Effect of Astragaloside IV On Precartilaginous Stem Cells to Promote Bone Development in Rats

Objective To explore the effect of astragaloside IV in promoting bone development by promoting the proliferation of precartilaginous stem cells. Methods To co-cultured the cells from the resting chondrocyte of growth plate and LaCroix of 24-hours old rats,and identified by FGFR-3 staining. Choosing astragaloside IV induce precartilaginous stem cells cultured in vitro, using Collagen type Ⅱ monoclonal antibody staining and MTT to test cell biological characteristics. Four 4 weeks old SD rats were selected and divided into an experimental group and control group, 24 rats in each group. The rats in the experimental group were injected with astragalus injection in a dose of 8.0g / kg once a day. The rats in the other group were injected with the same amount of normal saline. The 3rd and 5th week after feeding, 12 rats were killed, and the tibial length was measured by vernier caliper.Rusults The FGFR-3 staining was positive, which proved that the cultured cells were precartilaginous stem cells. Collagen typeⅡmonoclonal antibody staining is positive and the OD value detected by MTT test was higher, after astragaloside IV induced the precartilaginous stem cells. After astragaloside IV injection, the tibial length of experimental group measured by vernier caliper was significantly higher than that of the control group.Conclusion astragaloside IV can promote the proliferation and biological characteristics of precartilaginous stem cells, and then promote bone development.

MiR-132/212 promotes the growth of precartilaginous stem cells (PCSCs) by regulating Ihh/PTHrP signaling pathway

Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3′-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.