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[1]黃俊俊,史曉林,鄧祖躍.強骨飲對骨質(zhì)疏松性骨折愈合的影響及其作用機制[J].中醫(yī)正骨,2018,30(09):10-17.
　HUANG Junjun,SHI Xiaolin,DENG Zuyue.Effects of Qianggu Yin(強骨飲)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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強骨飲對骨質(zhì)疏松性骨折愈合的影響及其作用機制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期數(shù):
2018年09期
頁碼:
10-17
欄目:
基礎(chǔ)研究
出版日期:
2018-09-20

文章信息/Info

Title:
Effects of Qianggu Yin(強骨飲)on the healing of osteoporotic fracture and its mechanism of action
作者:
黃俊俊1; 史曉林1; 鄧祖躍2
1.浙江中醫(yī)藥大學附屬第二醫(yī)院,浙江 杭州 310005; 2.浙江省食品藥品檢驗研究院,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
關(guān)鍵詞:
骨質(zhì)疏松性骨折;  骨密度;  轉(zhuǎn)化生長因子β1;  白細胞介素1β;  腫瘤壞死因子α;  強骨飲;  大鼠;  動物實驗
Keywords:
osteoporotic fractures;  bone density;  transforming growth factor beta1;  interleukin-1beta;  tumor necrosis factor-alpha;  Qianggu Yin;  animal experimentation;  rats
摘要:
探討強骨飲對骨質(zhì)疏松性骨折愈合的影響及其作用機制。方法:將30只雌性SD大鼠隨機分成假手術(shù)/骨折組、骨質(zhì)疏松性骨折組、骨質(zhì)疏松性骨折強骨飲治療組,每組10只。骨質(zhì)疏松性骨折組、骨質(zhì)疏松性骨折強骨飲治療組大鼠摘除雙側(cè)卵巢建立骨質(zhì)疏松模型,假手術(shù)/骨折組大鼠未切除卵巢。骨質(zhì)疏松造模術(shù)后6周,雙能X線骨密度掃描儀測定3組大鼠股骨中段骨密度,并均于股骨中段截斷右側(cè)股骨后用克氏針內(nèi)固定,建立大鼠股骨中段骨折模型。骨折造模后,骨質(zhì)疏松性骨折強骨飲治療組大鼠以強骨飲濃縮液灌胃,假手術(shù)/骨折組、骨質(zhì)疏松性骨折組大鼠以等量生理鹽水灌胃,每日1次,連續(xù)6周。灌胃6周后,每只大鼠主動脈取血5 mL,分離血清,酶聯(lián)免疫法測定大鼠血清中腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)、白細胞介素-1β(interleukin-1β,IL-1β)和轉(zhuǎn)化生長因子-β1(transfer growth factor-β1,TGF-β1)水平。然后處死大鼠,完整取出右側(cè)股骨制成股骨標本,雙能X線骨密度掃描儀測定大鼠股骨中段骨密度; Micro-CT掃描儀觀察大鼠股骨中段骨折處骨痂的結(jié)構(gòu),分析、記錄骨小梁數(shù)量、骨小梁厚度、骨小梁分離度、骨體積分數(shù); 光學顯微鏡下觀察大鼠股骨中段骨折處骨痂的組織形態(tài)。結(jié)果:①骨質(zhì)疏松造模術(shù)后6周,3組大鼠股骨中段骨密度比較,組間差異有統(tǒng)計學意義[(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]; 骨質(zhì)疏松性骨折組、骨質(zhì)疏松性骨折強骨飲治療組股骨中段骨密度均低于假手術(shù)/骨折組(P=0.000,P=0.000); 骨質(zhì)疏松性骨折組股骨中段骨密度與骨質(zhì)疏松性骨折強骨飲治療組比較,差異無統(tǒng)計學意義(P=0.418); 證實骨質(zhì)疏松造模成功。灌胃6周后,3組大鼠股骨中段骨密度比較,組間差異有統(tǒng)計學意義[(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]; 假手術(shù)/骨折組股骨中段骨密度高于骨質(zhì)疏松性骨折組和骨質(zhì)疏松性骨折強骨飲治療組(P=0.000,P=0.000); 骨質(zhì)疏松性骨折組股骨中段骨密度低于骨質(zhì)疏松性骨折強骨飲治療組(P=0.003)。②灌胃6周后,3組大鼠股骨中段Micro-CT掃描示,假手術(shù)/骨折組骨折處骨膜反應明顯,骨痂膨大,骨皮質(zhì)厚實; 骨質(zhì)疏松性骨折組骨皮質(zhì)萎縮,骨痂體積小; 骨質(zhì)疏松性骨折強骨飲治療組骨膜反應明顯,骨痂體積較大,骨皮質(zhì)無明顯萎縮。3組大鼠股骨中段骨折處三維重建圖像顯示,假手術(shù)/骨折組骨折處骨痂生長明顯,骨小梁多,骨小梁結(jié)構(gòu)緊密; 骨質(zhì)疏松性骨折組骨痂少,骨小梁稀疏; 骨質(zhì)疏松性骨折強骨飲治療組比骨質(zhì)疏松性骨折組骨痂生長明顯,骨小梁多,排列有序。3組大鼠股骨中段骨折處骨痂的骨小梁數(shù)量、骨小梁厚度、骨小梁分離度、骨體積分數(shù)比較,組間差異均有統(tǒng)計學意義[(0.309±0.052)個·mm-1,(0.152±0.041)個·mm-1,(0.233±0.047)個·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)]; 假手術(shù)/骨折組骨痂骨小梁數(shù)量、骨小梁厚度及骨體積分數(shù)均高于骨質(zhì)疏松性骨折組和骨質(zhì)疏松性骨折強骨飲治療組(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.002),骨小梁分離度低于骨質(zhì)疏松性骨折組和骨質(zhì)疏松性骨折強骨飲治療組(P=0.000,P=0.000),骨質(zhì)疏松性骨折強骨飲治療組骨痂骨小梁數(shù)量、骨小梁厚度、骨體積分數(shù)高于骨質(zhì)疏松性骨折組(P=0.000,P=0.000,P=0.000),骨小梁分離度低于骨質(zhì)疏松性骨折組(P=0.000)。③灌胃6周后,3組大鼠血清TNF-α、IL-1β、TGF-β1水平比較,組間差異均有統(tǒng)計學意義[(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]; 假手術(shù)/骨折組大鼠血清中TNF-α和IL-1β水平均低于骨質(zhì)疏松性骨折組和骨質(zhì)疏松性骨折強骨飲治療組(P=0.000,P=0.000; P=0.000,P=0.000),TGF-β1水平高于骨質(zhì)疏松性骨折組和骨質(zhì)疏松性骨折強骨飲治療組(P=0.000,P=0.000); 骨質(zhì)疏松性骨折組大鼠血清中TNF-α和IL-1β水平均高于骨質(zhì)疏松性骨折強骨飲治療組(P=0.000,P=0.000),TGF-β1水平低于骨質(zhì)疏松性骨折強骨飲治療組(P=0.032)。④灌胃6周后,光學顯微鏡下觀察3組大鼠股骨中段骨折處骨痂組織可見,假手術(shù)/骨折組骨小梁較粗、分布較規(guī)則、排列有序、間距小,連接成拱橋狀,骨性骨痂取代軟骨性骨痂,小梁狀骨經(jīng)改建趨于成熟; 與假手術(shù)/骨折組相比,骨質(zhì)疏松性骨折組骨小梁較細,髓腔間隙較大,分布不規(guī)則、間距較寬,呈疏松化表現(xiàn),軟骨痂更明顯,成熟小梁狀骨較少,存在大量未鈣化軟骨細胞; 與骨質(zhì)疏松性骨折組相比,骨質(zhì)疏松性骨折強骨飲治療組骨小梁較粗、數(shù)量較多、間距較小,軟骨細胞骨化占比較高,更接近于假手術(shù)/骨折組。結(jié)論:強骨飲可促進骨質(zhì)疏松性骨折的愈合,其機制可能與提高血清中TGF-β1水平和降低TNF-α、IL-1β水平,從而影響骨代謝、增加骨密度、改善骨微結(jié)構(gòu)有關(guān)。
Abstract:
To explore the effects of Qianggu Yin(強骨飲,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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備注/Memo

備注/Memo:
基金項目:浙江省自然科學基金一般項目(LY14H270003)
更新日期/Last Update: 2018-09-20