姜黄素通过Wnt信号通路调节人牙髓干细胞的成牙本质分化
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摘要
背景:研究证实,姜黄素不仅可促进成体干细胞成骨分化还可通过激活Wnt信号通路促进神经干细胞的增殖与分化。但姜黄素能否通过激活Wnt信号通路促进人牙髓干细胞成牙本质分化尚未见报道。目的:探讨姜黄素对人牙髓干细胞增殖和成牙本质分化的影响及其可能机制。方法:将人牙髓干细胞(购于上海妍生实业有限公司)分为6组:正常对照组不进行干预,低、中、高浓度姜黄素组用50,250,500 nmol/L姜黄素干预,IWR-1组用1μmol/L Wnt信号通路抑制剂IWR-1干预,IWR-1+姜黄素组用1μmol/L IWR-1和500 nmol/L姜黄素干预。各组进行矿化诱导7 d和14 d后,实时定量PCR检测Wnt5a、碱性磷酸酶、牙本质涎磷蛋白的m RNA表达;Western blot检测Wnt5a、β-catenin、骨唾液酸蛋白、牙本质涎磷蛋白、骨钙蛋白、牙本质基质蛋白1水平;比色法检测细胞碱性磷酸酶活性;CCK-8法检测细胞增殖活性。结果与结论:①姜黄素能够提高牙髓干细胞增殖活性和碱性磷酸酶活性,且呈时间和剂量依赖性;②姜黄素能够提高牙髓干细胞中牙本质涎磷蛋白、碱性磷酸酶的m RNA表达以及牙本质涎磷蛋白、牙本质基质蛋白1、骨唾液酸蛋白和骨钙蛋白水平,且呈时间和剂量依赖性;③姜黄素能够提高牙髓干细胞中Wnt5a和β-catenin蛋白的表达,且呈时间和剂量依赖性;④IWR-1+姜黄素组人牙髓干细胞中Wnt5a m RNA表达以及Wnt5a、牙本质涎磷蛋白、牙本质基质蛋白1、骨唾液酸蛋白及骨钙蛋白水平和增殖活性高于正常对照组和IWR-1组(P <0.05);⑤以上结果表明,姜黄素通过激活Wnt信号通路促进人牙髓干细胞的增殖和成牙本质分化。
BACKGROUND: Studies have shown that curcumin not only promotes osteogenic differentiation of adult stem cells, but also promotes proliferation and differentiation of neural stem cells by activating the Wnt signaling pathway. However, there is no report on whether curcumin can promote odontoblast the differentiation of human dental pulp stem cells by activating the Wnt signaling pathway.OBJECTIVE: To investigate the effect of curcumin on the proliferation and odontogenic differentiation of human dental pulp stem cells and its possible mechanism.METHODS: Human dental pulp stem cells(purchased from Shanghai Yansheng Industrial Co., Ltd., China) were divided into six groups according to different intervention methods. The normal control group was not intervened. Low-, medium-and high-concentration curcumin groups were treated with 50, 250, and 500 nmol/L curcumin, respectively. IWR-1 group was intervened with 1 μmol/L Wnt signaling pathway inhibitor IWR-1. IWR-1+curcumin group were treated with 1 μmol/L IWR-1 and 500 nmol/L curcumin. After 7 and 14 days of mineralization, the mRNA levels of Wnt5 a, alkaline phosphatase and dentin sialophosphoprotein were detected by real-time quantitative PCR. The protein levels of Wnt5 a, β-catenin, bone sialic acid protein, dentin sialophosphoprotein, osteocalcin and dentin matrix protein 1 were detected by western blot. The activity of alkaline phosphatase was detected by colorimetry, and the proliferation activity of the cells was detected by cell counting kit-8 method.RESULTS AND CONCLUSION:(1) Curcumin had a time-dependent and dose-dependent effect on the proliferation of dental pulp stem cells.(2) Curcumin had a time-and dose-dependent effect on the mRNA expressions of dentin sialophosphoprotein and alkaline phosphatase, as well as the protein expressions of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein and osteocalcin in dental pulp stem cells.(3) Curcumin up-regulated the expressions of Wnt5 a and β-catenin proteins in dental pulp stem cells in a time-and dose-dependent manner.(4) Compared with the control and IWR-1 groups, the combination of curcumin and IWR-1 significantly increased the expressions of Wnt5 a mRNA and protein, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein and osteocalcin proteins in human dental pulp stem cells, and significantly promoted the proliferative ability of the cells(P < 0.05). These results suggest that curcumin promotes the proliferation and odontogenic differentiation of human dental pulp stem cells by activating the Wnt signaling pathway.
BACKGROUND: Studies have shown that curcumin not only promotes osteogenic differentiation of adult stem cells, but also promotes proliferation and differentiation of neural stem cells by activating the Wnt signaling pathway. However, there is no report on whether curcumin can promote odontoblast the differentiation of human dental pulp stem cells by activating the Wnt signaling pathway.OBJECTIVE: To investigate the effect of curcumin on the proliferation and odontogenic differentiation of human dental pulp stem cells and its possible mechanism.METHODS: Human dental pulp stem cells(purchased from Shanghai Yansheng Industrial Co., Ltd., China) were divided into six groups according to different intervention methods. The normal control group was not intervened. Low-, medium-and high-concentration curcumin groups were treated with 50, 250, and 500 nmol/L curcumin, respectively. IWR-1 group was intervened with 1 μmol/L Wnt signaling pathway inhibitor IWR-1. IWR-1+curcumin group were treated with 1 μmol/L IWR-1 and 500 nmol/L curcumin. After 7 and 14 days of mineralization, the mRNA levels of Wnt5 a, alkaline phosphatase and dentin sialophosphoprotein were detected by real-time quantitative PCR. The protein levels of Wnt5 a, β-catenin, bone sialic acid protein, dentin sialophosphoprotein, osteocalcin and dentin matrix protein 1 were detected by western blot. The activity of alkaline phosphatase was detected by colorimetry, and the proliferation activity of the cells was detected by cell counting kit-8 method.RESULTS AND CONCLUSION:(1) Curcumin had a time-dependent and dose-dependent effect on the proliferation of dental pulp stem cells.(2) Curcumin had a time-and dose-dependent effect on the mRNA expressions of dentin sialophosphoprotein and alkaline phosphatase, as well as the protein expressions of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein and osteocalcin in dental pulp stem cells.(3) Curcumin up-regulated the expressions of Wnt5 a and β-catenin proteins in dental pulp stem cells in a time-and dose-dependent manner.(4) Compared with the control and IWR-1 groups, the combination of curcumin and IWR-1 significantly increased the expressions of Wnt5 a mRNA and protein, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein and osteocalcin proteins in human dental pulp stem cells, and significantly promoted the proliferative ability of the cells(P < 0.05). These results suggest that curcumin promotes the proliferation and odontogenic differentiation of human dental pulp stem cells by activating the Wnt signaling pathway.
引文
[1]Suneelkumar C,Subha A,Gogala D.Effect of Preoperative Corticosteroids in Patients with Symptomatic Pulpitis on Postoperative Pain after Single-visit Root Canal Treatment:A Systematic Review and Meta-analysis.J Endod.2018;44(9):1347-1354.
[2]Shirvani A,Shamszadeh S,Eghbal MJ,et al.Effect of preoperative oral analgesics on pulpal anesthesia in patients with irreversible pulpitis-a systematic review and meta-analysis.Clin Oral Investig.2017;21(1):43-52.
[3]Yang X,Li L,Xiao L,et al.Recycle the dental fairy's package:overview of dental pulp stem cells.Stem Cell Res Ther.2018;9(1):347.
[4]Kaneko T,Gu B,Sone PP,et al.Dental Pulp Tissue Engineering Using Mesenchymal Stem Cells:a Review with a Protocol.Stem Cell Rev.2018;14(5):668-676.
[5]Graham CM,Kremer KL,Koblar SA,et al.Dental Pulp Stem CellsExploration in a Novel Animal Model:the Tasmanian Devil(Sarcophilus harrisii).Stem Cell Rev.2018;14(4):500-509.
[6]Hassan NT,AbdelAziz NA.Oral Mucosal Stem Cells,Human Immature Dental Pulp Stem Cells and Hair Follicle Bulge Stem Cells as Adult Stem Cells Able to Correct Limbal Stem Cell Deficiency.Curr Stem Cell Res Ther.2018;13(5):356-361.
[7]Anitua E,Troya M,Zalduendo M.Progress in the use of dental pulp stem cells in regenerative medicine.Cytotherapy.2018;20(4):479-498.
[8]杨雪超,李晓宁,王伟东,等.牙本质支架促进人牙髓干细胞体外增殖与分化能力的研究[J].牙体牙髓牙周病学杂志,2015,25(6):342-347.
[9]杨鑫,李思洁,赵玮.Wnt信号通路在调控牙髓干细胞多向分化及炎症损伤修复中的作用[J].国际口腔医学杂志,2018,45(3):286-290.
[10]陈婵婵,凌均棨.Wnt信号通路对牙齿发育和牙囊成骨向分化的调控作用[J].牙体牙髓牙周病学杂志,2015,25(3):175-178.
[11]Romanos GE.Biomolecular Cell-Signaling Mechanisms and Dental Implants:A Review on the Regulatory Molecular Biologic Patterns Under Functional and Immediate Loading.Int J Oral Maxillofac Implants.2016;31(4):939-951.
[12]Mc Cubrey JA,Lertpiriyapong K,Steelman LS,et al.Effects of resveratrol,curcumin,berberine and other nutraceuticals on aging,cancer development,cancer stem cells and microRNAs.Aging(Albany NY).2017;9(6):1477-1536.
[13]张庆美,李方正,姜忠玲,等.姜黄素对猪骨髓间充质干细胞增殖和成脂分化的影响[J].解剖学报,2014,45(6):793-799.
[14]陈飞,王皓香,向鑫,等.姜黄素对神经干细胞Wnt/β-catenin信号通路表达影响的离体研究[J].第三军医大学学报,2014,36(8):764-768.
[15]顾巧丽,蔡燕,黄晨,等.姜黄素调控大鼠骨髓间充质干细胞的成骨分化[J].中国组织工程研究,2012,16(27):5057-5061.
[16]Chauhan S,王盈.用于治疗牙周炎的载姜黄素可生物降解交联明胶薄膜的研制[J].中国医药工业杂志,2018,49(5):593.
[17]朱柯,潘芸,杨洋,等.姜黄素对牙周炎大鼠牙周支持组织炎症性破坏的保护作用[J].广东医学,2017,38(3):358-361.
[18]刘明月,胡伟平,王晓芬,等.低浓度β-甘油磷酸钠培养牙髓干细胞向成牙本质细胞分化及相关因子的表达[J].中国组织工程研究,2015,19(41):6688-6693.
[19]梁静,王君,唐传玲,等.低氧对H2O2预处理MC3T3-E1增殖和成骨分化功能的影响[J].中国组织工程研究,2017,21(4):505-511.
[20]Phan M,Conte F,Khandelwal KD,et al.Tooth agenesis and orofacial clefting:genetic brothers in arms.Hum Genet.2016;135(12):1299-1327.
[21]Mead B,Logan A,Berry M,et al.Concise Review:Dental Pulp Stem Cells:A Novel Cell Therapy for Retinal and Central Nervous System Repair.Stem Cells.2017;35(1):61-67.
[22]Nuti N,Corallo C,Chan BM,et al.Multipotent Differentiation of Human Dental Pulp Stem Cells:a Literature Review.Stem Cell Rev.2016;12(5):511-523.
[23]Ledesma-Martínez E,Mendoza-Nú?ez VM,Santiago-Osorio E.Mesenchymal Stem Cells Derived from Dental Pulp:A Review.Stem Cells Int.2016;2016:4709572.
[24]Shehzad A,Lee YS.Molecular mechanisms of curcumin action:signal transduction.Biofactors.2013;39(1):27-36.
[25]席晅,任小琼.姜黄素通过PI3K/AKT信号通路调控神经干细胞增殖和凋亡实验研究[J].新中医,2016,48(3):217-221.
[26]马晓晓,王春满,张高龙,等.姜黄素通过抑制糖皮质激素受体促进大鼠神经干细胞增殖[J].中国药理学与毒理学杂志,2015,29(02):202-207.
[27]刘剑锋,朱平,马守原,等.姜黄素通过血红素加氧酶-1信号通路促进脂肪源干细胞增殖和旁分泌研究[J].中国心血管杂志,2015,20(3):199-203.
[28]邬培红,华子义,赵秀娟.姜黄素促进大鼠骨髓间充质干细胞成骨分化中活性氧表达变化研究[J].中国医药导报,2013,10(18):24-26,29.
[29]陈飞,王皓香,向鑫,等.姜黄素对神经干细胞Wnt/β-catenin信号通路表达影响的离体研究[J].第三军医大学学报,2014,36(8):764-768.
[30]向鑫,袁继超,陈飞,等.姜黄素诱导内源性神经干细胞促进大鼠脊髓损伤后功能修复[J].第三军医大学学报,2014,36(9):883-887.
[31]Mah AT,Yan KS,Kuo CJ.Wnt pathway regulation of intestinal stem cells.J Physiol.2016;594(17):4837-4847.
[32]Yuan Z,Li Q,Luo S,et al.PPARγand Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.Curr Stem Cell Res Ther.2016;11(3):216-225.
[33]Sun TJ,Tao R,Han YQ,et al.Therapeutic potential of umbilical cord mesenchymal stem cells with Wnt/β-catenin signaling pathway pre-activated for the treatment of diabetic wounds.Eur Rev Med Pharmacol Sci.2014;18(17):2460-2464.
[34]Zhou Y,Kipps TJ,Zhang S.Wnt5a Signaling in Normal and Cancer Stem Cells.Stem Cells Int.2017;2017:5295286.
[35]Zhang X,Guo J,Zhou Y,et al.The roles of bone morphogenetic proteins and their signaling in the osteogenesis of adipose-derived stem cells.Tissue Eng Part B Rev.2014;20(1):84-92.
[36]Katoh M,Katoh M.STAT3-induced WNT5A signaling loop in embryonic stem cells,adult normal tissues,chronic persistent inflammation,rheumatoid arthritis and cancer(Review).Int J Mol Med.2007;19(2):273-278.
[37]柳鑫,郭倩,肖燕,等.Wnt5a对牙髓干细胞成骨/成牙本质向分化的影响[J].牙体牙髓牙周病学杂志,2018,28(6):311-320.
[38]包幸福,倪宇昕,李莹,等.Wnt5a在小鼠牙骨质发育中的表达特点及其对成牙骨质细胞分化的影响[J].吉林大学学报(医学版),2018,44(3):461-465,693.
[2]Shirvani A,Shamszadeh S,Eghbal MJ,et al.Effect of preoperative oral analgesics on pulpal anesthesia in patients with irreversible pulpitis-a systematic review and meta-analysis.Clin Oral Investig.2017;21(1):43-52.
[3]Yang X,Li L,Xiao L,et al.Recycle the dental fairy's package:overview of dental pulp stem cells.Stem Cell Res Ther.2018;9(1):347.
[4]Kaneko T,Gu B,Sone PP,et al.Dental Pulp Tissue Engineering Using Mesenchymal Stem Cells:a Review with a Protocol.Stem Cell Rev.2018;14(5):668-676.
[5]Graham CM,Kremer KL,Koblar SA,et al.Dental Pulp Stem CellsExploration in a Novel Animal Model:the Tasmanian Devil(Sarcophilus harrisii).Stem Cell Rev.2018;14(4):500-509.
[6]Hassan NT,AbdelAziz NA.Oral Mucosal Stem Cells,Human Immature Dental Pulp Stem Cells and Hair Follicle Bulge Stem Cells as Adult Stem Cells Able to Correct Limbal Stem Cell Deficiency.Curr Stem Cell Res Ther.2018;13(5):356-361.
[7]Anitua E,Troya M,Zalduendo M.Progress in the use of dental pulp stem cells in regenerative medicine.Cytotherapy.2018;20(4):479-498.
[8]杨雪超,李晓宁,王伟东,等.牙本质支架促进人牙髓干细胞体外增殖与分化能力的研究[J].牙体牙髓牙周病学杂志,2015,25(6):342-347.
[9]杨鑫,李思洁,赵玮.Wnt信号通路在调控牙髓干细胞多向分化及炎症损伤修复中的作用[J].国际口腔医学杂志,2018,45(3):286-290.
[10]陈婵婵,凌均棨.Wnt信号通路对牙齿发育和牙囊成骨向分化的调控作用[J].牙体牙髓牙周病学杂志,2015,25(3):175-178.
[11]Romanos GE.Biomolecular Cell-Signaling Mechanisms and Dental Implants:A Review on the Regulatory Molecular Biologic Patterns Under Functional and Immediate Loading.Int J Oral Maxillofac Implants.2016;31(4):939-951.
[12]Mc Cubrey JA,Lertpiriyapong K,Steelman LS,et al.Effects of resveratrol,curcumin,berberine and other nutraceuticals on aging,cancer development,cancer stem cells and microRNAs.Aging(Albany NY).2017;9(6):1477-1536.
[13]张庆美,李方正,姜忠玲,等.姜黄素对猪骨髓间充质干细胞增殖和成脂分化的影响[J].解剖学报,2014,45(6):793-799.
[14]陈飞,王皓香,向鑫,等.姜黄素对神经干细胞Wnt/β-catenin信号通路表达影响的离体研究[J].第三军医大学学报,2014,36(8):764-768.
[15]顾巧丽,蔡燕,黄晨,等.姜黄素调控大鼠骨髓间充质干细胞的成骨分化[J].中国组织工程研究,2012,16(27):5057-5061.
[16]Chauhan S,王盈.用于治疗牙周炎的载姜黄素可生物降解交联明胶薄膜的研制[J].中国医药工业杂志,2018,49(5):593.
[17]朱柯,潘芸,杨洋,等.姜黄素对牙周炎大鼠牙周支持组织炎症性破坏的保护作用[J].广东医学,2017,38(3):358-361.
[18]刘明月,胡伟平,王晓芬,等.低浓度β-甘油磷酸钠培养牙髓干细胞向成牙本质细胞分化及相关因子的表达[J].中国组织工程研究,2015,19(41):6688-6693.
[19]梁静,王君,唐传玲,等.低氧对H2O2预处理MC3T3-E1增殖和成骨分化功能的影响[J].中国组织工程研究,2017,21(4):505-511.
[20]Phan M,Conte F,Khandelwal KD,et al.Tooth agenesis and orofacial clefting:genetic brothers in arms.Hum Genet.2016;135(12):1299-1327.
[21]Mead B,Logan A,Berry M,et al.Concise Review:Dental Pulp Stem Cells:A Novel Cell Therapy for Retinal and Central Nervous System Repair.Stem Cells.2017;35(1):61-67.
[22]Nuti N,Corallo C,Chan BM,et al.Multipotent Differentiation of Human Dental Pulp Stem Cells:a Literature Review.Stem Cell Rev.2016;12(5):511-523.
[23]Ledesma-Martínez E,Mendoza-Nú?ez VM,Santiago-Osorio E.Mesenchymal Stem Cells Derived from Dental Pulp:A Review.Stem Cells Int.2016;2016:4709572.
[24]Shehzad A,Lee YS.Molecular mechanisms of curcumin action:signal transduction.Biofactors.2013;39(1):27-36.
[25]席晅,任小琼.姜黄素通过PI3K/AKT信号通路调控神经干细胞增殖和凋亡实验研究[J].新中医,2016,48(3):217-221.
[26]马晓晓,王春满,张高龙,等.姜黄素通过抑制糖皮质激素受体促进大鼠神经干细胞增殖[J].中国药理学与毒理学杂志,2015,29(02):202-207.
[27]刘剑锋,朱平,马守原,等.姜黄素通过血红素加氧酶-1信号通路促进脂肪源干细胞增殖和旁分泌研究[J].中国心血管杂志,2015,20(3):199-203.
[28]邬培红,华子义,赵秀娟.姜黄素促进大鼠骨髓间充质干细胞成骨分化中活性氧表达变化研究[J].中国医药导报,2013,10(18):24-26,29.
[29]陈飞,王皓香,向鑫,等.姜黄素对神经干细胞Wnt/β-catenin信号通路表达影响的离体研究[J].第三军医大学学报,2014,36(8):764-768.
[30]向鑫,袁继超,陈飞,等.姜黄素诱导内源性神经干细胞促进大鼠脊髓损伤后功能修复[J].第三军医大学学报,2014,36(9):883-887.
[31]Mah AT,Yan KS,Kuo CJ.Wnt pathway regulation of intestinal stem cells.J Physiol.2016;594(17):4837-4847.
[32]Yuan Z,Li Q,Luo S,et al.PPARγand Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.Curr Stem Cell Res Ther.2016;11(3):216-225.
[33]Sun TJ,Tao R,Han YQ,et al.Therapeutic potential of umbilical cord mesenchymal stem cells with Wnt/β-catenin signaling pathway pre-activated for the treatment of diabetic wounds.Eur Rev Med Pharmacol Sci.2014;18(17):2460-2464.
[34]Zhou Y,Kipps TJ,Zhang S.Wnt5a Signaling in Normal and Cancer Stem Cells.Stem Cells Int.2017;2017:5295286.
[35]Zhang X,Guo J,Zhou Y,et al.The roles of bone morphogenetic proteins and their signaling in the osteogenesis of adipose-derived stem cells.Tissue Eng Part B Rev.2014;20(1):84-92.
[36]Katoh M,Katoh M.STAT3-induced WNT5A signaling loop in embryonic stem cells,adult normal tissues,chronic persistent inflammation,rheumatoid arthritis and cancer(Review).Int J Mol Med.2007;19(2):273-278.
[37]柳鑫,郭倩,肖燕,等.Wnt5a对牙髓干细胞成骨/成牙本质向分化的影响[J].牙体牙髓牙周病学杂志,2018,28(6):311-320.
[38]包幸福,倪宇昕,李莹,等.Wnt5a在小鼠牙骨质发育中的表达特点及其对成牙骨质细胞分化的影响[J].吉林大学学报(医学版),2018,44(3):461-465,693.