[Experimental study on interleukin-10 secretion from M2 macrophages promoting osteogenic differentiation of rat bone...

Objective: To investigate the effect and underlying molecular mechanism of interleukin-10 (IL-10) secreted by M2 macrophages on the osteogenic differentiation ability of rat bone marrow mesenchymal stem cells (BMMSCs) after irradiation. Methods: Between February 2024 and April 2025, eight healthy male SD rats aged 2 to 3 weeks were selected, primary BMMSCs and macrophages from SD rats were cultured in vitro. Macrophages were polarized to the M2 phenotype, and their surface markers were identified by flow cytometry and immunofluorescence. The experiment was divided into four groups: normal BMSC control group (CON), 4 Gy irradiation group (4Gy), 4 Gy irradiation+M2 macrophage co-culture group (4Gy+8 μm), and 4 Gy irradiation+M2 macrophage co-culture+IL-10 neutralizing antibody group(4Gy+8 μm+m/r IL-10). An in vitro cellular irradiation model was established by exposing BMMSCs to X-ray radiation at a dose of 4 Gy. A non-contact co-culture system between M2 macrophages and irradiated BMMSCs was established using Transwell chambers with an 8 μm pore size. After 48 hours of co-culture, ELISA was used to detect the IL-10 concentration in the supernatant of each group, and Western blot was used to measure the expression levels of key osteogenic differentiation markers [alkaline phosphatase(ALP),Runt-related transcription factor 2(RUNX2),osteocalcin(OCN)] and STAT3 signaling pathway-related proteins (p-STAT3, STAT3) in BMMSCs. The specific role of IL-10 was verified by adding an IL-10 neutralizing antibody. Results: Cell counting kit-8 assay results showed that on day 7 post-irradiation, the absorbance value of the 4 Gy Irradiation group (0.241±0.093) was significantly lower than that of the control group (1.794±0.083) (t=21.63, P<0.001). Western blotting analysis indicated that the expression levels of osteogenic markers ALP and RUNX2 in the irradiation group (0.819±0.074, 0.785±0.074) were significantly lower than those in the control group (1.000±0.067, 1.000±0.056) (t=3.16, P=0.034; t=4.01, P=0.016, respectively). Immunofluorescence analysis revealed that the fluorescence intensities of RUNX2 and ALP in the irradiation group (19.932±1.291, 7.316±0.089) were markedly weaker compared to the control group (31.154±3.352, 30.789±1.455). After co-culture with M2 macrophages, the proportion of viable cells in the co-culture group [(77.800±1.758)%] was significantly higher than that in the Irradiation group [(61.933±2.732)%] (P<0.001). Furthermore, the IL-10 concentration in the supernatant of the co-culture group [(46.39±1.879) pg/ml] was significantly higher than that in the irradiation group [(7.530±0.239) pg/ml] (t=36.74, P<0.001). Western blot results demonstrated that the expression levels of ALP, RUNX2, and OCN in the co-culture group were significantly higher than those in the Irradiation group (t=8.07, P=0.015; t=5.01, P=0.038; t=3.07, P=0.918, respectively). Adding 1 μg/ml IL-10 neutralizing antibody to the co-culture system significantly reduced the IL-10 level in the supernatant [(6.521±0.460) pg/ml] compared to the co-culture group [(26.270±6.486) pg/ml] (t=5.06, P=0.037). The p-STAT3/STAT3 ratio in the neutralizing antibody group (0.840±0.071) was significantly lower than that in the co-culture group (1.289±0.156) (t=4.27, P=0.051), and the osteogenic differentiation capacity of BMMSCs was also notably attenuated. Conclusions: M2 macrophages can improve the osteogenic differentiation ability of BMMSCs after irradiation damage by secreting IL-10 and activating the STAT3 signaling pathway.