| Objective Based on the theory of kidney-bone-marrow-brain in traditional Chinese medicine, we investigated the mechanism of medullary supplementation in regulating neural stem cell-derived exosomes (NSCs-Exos) to intervene in postmenopausal osteoporosis (PMOP) based on the SEMA3A-NRP1 pathway. Methods Exogenous rat neural stem cells (NSCs) were cultured in vitro and intervened with the traditional Chinese medicine Busui DAN (BSD). NSCs-Exos were extracted after 14 days of cultivation. They were divided into the group of NSCs-derived exosomes and the group of exosomes from NSCs intervened with BSD. Subsequently, transmission electron microscopy (TEM), grain size analysis (NTA), and Alix, CD9, CD63 exosome marker protein assays were performed. Animal experiments: SD rats were modeled by ovariectomy (OVX) in addition to the Control group and divided into OVX, BSD, NSCs-Exos, NSCs-Exos (BSD), NSCs-Exos+BSD, and NSCs-Exos (BSD)+BSD groups. The drug administration group was administered by gavage. The rats in the exosome group was injected with the corresponding exosome through the tail vein. The serum, kidney, femurs, and brain were taken at the end of treatment. Bone mineral density, serum ALP/TRACP, and HE staining tests were performed to analyze the effects on bone mineral density and the structural morphology of the kidney, femurs, and brain. The effects on the expressions of SEMA3A, NRP1, Runx2, and β-catenin in the kidney, femurs, and brain were analyzed by using the qRT-PCR method, ELISA method, Western blotting method, and WES method. In the same period, the DIR small animal tracking experiments in vivo were carried out to observe the targeting of NSCs-Exos. Results NSCs-Exos extracted from in vitro experiments showed round particles, which were clearly visible. CD9, CD63 and Alix were expressed, which was consistent with the characteristics of exosome. Compared to the NSCs-Exos group, the NSCs-Exos (BSD) group had a fuller morphology and higher expressions of CD9, CD63, and Alix. Animal experiments: compared to those in the control group, rats in the OVX model group had disorganized trabecular structure, significantly lower femoral bone mineral density and serum ALP expression, elevated TRACP expression, and significantly lower expressions of SEMA3A, NRP1, Runx2, and β-catenin (P<0.05), indicating that the modeling had been successful. Compared to those in the OVX model group, the femoral bone mineral density in the treatment group was significantly higher, the trabeculae number and arrangement were improved, serum ALP expression elevated, TRACP expression reduced, and SEMA3A, NRP1, Runx2, and β-catenin expressions elevated (P<0.05). The best effect was found in the NSCs-Exos (BSD)+BSD group. In the DIR experiment, it was found that NSCs-Exos expressed stronger fluorescence signal in the kidney, bone, and brain tissues, which verified its targeting. Conclusion The medullary supplement improves bone metabolism through the regulation of SEMA3A-NRP1 pathway by NSCs-Exos to prevent and control PMOP. This demonstrates the scientific connotation of the theory of kidney-bone-medullary-brain axis. |