CN108685934B

CN108685934B - Application of geniposide in promoting generation of skeletal muscle fast muscle

Info

Publication number: CN108685934B
Application number: CN201810960478.7A
Authority: CN
Inventors: 王立; 李言; 潘海鸥; 钱海峰; 张晖; 齐希光
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-08-22
Filing date: 2018-08-22
Publication date: 2020-01-07
Anticipated expiration: 2038-08-22
Also published as: CN108685934A; WO2020037895A1

Abstract

The invention discloses an application of geniposide in promoting generation of skeletal muscle fast muscle, belonging to the field of natural product treatment. According to the invention, geniposide is used for drug administration treatment of mice, and the number of fast muscle fibers of an administration group is obviously more than that of a control group from the results of section staining and qPCR, so that skeletal muscle fiber typing is changed. According to the invention, geniposide is used for stimulating C2C12 myoblasts in an in vitro experiment, and from the immunofluorescence result, the geniposide treatment can increase the number of fast muscle fibers compared with a control group; further confirmation of mRNA levels, from the results of the concentration gradient and time gradient experiments, the treatment group caused a "slow → fast" switch in muscle fiber type. The geniposide is proved to have the effect of promoting the generation of skeletal muscle fast muscle, and has the potential of being applied to short-distance sports food.

Description

Application of geniposide in promoting generation of skeletal muscle fast muscle

Technical Field

The invention relates to an application of geniposide in promoting the generation of skeletal muscle fast muscle, belonging to the field of natural product treatment.

Background

Skeletal muscle is an organ of a human body directly participating in movement, accounts for about 40% -50% of the total weight of the human body, is the largest tissue of the human body, and can not normally play the functions of the human body when the movement and posture of the human body are maintained. Skeletal muscle, also known as striated muscle, is present in approximately 600 pieces of the human body. Skeletal muscle cells are fibrous, without branches, with distinct striations, numerous nuclei, and all located under the cell membrane. Within the muscle cell are numerous filamentous myofibrils aligned parallel to the long axis of the cell. The most basic functional unit of the motor system, also called motor unit, is composed of a motor neuron and a bundle of muscle fibers, which have similar, but not identical, structure and function. Many (from tens to hundreds) of motion units, each having its own particular and unique role, are assembled together to form a complete skeletal muscle. The movement units used for muscle assembly are specifically chosen to ensure that the muscles perform optimally all the requirements needed for the movement function. The heterogeneity of muscle fibers is the basis of strong adaptability of skeletal muscles, and the same skeletal muscle needs to adapt to different exercise requirements, such as persistent hypo-intense activity (maintaining body posture); intensity of movement in repeatability (movement of the body); and very high intensity activities (long jump, kick) for short periods of time. In addition, the nature of muscle fiber structure and function, also known as the muscle fiber phenotype.

Skeletal muscle fibers can be classified by morphology and function as fast muscle fibers and slow muscle fibers. The difference from the metabolic characteristics is that slow muscle fibers have high aerobic oxidation capacity, and are characterized by large number of mitochondria, large volume and high oxidase activity, ATP is continuously generated by relying on oxidative phosphorylation, lipid is the first choice energy supply raw material of the fibers, the triglyceride content is high, the capillary vessel is rich, and the myoglobin content is high. And the fast muscle fibers are activated when the muscles need to contract at high frequency for a short period of time. Fast muscle fibers have higher anaerobic metabolism capability, which is characterized in that the activity of enzymes participating in the anaerobic oxidation process in the muscle fibers is higher than that of slow muscle fibers, and the content of muscle glycogen is higher. The percentage of the number of different muscle fibers in the same muscle is referred to as the percentage composition of the muscle fiber type. The percentage composition of the two types of muscle fibers is closely related to some basic qualities. The percentage composition of slow muscles is related to general endurance and strength endurance; the percentage composition of fast muscles is related to speed and explosive force.

With the progress of research in recent years, skeletal muscle fiber types are determined by myosin heavy chain (MyHC) subtype. The MyHC gene family is also called as myosin heavy chain gene family, is one of the most important myofibrillar proteins in skeletal muscle fibers, is a main functional protein for muscle contraction, maintains the structural integrity of muscle cells, and has the gene expression as a main molecular marker for muscle fiber type division. Muscle contains myosin of different subtypes, i.e. exhibits different contractility and ATPase activity, and first the correspondence of a particular myofiber type to one of the four myofibrillar myosin heavy chain (MyHC) isomers was determined in humans and sentinel animals. To date, eight MyHC subtypes have been found in mammalian skeletal muscle, but adult skeletal muscle expresses only MyHC family types I, IIa, IIx and IIb, which correspond to 4 muscle fiber types of type I (slow oxidation), IIa (fast oxidation), IIx (intermediate) and IIb (fast glycolysis), respectively, and the metabolic types transition from oxidation to glycolysis, with the contraction rate increasing in order. During exercise, muscle fiber types may shift. Research shows that in a skeletal muscle acute contusion model, the expression of MyHC-IIb is increased, and the generation of new skeletal muscle can be promoted. Skeletal muscle with higher type IIb fiber content tends to have larger diameters. A high proportion of MyHC-IIb contributes to increased muscle mass. The increase in fast muscle mass may cause a decline in obesity and improve metabolic status by altering the fatty acid oxidation capacity in distant tissues, and strength training may be particularly beneficial to obese people. More studies have indicated that the oxidative to glycolytic metabolic shift in skeletal muscle has potential benefits in the diabetic state, increasing glucose homeostasis. MyHC-IIb is often absent in the elderly, suggesting that skeletal muscle composition is associated with age-related muscle loss and muscle disease. Although the maximum contraction rate of the muscle fiber is determined primarily by the ATPase of myosin, the maximum strength is determined primarily by the cross-sectional area of the muscle fiber.

The Gardenia fruit is mature fruit of Gardenia jasminoides Ellis of Gardenia of Rubiaceae, is mainly distributed in tropical and subtropical areas, and has yield accounting for about 90% of the total world yield in China. The gardenia fruit is the first medical and edible resource issued by the Ministry of health, is commonly used as a traditional Chinese medicine, a food additive, an edible pigment, oil pressing and the like, and has the effects of diminishing inflammation, easing pain, protecting liver and gallbladder, resisting oxidation and the like. The main active ingredients of the medicine comprise iridoid and crocin, wherein the iridoid mainly takes geniposide as the main component, and the crocin mainly takes crocin as the main component. Geniposide is one of the basic substances with pharmacological activity of fructus Gardeniae, the content is 3-9% according to the production area, and its structural formula is shown in figure 1. To date, geniposide has not been reported to have an effect on promoting skeletal muscle fast myogenesis.

Disclosure of Invention

The invention provides a compound or a composition for promoting skeletal muscle fast muscle generation, wherein the compound is geniposide, and the composition is a composition containing geniposide.

The skeletal muscle fast muscle is one of skeletal muscle fibers, namely type II muscle fibers, also called white muscle. The sugar metabolism of the fast skeletal muscle is mainly glycolysis. Skeletal muscle fast muscle is abundant in gastrocnemius and mainly plays a role in short-distance anaerobic exercise.

The promotion of skeletal muscle fast muscle generation means that type I muscle fibers (slow muscles) are reduced, type II muscle fibers (fast muscles) are increased, and/or the type of muscle fibers is converted, and/or the muscle mass is increased and the muscle fiber area is increased in myoblasts and skeletal muscle tissues in a certain dosage and time range. Promoting skeletal muscle fast muscle production is beneficial for playing a major role in short-term exercise.

The geniposide may be derived from: gardenia dregs, gardenia fruit, eucommia bark, rehmannia root, achyranthes root, spreading hedyotis herb, coptis root detoxification soup and the like.

The composition also comprises pharmaceutically acceptable auxiliary materials, such as a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an adhesive, a disintegrating agent, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a flavoring agent, an anti-adhesive, an integrating agent, an osmotic accelerator, a pH value regulator, a buffering agent, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, an encapsulating agent, a humectant, an absorbent, a diluting agent, a flocculating agent and a deflocculating agent, a filter aid, a release retardant and the like.

According to the invention, the geniposide standard substance is injected into the abdominal cavity to carry out drug delivery treatment on the mouse, and from the results of section dyeing and qPCR, the number of fast muscle fibers of a drug delivery group is obviously more than that of a control group, the expression of genes related to type I fibers is obviously reduced, the genes related to type II fibers are obviously increased, and the skeletal muscle fiber typing is changed. The invention is continuously verified in vitro experiments, the geniposide standard product is used for stimulating differentiated C2C12 myoblasts, and the geniposide treatment can increase the number of fast muscle fibers compared with a blank control group from the immunofluorescence result. Therefore, geniposide is believed to have a skeletal muscle fast myogenesis promoting effect.

Drawings

FIG. 1 chemical structural formula of geniposide.

Fig. 2 Hematoxylin and Eosin (HE) staining (n-3) (20X microscope) of mouse skeletal muscles gastrocnemius and soleus (GAS, SOL), Con being blank control group; geni is geniposide treatment group.

Fig. 3 shows Succinate Dehydrogenase (SDH) staining of GAS portion of mouse skeletal muscle (n ═ 3) (20 ×), Con for blank control; geni is geniposide treatment group. Statistics were performed on the proportion of type I muscle fibers in SDH-stained GAS results, data expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test).

Fig. 4(a) shows ATPase staining (PH 4.3) (n 3) (20X) of mouse skeletal muscle GAS sections, and statistics are given for the proportion of type I muscle fibers in the ATPase-stained GAS results; (B) ATPase staining (PH 4.3) (n 3) (20X) was performed on the mouse skeletal muscle SOL fraction, and statistics were performed on the proportion of type I muscle fibers in the results of ATPase staining SOL; (C) ATPase staining (PH 10.5) (n 3) (20X) was performed on the mouse skeletal muscle GAS fraction, and statistics were performed on the proportion of type I muscle fibers in the ATPase-stained GAS results; (D) the mouse skeletal muscle SOL fraction was ATPase stained (PH 10.5) (n 3) (20 ×), and the proportion of type I muscle fibers in the results of ATPase staining SOL was counted. Con is blank control group; geni is geniposide treated group, P <0.05 compared to control; p < 0.01; p <0.001 (T-test), each group was triplicated.

FIG. 5 real-time quantitative PCR (RT-qPCR) assay of mRNA expression levels of MyHC four subtypes, MyHC-I, MyHC-IIa, MyHC-IIx, MyHC-IIb, slow muscle marker genes MB and Tnni1, in mouse skeletal muscle GAS, data are expressed as mean. + -. standard error, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test). Con is blank control group; geni is geniposide treatment group.

FIG. 6C 2C12 cells were differentiated for 2 days, stimulated with 0.2mg/mL geniposide, and after 12h, the cells were fixed, immunofluorescent-detected with Anti-myostatin-fast (A) and Anti-myostatin-slow (B) antibodies, respectively, and fields of view were randomly photographed under a 10 Xmirror using a fluorescence microscope, where the selected fields of view are representative. Con is blank control group; geni is geniposide treatment group.

FIG. 7(A) C2C12 myotube cells were stimulated with geniposide at various concentrations (0,0.0125,0.025,0.05,0.1,0.2mg/mL) for 12h, RNA was extracted, and mRNA levels of MyHC-I, MyHC-IIa, MyHC-IIx, and MyHC-IIb were analyzed by RT-qPCR. (B) C2C12 myotube cells were treated with geniposide 0.4mg/mL for 0, 1, 2, 4, 8, 12h, RNA was extracted and mRNA levels of MyHC-I, MyHC-IIa, MyHC-IIx, and MyHC-IIb were analyzed by RT-qPCR. Con is blank control group; geni is geniposide treatment group. Data are expressed as mean ± sem, triplicate for each group. P <0.05 compared to control; p < 0.01; p <0.001 (T-test).

Detailed Description

Example 1 verification of the effect of geniposide on promoting skeletal muscle fast muscle production

1 method of experiment

1.1 animal experiments

The 6-8 week males used in the experiment were housed in SPF (specific pathogen free) grade animal houses, where the animals had free access to food and water (provided by the Shanghai Slek laboratory animal center). The animal room is kept at a constant temperature of 22 +/-3 ℃ and a relative humidity of 35 +/-5%, and 12 hours is a periodic day-night cycle. The use and handling of model animals were performed strictly in accordance with the protocols of the animal care and use committee of the institute of food science and technology, university of south China.

After the male mice had adapted to the new environment, they were randomly divided into two groups, a blank group and a treatment group. The blank group was not treated, and the treatment group was injected with geniposide standard (25mg/kg) daily according to body weight (fig. 1) for 21 consecutive days. After 21 days, all mice were sacrificed, Gastrocnemius (GAS), Soleus (SOL), and Tibialis Anterior (TA) muscles in the left and right hind limbs were taken, GAS and SOL in the right leg were fixed in gum tragacanth for rapid freezing and frozen sections for omics analysis, and the remaining tissues were immediately frozen with liquid nitrogen and stored at-80 ℃ for subsequent RNA extraction.

1.2 cell culture, cell treatment, immunofluorescence

Mouse myoblast of C2C12 was incubated at 37 ℃ with 5% CO₂Cultured in a DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% double antibody (P/S). When the C2C12 cells were induced to differentiate into myofibers, the FBS content in the cell culture medium was replaced with 2% Horse Serum (HS).

After the myoblasts had mostly differentiated into myotubes by day 4 of differentiation of the C2C12 cells, the C2C12 cells were treated with geniposide stimulation. Concentration gradient: the cells were stimulated with geniposide 0,0.0125,0.025,0.05,0.1,0.2mg/mL for 12h, respectively. Time gradient: C2C12 myotube cells were treated with 0.4mg/mL geniposide for 0, 1, 2, 4, 8, 12h, respectively.

And (3) immunofluorescence staining: after the cells were stimulated with 0.2mg/mL geniposide for 12h on day 2 of differentiation of C2C12 cells, the culture was washed off, fixed with methanol, and then rinsed 3 times with Phosphate Buffered Saline (PBS) for 10 minutes each. A3% Bovine Serum Albumin (BSA) solution in PBS was prepared, and polyethylene glycol octylphenyl ether (Triton X-100) was added to the solution to a final concentration of 0.1% to permeabilize the cells, and the cells were blocked at room temperature for 1 hour. PBS was washed 3 times. fast-Anti-myostatin-fast or slow-Anti-myostatin-slow diluted in the appropriate ratio was added and combined overnight at 4 ℃. After the primary antibody was bound, it was rinsed 3 times with PBS, and a secondary antibody was added to bind at room temperature for 1 hour, with the need to protect from light during the binding process. The nuclei were rinsed again, stained with 4', 6-diamidino-2-phenylindole (DAPI), and rinsed 3 times with PBS. The sample was observed under an inverted fluorescence microscope and a photograph was taken.

1.3RNA extraction and real-time quantitative fluorescent PCR

Total RNA was extracted from C2C12 cell and tissue samples using Trizol reagent and RNA concentration was determined using Nanodrop. First strand cDNA was synthesized using Prime Script RT System (Takara). Real-time quantitative PCR was performed on the ABI STEP-ONE7900 RT-PCR system. Triplicate runs were made for each sample.

TABLE 1 PT-PCR primer sequences

1.4 omics analysis

Hematoxylin and Eosin (HE), Succinate Dehydrogenase (SDH) and adenosine triphosphatase (ATPase) staining were used to observe the status of the placebo and geniposide treated groups in GAS and SOL tissue frozen sections. The sections were observed with an inverted optical microscope.

2 results of the experiment

2.1 geniposide can alter skeletal muscle fiber typing

As seen from HE staining in fig. 2, treatment with geniposide did not disrupt mouse skeletal muscle fiber morphology. SDH staining, dark blue for type I muscle fiber (slow muscle) and light blue for type II muscle fiber (fast muscle). As can be seen from FIG. 3, the proportion of geniposide group I muscle fibers in GAS was significantly reduced. We then performed typing of different muscles using ATPase staining technique and counted the proportion of type I muscle fibers (FIG. 4). Type I muscle fibers will appear black under acidic buffer conditions (pH 4.3) (fig. 4A and 4B), while type II muscle fibers will appear black under basic buffer conditions (pH 10.5) (fig. 4C and 4D). From the results, it can be seen intuitively that the treatment with geniposide can significantly reduce the number of slow muscle fibers in skeletal muscle fibers, whether in GAS or SOL.

We further examined gene expression changes in the relevant muscle fibers in GAS. The results of FIG. 5 show that geniposide can significantly inhibit the expression of slow muscle related genes (MyHC-I, MB, Tnni1) and promote the expression of fast muscle related genes (MyHC-IIa, MyHC-IIb, MyHC-IIx). Therefore, it is thought that geniposide can alter skeletal muscle fiber type in vivo, resulting in a "slow → fast" transition in muscle fiber type, helping to maintain the physiological properties of fast muscle fibers.

2.2 modulation of muscle fiber types in cell systems by geniposide

To further investigate the geniposide changes to the myofiber types, we applied immunofluorescence methods to label different types of myotubes. In fig. 6, geniposide stimulation in C2C12 differentiated for 2 days was able to significantly increase myotube numbers in the fast muscle type while decreasing myotube numbers in the slow muscle type. Moreover, the quantitative PCR experiment is repeated in the C2C12 cell, the geniposide stimulation is carried out on the cell by using a concentration gradient (figure 7A) and a time gradient (figure 7B), the result is basically consistent with the conclusion obtained by the in vivo experiment, and the slow muscle gene MyHC-I expression is obviously reduced and the fast muscle gene MyHC-IIb expression is obviously increased along with the increase of the geniposide concentration (0.2mg/mL) and the increase of the stimulation time (12 h). We conclude that geniposide can also cause a "slow → fast" switch in muscle fiber type in vitro.

Claims

1. The application of geniposide in preparing a medicament for promoting the generation of skeletal muscle fast muscle is characterized in that the skeletal muscle fast muscle is type II muscle fiber in skeletal muscle fiber; the promotion of skeletal muscle fast muscle production refers to an increase in type II muscle fibers in myoblasts and skeletal muscle tissue.

2. The use according to claim 1, wherein the sources of geniposide comprise: fructus Gardeniae dregs, fructus Gardeniae, Eucommiae cortex, rehmanniae radix, Achyranthis radix, herba Hedyotidis Diffusae, and Coptidis rhizoma detoxicating decoction.

3. The use of claim 1, wherein the medicament further comprises pharmaceutically acceptable excipients comprising solvents, propellants, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, sequestering agents, permeation enhancers, buffers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, flocculants and deflocculants, filter aids, release retardants.

4. Use according to claim 1, wherein the medicament is an aerosol or a dressing.

5. The use of claim 1, wherein the promotion of skeletal muscle fast muscle production is selected from at least one of a shift of other muscle fibers to type II muscle fibers, an increase in the mass of type II muscle fibers, or an increase in the area of type II muscle fibers.