Inhibition Of SET7 In Macrophages Promotes Bone Regeneration In Tooth Extraction Sockets Of OVX Mice Through Reducing Reactive Oxygen Species

Purpose: Osteoporosis is a chronic inflammatory condition that causes bone loss and delayed bone regeneration in patients.In clinical practice,osteoporosis may lead to delayed alveolar bone regeneration after tooth extraction.Pro-inflammatory macrophages play a dominant role in the inflammatory process of extraction socket healing.Estrogen deficiency leads to increased levels of reactive oxygen species(ROS),which promotes the development of inflammation.Epigenetic modifications have been shown to regulate the inflammatory response.Set domain containing lysine methyltransferase 7(SET7)acts as an epigenetic enzyme that drives monomethylation of histone H3 lysine K4(H3K4me1).And in other diseases such as diabetes,SET7 can regulate macrophage polarization through ROS-related signalings,then participate in the inflammatory process.In order to verify the effect of SET7 on extracted tooth sockets in the osteoporotic state,in this study we observed bone regeneration in extracted tooth sockets of osteoporotic(OVX)mice after inhibition of SET7 in proinflammatory macrophages.Methods: To verify the success of the modelling and the healing of the extraction sockets,we analysed the femur and maxilla of the mice by micro-CT scanning.To verify the effect of inhibiting SET7 in pro-inflammatory macrophages on reducing the inflammatory response,we used SiRNA to mimic the inflammatory microenvironment of osteoporosis using TNF-α stimulated cells after SET7 knockdown in the mouse macrophage line RAW264.7 cells.Real-time quantitative PCR(RT-qPCR),protein immunoblotting(Western Blot)and Immunofluorescence(IF)was used to detect the expression levels of proteins and genes associated with the inflammatory response and antioxidation.To verify the indirect effect of SET7 within pro-inflammatory macrophages on the osteogenic differentiation ability of mouse bone marrow MSCs,we used alkaline phosphate(ALP)staining.To detect the level of reactive oxygen species,observed the fluorescence intensity under confocal laser scanning microscope using DCFH-DA,which is a reactive oxygen species fluorescence probe.Results: micro-CT verified that the extraction sockets of the OVX mice had less new bone formation and higher expression of SET7 in pro-inflammatory macrophages,compared to the SHAM-operated(SHAM)group.Western Blot showed that after stimulation of RAW264.7 cells with TNF-α at a concentration of 5 ng/ml for 12 hours,the expression levels of H3K4 monomethylation,pro-inflammatory macrophageassociated factor i NOS,and pro-inflammatory factor IL-6 were lower in the SiSET7 group compared to the NC group;in addition,ELISA showed that the expression level of pro-inflammatory factor IL-1β was lower in the supernatant of SiSET7 group compared to that of the NC group.The supernatant collected after 12 hours of stimulation with TNF-α at a concentration of 5 ng/ml in the SiSET7/NC group RAW264.7 cells and the supernatant collected after 12 hours in the SiSET7/NC group RAW264.7 cells without stimulation were incubated with mineralization induction solution at a ratio of 1:8 for mBMSCs(NC-CON group,NC-TNF-α group,SiSET7-CON group,SiSET7-TNF-α group),and the results of ALP staining,RT-qPCR detection,and Western Blot showed that the expression levels of osteogenic-related genes ALP,RUNX2,OCN,and OPN and osteogenic-related proteins COL1A1,RUNX2,and OCN were higher in the SiSET7-TNF-α group than in the NC-TNF-α group,demonstrating that inhibition of SET7 could indirectly rescue the osteogenic differentiation ability of mBMSCs.PBS or 20 μM(R)-PFI-2 was injected locally into the extraction sockets on the third day after extraction,and the specimens were taken on the first and fourth day after surgery.Immunofluorescence staining of specimens on the fourth postoperative day for i NOS and SET7 showed that inhibition of SET7 led to a decrease in pro-inflammatory macrophages;meanwhile,immunohistochemical staining for H3K4me1 showed that inhibition of SET7 led to a decrease in H3K4me1 expression levels.H&E staining,MASSON staining and immunohistochemical staining of COL1A1 for specimens one week after surgery showed more new bone formation in the extraction sockets of OVX mice injected with 20 μM(R)-PFI-2,demonstrating that inhibition of SET7 promoted bone regeneration in the extraction sockets in osteoporosis.Tooth extracted sockets four days after surgery were examined for reactive oxygen species levels by DCFH-DA fluorescence probe,and it was found that osteoporotic mice injected with 20 μM(R)-PFI-2 had lower levels of reactive oxygen species than PBS-injected tooth extracted sockets.Western Blot results showed that the SiSET7 group with SET7 knockdown underwent TNF-α stimulation had more expression of antioxidant enzymes than the control NC group.Conclusion: Inhibition of SET7 in pro-inflammatory macrophages can promote bone regeneration in the tooth socket in OVX mice by reducing the level of reactive oxygen species,providing new strategies for the treatment of osteoporosis-related bone loss.