低频脉冲电磁场对大鼠成骨细胞骨形成的影响及作用机制
|
摘要
目的观察低频脉冲电磁场(PEMFs)对大鼠成骨细胞(ROBs)骨形成的影响,探讨了环磷酸腺苷(cAMP)/蛋白激酶A (PKA)/环磷腺苷效应结合蛋白(CREB)信号通路的作用机制。方法取新生Wistar大鼠颅骨,通过多次酶消化法获得ROBs并进行传代融合。采用50 Hz 0. 6 m T PEMFs处理3、6、9 d后,检测ROBs内的碱性磷酸酶(ALP)浓度;处理0、15、30、60、90、120 min后,检测ROBs内骨形成相关因子(RUNX2)和成骨相关转录因子(OSX)蛋白表达,cAMP浓度,p-PKA、p-CREB和CREB蛋白表达。观察p-CREB核转位情况。采用RNA干扰法干扰IFT88后,检测ROBs内RUNX2、OSX、p-PKA和p-CREB蛋白表达情况。结果 PEMFs处理ROBs 3、6、9 d后,ALP活性值分别为24. 356±4. 911、37. 688±2. 151和39. 922±5. 486,均明显高于相应对照组的18. 531±2. 401 (P=0. 0121)、33. 675±4. 366 (P=0. 0324)和36. 574±1. 339 (P=0. 0134)。PEMFs处理30 (P=0. 0042和P=0. 0058)、60 (P=0. 0097和P=0. 0079)、90 min (P=0. 0083和P=0. 0098)时,ROBs内的RUNX2和OSX活性均显著高于未处理组; PEMFs处理30(P=0. 0012)和60 min (P=0. 0035)时,ROBs内的cAMP浓度明显高于未处理时; PEMFs处理15 (P=0. 0018)、30 (P=0. 0087)、90 (P=0. 0250)和120 min (P=0. 0350)时,ROBs内的p-PKA水平明显高于未处理组; PEMFs处理15 (P=0. 0075)、30 (P=0. 0017)、60 (P=0. 0074)和90 min (P=0. 0096)时,ROBs内的p-CREB水平明显高于未处理组。PEMFs处理ROBs 15 min后,CREB发生磷酸化并聚集于细胞核内。将PKA、p-PKA与初级纤毛共定位染色,结果发现p-PKA定位于初级纤毛上。RNA干扰去除初级纤毛后,干扰组的p-PKA (F=78. 602,P=0. 0270)和p-CREB (F=76. 082,P=0. 0089)蛋白表达量及RUNX2 (F=41. 064,P=0. 0230)和OSX (F=57. 524,P=0. 0310)蛋白表达量均明显低于未干扰组。结论 PEMFs可能是通过初级纤毛相关的cAMP/PKA/CREB信号通路促进ROBs骨形成。
Objective To observe the effect of low-frequency pulsed electromagnetic fields( PEMFs)on bone formation in rat osteoblasts( ROBs) and explore the mechanism of action of the cyclic adenosine monophosphate( cAMP)/protein kinase A( PKA)/cyclic adenosine effect binding protein( CREB) signaling pathway. Methods The skulls of newborn Wistar rats were harvested,and the ROBs were obtained by multiple enzymatic digestion methods for subculture. After treatment with 50 Hz 0. 6 m T PEMFs for 3,6,and 9 days,the alkaline phosphatase( ALP) concentration in ROBs was detected; after 0,15,30,60,90,and 120 min,the expression of bone formation-related factor( RUNX2),the protein expression of osteogenesis-associated transcription factor( OSX),the cAMP concentration,as well as the protein expressions of p-PKA,p-CREB,and CREB were detected. The p-CREB nuclear translocation was observed. After interference with IFT88 by RNA interference,the expressions of RUNX2,OSX,p-PKA,and p-CREB protein in ROBs were detected. Results After treatment with PEMFs for 3,6,and 9 days,the ALP activity values in ROBs were 24. 356 ± 4. 911,37. 688 ±2. 151,and 39. 922 ± 5. 486, respectively, which were significantly higher than 18. 531 ± 2. 401( P =0. 0121),33. 675 ± 4. 366( P = 0. 0324),and 36. 574 ± 1. 339( P = 0. 0134) in the control groups. RUNX2 and OSX activities in ROBs were significantly higher than untreated group after PEMFs treatment for 30( P =0. 0042 and P = 0. 0058),60( P = 0. 0097 and P = 0. 0079),and 90 min( P = 0. 0083 and P = 0. 0098).After PEMFs treatment for 30( P = 0. 0012) and 60 min( P = 0. 0035),the cAMP concentrations in ROBs were significantly higher than that in untreated group. After PEMFs treatment for 15( P = 0. 0018),30( P =0. 0087),90( P = 0. 0250),and 120 min( P = 0. 0350),the p-PKA levels in ROBs were significantly higher than that in the untreated group. After PEMFs treatment for 15( P = 0. 0075),30( P = 0. 0017),60( P =0. 0074),and 90 min( P = 0. 0096),the level of p-CREB in the ROBs was significantly higher than in the untreated group. After PEMFs treatment of ROBs for 15 min,CREB phosphorylated and accumulated in the nuclei.PKA and p-PKA were co-localized with primary cilia and stained,and it was found that p-PKA was localized on the primary cilia. After the primary cilia was removed by RNA interference,the protein expression levels of pPKA( F = 78. 602,P = 0. 0270),p-CREB( F = 76. 082,P = 0. 0089),RUNX2( F = 41. 064,P = 0. 0230) and OSX( F = 57. 524,P = 0. 0310) were significantly lower than those of the non-interfered group. Conclusion PEMFs promote bone formation in ROBs by activating the primary cilia-associated cAMP/PKA/CREB signaling pathway.
Objective To observe the effect of low-frequency pulsed electromagnetic fields( PEMFs)on bone formation in rat osteoblasts( ROBs) and explore the mechanism of action of the cyclic adenosine monophosphate( cAMP)/protein kinase A( PKA)/cyclic adenosine effect binding protein( CREB) signaling pathway. Methods The skulls of newborn Wistar rats were harvested,and the ROBs were obtained by multiple enzymatic digestion methods for subculture. After treatment with 50 Hz 0. 6 m T PEMFs for 3,6,and 9 days,the alkaline phosphatase( ALP) concentration in ROBs was detected; after 0,15,30,60,90,and 120 min,the expression of bone formation-related factor( RUNX2),the protein expression of osteogenesis-associated transcription factor( OSX),the cAMP concentration,as well as the protein expressions of p-PKA,p-CREB,and CREB were detected. The p-CREB nuclear translocation was observed. After interference with IFT88 by RNA interference,the expressions of RUNX2,OSX,p-PKA,and p-CREB protein in ROBs were detected. Results After treatment with PEMFs for 3,6,and 9 days,the ALP activity values in ROBs were 24. 356 ± 4. 911,37. 688 ±2. 151,and 39. 922 ± 5. 486, respectively, which were significantly higher than 18. 531 ± 2. 401( P =0. 0121),33. 675 ± 4. 366( P = 0. 0324),and 36. 574 ± 1. 339( P = 0. 0134) in the control groups. RUNX2 and OSX activities in ROBs were significantly higher than untreated group after PEMFs treatment for 30( P =0. 0042 and P = 0. 0058),60( P = 0. 0097 and P = 0. 0079),and 90 min( P = 0. 0083 and P = 0. 0098).After PEMFs treatment for 30( P = 0. 0012) and 60 min( P = 0. 0035),the cAMP concentrations in ROBs were significantly higher than that in untreated group. After PEMFs treatment for 15( P = 0. 0018),30( P =0. 0087),90( P = 0. 0250),and 120 min( P = 0. 0350),the p-PKA levels in ROBs were significantly higher than that in the untreated group. After PEMFs treatment for 15( P = 0. 0075),30( P = 0. 0017),60( P =0. 0074),and 90 min( P = 0. 0096),the level of p-CREB in the ROBs was significantly higher than in the untreated group. After PEMFs treatment of ROBs for 15 min,CREB phosphorylated and accumulated in the nuclei.PKA and p-PKA were co-localized with primary cilia and stained,and it was found that p-PKA was localized on the primary cilia. After the primary cilia was removed by RNA interference,the protein expression levels of pPKA( F = 78. 602,P = 0. 0270),p-CREB( F = 76. 082,P = 0. 0089),RUNX2( F = 41. 064,P = 0. 0230) and OSX( F = 57. 524,P = 0. 0310) were significantly lower than those of the non-interfered group. Conclusion PEMFs promote bone formation in ROBs by activating the primary cilia-associated cAMP/PKA/CREB signaling pathway.
引文
[1]高堪达,俞永林,周建伟.脉冲电磁场治疗骨质疏松症研究进展[J].国外医学:骨科学分册,2004,25(5):307-309.
[2]方清清,李志忠,周建,等.低频脉冲电磁场通过cAMP/PKA信号通路促进成骨细胞分化的研究[J].南方医科大学学报,2016,36(11):1508-1513. DOI:10. 3969/j. issn. 1673-4254. 2016. 11. 10.
[3] Xie YF,Shi WG,Zhou J,et al. Pulsed electromagneticfields stimulate osteogenic differentiation and maturation of os-teoblasts by upregulating the expression of BMPRII localizedat the base of primary cilium[J]. Bone,2016,93:22-32. DOI:10. 1016/j. bone. 2016. 09. 008.
[4]罗二平,焦李成,申广浩,等.不同强度脉冲电磁场对兔股骨骨密度及生物力学特性的影响[J].生物医学工程学杂志,2005,22(6):1168-1170.
[5] Jing D,Shen G,Huang J,et al. Circadian rhythm affectsthe preventive role of pulsed electromagnetic fields on ovariec-tomy-induced osteoporosis in rats[J]. Bone,2010,46(2):487-495. DOI:10. 1016/j. bone. 2009. 09. 021.
[6]赵春燕,周瑞华,田永芝,等.个体化健康教育在脉冲电磁场治疗原发性骨质疏松症中的作用[J].中国骨质疏松杂志,2016(2):201-206. DOI:10. 3969/j. issn. 1006-7108. 2016. 02. 018.
[7] Fu YC,Lin CC,Chang JK,et al. A novel single pulsedelectromagnetic field stimulates osteogenesis of bone marrowmesenchymal stem cells and bone repair[J]. PLoS One,2014,9(3):e91581. DOI:10. 1371/journal. pone. 0091581.
[8] Lim K,Hexiu J,Kim J,et al. Effects of electromagneticfields on osteogenesis of human alveolar bone-derived mesen-chymal stem cells[J]. Biomed Res Int,2013,2013:296019.DOI:10. 1155/2013/296019.
[9] Hoey DA,Chen JC,Jacobs CR. The primary cilium as anovel extracellular sensor in bone[J]. Front Endocrinol(Lausanne),2012,3:75. DOI:10. 3389/fendo. 2012. 00075.
[10] Malone AM,Anderson CT,Tummala P,et al. Primary cili-a mediate mechanosensing in bone cells by a calcium-inde-pendent mechanism[J]. Proc Natl Acad Sci USA,2007,104(33):13325-13330. DOI:10. 1073/pnas. 0700636104.
[11]闫娟丽,周建,葛宝丰,等.脉冲电磁场对MC3T3-E1细胞增殖与分化的影响[J].中国骨质疏松杂志,2015,21(1):39-43. DOI:10. 3969/j. issn. 1006-7108201501008.
[12]谢艳芳,石文贵,王鸣刚,等.初级纤毛对蛇床子素促进成骨细胞成骨性分化的影响[J].中国现代应用药学,2016,33(7):837-841. DOI:10. 13748/j. cnki. issn1007-7693. 2016. 07. 001.
[13] Sadoghi P,Leithner A,Dorotka R,et al. Effect of pulsedelectromagnetic fields on the bioactivity of human osteoarthriticchondrocytes[J]. Orthopedics,2013,36(3):e360-e365.DOI:10. 3928/01477447-20130222-27.
[14] Delaine-Smith RM,Sittichokechaiwut A,Reilly GC. Prima-ry cilia respond to fluid shear stress and mediate flow-inducedcalcium deposition in osteoblasts[J]. Faseb J,2014,28(1):430-439. DOI:10. 1096/fj. 13-231894.
[15] Lepanto P,Davison C,Casanova G,et al. Characterizationof primary cilia during the differentiation of retinal ganglioncells in the zebrafish[J]. Neural Development,2016,11(1):1-22. DOI:10. 1186/s13064-016-0064-z.
[16] Jung B,Padula D,Burtscher I,et al. Pitchfork and gprasp2target smoothened to the primary cilium for hedgehog pathwayactivation[J]. PLo S One,2016,11(2):e0149477. DOI:10.1371/journal. pone. 0149477.
[17] Praetorius HA. The primary cilium as sensor of fluid flow:new building blocks to the model. A review in the theme:cell signaling:proteins,pathways and mechanisms[J]. AmJ Physiol Cell Physiol,2015,308(3):C198-C208. DOI:10.1152/ajpcell. 00336. 2014.
[2]方清清,李志忠,周建,等.低频脉冲电磁场通过cAMP/PKA信号通路促进成骨细胞分化的研究[J].南方医科大学学报,2016,36(11):1508-1513. DOI:10. 3969/j. issn. 1673-4254. 2016. 11. 10.
[3] Xie YF,Shi WG,Zhou J,et al. Pulsed electromagneticfields stimulate osteogenic differentiation and maturation of os-teoblasts by upregulating the expression of BMPRII localizedat the base of primary cilium[J]. Bone,2016,93:22-32. DOI:10. 1016/j. bone. 2016. 09. 008.
[4]罗二平,焦李成,申广浩,等.不同强度脉冲电磁场对兔股骨骨密度及生物力学特性的影响[J].生物医学工程学杂志,2005,22(6):1168-1170.
[5] Jing D,Shen G,Huang J,et al. Circadian rhythm affectsthe preventive role of pulsed electromagnetic fields on ovariec-tomy-induced osteoporosis in rats[J]. Bone,2010,46(2):487-495. DOI:10. 1016/j. bone. 2009. 09. 021.
[6]赵春燕,周瑞华,田永芝,等.个体化健康教育在脉冲电磁场治疗原发性骨质疏松症中的作用[J].中国骨质疏松杂志,2016(2):201-206. DOI:10. 3969/j. issn. 1006-7108. 2016. 02. 018.
[7] Fu YC,Lin CC,Chang JK,et al. A novel single pulsedelectromagnetic field stimulates osteogenesis of bone marrowmesenchymal stem cells and bone repair[J]. PLoS One,2014,9(3):e91581. DOI:10. 1371/journal. pone. 0091581.
[8] Lim K,Hexiu J,Kim J,et al. Effects of electromagneticfields on osteogenesis of human alveolar bone-derived mesen-chymal stem cells[J]. Biomed Res Int,2013,2013:296019.DOI:10. 1155/2013/296019.
[9] Hoey DA,Chen JC,Jacobs CR. The primary cilium as anovel extracellular sensor in bone[J]. Front Endocrinol(Lausanne),2012,3:75. DOI:10. 3389/fendo. 2012. 00075.
[10] Malone AM,Anderson CT,Tummala P,et al. Primary cili-a mediate mechanosensing in bone cells by a calcium-inde-pendent mechanism[J]. Proc Natl Acad Sci USA,2007,104(33):13325-13330. DOI:10. 1073/pnas. 0700636104.
[11]闫娟丽,周建,葛宝丰,等.脉冲电磁场对MC3T3-E1细胞增殖与分化的影响[J].中国骨质疏松杂志,2015,21(1):39-43. DOI:10. 3969/j. issn. 1006-7108201501008.
[12]谢艳芳,石文贵,王鸣刚,等.初级纤毛对蛇床子素促进成骨细胞成骨性分化的影响[J].中国现代应用药学,2016,33(7):837-841. DOI:10. 13748/j. cnki. issn1007-7693. 2016. 07. 001.
[13] Sadoghi P,Leithner A,Dorotka R,et al. Effect of pulsedelectromagnetic fields on the bioactivity of human osteoarthriticchondrocytes[J]. Orthopedics,2013,36(3):e360-e365.DOI:10. 3928/01477447-20130222-27.
[14] Delaine-Smith RM,Sittichokechaiwut A,Reilly GC. Prima-ry cilia respond to fluid shear stress and mediate flow-inducedcalcium deposition in osteoblasts[J]. Faseb J,2014,28(1):430-439. DOI:10. 1096/fj. 13-231894.
[15] Lepanto P,Davison C,Casanova G,et al. Characterizationof primary cilia during the differentiation of retinal ganglioncells in the zebrafish[J]. Neural Development,2016,11(1):1-22. DOI:10. 1186/s13064-016-0064-z.
[16] Jung B,Padula D,Burtscher I,et al. Pitchfork and gprasp2target smoothened to the primary cilium for hedgehog pathwayactivation[J]. PLo S One,2016,11(2):e0149477. DOI:10.1371/journal. pone. 0149477.
[17] Praetorius HA. The primary cilium as sensor of fluid flow:new building blocks to the model. A review in the theme:cell signaling:proteins,pathways and mechanisms[J]. AmJ Physiol Cell Physiol,2015,308(3):C198-C208. DOI:10.1152/ajpcell. 00336. 2014.