Panax Notoginseng Saponins Regulates Osteoblast-Derived Exosomes in a Hormonal Environment to Promote Osteogenic Differentiation of MC3T3-E1 Cells

Abstract

Background: Osteocellular necrosis due to prolonged or heavy glucocorticoid application plays an essential role in the steroid-induced necrosis of the femoral head (SONFH). Promoting osteoblast proliferation and osteogenic differentiation is an effective therapeutic strategy for SONFH. Panax Notoginseng Saponins (PNS) can increase osteoblasts proliferative activity and alleviate glucocorticoids inhibitory effect on the proliferation of MC3T3-E1 cells. As a nanoscale natural drug carrier, exosomes have unique advantages in improving drug efficacy and reducing side effects. This study aimed to test whether PNS could promote osteogenic differentiation of MC3T3-E1 cells via osteoclast-derived exosomes in a hormonal environment. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to screen the non-toxic concentrations of PNS action on RAW264.7 cells. In addition, the non-toxic concentration of dexamethasone (Dex) acting on RAW264.7 with MC3T3-E1 cells was screened using cell counting kit-8 (CCK-8) assay. RAW264.7 cells were induced to differentiate into osteoblasts by receptor activator for nuclear factor kappa-B ligand (RANKL) reagent and identified by tartrate resistant acid phosphatase (TRAP) staining. Model group exosomes (OC-Exos) and PNS intervention group exosomes (PNS+OC-Exos) in the Dex environment were obtained using ultra-high-speed centrifugation. MC3T3-E1 cells were sorted into blank control (NS) group, OC-Exos group, and PNS+OC-Exos group and added with phosphate buffered saline (PBS) solution, OC-Exos, and PNS+OC-Exos for intervention, respectively. In addition, the exosomes of the OC-Exos group and PNS+OC-Exos group were labeled by PKH67 dye. Subsequently, osteogenic changes were evaluated by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining. Finally, the expression levels of osteogenic markers such as ALP, osteocalcin (OCN), runt-related transcription factor2 (Runx2), and collagen type I alpha 1 (COL1A1) were determined in the three groups of MC3T3-E1 cells utilizing real time-polymerase chain reaction (RT-PCR) assay and Western blot assay. Results: TRAP staining highlighted that RAW264.7 was successfully induced to osteoclasts by RANKL, while PNS inhibited its differentiation. OC-Exos and PNS+OC-Exos were successfully extracted. In addition, PKH67 fluorescence staining confirmed the uptake of exosomes by MC3T3-E1 cells in both groups. The number of ALP activity and calcified nodules in MC3T3-E1 cells elevated in the PNS+OC-Exos group compared to the OC-Exos group. Gene and protein expression levels in the PNS+OC-Exos group significantly higher in ALP, OCN, Runx2 and COL1A1 than in the OC-Exos group (p < 0.05). Conclusion: PNS inhibited RANKL-induced differentiation of RAW264.7 cells to osteoblasts in a Dex environment. Furthermore, PNS reversed the inhibition of osteogenic differentiation of MC3T3-E1 cells by OC-Exos.