Low magnitude high frequency vibration promotes adipogenic differentiation of bone marrow stem cells via P38 MAPK signal

<div><p>Low magnitude high frequency vibration (LMHFV) has been mainly reported for its influence on the musculoskeletal system, particularly the bone tissue. In the bone structure, osteogenic activity is the main focus of study with regards to LMHFV. However, adipogenesis, another important mode of differentiation in the bone marrow cavity that might be affected by LMHFV, is much less researched. Furthermore, the molecular mechanism of how LMHFV influences adipogenesis still needs to be understood. Here, we tested the effect of LMHFV (0.3g, 40 Hz, amplitude: 50μm), 15min/d, on multipotent stem cells (MSCs), which are the common progenitors of osteogenic, chondrogenic, adipogenic and myogenic cells. It is previously shown that LMHFV promotes osteogenesis of MSCs. In this study, we further revealed its effect on adipo-differentiation of bone marrow stem cells (BMSCs) and studied the underlying signaling pathway. We found that when treated with LMHFV, the cells showed a higher expression of PPARγ, C/EBPα, adiponectin and showed more oil droplets. After vibration, the protein expression of PPARγ increased, and the phosphorylation of p38 MAPK was enhanced. After treating cells with SB203580, a specific p38 inhibitor, both the protein level of PPARγ illustrated by immunofluorescent staining and the oil droplets number, were decreased. Altogether, this indicates that p38 MAPK is activated during adipogenesis of BMSCs, and this is promoted by LMHFV. Our results demonstrating that specific parameters of LMHFV promotes adipogenesis of MSCs and enhances osteogenesis, highlights an unbeneficial side effect of vibration therapy used for preventing obesity and osteoporosis.</p></div

Western blot on day 8 indicated PPAR γ protein expression was enhanced under vibration: representative gel of PPAR γ (a) and quantification of PPARγ protein level (b); <i>P</i><0.01(**).

<p>Western blot on day 8 indicated PPAR γ protein expression was enhanced under vibration: representative gel of PPAR γ (a) and quantification of PPARγ protein level (b); <i>P</i><0.01(**).</p

Passage3 of BMSCs from Sprague Dawley rat under microscope(Olympus, Japan).

<p>Representative images:(<b>a</b>)scale bar = 100 μm,(<b>b</b>)scale bar = 50 μm, (<b>c</b>).scale bar = 50 μm.</p

LMHFV activated p38 MAPK.

<p>P-p38/p38 rose up by LMHFV on day 1, 4 and day 8:representative gel of p-p38 and p38 (a), and quantification of p-p38 protein level (b);<i>P</i><0.01(**).</p

Primers used for real-time RT-PCR.

<p>Primers used for real-time RT-PCR.</p

LMHFV increased the formation of lipid droplets during the adipogenic process of BMSCs.

<p>BMSCs after induction for 8 days. Staining is done by oil red o staining: control group cells (<b>a</b>)scale bar = 50 μm, (<b>b</b>)scale bar = 50 μm; vibration group cells(<b>c</b>) scale bar = 50 μm, (<b>d</b>)scale bar = 50 μm; (<b>e</b>) quantification of cells with oil droplets increased by vibration, <i>P</i><0.01(**).</p

mRNA expression of adipogenic genes were strengthened by LMHFV.

<p>Effects of LMHFV on adipogenic marker genes: when cells exposed to LMHFV, mRNA expression of (<b>a</b>)PPARγ, (<b>b</b>)C/EBPα and (<b>c</b>)adiponectin, were higher; <i>P</i><0.01(**).</p

(a)Vibration sensor with fixator for culture plates, and(b) vibration sensor with fixator for culture flasks.

<p>(a)Vibration sensor with fixator for culture plates, and(b) vibration sensor with fixator for culture flasks.</p

Activated p38 MAPK increased adipogenic gene expression of BMSCs.

<p>Protein level of PPAR γ on day 8 was detected by immunofluorescence staining (scale bar = 50 μm): (<b>a</b>)control and (<b>b</b>)control+SB; (<b>c</b>) vibration and(<b>d</b>)vibration+SB;(<b>e</b>)after inhibition, PPAR γ positive staining decreased, <i>P</i><0.05(*).</p