FIG. 5D). Similar inhibition of myotube formation by TNF α was found in primary myogenic cells of young C57BL/6 mice by magnetic cell sorting (MACS) (fig. 5E).
6. Morroniside for in vitro correction of TNF alpha induced myotube atrophy
C2C12 cells were treated with TNF α and induced to differentiate into myotubes with or without morroniside administration. The immunofluorescence staining results show that: the number of mature myotubes per unit area, the average number of nuclei per myotube and the relative diameter of myotubes in TNF α -treated C2C12 cells decreased during myotube formation compared to the vehicle group, while the above parameters after addition of low or high doses of morroniside to TNF α -treated cells were similar to the values in the vehicle group and significantly higher than the TNF α -treated group (fig. 6A and 6B). Thus, morroniside corrects TNF α -induced myotube atrophy in vitro.
7. Morroniside for preventing skeletal muscle atrophy caused by in vivo denervation
Adult male C57BL/6 mice were either sciatic nerve transection or Sham operated, and mice were randomized into Sham (Sham), denervation (Den), den + low dose morroniside (Mor-L), den + high dose morroniside group (Mor-H). The results show that: the weight of the denervated mice tended to decline over time, while the administration of low doses of morroniside tended to restore the mice weight to normal levels; high dose morroniside administration significantly increased the body weight of the denervated mice even on day 10 post-surgery compared to the denervated group (fig. 7A). Consistent with FIG. 5C, weights of Tibialis Anterior (TA), extensor Digitorum Longus (EDL), soleus (Soleus) and gastrocnemius (Gas) muscles were reduced in the denervated mice, but increased significantly in the high dose morroniside-treated denervated mice (FIG. 7A).
Pax7 is an established muscle stem cell (MuSC) marker that plays a key role in regulating MuSC proliferation. Immunofluorescent staining showed that the number of Pax7+ satellite cells was significantly lower in the denervated mouse tibialis anterior TA than in the sham-operated group, while the denervated mice treated with high doses of morroniside had similar number of Pax 7-positive skeletal muscle stem cells and significantly higher than the denervated mice (fig. 7B); thus, morroniside maintains skeletal muscle stem cell self-renewal.
The diversity of muscle fiber types is related to functional diversity, and changes in muscle fiber types affect the contractile, metabolic, and biochemical properties of muscles. The exact type of muscle fiber affected in TA after denervation is largely unknown. The average cross-sectional area (CSA) of the muscle fibers (fig. 7B), in particular the average CSA of the MyHC IIA, IIB and IIX muscle fibers (fig. 7C), the percentage of number of MyHC IIA muscle fibers (fig. 7C) was significantly lower in the tibialis anterior TA of the denervated mice than in the sham-operated group, while the denervated mice treated with high-dose morroniside were similar to the above parameters of the sham-operated mice and significantly higher than the denervated mice (fig. 7B-fig. 7C); thus, after denervation, morroniside maintained the type diversity and satiation status of mature muscle fibers.
The above results show that: the morroniside can relieve skeletal muscle atrophy caused by denervation in vivo by maintaining the self-renewal of skeletal muscle stem cells, promoting differentiation and maintaining the type diversity and plump state of mature muscle fibers.
8. Morroniside inhibits classical and non-classical NF-. Kappa.B signaling in TNF alpha-treated C2C12 myotubes and denervated mouse muscles
NF-. Kappa.B signaling is activated by two different pathways: the classical pathway and the non-classical pathway; wherein, classical NF-kB signal activation in satellite cells attenuates skeletal muscle regeneration after adult mice are injured; activation of non-classical NF- κ B impairs myoblast differentiation, muscle stem cell function and muscle regeneration in mice. Treating C2C12 cells with TNF α or TNF α plus morroniside during differentiation; as a result, it was found that: TNF α stimulated transcription and translation of classical NF-. Kappa.B RelA, p50 and non-classical NF-. Kappa.B RelB, p52 genes in C2C12 myotubes (FIG. 8A-FIG. 8C). Similarly, denervation-induced mice had higher mRNA and protein levels of RelA, p50, relB, and p52 in gastrocnemius than sham-operated mice (fig. 4D-fig. 4F). Morroniside treatment significantly reduced mRNA and protein levels of RelA, relB, and p52, but not p50, in C2C12 cells treated with TNF α compared to TNF α treatment alone (fig. 8A-8C). The levels of RelA, relB, and p52 mRNA were significantly reduced in the morroniside-treated denervated mice compared to the denervated mice, and the levels of RelA, p50, relB, and p52 protein were lower in the gastrocnemius than in the denervated mice (FIG. 8D-FIG. 8F).
9. Morroniside attenuates inflammatory mediators and zinc ion accumulation signals in muscles of C2C12 myotubes and denervated mice treated with TNF alpha
The results on the anti-inflammatory action of morroniside in muscle showed that: TNF α increased IL-6, IL-1b, CRP, NLRP3, PTGS2 mRNA levels and TNF α mRNA and protein levels in C2C12 myotubes (FIG. 9A-FIG. 9F, FIG. 9H). Similarly, denervation-induced mice had higher TNF α mRNA and protein levels in gastrocnemius than sham mice (fig. 9I-9J). Morroniside treatment significantly reduced the mRNA and/or protein levels of these inflammatory factors in TNF α -treated C2C12 myotubes and gastrocnemius muscle from denervated mice (fig. 9A-9F, fig. 9H-9J). Morroniside treatment significantly reduced ZIP14 mRNA levels in TNF α -treated C2C12 myotubes (fig. 9G).
The above results show that: morroniside can attenuate both classical (p 65/RelA, p 50) and non-classical (RelB, p 52) NF-. Kappa.B signaling and downstream inflammatory factors (IL 6, IL-1b, CRP, NIRP3, PTGS2, TNF. Alpha.) expression, while attenuating the zinc ion accumulation signal (ZIP 14) in TNF. Alpha. Treated C2C12 myotubes and/or in skeletal muscle samples from denervated mice.
10. Morroniside improves protein synthesis signaling pathways in TNF alpha-treated myotubes
An imbalance between protein synthesis and degradation results in skeletal muscle atrophy, with the IGF-1/IGF-1R/Akt and BMP pathways being the two key skeletal muscle protein synthesis pathways. The results of the effect of morroniside on the synthetic pathway of IGF-1/IGF-1R/Akt protein after TNF α treatment indicated that: TNF α reduced IGF-1mRNA expression, which was alleviated by morroniside treatment (FIG. 10A). Morroniside ameliorated TNF α -induced down-regulation of IGF-1R mRNA levels (fig. 10B). TNF α decreased the mRNA levels of IRS-1 adaptor protein (fig. 10C) and downstream PI3K regulatory subunit PIK3R1 (fig. 10D) and catalytic subunit PIK3CA (fig. 10E) as well as Akt1/2/3 (fig. 10F-fig. 10H). In TNF α -treated C2C12 myotubes, morroniside up-regulated IRS-1, PIK3R1, PIK3CA, and Akt1 (FIG. 10C-FIG. 10F).
In addition, the results of studies on the BMP protein synthesis pathway indicate that: TNF α reduced BMP ligand BMP14 (fig. 10I), mRNA expression of BMP type II receptor BMPR2 (fig. 10J), and recruitment levels of type 1 receptor ALK2/3 (fig. 10K-fig. 10L), which could be reversed by morroniside treatment (fig. 10I-fig. 10L). Morroniside tended to attenuate TNF α -induced increases in Smad6, a negative regulator of the BMP pathway (fig. 10M), and TNF α -induced decreases in Smad1 (fig. 10N). Morroniside significantly attenuated TNF α -induced reductions in Smad5/9mRNA levels (FIG. 10O-FIG. 10P).
The above results show that: the morroniside promotes IGF-1/IGF-1R/PI3K/Akt signal pathways and BMP14/BMPR2/ALK2/3/Smad5/9 signal pathways, reverses the inhibition effect of TNFa on protein synthesis in C2C12 myotubes, and further protects the C2C12 myotubes from TNF alpha-induced atrophy.
11. Morroniside inhibits proteasome and autophagy degradation signaling pathways in TNF alpha-treated C2C12 myotubes and/or denervated mouse muscles
The ubiquitin-protease system and autophagy-lysosome system induce muscle protein degradation. The Myostatin/activin pathway is involved in protein degradation. Myostatin/activin and GDF11 (BMP 11) bind to type II receptors (e.g., actRIIB/IIA) and recruit type I receptors ALK4/5/7, which activate the transcription factor Smad2/3 via Smad4, promoting ubiquitination-related protein degradation. Myostatin/activin ligands are inhibited extracellularly by the cytokine Follistatin. Smad7 down regulates the Myostatin/activin signaling pathway in the cytoplasm. Transcription factor forkhead box O3 (FoxO 3) can not only up-regulate ubiquitin-proteasome systems (Atrogin 1 and MuRF 1), but also activate autophagy-lysosome systems (BNIP 3 and LC 3).
The involvement of protein degradation signals in the protection mechanism of morroniside was studied, the protein degradation pathway of myostatin, as shown in fig. 11A, TNF α significantly reduced mRNA levels of myostatin pathway suppressor cytokine Follistatin and negative regulator Smad7, increased ligand myostatin, type I receptor ALK4/5/7, and effector Smad3mRNA expression, all of which were reversed by morroniside treatment in TNF α -treated C2C12 myotubes (fig. 11A).
To investigate whether morroniside inhibits the expression of FoxO3, atrogin1 and MuRF1 and the reduction of MyHC in muscle, qPCR and western blot were performed and as a result: in response to TNF α, foxO 3mRNA levels and transcription and translation of Atrogin1 and MuRF1 were upregulated in C2C12 myotubes (fig. 11B-11C). Similarly, the denervated mice had higher Atrogin1 and MuRF 1mRNA and protein expression in gastrocnemius than sham operated mice (FIG. 11D-FIG. 11E). Morroniside significantly reduced the mRNA and/or protein levels of these genes in TNF α -treated C2C12 myotubes and gastrocnemius muscle from denervated mice (fig. 11B-11E). In line with this, the level of MyHC protein in gastrocnemius muscle of TNF α treated C2C12 myotubes and denervated mice was significantly lower than in the control vehicle treated myotubes and sham operated mouse groups, while the level of MyHC in gastrocnemius samples from TNF α plus morroniside treated C2C12 myotubes and morroniside administered denervated mice was substantially similar to the level in control vehicle treated myotubes and sham operated mice and significantly higher than in TNF α treated C2C12 myotubes and gastrocnemius muscle from denervated mice, respectively (fig. 11F-11G).
To further investigate the mechanism by which morroniside prevents MyHC degradation, myHC ubiquitination levels were measured and found to be: they were higher in gastrocnemius in denervated mice than in sham-operated mice, but morroniside treatment significantly reduced these values compared to denervated mice (fig. 11H).
In addition, another protein degradation pathway (autophagy-lysosomal system) was also investigated; the results show that: the mRNA levels of autophagy-related proteins Bnip3, LC3a and LC3b were significantly increased by TNF α (FIG. 11I). Morroniside treatment significantly reduced TNF α -induced Bnip3, LC3a, and LC3b mRNA expression (fig. 11I).
The above results show that: in TNF α -treated C2C12 myotubes or denervated mouse muscles, morroniside attenuates the Myostatin/activin pathway (upregulation of Follistatin/Smad7, downregulation of Myostatin/Alk4/5/7/Smad 2/3), foxO3, and activation of downstream genes/proteins associated with ubiquitin-proteasome systems (Atrogin 1, muRF 1) and autophagy-lysosomal systems (Bnip 3, LC3a, LC3 b), thereby inhibiting degradation of myosin heavy chain MyHC, and consequently TNF α -induced myotube atrophy and denervation-induced muscle atrophy.
12. The morroniside has no obvious side effect on liver and kidney of mouse induced by denervation
Further researching whether the morroniside has any side effect on the mice, and finding out that: serum ALT and AST levels (two liver injury markers) were not significantly different in sham, denervation (Den) and Den + high dose morroniside (Mor) groups (fig. 12A). Also, morroniside did not significantly alter serum urea nitrogen and creatinine levels (two kidney injury markers) in the denervated mice compared to sham and denervated groups (fig. 12B). HE staining results showed that morroniside did not induce abnormal phenotypes such as inflammatory infiltration, degeneration or necrosis of liver cells and renal epithelial cells in liver and kidney of denervated mice, compared to sham and denervated groups (fig. 12C-12D). This indicates that the morroniside has no significant side effects on the liver and kidney of the denervation-induced muscular atrophy mice.
In summary, the above research results show that: the morroniside directly promotes the survival, proliferation and differentiation of skeletal muscle stem cells, and high-concentration morroniside (640 mu g/mL) has no obvious toxic or side effect on the skeletal muscle stem cells (figure 1); morroniside directly promoted skeletal muscle stem cell differentiation and promoted skeletal muscle regeneration after muscle injury (fig. 2); the morroniside directly promotes IGF-1/IGF-1R/PI3K/Akt and BMP14/BMPR2/ALK2/3/Smad6/5/9 signal pathways, and promotes protein synthesis of C2C12 myotubes (figure 3); morroniside directly inhibits activation of Myostatin/activin pathway, foxO3, ubiquitin-proteasome system, autophagy-lysosome system, and zinc ion aggregation signal-related genes in C2C12 myotubes (fig. 4); increased TNF α levels in denervation-induced atrophied muscle were shown to inhibit myotube formation in vitro (figure 5); morroniside corrected TNF α -induced myotube atrophy in vitro (figure 6); denervated mice receiving morroniside injection showed improvements in body weight, muscle mass, pax7+ satellite cell (skeletal muscle stem cell) number, muscle cross-sectional area (MyHC IIA, IIB, and IIX), and MyHC IIA + fiber percentage (fig. 7); morroniside can attenuate both classical (p 65/RelA, p 50) and non-classical (RelB, p 52) NF- κ B signaling and downstream inflammatory factors (IL 6, IL-1b, crp, nirp3, ptgs2, TNF α) as well as zinc ion accumulation signal (ZIP 14) expression in muscle samples from TNF α -treated C2C12 myotubes and/or from denervated mice (fig. 8 and 9); morroniside attenuated TNF α inhibition of IGF-1/IGF-1R/IRS-1/PI3K/Akt signaling pathway and BMP14/BMPR2/ALK2/3/Smad5/9 pathway, promoting protein synthesis in C2C12 myotubes (FIG. 10); in TNF α -treated C2C12 myotubes or denervated mouse muscles, morroniside attenuated the activation of Myostatin/activin pathway, foxO3, and downstream genes/proteins associated with the ubiquitin-protease system and autophagy-lysosome system (fig. 11); after morroniside treatment, no adverse reaction or significant change in appearance, behavior or diet was observed in the mice, no signs of redness or edema were seen in any part of the body, no significant toxicity or pathology was found in any organs including liver and kidney, and no adverse reaction was evident in the mice with morroniside (fig. 12). The morroniside-mediated activity of muscle proliferation, atrophy resistance and muscle injury resistance is related to promoting survival proliferation and differentiation of skeletal muscle stem cells, maintaining self-renewal of skeletal muscle stem cells and type diversity and plumpness of mature muscle fibers, inhibiting classical and non-classical NF-kB signal transduction, inflammatory mediators and zinc accumulation signals, proteasome and autophagy protein degradation pathways, and improving protein synthesis signal pathways (figure 13), and provides strong preliminary evidence for the muscle proliferation, atrophy resistance, muscle injury resistance, mechanism and safety of morroniside so as to support further development of health care products or drugs.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. Application of morroniside in preparing health products and/or medicines for increasing muscle, preventing and treating skeletal muscle atrophy and/or preventing and treating muscle injury is provided.
2. The use according to claim 1, wherein morroniside augments muscle, prevents skeletal muscle atrophy and/or prevents muscle damage by promoting survival proliferation and/or differentiation of skeletal muscle stem cells.
3. The use of claim 1, wherein the morroniside increases muscle mass, prevents skeletal muscle atrophy and/or prevents muscle damage by at least one of promoting skeletal muscle stem cell differentiation and promoting skeletal muscle regeneration; preferably, the skeletal muscle stem cell differentiation comprises differentiation of skeletal muscle stem cells into muscle fibers.
4. The use of claim 1, wherein the morroniside increases muscle mass, prevents skeletal muscle atrophy and/or prevents muscle injury by at least one of maintaining self-renewal of skeletal muscle stem cells, maintaining diversity of types of mature muscle fibers, and maintaining the satiation status of mature muscle fibers.
5. Use according to claim 1, characterized in that morroniside prevents skeletal muscle atrophy and/or prevents muscle damage by inhibiting classical and/or non-classical NF- κ B signalling; preferably, inhibiting classical NF- κ B signaling comprises inhibiting NF- κ B p/RelA signaling and/or inhibiting NF- κ B p signaling; inhibiting non-classical NF-. Kappa.B signaling includes inhibiting NF-. Kappa.B RelB signaling and/or inhibiting NF-. Kappa. B p52 signaling.
6. Use according to claim 1, characterized in that morroniside prevents skeletal muscle atrophy and/or prevents muscle damage by inhibiting the expression of inflammatory factors downstream of classical and/or non-classical NF- κ B signalling; preferably, the inflammatory factor comprises at least one of IL6, IL-1b, CRP, NIRP3, PTGS2 and TNF α.
7. The use of claim 1, wherein the morroniside augments muscle by one of activating IGF-1/IGF-1R/PI3K/Akt, activating BMP14/BMPR2/ALK2/3/Smad6/5/9 signaling pathways, and promoting myotube protein synthesis;
preferably, the morroniside can prevent and treat skeletal muscle atrophy and/or muscle injury by activating one of IGF-1/IGF-1R/IRS-1/PI3K/Akt, activating BMP14/BMPR2/ALK2/3/Smad5/9 signaling pathway and promoting myotube protein synthesis.
8. The use according to claim 1, wherein the morroniside increases muscle mass, prevents skeletal muscle atrophy and/or prevents muscle damage by at least one of inhibiting a Myostatin/activin protein degradation pathway, inhibiting activation of downstream genes/proteins associated with the ubiquitin-proteasome system, and inhibiting activation of downstream genes/proteins associated with the autophagy-lysosomal system;
preferably, inhibiting the Myostatin/activin protein degradation pathway comprises at least one of up-regulating Follistatin/Smad7 and down-regulating Myostatin/Bmp11/Alk4/5/7/Smad 2/3; inhibiting ubiquitin-proteasome system-associated downstream gene/protein activation includes inhibiting at least one of FoxO3, atrogin-1, and MuRF1 expression; inhibiting activation of a downstream gene/protein associated with the autophagy-lysosomal system includes inhibiting at least one of Bnip3, LC3A, and LC3B expression.
9. The use according to claim 1, wherein the morroniside increases muscle mass, prevents skeletal muscle atrophy and/or prevents muscle damage by inhibiting the zinc ion accumulation signal ZIP 14.
10. The use of claim 1, wherein augmenting muscle comprises preventing, ameliorating and/or treating a phenotype and/or disease associated with at least one of skeletal muscle growth, development, quality, strength and function; preventing skeletal muscle atrophy and/or preventing muscle damage including preventing, ameliorating and/or treating skeletal muscle atrophy-related diseases and/or muscle damage-related diseases;
preferably, the skeletal muscle atrophy-related disease includes muscular atrophy caused by at least one pathological condition of aging, rheumatoid arthritis, cachexia, cancer, disuse, absence of nerve, various muscular dystrophies including duchenne muscular dystrophy, muscle weakness, myositis, myopathy, crohn's disease, and diabetes;
preferably, the muscle injury-related disorder comprises muscle injury caused by at least one of exercise, trauma, bruise, strain, laceration, toxin, degenerative disease, and strain;
preferably, the purity of the morroniside is more than or equal to 98 percent;
preferably, the effective dose of the morroniside in muscle increasing, skeletal muscle atrophy preventing and/or muscle injury preventing is 0.8-4.7 μ g/kg/d, preferably 0.8-2.4 μ g/kg/d.
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