The Research On Function And Mechanism Of The Radiosensitive IGFBP-3 Protein In Blood

Ionizing radiation widely exists in the natural environment as well as the space environment.With the high-speed development of manned space flight in China,the hazard of space radiation to astronauts will become an inevitable problem.In addition,with the widespread use of nuclear energy and technologies in military,industry,medicine and natural sciences,more effective diagnostic and therapeutic measures are urgently needed for the health of occupational and non-occupational populations when accidental radiation events happen.Blood is one kind of minimally invasive and convenient collected biological sample,which contains abundant biomolecules and can be rapidly and precisely detected,making it an ideal source of biomarkers.Recent studies have reported a large number of radiosensitive biomolecules in blood,including RNA and protein,but fewer studies focused on high-LET（Linear energy transfer）rays such as heavy ion beams.Blood is a major component of the circulatory system,and various components in blood may originate from various organs,tissues or cells of the body,whereas the sources and production mechanisms of radiosensitive biomolecules have not been studied clearly.All of the above problems seriously hamper the clinical application of blood molecules as ionizing radiation biomarkers.In addition,if the blood radiation-sensitive molecules play a certain role in the radiation damage and repair process,or just indicate the stress responses to ionizing radiation?This is an important yet unresolved issue..To explore these problems,mice were irradiated with X-rays,protons or carbon ions,and the protein antibody microarray was performed to screen blood radiosensitive proteins at 24 hours postirradiation.Futhermore,ELISA（Enzyme linked immunosorbent assay）was used to validate the differentially expressed proteins.We found a key protein,IGFBP-3（Insulin-like growth factor binding protein-3）,which responses to the ionizing radiation significantly and stably.The responses was also verified in specimens from patients exposed to radiation.In brief,the suitability of IGFBP-3 in human samples was investigated using blood samples obtained before and after radiotherapy from patients who first received consolidative radiotherapy,following tumor resection surgery.To investigate the source of the elevated IGFBP-3 in blood after irradiation,the biological models of partial body irradiation mice and liver cells were established to study the main source of organs and cell types of blood IGFBP-3 protein.Furthermore,IGFBP-3 protein was found to significantly improve the survival of mice with acute radiation injury by tail vein injection of rh IGFBP-3（Recombinant human IGFBP-3 protein）before irradiation.Taking this as a clue,Igfbp3 gene overexpression and knockdown cell models were established using overexpression vectors and RNA interference technology to study the effects of IGFBP-3 expression on the radiation sensitivity of cells.We obtained results through these experiments and summarized in three hands as follows:（1）The blood IGFBP-3 was sensitive to both low-（X-ray,proton beams）and high-（carbon ion beams）LET radiation with a good dose-effect relationship and time-dependent stability and was able to respond to total body and partial body irradiation.Moreover,the responses of blood IGFBP-3 to ionizing radiation were scarcely influenced by gender and age.The radiosensitivity of blood IGFBP-3 was futher confirmed in blood samples from patients received radiotherapy.（2）In the major organs and tissues of TBI（total body irradiated）mice,the liver and lung showed an increase in expression of IGFBP-3,of which the IGFBP-3 in liver exhibited the largest fold change in expression changes.Both the pro-form（28.7 k Da）and glycosylated-form（40-45 k Da）of IGFBP-3 protein in the liver were obviously increased,whereas the IGFBP-3 protein in the blood was mainly the glycoprotein form with a molecular weight of 45 k Da.These results showed that the liver may be the main source of radiosensitive IGFBP-3 in vivo.IHC（Immunohistochemistry）results of the liver tissues of TBI mice showed that after exposure to ionizing radiation,the levels of IGFBP-3 in nonparenchymal cells in the hepatic sinusoids and hepatocyte nucleus increased significantly.Studies on the cell lines of three main cell types in liver revealed that IGFBP-3 protein levels increased significantly both in the intracellular and in the culture medium after irradiation only in Kuppfer cells（liver macrophages）.The Kupffer cell specific depletion agent Gd Cl₃significantly suppressed the increasing trend of IGFBP-3 induced by radiation in blood and liver.In summary,radiosensitive IGFBP-3 in blood is mainly derived from Kupffer cells in the liver.（3）Tail vein injection of rh IGFBP-3 before irradiation significantly rescues mice from lethal dose of irradiation.Through over expressing or knocking down of Igfbp3in Kupffer cells,we found that IGFBP-3 protein can significantly reduce the radiation damage effects on cells,improving cell viability,proliferation ability,DNA synthesis activity,and phagocytosis ability after irradiation.Furthermore,the co-expression of p53 and IGFBP-3 was found in liver tissues of irradiated mice and the Kupffer cells,and the responses of IGFBP-3 could be weakened by inhibition of Trp53.In addition,the detection of apoptosis and senescence of Kuppfer cells showed that IGFBP-3protein does not influence the apoptosis of irradiated cells,but can significantly promote cell senescence induced by ionizing radiation,indicating that IGFBP-3 may resist ionizing radiation damage by inducing cell senescence.In conclusion,our findings demonstrated that the circulating IGFBP-3 has a great potential as a convenient,effective and specific responsive biomarker for complex environmental radiation exposure.The assays of circulating IGFBP-3 will provide us rapid,convenient and effective methods for dose and toxicity assessment in space flight,occupational exposure or radiation disaster management.In addition,this study has demonstrated that regulation of blood IGFBP-3 protein level can alleviate radiation damage,indicating that blood IGFBP-3 protein has the potential as a key molecular target for radiation protection and IGFBP-3 has the potential to be developed as a radiation protection agent.Expanding on this,this study provides some new evidences for the academic view that the changes of biomolecules levels in blood induced by ionizing radiation may affect the process of body radiation injury and repair at the early stage or over a longer period of time,and is worth focusing on.