Study on the Mechanism of Injection Erycibe in Promoting Fracture Healing in Osteoporotic Rats

OBJECTIVE To study the promoting effect and mechanism of injection erycibe(IE) on fracture healing in osteoporotic rats. METHODS Castration and fracture surgery were used to establish a rat model of osteoporotic fracture. Fifty female healthy SD rats were randomly divided into sham operation group, model group and 1, 2, 4 mL·kg^-1 IE group. The rat femur bone mineral density, biomechanical indicators, serum calcium and alkaline phosphatase levels were detected. HE staining was used to observe the pathological changes of rat skeleton. The isolated bone marrow mesenchymal stem cells(MSCs) were intervened with drugs, which were divided into control group, 5, 10, 20 mg·L^-1IE group, induction group(using osteogenic induction medium), NC siRNA group, NCK1 siRNA group, IE+NC siRNA group and IE+NCK1 siRNA group. MTT method was used to detect the proliferation of MSCs. RT-qPCR was used to detect the mRNA levels of osteogenic differentiation related genes ALP, Runx2 and Osterix. Western blotting was used to detect the protein expression levels of NCK1 and p-AKT. Alizarin red staining was used to observe the number of calcium nodules in MSCs. RESULTS Compared with the model group, the bone mineral density, biomechanical indexes, and serum biochemical indexes of rats in 2, 4 mL·kg^-1 IE groups were significantly increased(P<0.05) in a dose-dependent manner, and 4 mL·kg^-1 IE group had the best effect. Compared with the control group, the proliferation of MSCs and the expression of osteogenic differentiation-related genes in 10, 20 mg·L^-1IE group were significantly increased(P<0.05 or P<0.01), and the expression levels of NCK1 and p-AKT proteins were significantly increased(P<0.05) and the amount of calcium nodules in MSCs was significantly increased in a dose-dependent manner. Moreover, the 20 mg·L^-1 IE group had the best effect. Compared with IE+NC siRNA group, the expression of bone differentiation-related genes ALP, Runx2 and Osterix mRNA were significantly decreased(P<0.01), the expression levels of NCK1 and p-AKT proteins were significantly decreased(P<0.01) in IE+NCK1 siRNA group. CONCLUSION EI can promote fracture healing in osteoporotic rats by promotes the expression of NCK1 protein and activates the AKT signaling pathway.