To investigate the effect and of hydrogen sulfide (H₂S) on the osteogenic differentiation of osteoblast precursor cells (MC3T3-E1) by regulating the polarization of macrophages (RAW264.7).

RAW264.7 cells were divided into four groups (n=10 in each group), including control group (untreated), M1 induction group [added with 100 ng·mL^-1 lipopolysaccharide (LPS)], M2 induction group (added with 20 ng·mL^-1 interleukin-4+20 ng·mL^-1 interleukin-10, IL-4+IL-10), GYY4137 intervention group (H₂S donor GYY4137 100 μmol·L^-1). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of M1 macrophage related factors including tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β) and inducible nitric oxide synthase (i-NOS) and M2 macrophage related factors including interleukin-10 (IL-10), arginase-1 (Arg-1) and mannose receptor (CD206), and to verify the effect of polarization induction and the intervention of GYY4137; cell counting kit-8 (CCK-8) was used to detect the activity of RAW264.7 cells treated with GYY4137 at different concentrations for 24 h and 72 h (6 wells in each group). The expression levels of Arg-1 and CD206 in control group and GYY4137 group (100 μmol·L^-1) were detected by flow cytometry (n=10 in each group). MC3T3-E1 cells were divided into four groups (n=10), which were blank group (without osteogenic induction medium), osteogenic control group (with osteogenic induction medium), co-culture group (co-cultured with RAW264.7 cells Transwell) and GYY4137 pretreatment co-culture group (co-cultured with 100 μmol·L^-1 GYY4137 pretreated RAW264.7 cells). The mRNA expression levels of osteogenic related genes including Runt-related transcription factor 2 (Runx-2), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2) and bone sialoprotein (BSP) mRNA were detected by RT-qPCR; alkaline phosphatase (ALP) staining and alizarin red staining were used to detect osteogenic differentiation.

The relative expression levels of TNF-α mRNA in control group, M1 induction group and GYY4137 intervention group were 1.56±0.22, 16.49±3.56 and 0.57±0.11, respectively; the relative expression levels of IL-1β mRNA were 1.06±0.24, 3.37±0.76 and 1.43±0.34, respectively. The above indexes in M1 induced group were significantly higher than those in control group, and the above indexes in GYY4137 intervention group were significantly lower than those in M1 induced group (all P<0.001). The relative expression levels of Arg-1 mRNA in GYY4137 group and control group were 1.38±0.36 and 0.91±0.12, respectively; the relative expression levels of CD206 mRNA were 17.43±2.84 and 1.14±0.21, respectively; the relative expression levels of CD206 protein were 0.89±0.25 and 0.20±0.04, respectively; the relative expression levels of i-NOS protein were 0.72±0.19 and 0.25±0.07, respectively; the relative expression levels of Arg-1 were 28.65±3.21 and 12.31±2.15, respectively; the relative expression levels of CD206 were 31.24±2.89 and 10.8±1.98, respectively. The above indexes in GYY4137 intervention group were significantly higher than those in control group, and the differences were statistically significant (P<0.05, P<0.001). The relative expression levels of Runx-2 mRNA in GYY4137 pretreatment and co-culture groups were 3.21±0.45 and 1.56±0.22, respectively; the relative expression levels of OPN mRNA were 2.89±0.38 and 1.43±0.19, respectively; the relative expression levels of BMP-2 mRNA were 3.56±0.52 and 1.67±0.25, respectively; the relative expression levels of BSP mRNA were 2.98±0.41 and 1.51±0.20, respectively. The above indexes in GYY4137 pretreatment co-culture group were significantly higher than those in co-culture group, and the differences were statistically significant (all P<0.05). The OD values of ALP in GYY4137 pretreatment co-culture group and osteogenic control group were (0.89±0.12) and (0.56±0.08) μm², respectively; the areas of alizarin red stained calcium nodules were (2 865.32±215.67) and (1 568.45±189.32) μm², respectively; the above indexes of GYY4137 pretreatment co-culture group were significantly higher than those of osteogenic control group, and the differences were statistically significant (all P<0.05).

100 μmol·L^-1 GYY4137 can enhance the osteogenic differentiation of MC3T3-E1 cells by promoting the polarization of RAW264.7 cells to M2 macrophages, up regulating the expression of osteogenic related genes.