黄芩素通过WNT/β-catenin信号通路促进大鼠腱骨愈合  

Baicalein promotes rat extra-articular tendon-to-bone transplanting healing through WNT/β-catenin signaling

作　　者：江华基 江小成 贺飞林 廖小卿[2] 张文涛[2] 张新涛[2] 

机构地区：[1]南方医科大学第三附属医院骨科,广东广州510000 [2]北京大学深圳医院运动医学与康复科,广东深圳510086

出　　处：《分子影像学杂志》2016年第3期297-301,共5页Journal of Molecular Imaging

基　　金：深圳市卫生计生系统科研项目(201302058)

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.

关 键 词：黄芩素 大鼠 腱骨愈合 肌腱干细胞 WNT/Β-CATENIN 

分 类 号：R285.5[医药卫生—中药学]