Objective:

2

To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of

Plastrum Testudinis

(PT) in the treatment of osteoporo

sis (OP).

Methods:

2

Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method

including sham group

ovariectomized group (OVX)

ovariectomized groups treated with high-

medium-

and low-dose PT (160

80

40 mg/kg per day

respectively)

with 6 rats in each group. Except for the sham group

the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment

bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry

Western blot

and quantitative polymerase chain reaction. In addition

Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently

PT extract was used to rescue the effects of Dkk-1 and miR-214

and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated.

Results:

2

PT-M and PT-L significantly reduced the weight gain in OVX rats (

P

<

0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats

and increased the expressions of bone formation-related factors including alkaline phosphatase

bone morphogenetic protein type 2

collagen type Ⅰ alpha 1

and runt-related transcription factor 2 when compared with the OVX group (

P

<

0.05 or

P

<

0.01). Meanwhile

different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats

and increased the mRNA or protein expressions of Wnt3a

β-catenin

glycogen synthase kinase-3β

and low-density lipoprotein receptor-related protein 5 (

P

<

0.05 or

P

<

0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition

the expression of miR-214 was decreased in OVX rats (

P

<

0.01)

and it was negatively correlated with the osteogenic differentiation of BMSCs (

P

<

0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (

P

<

0.05 or

P

<

0.01). Conversely

PT effectively counteracted the effect of miR-214 mimic

thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (

P

<

0.05 or

P

<

0.01).

Conclusion:

2

PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway

which may be related to inhibiting miR-214 in BMSCs.