| ObjectiveTo investigate the effects of 900 MHz radiofrequency on osteoporosis in mice after ovariectomy,and to explore the effects of 900 MHz radiofrequency on osteoclastogenesis as well as the underlying mechanism at the cellular level.Methods1.Effects of radiofrequency radiation on osteoporosis in ovariectomized mice(1)Establishment of osteoporosis animal modelTen 3-month-old female ICR mice were randomly divided into sham-operation(Sham)and ovariectomy(OVX)groups(n=5).Five weeks after operation,the left femurs of mice were collected,the bone mineral density(BMD)and bone microstructure indexes of the collected femurs were detected by Micro-CT,and the 3D model of mouse femurs were reconstructed according to the collected images.(2)Effects of different doses of radiofrequency radiation on osteoporosis in ovariectomized miceSixty 3-month-old female ICR mice were randomly divided into six groups(n=10):(a)Sham group(sham-operation mice were placed in radiofrequency radiation device,but without radiofrequency exposure,4 h/d for 30 d),(b)OVX group(ovariectomized mice were placed in radiofrequency radiation device,but without radiofrequency exposure,4 h/d for 30 d),(c)OVX+LRF group(ovariectomized mice were exposed to 900 MHz radiofrequency radiation at power density of 50 μW/cm~2,4 h/d for 30 d),(d)OVX+MRF group(ovariectomized mice were exposed to 900 MHz radiofrequency radiation at power density of 150 μW/cm~2,4 h/d for 30 d),(e)OVX+HRF(ovariectomized mice were exposed to 900 MHz radiofrequency radiation at power density of 450μW/cm~2,4 h/d for 30 d),(f)OVX+E2 group(ovariectomized mice were intramuscularly injected with estradiol benzoate at the dose of 0.04 mg·kg-1,once every two days for 30 d).After exposure,the mice were sacrificed,the serum samples of mice were collected,the levels of estrogen(E2),bone-specific alkaline phosphatase(BALP),osteocalcin(BGP),osteoprotegerin(OPG),type I collagen carboxy terminal telopeptide(CTX-I)and tartrate-resistant acid phosphatase(TRACP-5b)in serum were detected by Elisa method.The microstructure and morphology of left femur were observed by Micro-CT,the parameters of bone,including BMD,BV,TV,BV/TV,Tb.N,Tb.Th,and Tb.Sp were calculated directly from the reconstructed images.The gene expression of Runx2 and Nfatcl in right femur,which are considered as osteoblast and osteoclast differentiation transcription factors respectively,were determined by real-time PCR.The levels of OPG and RANKL proteins in left femur were detected by immunohistochemical staining.2.Effects of radiofrequency radiation on RANKL-induced osteoclastogenesis in RAW264.7 cells(1)Effects of different doses of radiofrequency radiation on proliferation,apoptosis and RANKL-induced osteoclast differentiation in RAW264.7 cellsRAW264.7 cells were cultured in DMEM with 10%FBS and divided into the following 6 groups:(a)Sham group(The cells cultured in regular DMEM were placed in the radiofrequency radiation device,but without radiofrequency radiation,4 h/d for 5 consecutive days),(b)RANKL group(The cells cultured in DMEM with 50 ng/ml RANKL were placed in the radiofrequency radiation device,but without radiofrequency radiation,4 h/d for 5 consecutive days),(c)LRF+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were irradiated with 900 MHz radiofrequency radiation at 50μW/cm~2 power intensity,4 h/d for 5 consecutive days),(d)MRF+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were irradiated with 900 MHz radiofrequency at 150 μW/cm~2 power intensity,4 h/d for 5 consecutive days),(e)HRF+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were irradiated with 900 MHz radiofrequency radiation at 450 μW/cm~2 power intensity,4 h/d for 5 consecutive days),(f)E2+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL and 10-7mol/L 17-β estradiol were placed in the radiofrequency radiation device,but without radiofrequency radiation,4 h/d for 5 consecutive days).Cells were collected post-exposure,cell viability was assayed using CCK-8,cell apoptosis were detected with flow cytometry,osteoclast differentiation was assayed with TRACP staining method.(2)Effects of 150 μW/cm~2 RF on the expression of genes related with RANKL-induced osteoclastogenesisAccording to the preceding results,900 MHz radiofrequency at dose of 150 μW/cm~2 exhibit the most obvious inhibitory effect on osteoclast differentiation,so the dose of 150μW/cm~2 was selected to study the acting mechanism.Cells were divided into the following 4 groups:(a)Sham group(The cells cultured in regular DMEM were sham-exposed,4 h/d for 5 consecutive days),(b)RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were sham-exposed,4 h/d for 5 consecutive days),(c)MRF group(The cells cultured in regular DMEM were irradiated with 900 MHz radiofrequency radiation at 150μW/cm~2 power intensity,4 h/d for 5 consecutive days),(d)MRF+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were irradiated with 900 MHz radiofrequency radiation at 150 μW/cm~2 power intensity,4 h/d for 5 consecutive days).Cells were collected after exposure,the expression levels of mRNA and protein of osteoclastogenesis-related genes were determined with RT-PCR and Western blot respectively.(3)Role of NF-κB activation in RF-induced inhibition of RANKL-induced osteoclastogenesis in RAW264.7 cellsRAW264.7 cells were divided into the following 5 groups:(a)Sham group(The cells cultured in regular DMEM were sham-exposed,4 h/d for 5 consecutive days),(b)RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were sham-exposed,4 h/d for 5 consecutive days),(c)BAY+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL and 10 ng/ml BAY11-7082 were sham-exposed,4 h/d for 5 consecutive days),(d)MRF group(The cells cultured in regular DMEM were irradiated with 900 MHz radiofrequency radiation at 150 μW/cm~2 power intensity,4 h/d for 5 consecutive days),(e)MRF+RANKL group(The cells cultured in DMEM containing 50 ng/ml RANKL were irradiated with 900 MHz radio frequency radiation at 150 μW/cm power intensity,4 h/d for 5 consecutive days).Cells were collected 4 hours post-exposure,the protein levels of NFκB in cytoplasm and nucleus,as well as the levels of NFATc1 and TRACP proteins in cells were determined with western blot analysis.Results1.(1)Compared with sham group,the BMD in OVX group was significantly decreased(P<0.05),the 3D model of femur showed that the trabecular structure inside was loose,and BV,BV/TV,Tb.Th,Tb.N,were significantly decreased while Tb.Sp was significantly increased(P<0.05),presenting typical symptoms of osteoporosis.The results indicated that resection of ovaries could lead to osteoporosis in mice.(2)Compared with sham group,the level of serum E2 in OVX group,OVX+LRF,OVX+MRF and OVX+HRF group were significantly decreased(P<0.05),while that was slightly increased in OVX+E2 group after estrogen supplementation,but still lower than that in sham group.Compared with sham group,the levels of serum BGP,OPG,CTX-I and TRACP-5b were significantly increased in OVX group(P<0.05),the BMD,BV,BV/TV,Tb.Th,Tb.N were significantly decreased while Tb.Sp and the expression of Nfatc1 mRNA were significantly increased(P<0.05),the expression of OPG and RANKL proteins in OVX group was significantly increased(P<0.05),indicating that ovariectomy could decrease serum estrogen levels and bone density,deteriorate bone micro structure and increase the serum level of bone remodeling molecular marker.Compared with OVX group,the levels of serum BGP in OVX+MRF and OVX+E2 groups were significantly increased while CTXI and TRACP-5b were significantly decreased(P<0.05),the level of Runx2 mRNA in right femur was significantly increased while the level of Nfatc1 mRNA was significantly decreased(P<0.05),the BMD,BV,BV/TV,Tb.Th,Tb.N and OPG were increased while Tb.Sp and RANKL were decreased.Among three RF groups,OVX+MRF group showed the most significant changes(P<0.05).These results suggest that radiofrequency radiation and estrogen supplement could relieve the symptoms of osteoporosis caused by ovariectomy.2.(1)Compared with sham group,there were no significant changes in cell proliferation in RANKL group,LRF+RANKL group and HRF+RANKL group,while the cell proliferation in MRF+RANKL group and E2+RANKL group was significantly decreased(P<0.05).Compared with sham group,the apoptosis level in RANKL group,three RF groups and E2 group were significantly increased(P<0.05).Among three RF groups,the apoptosis level in MRF+RANKL group was mostly increased(P<0.05).Compared with Sham group,TRACP-positive multinuclear cells in RANKL group was increased significantly(P<0.05),suggesting that osteoclastogenesis is active under RANKL treatment.Compared with the RANKL group,TRACP-positive multinuclear cells in three RF groups and E2 group was decreased.Among three RF groups,TRACP-positive multinuclear cells in MRF+RANKL group was less than that in LRF+RANKL group and HRF+RANKL group,(P<0.05),indicating that treatment with MRF and E2 could promote apoptosis and inhibit the osteoclastogenesis in RAW264.7 cells induced by RANKL.(2)Compared with sham group,the expression of mRNA levels of Rank,Nfatc1,TRACP and protein levels of RANK,NFATc1,TRACP in RANKL group were significantly increased(P<0.05),indicating that RANKL could induce osteoclastogenesis in RAW264.7 cells.Compared with RANKL group,the expression levels of above genes and proteins in MRF group and MRF+RANKL group were significantly decreased(P<0.05),indicating that MRF could inhibit the expression of genes related with osteoclastogenesis in RAW264.7 cells.(3)Compared with sham group,the level of NF-κB protein in cytoplasm was decreased while that in nucleus was increased in RANKL group 4 h post-radiofrequency radiation(P<0.05),indicating that RANKL could activate NF-κB and transport it to nucleus.Compared with RANKL group,the level of NF-κB protein in cytoplasm was increased while that was decreased in nucleus in BAY+RANKL group(P<0.05),indicating that BAY117082 could inhibit the nuclear translocation of NF-κB;Compared with RANKL group,the level of NF-κB protein in cytoplasm was increased while that was decreased in nucleus in MRF+RANKL group(P<0.05),suggesting that MRF could inhibit the nuclear translocation of NF-κB.When nuclear translocation of NF-κB was inhibited by MRF(150 μW/cm~2 RF),the expression levels of osteoclastogenesis related proteins NFATc1 and TRACP were significantly decreased(P<0.05),indicating that in the process of osteoclastogenesis induced by RANKL,MRF could inhibit the RANKL-induced activation of NF-κB,and then inhibit the expression of osteoclastogenesis-related genes which are regulated by NF-κB.Conclusion1.150 μW/cm~2 900 MHz radiofrequency radiation could increase BMD,improve bone microstructure,promote bone formation and inhibit bone resorption in ovariectomized mice,and has therapeutic effect on osteoporosis caused by ovariectomized mice.2.150 μW/cm~2 900 MHz radiofrequency radiation could inhibit osteoclastogenesis by inhibiting NF-κB nuclear translocation and then inhibit the protein expression of NFATc1 and TRACP regulated by NF-κB. |
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