Cytokine Expression In Bone Marrow Mesenchymal Stem Cell Supernatant Under Inflammatory Microenvironment And Its Effect On Osteogenic Differentiation

Objective: To investigate the expression of cytokines in the supernatant of bone marrow mesenchymal stem cells（BMMSCs）under inflammatory environment and its effect on osteo-differentiation of BMMSCs.Methods: Western blot was used to detect the expression of osteogenic-related proteins in BMMSCs under different culture conditions in normal and inflammatory environments;ELISA was used to detect the expression of different cytokines in the supernatant of BMMSCs under normal and inflammatory environments and to compare the changes in the content of each cytokine;Each cytokine was added under osteogenic conditions,and the expression of osteogenic differentiation-related proteins within each group of BMMSCs was detected by Western blot.Results:1.The expression levels of osteogenesis-related proteins ALP,OCN and Runx2 were higher in the osteogenesis induction group and the osteogenesis induction group under the effect of TNF-α at 7 days of induction than in the control group and the TNF-αgroup,respectively,and the differences were statistically significant（P<0.05）;The differences in ALP and OCN expression in the TNF-α group compared with the control group were not statistically significant（P>0.05）,while the expression of Runx2 was slightly higher than that in the normal group,and the difference was statistically significant（P<0.05）;The expression levels of ALP in the TNF-α-induced osteogenesis group were higher than those in the osteogenesis-induced group,while the expression levels of OCN and Runx2 were lower than those in the osteogenesis-induced group,and the differences were all statistically significant（P<0.05）.2.The highest concentration of IL-8 was found in the supernatant of BMMSCs under either normal conditions or under the effect of TNF-α;the concentrations of IL-6,IL-8,IL-1β,TGF-β1,and TGF-β2 cytokines were increased in the supernatant of BMMSCs under the effect of TNF-α compared with those in the control group,and the differences were statistically significant（P<0.05）;although different cytokines were increased in the TNF-α group compared with the control group,the increase in the expression levels of different cytokines after TNF-α stimulation was more different,and the expression levels of TGF-β1 and TGF-β2 increased more after TNF-α action,with TGF-β1 being the most significant,while the expression of IL-6,IL-8,and IL-1β increased less after TNF-α stimulation.3.During the osteogenesis induction of BMMSCs,when IL-6,IL-8,IL-1β,TGF-β1 and TGF-β2 were added respectively,the ALP protein expression in the IL-6,IL-8 and IL-1β groups was not statistically different from that in the control group（P>0.05）,while the TGF-β1 and TGF-β2 groups were both higher than the control group,with the highest ALP protein expression in the TGF-β2 group,and the difference was statistically significant（P<0.05）;Runx2 protein expression in IL-1β and TGF-β2 groups was not statistically different from the control group,while Runx2 protein expression in IL-8group was lower than the control group,Runx2 protein expression in IL-6 and TGF-β1groups was higher than the control group,among which Runx2 protein expression in TGF-β1 group was the highest,and the differences were statistically significant（P<0.05）;OCN protein expression was higher in IL-6,IL-8,IL-1β,TGF-β1,and TGF-β2 groups compared with the control group,and the differences were statistically significant（P<0.05）,with the highest OCN protein expression in the TGF-β1 group and no statistical difference in OCN protein expression among IL-6,IL-8,and IL-1β groups.Conclusion:1.The expression of osteogenesis-related proteins was higher in BMMSCs under osteogenesis-inducing conditions compared with normal conditions;the osteogenic ability of BMMSCs was not significantly diminished by TNF-α under either normal or osteogenic conditions.2.After inflammatory stimulation,BMMSCs can secrete different cytokines involved in the anti-inflammatory process,among which the TGF-β family,especially TGF-β1,plays an important role in this process.3.Compared with IL-6,IL-8,IL-1β,and TGF-β2 groups,BMMSCs in the TGF-β1group had the highest expression of osteogenic-related proteins.BMMSCs participate in the anti-inflammatory process and promote osteogenic differentiation of BMMSCs mainly through the secretion of TGF-β1 during osteogenesis.