The Role Of Growth Arrest-specific 6(Gas6) In The Repair Of Radiation-induced Osteoblast Precursor Cell Injury

Objective:Osteoradionecrosis of the jaws(ORNJ)is a serious complication of radiotherapy for oral and maxillofacial malignancies.Currently,there is no gold standard treatment and consensus guidelines exist for ORNJ.Although with the improvement of dental preventive health care and radiation technology,the incidence of radiation osteonecrosis of the jaw has decreased significantly in recent years,due to its difficulty in early diagnosis and treatment,we still need to further study its complex mechanism in order to provide new strategies for clinical treatment.The purpose of this study was to study the function of growth arrest specific-gene 6(Gas6)in the apoptosis and proliferation of osteoblast precursor cell line(MC3T3-E1)after irradiation.RNA interference technology and receptor blockers were used to study the role of Gas6 in apoptosis and proliferation of osteoblast precursor cells after radiation injury.Methods:1.CCK8 was used to detect the proliferation of MC3T3-E1 at different irradiation doses and at different time points in order to construct the MC3T3-E1 cell irradiation damage model.2.Cells were stained with Annexin V/PI,and the effects of irradiation on cell apoptosis and proliferation of MC3T3-E1 were detected by flow cytometry.3.Enzyme linked immunosorbent assay(ELISA)was used to detect the changes of Gas6 in the bone marrow(BM)supernatant of irradiated mice to verify the previous experimental findings of this group.Detecting the expression of Gas6 in healthy MC3T3-E1 by methods such as polymerase chain reaction(PCR)and immunofluorescence(IF).The secretion of Gas6 protein and the expression of Gas6 gene in MC3T3-E1 after irradiation were detected by Quantitative real time polymerase chain reaction(q RT-PCR)and enzyme linked immunosorbent assay(ELISA).4.The interference efficiency of Gas6 small interfering RNA(si RNA)was identified.Then,MC3T3-E1 was transfected with Gas6 si RNA to silence the expression of Gas6,and its effect on MC3T3-E1 proliferation after 9Gy γ-ray irradiation was detected by using Brdu incorporation experiment and flow cytometry;5.Irradiation-induced MC3T3-E1 damage model was treated with R428(an Axl inhibitor),and its effect on MC3T3-E1 proliferation was detected by using Brdu incorporation experiment and flow cytometry.Results:1.The data of CCK8 experiment showed that compared with the non-irradiated group at 24 hours,the cell proliferation ability of the 3Gy,6Gy,and 9Gy groups was significantly reduced(P<0.0001).At 48 hours,the cell proliferation ability of the 3Gy group was significantly reduced(P=0.001),and the 6Gy and 9Gy groups also decreased significantly(P<0.0001).At 72 hours,the cell proliferation ability of the 3Gy group decreased significantly(P=0.0047),and the 6Gy and 9Gy group was significantly reduced(P<0.0001).The irradiation dose was 9Gy and the detection time was set to 24 h can meet the needs of the next vitro experiments.2.The results of flow cytometry analysis showed that the ratio of early and late apoptosis was increased after 9Gy γ-ray irradiation,and the apoptosis rate increased from 9.08% ± 1.69% to 15.78% ± 0.42%(P<0.01).It showed that 9Gy γ-ray irradiation could significantly induce MC3T3-E1 apoptosis,and irradiationinduced MC3T3-E1 damage model was successfully established.3.Compared with the non-irradiated group(-IR),the concentration of Gas6 protein in the BM supernatant of the irradiated mice increased significantly after 24 h.Agarose gel electrophoresis and immunofluorescence images showed that Gas6 is expressed in healthy MC3T3-E1.The results of q RT-PCR and ELISA experiments showed that 9Gy γ-ray irradiation could cause a significant increase in Gas6 secretion and expression in MC3T3-E1.(P<0.01).4.The results of Brd U flow cytometry showed that 9Gy γ-ray irradiation significantly reduced the Brd U incorporation rate in MC3T3-E1 cells(P<0.01),and the silence of Gas6 inhibited the proliferation of osteoblast precursor cells damaged by irradiation(P<0.05);5.The data of Brd U flow cytometry showed that the Axl receptor inhibitor R428 could significantly inhibit the proliferation of irradiation-induced MC3T3-E1(P<0.01),suggesting that Gas6/Axl axis plays an important role in it.Conclusion:1.Gas6 can promote the proliferation of osteoblast precursor cells(MC3T3-E1)after radiation injury.2.The Gas6/Axl axis may play a positive regulatory role in the proliferation of osteoblast precursor cells after radiation injury.3.Gas6/Axl is likely to become a potentially effective therapeutic target in enhancing bone regeneration in the treatment of radiation bone injury.