黄芩素通过WNT/β-catenin信号通路促进大鼠腱骨愈合
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摘要
目的探讨黄芩素对大鼠腱骨愈合的作用及相关分子机制。方法取40只8周龄的雄性SD大鼠,随机分为对照组(n=20)和实验组(n=20)。两组动物均行大鼠腱骨愈合模型造模。实验组大鼠灌胃黄芩素10 mg/(kg·d),对照组灌胃等量的生理盐水。术后3周、6周各处死10只大鼠并取材,通过生物力学、组织学观察各组腱骨愈合的程度。此外,体外培养原代大鼠肌腱干细胞,加入不同浓度(0、0.1、1、10μmol/L)的黄芩素处理,14 d后分别进行ALP、茜素红染色。Q-PCR、Western Blot检测Runx2、OSX、OCN、一型胶原以及β-catenin的变化。随后,在肌腱干细胞中加入10μmol/L的黄芩素,并且加入WNT/β-catenin通路抑制剂DKK-1共培养,14 d后收集蛋白行Western Blot检测,观察β-catenin、Runx2以及OCN蛋白表达的变化。结果生物力学结果显示术后3周和术后6周时实验组的腱骨愈合程度明显高于对照组,差异有统计学意义(P<0.05)。组织学结果显示实验组在术后3周和6周时腱骨界面细胞成熟程度更高,sharpey纤维生长密集度与间质钙化程度更高。ALP、茜素红染色、Q-PCR和Western Blot结果显示不同浓度黄芩素均能提高肌腱干细胞的ALP活性,增加矿化结节形成、提高Runx2、OSX、OCN、一型胶原和β-catenin的表达。而β-catenin抑制剂DKK-1后能显著降低黄芩素的这种促成骨作用。结论黄芩素可以有效促进腱骨界面细胞成熟与腱骨界面的胶原分泌,加速腱骨愈合并提高腱骨愈合质量,这种促进作用呈现WNT/β-catenin信号依赖性。
Objective To investigate the effect of baicalein on tendon healing in bone tunnel and reveal its related molecular mechanism.Methods In vivo,40 Sprague Dawley male rats were used to establish rat extra-articular tendon-to-bone transplanting healing,then randomly divided into 2 groups.Experiment group was treated with baicalein 10 mg/(kg·d),while control group was treated by normal saline(0.9%) with the same volume.Biomechanical testing and histological detection were tested at 3 weeks and 6 weeks after surgery.In vitro,tendom stem cells were isolated from rat Achilles tendon,and being treated with various concentrations of baicalein for 14 days.The expression of alkaline phosphatase( ALP)and mineralized nodule was detected by ALP activity/staining assays and Alizarin red staining respectively,while the expression of Runx2,OSX,OCN,Collagen I were tested by Q-PCR and the expression of Runx2,OSX,OCN and β-catenin were evaluated by Western Blot assay.Results Tendon bone healing strength of experiment group was obviously stronger than control group in 3weeks as well as in 6 weeks.In experiment group,there were more mature fibroblasts,more Sharphey fibers,and larger new bone formation area compared with control group.After intervention for 14 days,the expression of ALP,Runx2,OCN and VEGF were up-regulated,and WNT/β-catenin signaling were enhanced in vitro.Conclusion Baicalein can promotes osteogenic differentiation of tendon stem cells through WNT/β-catenin signaling in vitro,and stimulate tendon-bone healing in bone tunnel and enhance the connection between tendon and bone.
Objective To investigate the effect of baicalein on tendon healing in bone tunnel and reveal its related molecular mechanism.Methods In vivo,40 Sprague Dawley male rats were used to establish rat extra-articular tendon-to-bone transplanting healing,then randomly divided into 2 groups.Experiment group was treated with baicalein 10 mg/(kg·d),while control group was treated by normal saline(0.9%) with the same volume.Biomechanical testing and histological detection were tested at 3 weeks and 6 weeks after surgery.In vitro,tendom stem cells were isolated from rat Achilles tendon,and being treated with various concentrations of baicalein for 14 days.The expression of alkaline phosphatase( ALP)and mineralized nodule was detected by ALP activity/staining assays and Alizarin red staining respectively,while the expression of Runx2,OSX,OCN,Collagen I were tested by Q-PCR and the expression of Runx2,OSX,OCN and β-catenin were evaluated by Western Blot assay.Results Tendon bone healing strength of experiment group was obviously stronger than control group in 3weeks as well as in 6 weeks.In experiment group,there were more mature fibroblasts,more Sharphey fibers,and larger new bone formation area compared with control group.After intervention for 14 days,the expression of ALP,Runx2,OCN and VEGF were up-regulated,and WNT/β-catenin signaling were enhanced in vitro.Conclusion Baicalein can promotes osteogenic differentiation of tendon stem cells through WNT/β-catenin signaling in vitro,and stimulate tendon-bone healing in bone tunnel and enhance the connection between tendon and bone.
引文
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[12]徐伟丽,牛玲玲,王文侠,等.经典Wnt信号通路对骨代谢的调节作用[J].中国骨质疏松杂志,2016(3):376-80.
[13]李晨睿,孟志远,牛银波,等.黄芩苷通过Wnt/β-catenin信号通路对大鼠骨髓间充质干细胞成骨分化的促进作用[J].中国药理学通报,2015(7):919-24.
[14]Liu XZ,Chen W,Zhou Y,et al.Mechanical tension promotes the osteogenic differentiation of rat tendon-derived stem cells through the Wnt5a/Wnt5b/JNK signaling pathway[J].Cell Physiol Biochem,2015,36(2):517-30.
[2]Kim JM,Lee SU,Kim YS,et al.Baicalein stimulates osteoblast differentiation via coordinating activation of MAP kinases and transcription factors[J].J Cell Biochem,2008,104(5):1906-17.
[3]Kim MH,Ryu SY,Bae MA,et al.Baicalein inhibits osteoclast differentiation and induces mature osteoclast apoptosis[J].Food Chem Toxicol,2008,46(11):3375-82.
[4]Zhang JY,Wang JH.Characterization of differential properties of rabbit tendon stem cells and tenocytes[J].BMC Musculoskelet Disord,2010,11(5):10-3.
[5]张磊,李智尧,孙晋,等.三七总皂苷促进腱骨愈合的实验研究[J].中国骨伤,2011,24(2):132-6.
[6]陆军,Jr RV.大鼠关节外骨隧道-游离肌腱移植模型的建立[J].中华骨科杂志,2014(8):864-71.
[7]Lui PP.Markers for the identification of tendon-derived stem cells in vitro and tendon stem cells in situ-update and future development[J].Stem Cell Res Ther,2015,6(11):106-9.
[8]Li SF,Tang JJ,Chen J,et al.Regulation of bone formation by baicalein via the m TORC1 pathway[J].Drug Des Devel Ther,2015,9(4):5169-83.
[9]兰波,刘亭,谢玉敏,等.两种杜仲黄酮类化合物对成骨细胞OPG/RANKL及成骨相关转录因子的影响[J].中国实验方剂学杂志,2014(22):180-4.
[10]Lui PP,Wong YM.Higher BMP/Smad sensitivity of tendon-derived stem cells(TDSCs)isolated from the collagenase-induced tendon injury model:possible mechanism for their altered fate in vitro[J].BMC Musculoskelet Disord,2013,14(8):248-52.
[11]Lui PP,Chan LS,Cheuk YC,et al.Expression of bone morphogenetic protein-2 in the chondrogenic and ossifying sites of calcific tendinopathy and traumatic tendon injury rat models[J].J Orthop Surg Res,2009,4(6):27-30.
[12]徐伟丽,牛玲玲,王文侠,等.经典Wnt信号通路对骨代谢的调节作用[J].中国骨质疏松杂志,2016(3):376-80.
[13]李晨睿,孟志远,牛银波,等.黄芩苷通过Wnt/β-catenin信号通路对大鼠骨髓间充质干细胞成骨分化的促进作用[J].中国药理学通报,2015(7):919-24.
[14]Liu XZ,Chen W,Zhou Y,et al.Mechanical tension promotes the osteogenic differentiation of rat tendon-derived stem cells through the Wnt5a/Wnt5b/JNK signaling pathway[J].Cell Physiol Biochem,2015,36(2):517-30.