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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:

第29卷

期數(shù):

2017年10期

頁碼:

1-12

欄目:

基礎(chǔ)研究

出版日期:

2017-10-20

文章信息/Info

Title:

Study on mechanism of action of Jiegu Xujin Wan(接骨續(xù)筋丸)in promoting fracture healing in rats

作者:

劉震¹; 于訓(xùn)意²; 曹長征²; 付偉¹; 蘇紀(jì)權(quán)¹; 馬賢德³; 侯德才²

1.遼寧省海城市正骨醫(yī)院,遼寧 海城 114200; 2.遼寧中醫(yī)藥大學(xué)附屬醫(yī)院,遼寧 沈陽 110032; 3.遼寧中醫(yī)藥大學(xué),遼寧 沈陽 110032

Author(s):

LIU Zhen¹; YU Xunyi²; CAO Changzheng²; FU Wei¹; SU Jiquan¹; MA Xiande³; HOU Decai²

1.Haicheng Orthopedic-Traumatological Hospital,Haicheng 114200,Liaoning,China 2.The Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning,China 3.Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning,China

關(guān)鍵詞:

骨折愈合;  接骨續(xù)筋丸;  成骨細(xì)胞;  破骨細(xì)胞;  堿性磷酸酶;  骨形態(tài)發(fā)生蛋白質(zhì)2;  血管內(nèi)皮生長因子類;  鈣;  磷;  大鼠;  動(dòng)物實(shí)驗(yàn)

Keywords:

Key words fracture healing;  Jiegu Xujin Wan; osteoblasts;  osteoclasts;  alkaline phosphatase;  bone morphogenetic protein2;  vascular endothelial growth factors;  calcium;  phosphorus;  rats;  animal experimentation

摘要:

目的:探討接骨續(xù)筋丸促進(jìn)大鼠骨折愈合的作用機(jī)制。方法:將72只3月齡雄性SD大鼠隨機(jī)分為假手術(shù)組、模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組,每組18只。截?cái)嗄Ｐ蛯φ战M、接骨七厘膠囊組、接骨續(xù)筋丸組大鼠股骨干中段,制作股骨開放性骨折模型; 假手術(shù)組僅暴露股骨干,不做截骨。造模后第2天開始灌胃,接骨續(xù)筋丸組以3 g·kg^-1劑量的接骨續(xù)筋丸混懸液灌胃,接骨七厘膠囊組以0.52 g·kg^-1劑量的接骨七厘膠囊混懸液灌胃,模型對照組和假手術(shù)組給予蒸餾水灌胃; 每天灌胃1次,各組連續(xù)灌胃21 d。分別于藥物干預(yù)7 d、14 d、21 d后取材,各組隨機(jī)選取6只大鼠從腹主動(dòng)脈抽血,經(jīng)靜置、離心后吸取上層血清,保存待檢; 抽血完成后,取出大鼠左側(cè)股骨干,制備骨折端股骨石蠟標(biāo)本。分別采用鈣(calcium,Ca)、磷(phosphorus,P)、血清堿性磷酸酶(alkaline phosphatase,ALP)測試盒測定大鼠血清Ca、P、ALP含量,采用蘇木精-伊紅(HE)染色觀察骨組織形態(tài)學(xué)變化,采用免疫組化法檢測骨折端骨組織中骨形態(tài)發(fā)生蛋白2(bone morphogenetic protein 2,BMP-2)和血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)的表達(dá)。結(jié)果:①藥物干預(yù)后血清Ca含量。藥物干預(yù)7 d、14 d、21 d 后,4組大鼠血清Ca含量比較,組間差異均無統(tǒng)計(jì)學(xué)意義[(1.80±1.03)mmol·L^-1,(1.88±0.80)mmol·L^-1,(1.91±0.68)mmol·L^-1,(1.83±0.62)mmol·L^-1,F=0.023,P=0.995;(1.77±0.89)mmol·L^-1,(1.77±0.73)mmol·L^-1,(1.88±1.18)mmol·L^-1,(1.74±0.89)mmol·L^-1,F=0.025,P=0.095;(1.72±0.98)mmol·L^-1,(1.74±0.61)mmol·L^-1,(1.80±0.90)mmol·L^-1,(1.69±0.79)mmol·L^-1,F=0.019,P=0.996]。②藥物干預(yù)后血清P含量。藥物干預(yù)7 d后,4組大鼠血清P含量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.89±0.56)mmol·L^-1,(1.97±0.53)mmol·L^-1,(3.23±0.90)mmol·L^-1,(4.24±0.68)mmol·L^-1,F=16.041,P=0.000]; 組間兩兩比較,假手術(shù)組與模型對照組的差異無統(tǒng)計(jì)學(xué)意義(P=0.846),假手術(shù)組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.003),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.005),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.018)。藥物干預(yù)14 d后,4組大鼠血清P含量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.81±0.48)mmol·L^-1,(2.65±0.36)mmol·L^-1,(3.73±0.77)mmol·L^-1,(4.37±0.93)mmol·L^-1,F=16.998,P=0.000]; 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.045,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.012),接骨七厘膠囊組與接骨續(xù)筋丸組的差異無統(tǒng)計(jì)學(xué)意義(P=0.116)。藥物干預(yù)21 d后,4組大鼠血清P含量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.82±0.40)mmol·L^-1,(2.15±0.50)mmol·L^-1,(3.35±0.62)mmol·L^-1,(4.21±0.70)mmol·L^-1,F=22.663,P=0.000]; 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.036,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.002),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.016)。③藥物干預(yù)后血清ALP含量。藥物干預(yù)7 d后,4組大鼠血清ALP含量比較,差異有統(tǒng)計(jì)學(xué)意義[(49.71±14.67)U·L^-1,(93.75±15.11)U·L^-1,(125.00±22.89)U·L^-1,(145.49±20.79)U·L^-1,F=29.797,P=0.000]; 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000,P=0.001),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.001,P=0.000),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.009)。藥物干預(yù)14 d后,4組大鼠血清ALP含量比較,差異有統(tǒng)計(jì)學(xué)意義[(41.57±10.69)U·L^-1,( 91.13±10.76)U·L^-1,(111.77±19.66)U·L^-1,(149.42±12.71)U·L^-1,F=62.354,P=0.000]; 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.019)。藥物干預(yù)21 d后,4組大鼠血清ALP含量比較,差異有統(tǒng)計(jì)學(xué)意義[(42.73±14.94)U·L^-1,(77.33±15.54)U·L^-1,(95.49±26.12)U·L^-1,(124.85±25.34)U·L^-1,F=15.847,P=0.000]; 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000,P=0.010),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.026,P=0.001); 接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.015)。④藥物干預(yù)后骨折端骨組織形態(tài)。藥物干預(yù)7 d后,模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組骨 折端骨組織中可見少量成骨細(xì)胞聚集; 藥物干預(yù)14 d后成骨細(xì)胞顯著增多,且接骨續(xù)筋丸組及接骨七厘膠囊組的成骨細(xì)胞數(shù)量較模型對照組明顯增多; 藥物干預(yù)21 d后成骨細(xì)胞開始減少; 3組破骨細(xì)胞在藥物干預(yù)21 d后出現(xiàn)增長趨勢。⑤藥物干預(yù)后骨折端骨組織中BMP-2的表達(dá)量。藥物干預(yù)7 d后,4組大鼠骨折端骨組織中BMP-2表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(0.10±0.01,0.14±0.02,0.23±0.03,0.27±0.03,F=59.960,P=0.000); 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.018,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.015)。藥物干預(yù)14 d后,4組大鼠骨折端骨組織中BMP-2表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(0.12±0.02,0.15±0.02,0.24±0.03,0.28±0.03,F=47.588,P=0.000); 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.047,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.006)。藥物干預(yù)21 d后,4組大鼠骨折端骨組織中BMP-2表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(0.11±0.02,0.15±0.02,0.24±0.03,0.28±0.02,F=80.017,P=0.000); 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.004,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.004)。⑥藥物干預(yù)后骨折端骨組織中VEGF的表達(dá)量。藥物干預(yù)7 d后,4組大鼠骨折端骨組織中VEGF表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(0.11±0.02,0.14±0.02,0.26±0.01,0.27±0.04,F=69.567,P=0.000); 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.015,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組與接骨續(xù)筋丸組的差異無統(tǒng)計(jì)學(xué)意義(P=0.905)。藥物干預(yù)14 d后,4組大鼠骨折端骨組織中VEGF表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(0.11±0.02,0.15±0.02,0.25±0.03,0.28±0.02,F=72.334,P=0.000); 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.026,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組與接骨續(xù)筋丸組的差異無統(tǒng)計(jì)學(xué)意義(P=0.071)。藥物干預(yù)21 d后,4組大鼠骨折端骨組織中VEGF表達(dá)量比較,差異有統(tǒng)計(jì)學(xué)意義(0.11±0.02,0.15±0.02,0.24±0.02,0.27±0.05,F=39.739,P=0.000); 組間兩兩比較,假手術(shù)組低于模型對照組、接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000,P=0.000),模型對照組低于接骨七厘膠囊組、接骨續(xù)筋丸組(P=0.000,P=0.000),接骨七厘膠囊組低于接骨續(xù)筋丸組(P=0.029)。結(jié)論:接骨續(xù)筋丸促進(jìn)大鼠骨折愈合的作用機(jī)制可能是通過提高大鼠血清P、ALP的含量,促進(jìn)其骨組織中BMP-2及VEGF的表達(dá),從而促進(jìn)成骨細(xì)胞增殖,但其具體作用機(jī)制有待進(jìn)一步深入研究。

Abstract:

ABSTRACT Objective:To explore the mechanism of action of Jiegu Xujin Wan(接骨續(xù)筋丸,JGXJW)in promoting fracture healing in rats.Methods:Seventy-two 3-month-old male SD rats were randomly divided into sham-operated group,model group,Jiegu Qili Jiaonang(接骨七厘膠囊,JGQLJN)group and JGXJW group,18 cases in each group.The middle femoral shafts of rats in model group,JGQLJN group and JGXJW group were cut off to build open femoral fracture models,while the surgeries were performed on the rats in sham-operated group to expose their femoral shafts only.Since the 2nd day after the modeling operation,the rats in JGXJW group,JGQLJN group,model group and sham-operated group were intragastric administrated with JGXJW suspension(3 g/kg),JGQLJN suspension(0.52 g/kg)and distilled water respectively,once a day for 21 consecutive days.At 7,14 and 21 days after the beginning of drug intervention,6 rats were randomly selected from each group,and their blood were fetched out from aorta abdominalis.The upper serum was sucked from the blood samples after standing and centrifuging for further inspection.Then the rats were sacrificed and their left femoral shafts were fetched out for making femoral paraffin specimens.The serum contents of calcium(Ca),phosphorus(P)and alkaline phosphatase(ALP)were determined by using Ca,P and ALP assay kit.The morphological changes of bone tissues were observed after hematoxylin-eosin(HE)staining,and the expression of bone morphogenetic protein 2(BMP-2)and vascular endothelial growth factor(VEGF)in bone tissues of broken ends of fractured bone were detected by immunohistochemical method respectively.Results:There was no statistical difference in serum content of Ca between the 4 groups at 7,14 and 21 days after the beginning of drug intervention(1.80+/-1.03,1.88+/-0.80,1.91+/-0.68,1.83+/-0.62 mmol/l,F=0.023,P=0.995; 1.77+/-0.89,1.77+/-0.73,1.88+/-1.18,1.74+/-0.89 mmol/l,F=0.025,P=0.095; 1.72+/-0.98,1.74+/-0.61,1.80+/-0.90,1.69+/-0.79 mmol/l,F=0.019,P=0.996).At 7 days after the beginning of drug intervention,there was statistical difference in serum content of P between the 4 groups(1.89+/-0.56,1.97+/-0.53,3.23+/-0.90,4.24+/-0.68 mmol/l,F=16.041,P=0.000).Further pairwise comparison showed that there was no statistical difference in the serum content of P between sham-operated group and model group(P=0.846),and the serum content of P was lower in sham-operated group compared to JGQLJN group and JGXJW group(P=0.000,P=0.003),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.005),and was lower in JGQLJN group compared to JGXJW group(P=0.018).At 14 days after the beginning of drug intervention,there was statistical difference in serum content of P between the 4 groups(1.81+/-0.48,2.65+/-0.36,3.73+/-0.77,4.37+/-0.93 mmol/l,F=16.998,P=0.000).Further pairwise comparison showed that the serum content of P was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.045,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.012),and there was no statistical difference in serum content of P between JGQLJN group and JGXJW group(P=0.116).At 21 days after the beginning of drug intervention,there was statistical difference in serum content of P between the 4 groups(1.82+/-0.40,2.15+/-0.50,3.35+/-0.62,4.21+/-0.70 mmol/l,F=22.663,P=0.000).Further pairwise comparison showed that the serum content of P was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.036,P=0.000,P=0.000),and was lower in model group compard to JGQLJN group and JGXJW group(P=0.000,P=0.002),and was lower in JGQLJN group compared to JGXJW group(P=0.016).At 7 days after the beginning of drug intervention,there was statistical difference in serum content of ALP between the 4 groups(49.71+/-14.67,93.75+/-15.11,125.00+/-22.89,145.49+/-20.79 U/L,F=29.797,P=0.000).Further pairwise comparison showed that the serum content of ALP was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.000,P=0.000,P=0.001),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.001,P=0.000),and was lower in JGQLJN group compared to JGXJW group(P=0.009).At 14 days after the beginning of drug intervention,there was statistical difference in serum content of ALP between the 4 groups(41.57+/-10.69,91.13+/-10.76,111.77+/-19.66,149.42+/-12.71 U/L,F=62.354,P=0.000).Further pairwise comparison showed that the serum content of ALP was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.000,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and was lower in JGQLJN group compared to JGXJW group(P=0.019).At 21 days after the beginning of drug intervention,there was statistical difference in serum content of ALP between the 4 groups(42.73+/-14.94,77.33+/-15.54,95.49+/-26.12,124.85+/-25.34 U/L,F=15.847,P=0.000).Further pairwise comparison showed that the serum content of ALP was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.000,P=0.000,P=0.010),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.026,P=0.001),and was lower in JGQLJN group compared to JGXJW group(P=0.015).At 7 days after the beginning of drug intervention,a small number of osteoblasts can be found in bone tissues of broken ends of fractured bone in model group,JGQLJN group and JGXJW group.At 14 days after the beginning of drug intervention,there was a notable increase in the number of osteoblasts,and the number of osteoblasts were greater in JGXJW group and JGQLJN group compared to model group.At 21 days after the beginning of drug intervention,the number of osteoblasts began to decrease while the number of osteoclasts began to increase.At 7 days after the beginning of drug intervention,there was statistical difference in the expression of BMP-2 in bone tissues of broken ends of fractured bone between the 4 groups(0.10+/-0.01,0.14+/-0.02,0.23+/-0.03,0.27+/-0.03,F=59.960,P=0.000).Further pairwise comparison showed that the expression of BMP-2 in bone tissues was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.018,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and was lower in JGQLJN group compared to JGXJW group(P=0.015).At 14 days after the beginning of drug intervention,there was statistical difference in the expression of BMP-2 in bone tissues of broken ends of fractured bone between the 4 groups(0.12+/-0.02,0.15+/-0.02,0.24+/-0.03,0.28+/-0.03,F=47.588,P=0.000).Further pairwise comparison showed that the expression of BMP-2 in bone tissues was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.047,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and was lower in JGQLJN group compared to JGXJW group(P=0.006).At 21 days after the beginning of drug intervention,there was statistical difference in the expression of BMP-2 in bone tissues of broken ends of fractured bone between the 4 groups(0.11+/-0.02,0.15+/-0.02,0.24+/-0.03,0.28+/-0.02,F=80.017,P=0.000).Further pairwise comparison showed that the expression of BMP-2 in bone tissues was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.004,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and was lower in JGQLJN group compared to JGXJW group(P=0.004).At 7 days after the beginning of drug intervention,there was statistical difference in the expression of VEGF in bone tissues of broken ends of fractured bone between the 4 groups(0.11+/-0.02,0.14+/-0.02,0.26+/-0.01,0.27+/-0.04,F=69.567,P=0.000).Further pairwise comparison showed that the expression of VEGF in bone tissues was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.015,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and there was no statistical difference between JGQLJN group and JGXJW group(P=0.905).At 14 days after the beginning of drug intervention,there was statistical difference in the expression of VEGF in bone tissues of broken ends of fractured bone between the 4 groups(0.11+/-0.02,0.15+/-0.02,0.25+/-0.03,0.28+/-0.02,F=72.334,P=0.000).Further pairwise comparison showed that the expression of VEGF in bone tissues was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.026,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and there was no statistical difference between JGQLJN group and JGXJW group(P=0.071).At 21 days after the beginning of drug intervention,there was statistical difference in the expression of VEGF in bone tissues of broken ends of fractured bone between the 4 groups(0.11+/-0.02,0.15+/-0.02,0.24+/-0.02,0.27+/-0.05,F=39.739,P=0.000).Further pairwise comparison showed that the expression of VEGF in bone tissues was lower in sham-operated group compared to model group,JGQLJN group and JGXJW group(P=0.000,P=0.000,P=0.000),and was lower in model group compared to JGQLJN group and JGXJW group(P=0.000,P=0.000),and was lower in JGQLJN group compared to JGXJW group(P=0.029).Conclusion:JGXJW can increase the serum contents of P and ALP and promote the expression of BMP-2 and VEGF in bone tissues to promote the osteoblast proliferation,which may be the mechanisms of action for promoting fracture healing in rats.However,its specific mechanism of action needs to be further studied.

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通訊作者:侯德才 E-mail:[email protected]

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更新日期/Last Update: 2018-03-10