Hydrogen sulfide promotes osteogenic differentiation of MC3TC-E1 cells by regulating macrophage polarization

Objective To investigate the influence of hydrogen sulfide (H2S, GYY4137 as a H2S donor) on the osteogenic differentiation of osteoprogenitor cells (MC3T3-E1) by regulating the polarization of macrophages (RAW264.7).Methods GYY4137 was applied to intervene macrophages (RAW264.7) and compared with M0 macrophages, lipopolysaccharide (LPS)-induced M1 macrophages, and IL4+IL10-induced M2 macrophages. The expression of mRNA and proteins related to M1 and M2 macrophages was detected by RT-qPCR and Western blot to ascertain the successful induction of M1 and M2 macrophages and the impact of GYY4137 on the polarization of RAW264.7 cells. To further explore the effect of H2S on M1 macrophages, control groups (Control group), M1 control group (LPS group), GYY4137 group, and GYY4137+LPS group were established, and the expression of M1 and M2 macrophage-related molecules was examined using RT-qPCR, Western blot, and flow cytometry. Osteoprogenitor cells (MC3T3-E1) were co-cultured with RAW264.7 cells; blank group (MC3T3-E1 cells without osteogenic induction medium), osteogenic control group (MC3T3-E1 cells with osteogenic induction), co-culture group of RAW264.7 cells with MC3T3-E1 cells, and co-culture group of GYY4137-preconditioned RAW264.7 cells with MC3T3-E1 cells were set up. The expression of Runt-related transcription factor 2 (Runx-2), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2), and bone sialoprotein (BSP) mRNA was detected by RT-qPCR. After osteogenic induction, alkaline phosphatase (ALP) staining and alizarin red staining were performed to assess the osteogenic differentiation capacity of the cells.Results Compared with the Control group, the expression of M2 macrophage-related mRNA and proteins in the GYY4137 group was significantly increased (P<0.05), and it was found that GYY4137 inhibited the expression of IL-1β and i-NOS in LPS-induced M1 macrophages (P<0.05). In the co-culture system with MC3T3-E1 cells, compared with the blank group, the relative expression of Runx-2, OPN, BMP-2, and BSP mRNA in the osteogenic control group was increased (P<0.05); compared with the other groups, the levels of Runx-2, OPN, BMP-2, and BSP mRNA in the co-culture group of GYY4137-preconditioned RAW264.7 cells with MC3T3-E1 cells were significantly elevated (P<0.05); ALP and alizarin red staining further confirmed that the addition of GYY4137 enhanced the osteogenic differentiation capacity of MC3T3-E1 cells (P<0.05).Conclusion GYY4137 promotes the osteogenic differentiation of MC3T3-E1 cells by regulating the polarization of RAW264.7 cells towards M2 macrophages.