The role of Notch signaling pathway in bone immune regulation of osteogenic differentiation of macrophages and mesenchymal stem cells
Authors: shen xiaoyu , Fu Dehao , Yao Qiang , chen jianchao , Qi Xiangbei 
Published: September 2023

Abstract
Backgroud:During various stages of fracture healing, macrophages control mesenchymal stem cells' (BMSCs') proliferative behavior and osteogenic differentiation through varying polarization states. BMSCs also regulate their own osteogenic differentiation through the polarization state of macrophages to meet the requirements of tissue repair and osteogenic environment. A crucial role in cell proliferation, differentiation, and death is played by the evolutionarily conserved Notch signaling system. It also plays an important role in the osteogenic differentiation and regulation of macrophage polarization of BMSCs. The NOTCH signaling pathway typically plays a role in information exchange through direct contact between cells. Therefore, the Notch signaling pathway is involved in information exchange during direct contact between macrophages and BMSCs.
Methods: A co culture system of mouse monocytic megacytic leukemia cell line (RAW264.7) and BMSC was established. RAW264.7 cells in logarithmic growth phase were divided into M0 group (unpolarized) and M1 group (LPS+INF γ induction), M2 group (IL4+IL13 induction), polarization status was detected by flow cytometry, and then BMSC were added to detect the Notch signaling pathway and the expression levels of RUNX2 gene and protein at different time points in each group. To further validate the role of the NOTCH signaling pathway in osteogenesis, we chose to apply the NOTCH signaling blocker RO4929097 to the co culture system of M2 and BMSC.According to whether blockers were used or not, they were divided into control group, M2 group, M2+blocker group, and blocker group. The transmission of the NOTCH signaling pathway in the interaction between M2 and BMSC as well as the production of Hes1 linked to the osteogenic gene RUNX2 were observed by blocking the NOTCH signaling pathway's conduction. At the same time, we detected the polarization of RAW264.7 cells in Mo and M1 groups to determine whether there was a change in the polarization state of RAW264.7 cells after the addition of BMSC.
Results:PCR and WB results showed that the NOTCH signaling pathway and osteogenic specific RUNX2 related protein and gene expression were basically synchronized: the expression of Jagged1 and Notch1 in M2 group was higher than that in M0 and M1 groups (p<0.05), while the expression level of M0 group was higher than that in M1 group (p<0.05). Hes1, as an associated gene and protein of Notch signaling pathway and Runx2, had the highest expression level with RUNX2 in M2 group (p<0.05), followed by M0, and the lowest in M1 group.This revealed that the Notch signaling pathway is involved in the bone immune regulatory effect between RAW264.7 and BMSC. After administering the NOTCH signaling blocker RO4929097, the M2 group had the highest expression of Notch signaling pathway related protein genes (p<0.05), followed by the control group (<0.05), and the blocker group had the lowest expression level (p<0.05), indicating a higher expression of the NOTCH signaling pathway between M2 cells and BMSC. The M2+blocker group had a higher expression level than the blocker group, suggesting that there are other pathways between M2 and BMSC that affect the conduction of the NOTCH signaling pathway. BMSC and RAW264.7 were co cultured, and flow cytometry analysis showed that the proportion of M2 like cells in the M0 group was higher than that in the M1 group.
Conclusion: In the co culture system of macrophages and BMSC,the Notch signaling pathway promotes macrophage polarization towards M2 type, thereby regulating the osteogenic differentiation of BMSC and participating in the bone immune regulation of macrophages and mesenchymal stem cells.