淫羊藿苷对体外牙周膜细胞增殖、成骨分化以及成骨和破骨因子表达的影响
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摘要
目的:明确淫羊藿苷对于体外分离培养的人牙周膜细胞的增殖、成骨分化以及成骨和破骨因子表达的影响。方法:原代分离培养人牙周膜细胞,实验组细胞加入0.01 mg/L淫羊藿苷溶液。采用MTT法检测牙周膜细胞的增殖速率,采用碱性磷酸酶活性检测试剂盒评估牙周膜细胞的成骨分化能力,分别通过real-time PCR和Western印迹检测成骨相关的OCN、Runx2、BMP2以及破骨相关的RANKL的基因和蛋白表达。结果:MTT结果揭示,淫羊藿苷显著提高了人牙周膜细胞的增殖速率(P<0.05),也显著促进了碱性磷酸酶活性的提升(P<0.05),揭示其对于牙周膜细胞的成骨分化具有促进作用;real-time PCR和Western印迹结果均表明,淫羊藿苷能够上调OCN、Runx2和BMP2的基因和蛋白表达(P<0.05),并下调RANKL的基因和蛋白表达(P<0.05)。结论:淫羊藿苷能够促进体外牙周膜细胞的增殖、成骨分化潜能和成骨功能活性。本研究有望为牙周炎治疗以及基于组织工程技术的牙周组织的损伤修复提供新思路。
Objective: To identify the effect of icariin on the proliferation, osteogenic differentiation, and molecule expression associated with osteoblastogenesis and osteoclastogenesis in periodontal ligament cells. Methods:The primary human periodontal ligament cells were extracted and cultured. The cells in the experimental group were treated with 0.01 mg/L icariin solution. The cell proliferation rate was measured via the MTT assays. The ability of osteogenic differentiation was determined using the alkaline phosphatase kits. The gene and protein expression of osteoblastogenesis-related OCN, Runx2 and BMP2 and osteoclastogenesis-related RANKL were determined using the real-time PCR and Western blotting technique, respectively. Results: Icariin significantly increased the proliferation rate of human periodontal ligament cells as revealed by the MTT assays(P<0.05). Icariin also significantly stimulated the increase of the alkaline phosphatase activity(P<0.05), demonstrating the promoting effects on osteogenic differentiation of periodontal ligament cells. The real-time PCR and Western blotting results showed that icrariin up-regulated the OCN, Runx2 and BMP2 gene and protein expression, and down-regulated the RANKL gene and protein expression(P<0.05). Conclusion: Icariin can promote the proliferation, the potential of osteogenic differentiation, and osteogenic activity and function. This study may provide new strategy for the periodontitis treatment and periodontal regeneration based on tissue engineering.
Objective: To identify the effect of icariin on the proliferation, osteogenic differentiation, and molecule expression associated with osteoblastogenesis and osteoclastogenesis in periodontal ligament cells. Methods:The primary human periodontal ligament cells were extracted and cultured. The cells in the experimental group were treated with 0.01 mg/L icariin solution. The cell proliferation rate was measured via the MTT assays. The ability of osteogenic differentiation was determined using the alkaline phosphatase kits. The gene and protein expression of osteoblastogenesis-related OCN, Runx2 and BMP2 and osteoclastogenesis-related RANKL were determined using the real-time PCR and Western blotting technique, respectively. Results: Icariin significantly increased the proliferation rate of human periodontal ligament cells as revealed by the MTT assays(P<0.05). Icariin also significantly stimulated the increase of the alkaline phosphatase activity(P<0.05), demonstrating the promoting effects on osteogenic differentiation of periodontal ligament cells. The real-time PCR and Western blotting results showed that icrariin up-regulated the OCN, Runx2 and BMP2 gene and protein expression, and down-regulated the RANKL gene and protein expression(P<0.05). Conclusion: Icariin can promote the proliferation, the potential of osteogenic differentiation, and osteogenic activity and function. This study may provide new strategy for the periodontitis treatment and periodontal regeneration based on tissue engineering.
引文
[1]Hajishengallis G.Periodontitis:from microbial immune subversion to systemic inflammation[J].Nat Rev Immunol,2015,15(1):30-44.
[2]Zhu W,Liang M.Periodontal ligament stem cells:current status,concerns,and future prospects[J].Stem Cells Int,2015,2015:972313.
[3]Li C,Li Q,Mei Q,et al.Pharmacological effects and pharmacokinetic properties of icariin,the major bioactive component in Herba Epimedii[J].Life Sci,2015Apr 1,126:57-68.
[4]Tang Y,Jacobi A,Vater C,et al.Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells[J].Stem Cells,2015,33(6):1863-1877.
[5]Tang D,Ju C,Liu Y,et al.Therapeutic effect of icariin combined with stem cells on postmenopausal osteoporosis in rats[J].J Bone Miner Metab,2018,36(2):180-188.
[6]Wang Q S,Wang G F,Lu Y R,et al.The combination of icariin and constrained dynamic loading stimulation attenuates bone loss in ovariectomy-induced osteoporotic mice[J].J Orthop Res,2018,36(5):1415-1424.
[7]Xu J H,Yao M,Ye J,et al.Bone mass improved effect of icariin for postmenopausal osteoporosis in ovariectomy-induced rats:a meta-analysis and systematic review[J].Menopause,2016,23(10):1152-1157.
[8]Wang P,Zhang F,He Q,et al.Flavonoid compound icariin activates hypoxia inducible factor-1αin chondrocytes and promotes articular cartilage repair[J].PLoS One,2016,11(2):e0148372.
[9]Tan H L,Chan K G,Pusparajah P,et al.Anti-cancer properties of the naturally occurring aphrodisiacs:icariin and its derivatives[J].Front Pharmacol,2016,7:191.
[10]Wang Z,Wang D,Yang D,et al.The effect of icariin on bone metabolism and its potential clinical application[J].Osteoporos Int,2018,29(3):535-544.
[11]Cao H,Zhang Y,Qian W,et al.Effect of icariin on fracture healing in an ovariectomized rat model of osteoporosis[J].Exp Ther Med,2017,13(5):2399-2404.
[12]Wu Y,Cao L,Xia L,et al.Evaluation of osteogenesis and angiogenesis of icariin in local controlled release and systemic delivery for calvarial defect in ovariectomized rats[J].Sci Rep,2017,7(1):5077.
[13]Wu Y,Xia L,Zhou Y,et al.Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner[J].Cell Prolif,2015,48(3):375-384.
[14]Luo G,Gu F,Zhang Y,et al.Icariside II promotes osteogenic differentiation of bone marrow stromal cells in beagle canine[J].Int J Clin Exp Pathol,2015,8(5):4367-4377.
[15]Ho M X,Poon C C,Wong K C,et al.Icariin,but not genistein,exerts osteogenic and anti-apoptotic effects in osteoblastic cells by selective activation of non-genomic ERαsignaling[J].Front Pharmacol,2018,9:474.
[16]Hsieh T P,Sheu S Y,Sun J S,et al.Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1αand PGE(2)synthesis[J].Phytomedicine,2011,18(2-3):176-185.
[17]Gundberg C M,Looker A C,Nieman S D,et al.Patterns of osteocalcin and bone specific alkaline phosphatase by age,gender,and race or ethnicity[J].Bone,2002,31(6):703-708.
[18]Pittas A G,Harris S S,Eliades M,et al.Association between serum osteocalcin and markers of metabolic phenotype[J].J Clin Endocrinol Metab,2009,94(3):827-832.
[19]Komori T.Regulation of osteoblast differentiation by Runx2[J].Adv Exp Med Biol,2010,658:43-49.
[20]Chen G,Deng C,Li Y P.TGF-βand BMP signaling in osteoblast differentiation and bone formation[J].Int J Biol Sci,2012,8(2):272-288.
[21]Martin T J,Sims N A.RANKL/OPG,critical role in bone physiology[J].Rev Endocr Metab Disord,2015,16(2):131-139.
[2]Zhu W,Liang M.Periodontal ligament stem cells:current status,concerns,and future prospects[J].Stem Cells Int,2015,2015:972313.
[3]Li C,Li Q,Mei Q,et al.Pharmacological effects and pharmacokinetic properties of icariin,the major bioactive component in Herba Epimedii[J].Life Sci,2015Apr 1,126:57-68.
[4]Tang Y,Jacobi A,Vater C,et al.Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells[J].Stem Cells,2015,33(6):1863-1877.
[5]Tang D,Ju C,Liu Y,et al.Therapeutic effect of icariin combined with stem cells on postmenopausal osteoporosis in rats[J].J Bone Miner Metab,2018,36(2):180-188.
[6]Wang Q S,Wang G F,Lu Y R,et al.The combination of icariin and constrained dynamic loading stimulation attenuates bone loss in ovariectomy-induced osteoporotic mice[J].J Orthop Res,2018,36(5):1415-1424.
[7]Xu J H,Yao M,Ye J,et al.Bone mass improved effect of icariin for postmenopausal osteoporosis in ovariectomy-induced rats:a meta-analysis and systematic review[J].Menopause,2016,23(10):1152-1157.
[8]Wang P,Zhang F,He Q,et al.Flavonoid compound icariin activates hypoxia inducible factor-1αin chondrocytes and promotes articular cartilage repair[J].PLoS One,2016,11(2):e0148372.
[9]Tan H L,Chan K G,Pusparajah P,et al.Anti-cancer properties of the naturally occurring aphrodisiacs:icariin and its derivatives[J].Front Pharmacol,2016,7:191.
[10]Wang Z,Wang D,Yang D,et al.The effect of icariin on bone metabolism and its potential clinical application[J].Osteoporos Int,2018,29(3):535-544.
[11]Cao H,Zhang Y,Qian W,et al.Effect of icariin on fracture healing in an ovariectomized rat model of osteoporosis[J].Exp Ther Med,2017,13(5):2399-2404.
[12]Wu Y,Cao L,Xia L,et al.Evaluation of osteogenesis and angiogenesis of icariin in local controlled release and systemic delivery for calvarial defect in ovariectomized rats[J].Sci Rep,2017,7(1):5077.
[13]Wu Y,Xia L,Zhou Y,et al.Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner[J].Cell Prolif,2015,48(3):375-384.
[14]Luo G,Gu F,Zhang Y,et al.Icariside II promotes osteogenic differentiation of bone marrow stromal cells in beagle canine[J].Int J Clin Exp Pathol,2015,8(5):4367-4377.
[15]Ho M X,Poon C C,Wong K C,et al.Icariin,but not genistein,exerts osteogenic and anti-apoptotic effects in osteoblastic cells by selective activation of non-genomic ERαsignaling[J].Front Pharmacol,2018,9:474.
[16]Hsieh T P,Sheu S Y,Sun J S,et al.Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1αand PGE(2)synthesis[J].Phytomedicine,2011,18(2-3):176-185.
[17]Gundberg C M,Looker A C,Nieman S D,et al.Patterns of osteocalcin and bone specific alkaline phosphatase by age,gender,and race or ethnicity[J].Bone,2002,31(6):703-708.
[18]Pittas A G,Harris S S,Eliades M,et al.Association between serum osteocalcin and markers of metabolic phenotype[J].J Clin Endocrinol Metab,2009,94(3):827-832.
[19]Komori T.Regulation of osteoblast differentiation by Runx2[J].Adv Exp Med Biol,2010,658:43-49.
[20]Chen G,Deng C,Li Y P.TGF-βand BMP signaling in osteoblast differentiation and bone formation[J].Int J Biol Sci,2012,8(2):272-288.
[21]Martin T J,Sims N A.RANKL/OPG,critical role in bone physiology[J].Rev Endocr Metab Disord,2015,16(2):131-139.