Protein Chip Analysis On Culture Supernatant Of Mouse Osteoblasts Treated By X-ray Irradiation

Background and ObjectiveRadiotherapy is an important therapeutic approach for oral maxillofacial-head and neck malignant tumors.However,radiotherapy kills tumors and damages normal tissues simultaneously,leading to bone metabolic disorders,osteoporosis,poor bone healing,etc.Among them,osteoradionecrosis of the jaws is one of the serious complications,and its pathogenesis is not yet clear.Furthermore,physical stimuli such as ionizing radiation can cause bone remodeling.Besides,bone formation of osteoblasts and bone resorption of osteoclasts maintain the dynamic balance of bone.Changes in morphology,phenotype,gene and protein expression may developed in cells exposed to ionizing radiation.So far,there is no in-depth experimental study to confirm some existed problems,for example,how radiation affects osteoblasts proliferation and differentiation as well as expression of osteoblasts-related genes,whether there are differences in the proteins secreted by osteoblasts after exposure to different doses of radiation,whether there are specific proteins for radiation damage.which biological processes and signal transduction pathways are involved in these differential proteins.Accordingly,in order to address the above research gap,the present study was carried out to investigate the effects of irradiation under different doses on osteoblasts proliferation and differentiation as well as secreted proteins,to screen specific proteins that might be involved in radiation damage,and to preliminarily explore the biological processes and signal transduction pathways involved so as to provide a reference for better understanding the biological effects of radiation on bone tissues.Methods1.Mouse osteoblasts MC3T3-E1 cultured in vitro were collected and divided into 5 groups according to different irradiation dose:0 Gy group,2 Gy group,4 Gy group,8 Gy group and 16 Gy group.X-ray irradiation with a 6 MV linear accelerator was performed to observe the changes of cell morphology after irradiation.CCK8 assay was conducted to detect cell proliferation.ALP activity in cells of 0 Gy group,2 Gy group,4 Gy group and 8 Gy group was detected 7 days after irradiation.Besides,the mRNA expression levels of OPG,OCN,Runx2and ALP were detected by qPCR.2.Cell supernatants of 0 Gy group,2 Gy group,4 Gy group and 8 Gy group were collected 7 days after irradiation.High-throughput protein chip was then used to detect the expression of secreted proteins.GO and KEGG pathway were involved to analyze the biological information of differentially expressed proteins.Subsequently,differentially expressed proteins showing dose dependent up-regulation were screened and then verified by ELISA.Results1.After irradiation,there were swollen cell body of osteoblasts,enlarged nucleus and increased vacuoles in the cytoplasm,especially the presence of black granules near the nuclear area,accompanied by decreased adherence ability and elevated amount of floating cells.Besides,the above changes became increasing more obvious with the increase of radiation dose.In accordance with the detected proliferation results of osteoblasts by CCK8 assay,the proliferation of osteoblasts in four irradiation groups decreased in a dose-dependent manner at 3 d,5 d and 7 d after irradiation(P<0.05).Furthermore,7 d after irradiation,intracellular ALP activity was lower in each irradiation group than that in control group,and the difference was statistically significant(P<0.05).No significant change was found in mRNA expression levels of osteogenesis-related genes under 2 Gy irradiation.The expression levels of OPG,Runx2 and ALP genes in irradiation groups above 4 Gy were significantly lower than those in control group,with statistical difference(P<0.05).Meanwhile,the expression level of OCN gene was down-regulated under 4 Gy irradiation and up-regulated remarkably under 8 Gy irradiation(P<0.05).2.A total of 36 differentially expressed proteins were found in the supernatant after the irradiation of osteoblasts.GO and KEGG Pathway analysis showed that after X-ray irradiation of osteoblasts,multiple phenomena were observed,such as activation of various inflammatory cells and immune cells,enhancement in cell proliferation,differentiation,migration and chemotaxis,and obvious enrichment in cytokine-cytokine receptor interaction,cell adhesion signal,angiogenesis signal,TNF signal pathway,JAK/STAT signal pathway,ERK1 and ERK2 signal pathways,IL-17 signal pathway,etc.Among them,the expression levels of IL-22 and TremLl were different under three radiation doses,and both of which were up-regulated.Meanwhile,the expression of RANTES was up-regulated in a dose-dependent manner.ELISA confirmed that the expression of RANTES was up-regulated significantly after irradiation of osteoblasts(P<0.05).Conclusions:1.X-ray irradiation exerts an inhibitory effect on the proliferation,differentiation and matrix mineralization of osteoblasts,thereby interfering with bone formation.2.The proteins expressed by osteoblasts after X-ray irradiation involve acute inflammation,immune response,oxidative stress,defensive response,accompanied by tissue repair and other related biological processes and signaling pathways.3.RANTES is speculated in our study to be a key factor in a series of biological processes such as inflammation,chemotaxis and repair of osteoblasts after radiation damage.However,its specific mechanism remains to be further clarified.