[Mechanism of Zhenggu Wan in promoting fracture healing based on BMP-2/Smad1/Runx2/Osterix signaling pathway].

This study aims to investigate the mechanism of action of Zhenggu Wan(ZGW) in promoting fracture healing via regulating the bone morphogenetic protein-2(BMP-2)/recombinant Smad 1 protein(Smad1)/runt-related transcription factor 2(Runx2)/osteoblast-specific transcription factor(Osterix) signaling pathway. Femoral fracture models of 54 SD rats were established and randomly divided into six groups: control group, model group, low-dose ZGW group(315 mg·kg~(-1)), high-dose ZGW group(630 mg·kg~(-1)), Xianling Gubao Capsule group(315 mg·kg~(-1)), and the group with high-dose ZGW and noggin(BMP specific inhibitor). And they were administered intragastrically for 28 days. UMR-106 rat osteoblasts were treated with ZGW-containing serum and ZGW-containing serum combined with BMP-specific inhibitor noggin. The contents of bone formation markers osteocalcin(BGP), type Ⅰ collagen amino-terminal propeptide(PINP), bone resorption markers type Ⅰ collagen C-terminal telopeptide(CTX-1), and type Ⅰ collagen N-terminal cross-linked telopeptide(NTX-1) in rats' serum were detected by enzyme-linked immunosorbent assay(ELISA). The pathological morphological changes of the femoral fracture end tissue were assessed via hematoxylin and eosin(HE) staining. The expressions of osteogenic markers BGP and osteopontin(OPN) in both fracture end tissues and UMR-106 cells were analyzed by immunofluorescence. The protein expressions of BMP-2, Smad1, Runx2, and Osterix in both fracture end tissues and UMR-106 cells were determined by Western blot. The results demonstrate that compared to those in the model group, the contents of BGP and PINP in rats' serum of the model group are significantly decreased, and the contents of CTX-1 and NTX-1 are significantly increased. The pathological results show that the trabecula of fracture end tissue is sparse, slender, and arranged irregularly, and the pathway protein expressions of BGP, OPN, BMP-2, Smad1, Runx2, and Osterix in tissues and cells are significantly decreased. After the intervention of ZGW, the contents of BGP and PINP in serum are significantly increased; the contents of CTX-1 and NTX-1 are significantly decreased; different numbers of chondrocytes and osteoblasts appear in the fracture end tissues, and the bone trabecula transforms into compact bone more rapidly; the expressions of BGP, OPN, BMP-2, Smad1, Runx2, and Osterix in tissues and cells are significantly increased. In conclusion, ZGW can promote fracture healing and improve bone metabolism by upregulating BMP-2/Smad1/Runx2/Osterix signaling pathway.