[1]黄俊俊,史晓林,邓祖跃.强骨饮对骨质疏松性骨折愈合的影响及其作用机制[J].中医正骨,2018,30(09):10-17.
　HUANG Junjun,SHI Xiaolin,DENG Zuyue.Effects of Qianggu Yin(强骨饮)on the healing of osteoporotic fracture and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(09):10-17.
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强骨饮对骨质疏松性骨折愈合的影响及其作用机制()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期数:
2018年09期
页码:
10-17
栏目:
基础研究
出版日期:
2018-09-20

文章信息/Info

Title:
Effects of Qianggu Yin(强骨饮)on the healing of osteoporotic fracture and its mechanism of action
作者:
黄俊俊1; 史晓林1; 邓祖跃2
1.浙江中医药大学附属第二医院,浙江 杭州 310005; 2.浙江省食品药品检验研究院,浙江 杭州 310052
Author(s):
HUANG Junjun1; SHI Xiaolin1; DENG Zuyue2
1.The Second Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine,Hangzhou 310005,Zhejiang,China 2.Zhejiang institute for food and drug control,Hangzhou 310052,Zhejiang,China
关键词:
骨质疏松性骨折;  骨密度;  转化生长因子β1;  白细胞介素1β;  肿瘤坏死因子α;  强骨饮;  大鼠;  动物实验
Keywords:
osteoporotic fractures;  bone density;  transforming growth factor beta1;  interleukin-1beta;  tumor necrosis factor-alpha;  Qianggu Yin;  animal experimentation;  rats
摘要:
探讨强骨饮对骨质疏松性骨折愈合的影响及其作用机制。方法:将30只雌性SD大鼠随机分成假手术/骨折组、骨质疏松性骨折组、骨质疏松性骨折强骨饮治疗组,每组10只。骨质疏松性骨折组、骨质疏松性骨折强骨饮治疗组大鼠摘除双侧卵巢建立骨质疏松模型,假手术/骨折组大鼠未切除卵巢。骨质疏松造模术后6周,双能X线骨密度扫描仪测定3组大鼠股骨中段骨密度,并均于股骨中段截断右侧股骨后用克氏针内固定,建立大鼠股骨中段骨折模型。骨折造模后,骨质疏松性骨折强骨饮治疗组大鼠以强骨饮浓缩液灌胃,假手术/骨折组、骨质疏松性骨折组大鼠以等量生理盐水灌胃,每日1次,连续6周。灌胃6周后,每只大鼠主动脉取血5 mL,分离血清,酶联免疫法测定大鼠血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)和转化生长因子-β1(transfer growth factor-β1,TGF-β1)水平。然后处死大鼠,完整取出右侧股骨制成股骨标本,双能X线骨密度扫描仪测定大鼠股骨中段骨密度; Micro-CT扫描仪观察大鼠股骨中段骨折处骨痂的结构,分析、记录骨小梁数量、骨小梁厚度、骨小梁分离度、骨体积分数; 光学显微镜下观察大鼠股骨中段骨折处骨痂的组织形态。结果:①骨质疏松造模术后6周,3组大鼠股骨中段骨密度比较,组间差异有统计学意义[(0.348±0.048)g·cm-2,(0.218±0.047)g·cm-2,(0.221±0.052)g·cm-2; F=22.590,P=0.000]; 骨质疏松性骨折组、骨质疏松性骨折强骨饮治疗组股骨中段骨密度均低于假手术/骨折组(P=0.000,P=0.000); 骨质疏松性骨折组股骨中段骨密度与骨质疏松性骨折强骨饮治疗组比较,差异无统计学意义(P=0.418); 证实骨质疏松造模成功。灌胃6周后,3组大鼠股骨中段骨密度比较,组间差异有统计学意义[(0.258±0.039)g·cm-2,(0.202±0.021)g·cm-2,(0.227±0.038)g·cm-2; F=25.957,P=0.000]; 假手术/骨折组股骨中段骨密度高于骨质疏松性骨折组和骨质疏松性骨折强骨饮治疗组(P=0.000,P=0.000); 骨质疏松性骨折组股骨中段骨密度低于骨质疏松性骨折强骨饮治疗组(P=0.003)。②灌胃6周后,3组大鼠股骨中段Micro-CT扫描示,假手术/骨折组骨折处骨膜反应明显,骨痂膨大,骨皮质厚实; 骨质疏松性骨折组骨皮质萎缩,骨痂体积小; 骨质疏松性骨折强骨饮治疗组骨膜反应明显,骨痂体积较大,骨皮质无明显萎缩。3组大鼠股骨中段骨折处三维重建图像显示,假手术/骨折组骨折处骨痂生长明显,骨小梁多,骨小梁结构紧密; 骨质疏松性骨折组骨痂少,骨小梁稀疏; 骨质疏松性骨折强骨饮治疗组比骨质疏松性骨折组骨痂生长明显,骨小梁多,排列有序。3组大鼠股骨中段骨折处骨痂的骨小梁数量、骨小梁厚度、骨小梁分离度、骨体积分数比较,组间差异均有统计学意义[(0.309±0.052)个·mm-1,(0.152±0.041)个·mm-1,(0.233±0.047)个·mm-1,F=11.583,P=0.000;(0.375±0.055)mm,(0.206±0.036)mm,(0.296±0.043)mm,F=18.590,P=0.000;(3.489±0.367)mm,(4.427±0.191)mm,(3.768±0.269)mm,F=28.559,P=0.000;(55.52±7.24)%,(38.31±5.12)%,(50.96±6.15)%,F=12.445,P=0.000)]; 假手术/骨折组骨痂骨小梁数量、骨小梁厚度及骨体积分数均高于骨质疏松性骨折组和骨质疏松性骨折强骨饮治疗组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.002),骨小梁分离度低于骨质疏松性骨折组和骨质疏松性骨折强骨饮治疗组(P=0.000,P=0.000),骨质疏松性骨折强骨饮治疗组骨痂骨小梁数量、骨小梁厚度、骨体积分数高于骨质疏松性骨折组(P=0.000,P=0.000,P=0.000),骨小梁分离度低于骨质疏松性骨折组(P=0.000)。③灌胃6周后,3组大鼠血清TNF-α、IL-1β、TGF-β1水平比较,组间差异均有统计学意义[(94.52±10.13)ng·L-1,(144.02±17.71)ng·L-1,(112.82±12.16)ng·L-1,F=14.494,P=0.000;(112.05±24.59)ng·L-1,(176.83±35.92)ng·L-1,(129.93±29.04)ng·L-1,F=9.494,P=0.000;(35.49±4.02)ng·L-1,(29.03±3.19)ng·L-1,(32.82±3.54)ng·L-1,F=18.957,P=0.000]; 假手术/骨折组大鼠血清中TNF-α和IL-1β水平均低于骨质疏松性骨折组和骨质疏松性骨折强骨饮治疗组(P=0.000,P=0.000; P=0.000,P=0.000),TGF-β1水平高于骨质疏松性骨折组和骨质疏松性骨折强骨饮治疗组(P=0.000,P=0.000); 骨质疏松性骨折组大鼠血清中TNF-α和IL-1β水平均高于骨质疏松性骨折强骨饮治疗组(P=0.000,P=0.000),TGF-β1水平低于骨质疏松性骨折强骨饮治疗组(P=0.032)。④灌胃6周后,光学显微镜下观察3组大鼠股骨中段骨折处骨痂组织可见,假手术/骨折组骨小梁较粗、分布较规则、排列有序、间距小,连接成拱桥状,骨性骨痂取代软骨性骨痂,小梁状骨经改建趋于成熟; 与假手术/骨折组相比,骨质疏松性骨折组骨小梁较细,髓腔间隙较大,分布不规则、间距较宽,呈疏松化表现,软骨痂更明显,成熟小梁状骨较少,存在大量未钙化软骨细胞; 与骨质疏松性骨折组相比,骨质疏松性骨折强骨饮治疗组骨小梁较粗、数量较多、间距较小,软骨细胞骨化占比较高,更接近于假手术/骨折组。结论:强骨饮可促进骨质疏松性骨折的愈合,其机制可能与提高血清中TGF-β1水平和降低TNF-α、IL-1β水平,从而影响骨代谢、增加骨密度、改善骨微结构有关。
Abstract:
To explore the effects of Qianggu Yin(强骨饮,QGY)on the healing of osteoporotic fracture and its mechanism of action.Methods:Thirty female SD rats were randomly divided into sham-operated/fractured group,osteoporotic fracture group and QGY treatment group,10 cases in each group.The bilateral ovariectomy were performed on rats in osteoporotic fracture group and QGY treatment group to build the osteoporosis rat models,while the rats in sham-operated/fractured group were not given ovariectomy.At 6 weeks after osteoporosis modeling operation,the bone densities of middle-segment femurs were detected by using dual-energy X-ray absorptiometry(DEXA)in rats of the 3 groups,and their right femurs were cut off at middle segment and fixed with Kirschner wires to built middle-segment femoral fracture models.After modeling,the rats in QGY treatment group were intragastric administrated with QGY concentrated solution,and the rats in sham-operated/fractured group and osteoporotic fracture group were intragastric administrated with isodose normal saline,once a day for consecutive 6 weeks.After 6-week intragastric administration,the blood(5 mL)were fetched out from aorta of each rat and the serum were isolated from the blood.The serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and transfer growth factor-β1(TGF-β1)were measured by using enzyme linked immunosorbent assays.Then the rats were executed and their right femurs were fetched out for making specimens.The bone densities of middle-segment femurs were detected by using DEXA.The structure of bony callus at fractured site of middle-segment femurs were observed by using Micro-CT scanner,and trabecular number(Tb.N),trabecular thickness(Tb.Th),trabecular separation(Tb.Sp)and bone volume fraction were analyzed and recorded.The histomorphological changes of bony callus at fractured site of middle-segment femurs were observed under the optical microscope.Results:At 6 weeks after osteoporosis modeling operation,there was statistical difference in the bone density of middle-segment femur between the 3 groups(0.348+/-0.048,0.218+/-0.047,0.221+/-0.052 g/cm(-2); F=22.590,P=0.000).The bone densities of middle-segment femurs were lower in osteoporotic fracture group and QGY treatment group compared to sham-operated/fractured group(P=0.000,P=0.000),and there was no statistical difference in the bone density of middle-segment femur between osteoporotic fracture group and QGY treatment group(P=0.418).After successful modeling and 6-week intragastric administration,there was statistical difference in the bone density of middle-segment femur between the 3 groups(0.258+/-0.039,0.202+/-0.021,0.227+/-0.038 g/cm(-2); F=25.957,P=0.000).The bone density of middle-segment femur was higher in sham-operated/fractured group compared to osteoporotic fracture group and QGY treatment group,and was lower in osteoporotic fracture group compared to QGY treatment group(P=0.000,P=0.000; P=0.003).After 6-week intragastric administration,the results of Micro-CT scanning on middle-segment femurs showed that(1)obvious periosteal reaction,dilatate bony callus and thick cortical bone were found at fractured site in sham-operated/fractured group;(2)auantic cortical bone and small bony callus were found in osteoporotic fracture group;(3)obvious periosteal reaction,large bony callus and mild auantic cortical bone were found in QGY treatment group.The three-dimensional reconstruction images of fractured sites of middle-segment femurs showed that(1)well-grown bony callus and a great quantity of bone trabecula with compact structure were found at the fractured site in operated/fractured group;(2)sparse and less bone trabecula were found in osteoporotic fracture group;(3)compared to osteoporotic fracture group,more bony callus and regularly arranged bone trabecula were found in QGY treatment group.There was statistical difference in Tb.N,Tb.Th,Tb.Sp and bone volume fraction of bony callus at fractured site of middle-segment femur between the 3 groups(0.309+/-0.052,0.152+/-0.041,0.233+/-0.047 piece/mm,F=11.583,P=0.000; 0.375+/-0.055,0.206+/-0.036,0.296+/-0.043 mm,F=18.590,P=0.000; 3.489+/-0.367,4.427+/-0.191,3.768+/-0.269 mm,F=28.559,P=0.000; 55.52+/-7.24,38.31+/-5.12,50.96+/-6.15%,F=12.445,P=0.000).The Tb.N,Tb.Th and bone volume fraction of bony callus were higher and the Tb.Sp was lower in sham-operated/fractured group compared to osteoporotic fracture group and QGY treatment group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.002; P=0.000,P=0.000).The Tb.N,Tb.Th and bone volume fraction of bony callus were higher and the Tb.Sp was lower in QGY treatment group compared to osteoporotic fracture group(P=0.000,P=0.000,P=0.000; P=0.000).After 6-week intragastric administration,there was statistical difference in serum levels of TNF-α,IL-1β and TGF-β1 between the 3 groups(94.52+/-10.13.144.02+/-17.71.112.82+/-12.16 ng/L,F=14.494,P=0.000; 112.05+/-24.59,176.83+/-35.92,129.93+/-29.04 ng/L,F=9.494,P=0.000; 35.49+/-4.02,29.03+/-3.19,32.82+/-3.54 ng/L,F=18.957,P=0.000).The serum levels of TNF-α and IL-1β were lower and the serum level of TGF-β1 was higher in sham-operated/fractured group compared to osteoporotic fracture group and QGY treatment group(P=0.000,P=0.000; P=0.000,P=0.000; P=0.000,P=0.000).The serum levels of TNF-α and IL-1β were higher and the serum level of TGF-β1 was lower in osteoporotic fracture group compared to QGY treatment group(P=0.000,P=0.000; P=0.032).After 6-week intragastric administration,the results of observation on bony callus at fractured site of middle-segment femurs under optical microscope showed that(1)thicker,regularly and compacted arranged and arch-bridge-shaped connected bone trabecula were found in the bony callus in sham-operated/fractured group,and the cartilaginous callus were replaced by bony callus and the trabecular bone became matured after remodeling;(2)thin,irregular and sparse bone trabecula and large intramedullary space were found in bony callus in osteoporotic fracture group,and more remarkable cartilaginous callus,less matured trabecular bone and large numbers of uncalcified chondrocytes were found in the bony callus;(3)compared to osteoporotic fracture group,thicker,more compacted and more numerous bone trabecula and higher proportion of ossified chondrocytes were found in the bony callus in QGY treatment group,which was more closer to sham-operated/fractured group.Conclusion:QGY can promote the healing of osteoporotic fractures,and it can promote bone metabolism,increase bone density and improve bone microstructure through up-regulating the serum level of TGF-β1 and down-regulating the serum levels of TNF-α and IL-1β,which may be the mechanism of action.

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备注/Memo

备注/Memo:
基金项目:浙江省自然科学基金一般项目(LY14H270003)
更新日期/Last Update: 2018-09-20