| Objective:To investigate the effects of adaptive response induced by low dose radiofrequency radiation on bone microstructure and bone metabolism in mice,and to explore the effect of adaptive response induced by low dose radiofrequency radiation on osteoblast differentiation and the underlying mechanism at the cellular level.Methods:1.Effects of adaptive response induced by low dose radiofrequency radiation on bone microstructure and bone metabolism in mice.Fourty healthy male C57BL/6J mice were randomly divided into the following 4 groups after adaptive feeding for one week:(a)Sham group(mice were placed in GTEM device,but without radiofrequency exposure,4h/d for 14 days),(b)RF group(mice were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 14 days),(c)XR group(mice were irradiated with 6 Gy X-rays radiation at a dose rate of 1.103 Gy/min 4h post-sham exposure),(d)RF+XR group(mice were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 14 days,then subjectd to 6 Gy X-rays radiation at a dose rate of 1.103 Gy/min 4h post-radiofrequency exposure).Seven days after exposure,the blood was collected after anaesthesia with sodium pentobarbital,then the mice were sacrificed,the bilateral femurs of the mice were separated.The blood samples and bone tissues were stored at-80℃.Micro-computed tomography(Micro-CT)was used to analyze the microstructure of the right femur of mice,and the concentration of bone metabolism-related molecule in serum was determined.2.Effect of adaptive response induced by low dose radiofrequency radiation on osteoblasts.MC3T3-E1 osteoblasts were divided into the following 4 groups:(a)Sham group(cells were kept in GTEM device without radiofrequency exposure),(b)RF group(cells were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days),(c)XR group(cells were irradiated with 6 Gy X-rays radiation 4h post-sham exposure),(d)RF+XR group(cells were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days,and subjectd to 6 Gy X-rays radiation 4h post-radiofrequency exposure).CCK-8 and EdU assays were used to measure cell proliferation ability.Cell apoptosis was measured with flow cytometry.The activity of ALP was detected using alkaline phosphatase assay kit.The expression levels of osteoblast differentiation-related molecules were determined with RT-PCR.3.Effecting mechanism of adaptive response induced by low dose radiofrequency radiation on osteoblast differentiation.(1)The role of ROS in adaptive response induced by low dose radiofrequency radiation.MC3T3-E1 osteoblasts were divided into the following 8 groups:(a)sham group(cells were kept in GTEM device without radiofrequency exposure),(b)RF group(cells were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days),(c)XR group(cells were subjectd to 6 Gy X-rays radiation 4h post-sham exposure),(d)RF+XR group(cells were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days,and irradiated with 6 Gy X-rays 4h post-radiofrequency exposure),(e)melatonin group(cells were treated with 500nM melatonin for 4h),(f)melatonin+RF group(cells were treated with 500nM melatonin 4h prior to 150μW/cm2 900MHz radiofrequency radiation,4 h/d for 5 days),(g)melatonin+XR group(cells were treated with 500nM melatonin 4h prior to 6 Gy X-rays radiation),(h)melatonin+RF+XR group(cells were treated with 500nM melatonin for 4h,then irradiated with 150μW/cm2900MHz radiofrequency,4h/d for 5 days,then irradiated with 6 Gy X-rays 4h postradiofrequency exposure).Cells were collected at different time points after exposure.The level of ROS was measured with flow cytometry,and the activity of ALP was determined with alkaline phosphatase assay kits.(2)Low-dose radiofrequency radiation regulates NF-κB expression via ROS.MC3T3-E1 osteoblasts were divided into the following groups:(a)sham group(cells were kept in GTEM device without radiofrequency exposure),(b)RF group(cells were irradiated with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days),(c)melatonin+RF group(cells were treated with 500nM melatonin 4h prior to 150μW/cm2900MHz radiofrequency,4h/d for 5 days),(d)XR group(cells were exposed to 6 Gy Xrays radiation 4h post-sham exposure),(e)RF+XR group(cells were exposured with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days,and then irradiated with 6 Gy X-rays 4h post-radiofrequency exposure),(f)melatonin+RF+XR group(cells were treated with 500nM melatonin for 4h,then irradiated with 150μW/cm2 900MHz radiofrequency,4h/d for 5 days,and subjected to 6 Gy X-rays radiation 4h postradiofrequency exposure).Cells were collected at different time points after exposure,the expression level of NF-κB protein in the cytoplasm and nucleus were determined with western blot analysis.(3)The role of NF-κB in adaptive response induced by low dose radiofrequency radiation.MC3T3-E1 osteoblasts were divided into the following groups:(a)sham group(cells were kept in GTEM device without radiofrequency exposure),(b)RF group(cells were irradiated with 150μW/em2 900MHz radiofrequency radiation,4h/d for 5 days),(c)XR group(cells were exposed to 6 Gy X-rays radiation 4h post-sham exposure),(d)RF+XR group(cells were exposured with 150μW/cm2 900MHz radiofrequency radiation,4h/d for 5 days,and then irradiated with 6 Gy X-rays 4h post-radiofrequency exposure),(e)BAY group(cells were treated with 10μM BAY11-7082 for 1h),(f)BAY+RF+XR group(cells were treated with 10μM BAY11-7082 for 1h,then irradiated with 150μW/cm2900MHz radiofrequency,4h/d for 5 days,and irradiated with 6 Gy X-rays 4h postradiofrequency exposure).The ALP activity of osteoblasts was determined with alkaline phosphatase assay kit.Results:1.Effects of adaptive response induced by low dose radiofrequency radiation on bone microstructure and bone metabolism in mice.(1)Compared with Sham group,the mice femoral cancellous BMD,BV/TV,Tb.N and Tb.Th of femur cancellous bone of mice in XR group were significantly decreased(P<0.05),Tb.Sp and DA were significantly increased(P<0.05),but SMI was not significantly changed(P>0.05),Compared with XR group,the BMD,BV/TV and Tb.Th in RF+XR group were significantly increased(P<0.05)and Tb.Sp was significantly decreased(P<0.05).(2)Compared with sham group,the level of OPG was decreased significantly(P<0.05)and TRACP-5b was increased significantly(P<0.05)in XR group.Compared with XR group,the level of OPG in RF+XR group was increased significantly(P<0.05)and TRACP-5b was significantly decreased(P<0.05).2.Effect of adaptive response induced by low dose radiofrequency radiation on osteoblasts.(1)Compared with Sham group,the cell proliferation ability in XR group was significantly decreased(P<0.05)and the cell apoptosis was significantly increased(P<0.05).Compared with XR group,the cell proliferation ability in RF+XR group was significantly increased(P<0.05)and cell apoptosis was significantly decreased(P<0.05).(2)Compared with Sham group,the alkaline phosphatase activity,the expression level of ALP and Runx2 mRNA in XR group were significantly decreased(P<0.05).Compared with XR group,the alkaline phosphatase activity,the expression level of ALP and Runx2 mRNA in RF+XR group were significantly increased(P<0.05).3.Effecting mechanism of adaptive response induced by low dose radiofrequency radiation on osteoblast differentiation.(1)Compared with Sham group,the ROS level in RF group increased significantly at 30min and 4h post-radiofrequency radiation(P<0.05),but did not differ significantly at 24h post-radiofrequency radiation(P>0.05).Compared with RF+XR group,the activity of ALP in melatonin+RF+XR group was significantly decreased(P<0.05).(2)Compared with Sham group,the expression level of NF-κB protein in nucleus increased significantly(P<0.05)in RF group while NF-κB protein in cytoplasm decreased significantly(P<0.05),but did not differ significantly at 24h post-radiofrequency radiation(P>0.05).Compared with the RF group,the expression level of NF-κB protein in cytoplasm was significantly increased(P<0.05),and NF-κB protein in nucleus was significantly decreased(P<0.05)in melatonin+RF group.Compared with RF+XR group,the expression level of cytoplasmic NF-κB protein in melatonin+RF+XR group was significantly decreased(P<0.05),and nuclear NF-κB protein was significantly increased(P<0.05).(3)Compared with RF+XR group,the activity of ALP in BAY117082+RF+XR group was significantly decreased(P<0.05).Conclusions:1.Pre-exposure to low-dose radiofrequency radiation could reduce microstructural damage of femur cancellous bone and bone metabolic abnormalities in mice induced by subsequent high-dose X-ray exposure.2.Pre-exposure to low-dose radiofrequency radiation could induce adaptive response and reduce the inhibition of osteoblast differentiation induced by X-ray radiation.3.Low dose radiofrequency radiation could induce adaptive response via altering NF-κB expression mediated by ROS production. |
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