Role Of HMGB1 In Radiation-Induced Injury And Underlying Mechnaisms

It’s well known that ionizing radiation can cause damages via affecting cytokines, enzymes and gene expression—some of which are directly reflect the extent of radiation-induce damage. Due to the development and application of new technology in radiobiology, increasing studies focus on the alterations of genes which are possibly related to radiation-induced injury. High-mobility group box 1 (HMGB1), a chromatin associated nuclear protein and extracellular damage associated molecular pattern molecular (DAMP), is an evolutionarily ancient and critical regulator of cell survival. The study described in this thesis was designed to explore the in vitro and in vivo effects of ionizing radiation on HMGB1 expression and release as well as the impact of alterations of HMGB1 expression levels on irradiation-induced injury and cell radiosensitivity.The findings described in this thesis were described as three sections:(1) Translocation and release of HMGB1 in GM and 16HBE cells exposed to X-ray irradiation. The HMGB1 protein released to the culture medium and expression of the HMGB1 protein in both cell nuclei and cytoplasm after irradiation were detected by Western blot and immune-fluorescence assay. The HMGB1 in the serum of SD rats with whole-body irradiation and in the serum of the patients accepted radiotherapy were detected by an Enzyme-linked immune-sorbent assay. Irradiation with 8Gy of 6MV-X-rays caused a significant translocation of HMGB1 protein from nucleus to cytoplasm in both GM and 16HBE cell lines. A release of HMGB1 were observed in 12 cell lines, including normal and tumor cell lines, after irradiation, which was in a dose-dependent and time-dependent manner.There was a lower HMGB1 in the peripheral blood of SD rats, whole body irradiation led to a dose-dependent release of HMGB1 way. When whole body irradiation with 6Gy of 6MV-X-rays, the HMGB1 protein in the peripheral blood of SD rats exhibited an early increase and reached a peak at 24h, then slowly declined, and still was detectable one week after irradiation.(2). An increased amount of y-H2AX foci in the nuclear was found in either GM or 16HBE cells received 4Gy of 6MV-X-ray irradiation. Decreased survival and increased DNA double-strand breaks were observed in GM or 16HBE cells which were cultured with the conditioned medium from irradiated GM or 16HBE cells, as demonstrated by MTT viability assay and immune-fluorescence staining. On the contrary, the conditioned medium failed to do so when the anti-HMGB1 monoclonal antibody was added into the conditioned medium. A prolong survival of C57 mice was found when these animals were pre-administrated with a specific HMGB1 inhibitor, ethyl pyruvate (EP) before radiation. In addition, an increased RAGE expression was verified in both GM and 16HBE cells after irradiation. The protein interaction of HMGB1 with RAGE was identified in GM and 16HBE cells by immune-precipitation and Western blot assay. The phosphorylated ERK and JNK protein were increased in GM and 16HBE cells after irradiation; and similar results were found in the cells cultured in the conditioned medium. Conversely, the increased phosphorylation was reduced by anti-HMGB1 antibody.(3). A significant reduction of the HMGB1 protein and mRNA was obtained in cells transfected with HMGB1 siRNA, in GM and 16HBE cell lines, as detected by Western blot and RT-PCR assay. Without any change of morphology, an S arrest of cell cycle and more y-H2AX foci caused by radiation were observed in the cells with HMGB1 siRNA, as observed using flow cytometry assay and immune-flourescence staining. Also, a reduced radiosensitivity was seen in the HMGB1 siRNA, as analyzed with a clonogenic survival assay. This HMGB1 siRNA-mediated radioresistance was accompanied with a increased expression of Ku-70 protein, FEN-1, XRCC1 protein, activation of NF-KB, DNA-PKC and phosphorylated AKT.In conclusion, radiation, at least X-rays, causes a translocation of HMGB1 protein from nucleus to cytoplasm and triggers extracellular release of the HMGB 1 protein via a time- and dose-dependent fashion. The participation of HMGB1 in the irradiation-induced injury may be at least through the interaction of extracellular HMGB1 and RAGE, activation of MAPK and NF-KB pathway. Down-regulation of HMGB1 leads to radioresistance, the underlying mechanism may be related to the regulation of Ku-70, FEN-1 and XRCC1 protein expression as well as the NF-κB and AKT signaling pathway activated by HMGB1.