Page 21 - A. Aging

Long-term administration of red ginseng non-saponin fraction rescues the loss of skeletal muscle mass and function associated with aging in
mice
Da-Eun Cho¹, Gwang-Muk Choi¹, Hyein Song², Mijung Yeom³, Bombi Lee³, Dae Hyun Hahm 1,3,4*
¹Department of Biomedical Sciences, Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea
²Department of Medicinal Biosciece, Nonsan Interdisciplinary & Creative Studies Campus, Konyang Univeristy, nonsan-si, Chungcheongnam-do, 32992, Repulic of Korea
³Acupuncture and Meridian Science Research Center, Kyung Hee University, Seoul, 02447, Republic of Korea
⁴Department of Physiology, College of Medicine, Kyung Hee University, Seoul, 02447, Republic of Korea
ABSTRACT AIM
Korean red ginseng has been known to relieve fatigue and enhance the physical performance especially in the elderly. Anti-
sarcopenic effect of its non-saponin fraction (NS) was investigated in 22-month-old mice, administered with 0.08% NS chow 1. To examine whether red ginseng non-saponin fraction (NS) can alleviate the loss of muscle mass and strength in the aged mice.
diet for another 4 months. Aging-associated weight loss of hindlimb skeletal muscle such as tibials anterior, extensor 2. To verify whether NS can alleviate the shift of fast-to slow-twitch muscle type strength in the aged mice.
digitorum longus (EDL), gastrocnemius and soleus was significantly alleviated by NS treatment. The cross-sectional areas of
soleus, decreased by 40% in aged mice raised from 22 to 26 months, were mitigated to 12.5%, and a shift of fast-to slow- 3. To investigate the effects of NS on senescence characteristics such as intracellular lipid accumulation, increased amount of β-
twitch muscle type, observed in soleus and EDL was also retarded. In order to elicit a senescence in C2C12 myoblasts, the galactosidase protein from lysosome, and reduced cell proliferative capacity in C2-ceramide-elicited cellular senescence model of
C2C12 myoblasts.
cells were treated with C2-ceramide, and NS 1h earlier. NS significantly alleviated C2-ceramide-induced senescence
indicated by intracellular lipid accumulation, increased amount of lysosomal β-galactosidase, cell cycle retardation, and 4. To examine whether NS can inhibit C2-ceramide-stimulated mRNA expression of cell cycle-associated genes such as p21 and
reduced cell proliferative capacity. NS restored C2-ceramide-stimulated expression of p21, p57, p16, MuRF1 and atrogin-1, p57, and muscle-specific ubiquitin ligases such as MuRF-1 and MAFbx/atrogin-1 in C2C12 myoblasts.
and the suppression of Pax-7 in a dose dependent manner. NS thus might be invaluable for the development of therapeutic 5. To examine whether NS can inhibit C2-ceramide-downregulated expression of Pax-7 mRNA in C2C12 myoblast.
medicines to rescue sarcopenia and its accompanying muscle dysfunction.
METHODS

• Laboratory chow diet containing 0.08% NS (800 mg/kg-chow) was manufactured and offered to 22- • DMSO: dimethyl sulfoxide. C2-ceramide was dissolved in 0.1% DMSO, Naïve: non-treated
month-old C57B/L6 mice daily for another 4 months. myoblast cells, NS: non-saponin fraction of red ginseng treated myoblast cells
• 26CO: a group of 22-month-old mice fed a standard chew diet during 4 months. 26NS: a group of 22-
month-oldmice fed a standard chew diet mixed NS during 4 months. 22CO: a group of 18 month-old-mice
fed a standard chew diet during 4 months.
RESULTS

Figure 5. Effect of NS on the
Figure 1. Time-course patterns of C2C12 myoblast cell viability.
body weight (A), food intake (B),
grip strength test (C) and hanging
wire test (E) in NS-administrated Figure 6. Trypan blue exclusion
mice.
assay showing ceramide-induced
cellular senescence in C2C12
myoblasts. The cells were stained
with trypan blue immediately
following (A, 0h-post) or 72h (B,
72h-post) after the treatment
periods. T: total cell numbers (blue-
stained and non-stained), D: dead
cell numbers (non-stained).
Figure 2. Muscle weights/body weight (BW) of tibials anterior (TA), extensor digitorum longus (EDL) (B), gastrocnemius
(GN) (C) and soleus (SOL) (D).
Figure 3. (A) Representative
photomicrograph of H&E staining
of soleus muscle sections. Scale
bar = 50 μm (Left panel), 100 μm Figure 7. Microscopic images (A) of sequentially NS-treated, ceramide-treated
(Right panel). (B) Distribution of and β-galactosidase-stained C2C12 myoblasts, and the bar graph showing the
CSA sizes (C) Quantitative cellular concentration of β-galactosidase (B). Naïve myoblasts (a) were treated
estimation of myofibers fiber with different concentrations of NS (0 μg/ml-: b; 1000 μg/ml-RGNS : d) for 9h,
cross-sectional area of soleus and 50mM C2-ceramide was added 1h after the treatment. After all treatments,
muscle. (D) The distribution of the cells were stained for β-galactosidase and quantified from captured images
myofiber sizes for each group. (E) (10X). Scale bar = 100 μm
Representative soleus (SOL)
immunofluorescent cross-
sectional images based on myosin 150
heavy chain immunofluorescent
staining (blue = typeⅠ, green = 100
typeⅡA, black = typeⅡX). (F) Brdu Incorporation (Abs. at 450 nm) ## ## *
Fiber type distribution pattern
(% of total fibers) 50 Figure 8. Inhibitory effect of NS on ceramide-induced retardation of C2C12
0 myoblast cell proliferation. The 8h-incubated cells with ceramide were pulsed
Naive DMSO 0 100 1000
with BrDU, then underwent an ELISA-based assay for BrDU incorporation into
NS (µg/ml) DNA, and then read at 450 nm on a plate reader.
C2-ceramide (50 µM)
Figure 4. Microscopic images showing lipid accumulation visualized by Oil Red O staining (2.0%, 1h) (A) and β- Figure 9. Effect of NS on ceramide-stimulated expression of a atrogin-1 (A), MuRF-1 (B), p21 (C), p57 (D), p16 (E) and
galactosidase staining (B) in non-treated (a and a’) or C2-ceramide-treated (b, b’) C2C12 myoblasts. Scale bar = 50 μm Pax-7 (F) in C2C12 myoblast cells.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
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