Pharmaceutical composition for improving, preventing or treating muscle related disease comprising ginsenoside Rh2

Abstract

The present invention provides a pharmaceutical composition or food composition for preventing, alleviating or treating muscular diseases comprising ginsenoside Rh2 as an active ingredient.

Claims

1. A method of treating a muscular disease, comprising administering an effective amount of ginsenoside Rh2 represented by the following Formula 1 an active ingredient, to a subject in need: ##STR00003## wherein the muscular disease is muscular atrophy.

2. The method according to claim 1, wherein the ginsenoside Rh2 is extracted from ginseng (Panax ginseng).

3. The method according to claim 1, wherein the ginsenoside Rh2 decreases the width of the myotube.

4. The method according to claim 1, wherein the ginsenoside Rh2 inhibits myostatin-induced atrophy.

5. The method according to claim 1, wherein the ginsenoside Rh2 increases the activity of the transcription factor MyoD by reduction of Smad2 phosphorylation, and MAFbx expression level.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a microscope image showing that C2C12 myoblasts cultured with Horse Serum differentiate into myotube cells.

(2) FIG. 2 is a graph showing the reduced width of differentiated myotube cells shown in FIG. 1 added with various concentrations of MSTN

(3) FIG. 3 shows the inhibitory effect of ginsenoside Rh2 on the reduction of myotube cell width in the MSTN-induced atrophic myotube cells. The left side is a photomicrograph showing the width change of myotube cell at the treatment condition of MSTN and Rh2, and the right side is a graph quantifying the width reduction effect according to the concentration of ginsenoside Rh2.

(4) FIG. 4 shows the inhibitory effect of ginsenoside Rh2 on MyoD degradation pathway in the MSTN-induced atrophic myotube cells. The increase and decrease in the expression level of the proteins related to MyoD degradation pathway were quantified by the Western blotting as the increased concentration of ginsenoside Rh2.

(5) FIG. 5 shows the inhibition mechanism of MyoD degradation by ginsenoside Rh2 in MSTN-induced atrophic myotube cells.

MODE FOR INVENTION

(6) Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Experimental Example 1: C2C12 Myotube Cells Differentiation and Induction of Muscular Atrophy Cell Model

(7) The model of muscular atrophy was constructed by differentiating mouse myoblast cell lines (C2C12 ATCC CRL-1772) into myotube cells followed by treating with MSTN.

(8) Specifically, the cell lines were cultured in DMEM (Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin (Sigma) at 37° C. in 5% CO2. For differentiating the cell lines into myotube cells, the cells were grown to 80-90% confluence and then cultured in DMEM supplemented with 2% (v/v) horse serum (Horse Serum, HS, Gibco). To verify the differentiation into myotube cells, the merge of multiple myoblasts to one long myotube cells were observed by the microscopic images. FIG. 1 shows microscopic images with differentiation of C2C12 myoblasts to myotube cells by incubation with horse serum.

(9) In order to induce an atrophy model, differentiated myotube cells were cultured in serum-free DMEM medium for 3 hours, and then MSTN was treated with 0.4 μg/ml, 0.6 μl/ml, 0.8 μg/ml and 1.2 μg respectively for 24 hours. MSTN-untreated cells were used as control.

(10) To determine whether the atrophy model was successfully induced, changes in the width of the myotube cells after induction of atrophy were measured. C2C12 myotube cells were fixed with 4% (w/v) paraformaldehyde (Biosesang) for 15 minutes and then treated with 0.2% (v/v) Triton X-100, and blocked with 5% (w/v) BSA for 6 h. The cells were then stained with Myosin Heavy Chain (MHC) antibody (Santa Cruz, sc-20641, 1: 1000) for 12 h at 4° C. and then incubated with Alexa Fluor 488 conjugated secondary antibody at room temperature for 4 h. Images were acquired via HCS equipment (Molecular Devices) and quantified using ImageJ software. The width of myotube cells was quantified by measuring a total of more than 100 myotube cell widths in randomly selected five regions. The width per myotube was determined as the average of three independent measurements.

(11) FIG. 2 is a graph showing relative widths of myotubes depending on treating concentration of MSTN comparing with control group. The width of control group was showed as 1.0. As a result, the width of myotube treated with 0.4 ug/ml of MSTN was greatly decreased (about 45% compared to the normal), and the width of myotube gradually decreased as the concentration of MSTN increased (about 60% to the normal, FIG. 2). Because of the similar tendency of width reduction at concentrations over 0.4 μg/ml of MSTN treatment group, the present inventors decided MSTN concentration for inducing atrophy model in subsequent experiments to 0.4 μg/ml. The results were shown in FIG. 2.

Experimental Example 2: Confirmation of Cell Size Promotion in Atrophic Myotubes by Rh2

(12) To investigate the effect of Rh2 on MSTN-induced atrophy, myotubes were treated with 0.4 ug/ml MSTN alone for 24 hours or treated with combination of 0.4 ug/ml MSTN and 1 ug/ml or 10 ug/ml Rh2 (Sigma, 73658) for 24 hours. In order to measure the change of the myotube width, the myotube cells were stained with MHC antibody as in the above Experimental Example 1, and cell images were obtained using HCS, and the width of myotube was quantified using ImageJ.

(13) The left side of FIG. 3 is showing the stained cell image, and the right side is a graph of the relative value of the control group by quantifying the width of myotube. Experimental results showed that ginsenoside Rh2 inhibits the decrease of width of myotube induced by MSTN and maintains the myotube width above normal level (FIG. 3). The width of myotube treated with MSTN was reduced about 50% compare to normal cells, and when the cells were treated with combination of MSTN and 1 ug/ml Rh2, the width of myotube was about 70% of that of normal cells. When cells were treated with combination of 10 ug/ml Rh2 and MSTN, the width was maintained above 100% of that of normal. These results confirm that Rh2 inhibits MSTN induced atrophy depending on the concentration. The results are shown in FIG. 3.

Experimental Example 3: Confirmation of the Inhibitory Effect of Rh2 on MyoD Degradation Pathway in Atrophic Myotube Cells

(14) MSTN is known to induce atrophy by increasing UPP-mediated protein degradation through activation of the ActRIIB-Smad2 signaling pathway. Phosphorylated Smad2 (pSmad2) activates this UPP-mediated proteolytic signaling. Activated UPP is involved in the degradation of MyoD and is regulated by the E3 ligase, MAFbx. To investigate the mechanism of Rh2 in this signal transduction, changes of the amount of Rh2-induced phosphorylation of Smad2 and the amount of MAFbx and MyoD expression were determined. For this purpose, myotube cells were treated with 0.4 μg/ml MSTN alone, or with combination of 0.4 μg/ml MSTN and 1 μg/ml or 10 μg/ml Rh2. Western blotting was performed using pSmad2 antibody, MAFbx antibody, and MyoD antibody respectively.

(15) The bands of Western blot and the relative fold change values of pSmad2/Smad2, MAFbx/GAPDH and MyoD/GAPDH in each treatment conditions were shown in FIG. 4. As shown in FIG. 4, the amount of pSmad2 and MAFbx increased to about 50% compared with normal condition, and the amount of MyoD decreased to about 30% of the normal condition, in the case of MSTN alone treatment. When 10 ug/ml Rh2 was treated with MSTN, the amounts of pSmad2, MAFbx and MyoD were kept at the same level as normal conditions.

(16) These results suggest that Rh2 increases the amount of MyoD and reduces the phosphorylation of Smad2 as well as expression of MAFbx, the MyoD ubiquitinases, and the upregulator of UPP activation respectively. Therefore, the inhibition mechanism of Rh2 on MSTN-ActRIIB pathway can be interpreted as inhibition of MAFbx and MyoD degradation through inhibition of Smad2 phosphorylation. The mechanism of Rh2 to inhibit MyoD degradation in the cell model of atrophy is shown in FIG. 5.