黄芩素对人牙周膜细胞骨向分化能力的影响
|
摘要
目的:探讨中药黄芩提取物黄芩素对人牙周膜细胞(h PDLCs)增殖和成骨分化功能的影响。方法:原代培养h PDLCs,向h PDLCs中分别加入浓度为0.3125、0.625、1.25μmol/L的黄芩素作为实验组,对照组不加黄芩素。MTT法检测黄芩素对h PDLCs增殖的影响后,分别采用碱性磷酸酶(ALP)活性测定、茜素红S染色、Real-time PCR和Western Blot法研究黄芩素对h PDLCs ALP活性、矿化结节形成、I型胶原(Col-I)m RNA和蛋白表达的影响。结果:与对照组相比,各浓度黄芩素对h PDLCs增殖均无明显影响;各浓度黄芩素组均表现为ALP活性上升;同时骨向诱导21 d后可见黄芩素组矿化结节形成增加,定量分析结果显示1.25μmol/L黄芩素组形成矿化结节数量最多;Real-time PCR和Western Blot结果显示各浓度黄芩素组Col-I m RNA和蛋白表达均有上升,7 d时随浓度的增加其表达上升,1.25μmol/L黄芩素组达到最高水平。结论:黄芩素能促进h PDLCs的骨向分化能力,具有进一步研究其辅助牙周再生治疗的价值。
Objective: To evaluate the effects of baicalein extracted from Scutellaria baicalensis Georgi on the function of proliferation and osteogenesis of human periodontal ligament cells(h PDLCs). Methods: Baicalein at a concentration of 0.3125, 0.625, 1.25 μmol/L was added into h PDLCs primary cultured in vitro accordingly as experimental group, the one with no baicalein as blank control group. The effect of proliferation of h PDLCs was assessed through MTT assay. Alkaline phosphatase(ALP) activity assay, Alizarin red S staining, RT-q PCR and Western Blot were used to observe the ALP activity, formation of mineral nodules, m RNA and protein expression of collagenase-Ι(Col-Ι) of h PDLCs, respectively. Results: Compared to the control group, baicalein at different concentrations had no significant influence on the proliferation of h PDLCs. Baicalein was shown to enhance the ALP activity of h PDLCs. After 21 days of osteogenic induction, more mineral nodules could be observed compared to the control group, which increased in a dose-dependent manner at the concentration of 0.3125-1.25 μmol/L baicalein according to the results of quantification. The m RNA and protein level of Col-Ι increased by baicalein in different concentration, which reached maximum at 7 days, appearring in a dosedependent manner from 0.3125 to 1.25 μmol/L using Western Blot and Real-time PCR analysis. Conclusion: Baicalein can promote h PDLCs to differentiate into osteoblasts, which is of great value for further study acting as the adjuvant drug for periodontal regenerative therapy.
Objective: To evaluate the effects of baicalein extracted from Scutellaria baicalensis Georgi on the function of proliferation and osteogenesis of human periodontal ligament cells(h PDLCs). Methods: Baicalein at a concentration of 0.3125, 0.625, 1.25 μmol/L was added into h PDLCs primary cultured in vitro accordingly as experimental group, the one with no baicalein as blank control group. The effect of proliferation of h PDLCs was assessed through MTT assay. Alkaline phosphatase(ALP) activity assay, Alizarin red S staining, RT-q PCR and Western Blot were used to observe the ALP activity, formation of mineral nodules, m RNA and protein expression of collagenase-Ι(Col-Ι) of h PDLCs, respectively. Results: Compared to the control group, baicalein at different concentrations had no significant influence on the proliferation of h PDLCs. Baicalein was shown to enhance the ALP activity of h PDLCs. After 21 days of osteogenic induction, more mineral nodules could be observed compared to the control group, which increased in a dose-dependent manner at the concentration of 0.3125-1.25 μmol/L baicalein according to the results of quantification. The m RNA and protein level of Col-Ι increased by baicalein in different concentration, which reached maximum at 7 days, appearring in a dosedependent manner from 0.3125 to 1.25 μmol/L using Western Blot and Real-time PCR analysis. Conclusion: Baicalein can promote h PDLCs to differentiate into osteoblasts, which is of great value for further study acting as the adjuvant drug for periodontal regenerative therapy.
引文
[1]曹采方.牙周病学[M].北京:人民卫生出版社,2003:169-173.
[2]ISAKA J,OHAZAMA A,KOBAYASHI M,et al.Participation of periodontal ligament cells with regeneration of alveolar bone[J].J Periodontol,2001,72(3):314-323.
[3]吴亚菲,赵蕾.牙周病药物治疗及进展[J].中国医药导刊,2006,8(4):285-288.
[4]唐琪,陈莉丽,严杰.骨碎补提取物促小鼠成骨细胞株MC3T3-E1细胞增殖、分化和钙化作用的研究[J].中国中药杂志,2004,29(2):164-168.
[5]唐琪,王仁飞,王维倩,等.骨碎补提取液及柚皮甙对小鼠成骨细胞MC3T3-E1基质矿化作用影响的比较研究[J].口腔医学,2009,29(8):403-404,408.
[6]SHANG X,HE X,HE X,et al.The genus Scutellaria an ethnopharmacological and phytochemical review[J].J Ethnopharmacol,2010,128(2):279-313.
[7]FAN G W,ZHANG Y,JIANG X,et al.Anti-infl ammatory activity of baicalein in LPS-stimulated RAW264.7 macrophages via estrogen receptor and NF-kappa B-dependent pathways[J].Infl ammation,2013,36(6):1584-1591.
[8]LI-WEBER M.New therapeutic aspects of flavones:the anticancer properties of Scutellaria and its main active constituents Wogonin,Baicalein and Baicalin[J].Cancer Treat Rev,2009,35(1):57-68.
[9]FU Y,LUO J,JIA Z,et al.Baicalein protects against type2 diabetes via promoting islet beta-cell function in obese diabetic mice[J].Int J Endocrinol,2014,2014:846742.
[10]LIN H Y,SHEN S C,LIN C W,et al.Baicalein inhibition of hydrogen peroxide-induced apoptosis via ROS-dependent heme oxygenase 1 gene expression[J].Biochim Biophys Acta,2007,1773(7):1073-1086.
[11]YUN B Y,ZHOU L,XIE K P,et al.Antibacterial activity and mechanism of baicalein[J].Yao Xue Xue Bao,2012,47(12):1587-1592.
[12]梁英,韩鲁佳.黄芩中黄芩甙和黄芩素提取、分离纯化的研究进展[J].农业工程学报,2003,19(z1):218-222.
[13]KIM J M,LEE S U,KIM Y S,et al.Baicalein stimulates osteoblast differentiation via coordinating activation of MAP kinases and transcription factors[J].J Cell Biochem,2008,104(5):1906-1917.
[14]HAN J,MENICANIN D,GRONTHOS S,et al.Stem cells,tissue engineering and periodontal regeneration[J].Aust Dent J,2014,59(Suppl 1):117-130.
[15]张蕴惠.牙周组织再生[J].中华口腔医学杂志,1998,33(4):59-60.
[16]LEE J H,UM S,JANG J H,et al.Effects of VEGF and FGF-2 on proliferation and differentiation of human periodontal ligament stem cells[J].Cell Tissue Res,2012,348(3):475-484.
[17]ACIL Y,YANG F,GULSES A,et al.Isolation,characterization and investigation of differentiation potential of human periodontal ligament cells and dental follicle progenitor cells and their response to BMP-7 in vitro[J].Odontology,2015.DOI:10.1007/s10266-015-0198-1.
[18]LOSSDORFER S,SUN M,GOTZ W,et al.Enamel matrix derivative promotes human periodontal ligament cell differentiation and osteoprotegerin production in vitro[J].J Dent Res,2007,86(10):980-985.
[19]VAN DER PAUW M T,VAN DEN BOS T,EVERTS V,et al.Enamel matrix-derived protein stimulates attachment of periodontal ligament fi broblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fi broblasts[J].J Periodontol,2000,71(1):31-43.
[20]NAGANO T,IWATA T,OGATA Y,et al.Effect of heat treatment on bioactivities of enamel matrix derivatives in human periodontal ligament(HPDL)cells[J].J Periodontal Res,2004,39(4):249-256.
[21]LEE S K,CHOI H I,YANG Y S,et al.Nitric oxide modulates osteoblastic differentiation with heme oxygenase-1 via the mitogen activated protein kinase and nuclear factor-kappa B pathways in human periodontal ligament cells[J].Biol Pharm Bull,2009,32(8):1328-1334.
[22]辛文妤,宋俊科,何国荣,等.黄芩素和黄芩苷的药理作用及机制研究进展[J].中国新药杂志,2013,22(6):647-653,659.
[23]李敏,杨瑞芳.黄芩药理学研究进展[J].临床和实验医学杂志,2009,8(1):137-138.
[24]CHUNG C P,PARK J B,BAE K H.Pharmacological effects of methanolic extract from the root of Scutellaria baicalensis and its fl avonoids on human gingival fi broblast[J].Planta Med,1995,61(2):150-153.
[25]LIU J,WANG S,SUN J,et al.Screening of osteoanagenesisactive compounds from Scutellaria baicalensis Georgi by h PDLC/CMC-online-HPLC/MS[J].Fitoterapia,2014,93:105-114.
[26]蔡霞,李成章,曹正国,等.黄芩苷对大鼠实验性牙周炎的保护效应及作用机制研究[J].中华口腔医学杂志,2008,43(5):281-285.
[27]KIM M H,RYU S Y,BAE M A,et al.Baicalein inhibits osteoclast differentiation and induces mature osteoclast apoptosis[J].Food Chem Toxicol,2008,46(11):3375-3382.
[28]BELLOWS C G,AUBIN J E,HEERSCHE J N.Initiation and progression of mineralization of bone nodules formed in vitro:the role of alkaline phosphatase and organic phosphate[J].Bone Miner,1991,14(1):27-40.
[29]JIAO L,CAO D P,QIN L P,et al.Antiosteoporotic activity of phenolic compounds from Curculigo orchioides[J].Phytomedicine,2009,16(9):874-881.
[30]郭海玲,王翔,徐宇,等.黄芪调控体外培养大鼠成骨细胞I型胶原蛋白的表达[J].中国组织工程研究与临床康复,2010,14(7):1257-1261.
[31]WANG Z H,HUANG J,MA X C,et al.Phenolic glycosides from Curculigo orchioides Gaertn[J].Fitoterapia,2013,86:64-69.
[2]ISAKA J,OHAZAMA A,KOBAYASHI M,et al.Participation of periodontal ligament cells with regeneration of alveolar bone[J].J Periodontol,2001,72(3):314-323.
[3]吴亚菲,赵蕾.牙周病药物治疗及进展[J].中国医药导刊,2006,8(4):285-288.
[4]唐琪,陈莉丽,严杰.骨碎补提取物促小鼠成骨细胞株MC3T3-E1细胞增殖、分化和钙化作用的研究[J].中国中药杂志,2004,29(2):164-168.
[5]唐琪,王仁飞,王维倩,等.骨碎补提取液及柚皮甙对小鼠成骨细胞MC3T3-E1基质矿化作用影响的比较研究[J].口腔医学,2009,29(8):403-404,408.
[6]SHANG X,HE X,HE X,et al.The genus Scutellaria an ethnopharmacological and phytochemical review[J].J Ethnopharmacol,2010,128(2):279-313.
[7]FAN G W,ZHANG Y,JIANG X,et al.Anti-infl ammatory activity of baicalein in LPS-stimulated RAW264.7 macrophages via estrogen receptor and NF-kappa B-dependent pathways[J].Infl ammation,2013,36(6):1584-1591.
[8]LI-WEBER M.New therapeutic aspects of flavones:the anticancer properties of Scutellaria and its main active constituents Wogonin,Baicalein and Baicalin[J].Cancer Treat Rev,2009,35(1):57-68.
[9]FU Y,LUO J,JIA Z,et al.Baicalein protects against type2 diabetes via promoting islet beta-cell function in obese diabetic mice[J].Int J Endocrinol,2014,2014:846742.
[10]LIN H Y,SHEN S C,LIN C W,et al.Baicalein inhibition of hydrogen peroxide-induced apoptosis via ROS-dependent heme oxygenase 1 gene expression[J].Biochim Biophys Acta,2007,1773(7):1073-1086.
[11]YUN B Y,ZHOU L,XIE K P,et al.Antibacterial activity and mechanism of baicalein[J].Yao Xue Xue Bao,2012,47(12):1587-1592.
[12]梁英,韩鲁佳.黄芩中黄芩甙和黄芩素提取、分离纯化的研究进展[J].农业工程学报,2003,19(z1):218-222.
[13]KIM J M,LEE S U,KIM Y S,et al.Baicalein stimulates osteoblast differentiation via coordinating activation of MAP kinases and transcription factors[J].J Cell Biochem,2008,104(5):1906-1917.
[14]HAN J,MENICANIN D,GRONTHOS S,et al.Stem cells,tissue engineering and periodontal regeneration[J].Aust Dent J,2014,59(Suppl 1):117-130.
[15]张蕴惠.牙周组织再生[J].中华口腔医学杂志,1998,33(4):59-60.
[16]LEE J H,UM S,JANG J H,et al.Effects of VEGF and FGF-2 on proliferation and differentiation of human periodontal ligament stem cells[J].Cell Tissue Res,2012,348(3):475-484.
[17]ACIL Y,YANG F,GULSES A,et al.Isolation,characterization and investigation of differentiation potential of human periodontal ligament cells and dental follicle progenitor cells and their response to BMP-7 in vitro[J].Odontology,2015.DOI:10.1007/s10266-015-0198-1.
[18]LOSSDORFER S,SUN M,GOTZ W,et al.Enamel matrix derivative promotes human periodontal ligament cell differentiation and osteoprotegerin production in vitro[J].J Dent Res,2007,86(10):980-985.
[19]VAN DER PAUW M T,VAN DEN BOS T,EVERTS V,et al.Enamel matrix-derived protein stimulates attachment of periodontal ligament fi broblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fi broblasts[J].J Periodontol,2000,71(1):31-43.
[20]NAGANO T,IWATA T,OGATA Y,et al.Effect of heat treatment on bioactivities of enamel matrix derivatives in human periodontal ligament(HPDL)cells[J].J Periodontal Res,2004,39(4):249-256.
[21]LEE S K,CHOI H I,YANG Y S,et al.Nitric oxide modulates osteoblastic differentiation with heme oxygenase-1 via the mitogen activated protein kinase and nuclear factor-kappa B pathways in human periodontal ligament cells[J].Biol Pharm Bull,2009,32(8):1328-1334.
[22]辛文妤,宋俊科,何国荣,等.黄芩素和黄芩苷的药理作用及机制研究进展[J].中国新药杂志,2013,22(6):647-653,659.
[23]李敏,杨瑞芳.黄芩药理学研究进展[J].临床和实验医学杂志,2009,8(1):137-138.
[24]CHUNG C P,PARK J B,BAE K H.Pharmacological effects of methanolic extract from the root of Scutellaria baicalensis and its fl avonoids on human gingival fi broblast[J].Planta Med,1995,61(2):150-153.
[25]LIU J,WANG S,SUN J,et al.Screening of osteoanagenesisactive compounds from Scutellaria baicalensis Georgi by h PDLC/CMC-online-HPLC/MS[J].Fitoterapia,2014,93:105-114.
[26]蔡霞,李成章,曹正国,等.黄芩苷对大鼠实验性牙周炎的保护效应及作用机制研究[J].中华口腔医学杂志,2008,43(5):281-285.
[27]KIM M H,RYU S Y,BAE M A,et al.Baicalein inhibits osteoclast differentiation and induces mature osteoclast apoptosis[J].Food Chem Toxicol,2008,46(11):3375-3382.
[28]BELLOWS C G,AUBIN J E,HEERSCHE J N.Initiation and progression of mineralization of bone nodules formed in vitro:the role of alkaline phosphatase and organic phosphate[J].Bone Miner,1991,14(1):27-40.
[29]JIAO L,CAO D P,QIN L P,et al.Antiosteoporotic activity of phenolic compounds from Curculigo orchioides[J].Phytomedicine,2009,16(9):874-881.
[30]郭海玲,王翔,徐宇,等.黄芪调控体外培养大鼠成骨细胞I型胶原蛋白的表达[J].中国组织工程研究与临床康复,2010,14(7):1257-1261.
[31]WANG Z H,HUANG J,MA X C,et al.Phenolic glycosides from Curculigo orchioides Gaertn[J].Fitoterapia,2013,86:64-69.