Study On Effect And Mechanism Of γ Ionizing Radiation On Osteoclastic Metabolism

Objective: To investigate the effect of γ ionizing radiation on osteoclasticmetabolism and, understand the relevant dose of radiation，preliminary explore theeffect mechanism. The purposes are for further understanding the pathway of ionizingradiation changes bone metabolism and, try to provide a new deliberation to prevent andcure the bone damage induced by ionizing radiation.Methods: The mainly work of the study included four sections. Section1) Toinvestigate the effect of a single whole-body2Gy γ-ray radiation on mice osteoclasticmetabolism and bone resorption, male C57BL/6mice were divided randomly intocontrol group or γ-ray exposed group. Double antibody sandwich ELISA wereperformed to determine levels of TRAP-5b,CTX-1,sRANKL and TNF-α in mice serum,Ca²⁺level was determined by methylthymol blue colorimetric method, TRAP stainingwas performed on mice bone histopathology to detect osteoclast. Section2) Culturesystems of osteoclast in vitro were established. Osteoclasts were induced fromosteoclast precursors in bone marrow cells at the maintaining of conditional medium ofBMSCs and also developed from RAW264.7cells in the presence of mouserecombinant sRANKL. Section3) To investigate the effect of γ-ray on osteoclastdevelops from RAW264.7cell, cells exposed to γ-ray radiation in the processdeveloping into osteoclast, quantity of osteoclast and osteoclastic metabolism markerTRAP-5b were detected, intracellular ROS and Ca²⁺were also determined by flowcytometry. Section4) To understand the effect of γ-ray on the regulation of BMSCs toosteoclast differentiation, male C57BL/6mice received a single γ-ray radiation atdifferent dose; RANKL mRNA and OPG mRNA in bone marrow cells were detected onpost-radiation day1,2,3and7by a single-step RT-PCR. BMSCs were irradiated with0～6Gy γ-ray radiation in vitro, expression of RANKL protein of BMSCs was detectedby ELISA or Laser Scanning Confocal Microscope post-radiation, and osteoclasts weredeveloped from BMMNCs at the maintaining of conditional medium of irradiatedBMSCs. Results: The results of each section as follow:（1） On post-radiation day4, levels of TRAP-5b,CTX-1,sRANKL and TNF-α inγ-ray exposed group mice serum were raised by37.30%,47.14%,19.55%and17.67%respectively compared with control group（P<0.05）, osteoclasts in irradiated groupshowed active metabolism; On post-radiation day90, levels of TRAP-5b and CTX-1inγ-ray exposed group were still significantly higher than control group（P<0.01）.（2） Two of the procedures developed typical osteoclast which is TRAP positive andmultinuclear cells fusing from osteoclast precursors. The quantity and size of osteoclastsderived from RAW264.7stimulated by exogenous sRANKL were more and bigger thanthose derived from BMMNCs stimulated by conditional medium of BMSCs.（3） Quantity of osteoclasts were significantly increased in all irradiated groupscompared with control group （P<0.001）. TRAP-5b level in osteoclasts culture mediumwas positive correlated to dose of radiation （r²=0.919, P=0.041）. Intracellular ROS andCa²⁺also increased post-radiation.（4） Before3days post-radiation, RANKL/OPG mRNA ratios of bone marrow cellsof irradiated mice were significantly higher compared with control mice （P<0.01）,which rose depended on the increased expression of RANKL mRNA. γ-ray earlyfacilitated expression of RANKL protein of BMSCs in the time-and dose-dependedmanner, RANKL protein up-expressed more urgent as dose increased. In consequence,quantity and TRAP-5b of osteoclasts derived from BMMNCs also rose in the irradiatedgroup, particularly at2Gy irradiated dose.Conclusions: A single whole-body2Gy γ-ray radiation on mice activatesosteoclastic metabolism and increases bone resorption，which contributes an importantrole in ionizing radiation-induced bone damage. The mechanism of γ-ray activatesosteoclastic metabolism includes:（1） γ-ray elevates the intracellular second messenger ROS and Ca²⁺that relevant toevoke osteoclast， enhances osteoclastogentic potential of osteoclast precursor indose-depended manner.（2） γ-ray early facilities expression of RANKL protein of BMSCs in the time-anddose-depended manner, RANKL protein up-expressed more urgent as dose increased，consequently, γ-ray displays promotion to osteoclastic metabolism indirectly,particularly at2Gy irradiated dose the facilitation is significantly in vitro. （3） Elevation of inflammatory factors such as TNF-α induced by γ-ray maysynergistically with RANKL or directly stimulate the metabolism of osteoclast.