【摘要】 目的:探讨接骨续筋丸促进大鼠骨折愈合的作用机制。方法:将72只3月龄雄性SD大鼠随机分为假手术组、模型对照组、接骨七厘胶囊组、接骨续筋丸组,每组18只。截断模型对照组、接骨七厘胶囊组、接骨续筋丸组大鼠股骨干中段,制作股骨开放性骨折模型;假手术组仅暴露股骨干,不做截骨。造模后第2天开始灌胃,接骨续筋丸组以3 g·kg^-1剂量的接骨续筋丸混悬液灌胃,接骨七厘胶囊组以0.52 g·kg^-1剂量的接骨七厘胶囊混悬液灌胃,模型对照组和假手术组给予蒸馏水灌胃;每天灌胃1次,各组连续灌胃21 d。分别于药物干预7 d、14 d、21 d后取材,各组随机选取6只大鼠从腹主动脉抽血,经静置、离心后吸取上层血清,保存待检;抽血完成后,取出大鼠左侧股骨干,制备骨折端股骨石蜡标本。分别采用钙（calcium,Ca）、磷（phosphorus,P）、血清碱性磷酸酶（alkaline phosphatase,ALP）测试盒测定大鼠血清Ca、P、ALP含量,采用苏木精-伊红（HE）染色观察骨组织形态学变化,采用免疫组化法检测骨折端骨组织中骨形态发生蛋白2（bone morphogenetic protein 2,BMP-2）和血管内皮生长因子（vascular endothelial growth factor,VEGF）的表达。结果:（1）药物干预后血清Ca含量。药物干预7 d、14 d、21 d后,4组大鼠血清Ca含量比较,组间差异均无统计学意义[（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）药物干预后血清P含量。药物干预7 d后,4组大鼠血清P含量比较,差异有统计学意义[（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];组间两两比较,假手术组与模型对照组的差异无统计学意义（P=0.846）,假手术组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.003）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.005）,接骨七厘胶囊组低于接骨续筋丸组（P=0.018）。药物干预14 d后,4组大鼠血清P含量比较,差异有统计学意义[（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];组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.045,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.012）,接骨七厘胶囊组与接骨续筋丸组的差异无统计学意义（P=0.116）。药物干预21 d后,4组大鼠血清P含量比较,差异有统计学意义[（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];组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.036,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.002）,接骨七厘胶囊组低于接骨续筋丸组（P=0.016）。（3）药物干预后血清ALP含量。药物干预7 d后,4组大鼠血清ALP含量比较,差异有统计学意义[（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];组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000,P=0.001）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.001,P=0.000）,接骨七厘胶囊组低于接骨续筋丸组（P=0.009）。药物干预14 d后,4组大鼠血清ALP含量比较,差异有统计学意义[（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];组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组低于接骨续筋丸组（P=0.019）。药物干预21 d后,4组大鼠血清ALP含量比较,差异有统计学意义[（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];组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000,P=0.010）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.026,P=0.001）;接骨七厘胶囊组低于接骨续筋丸组（P=0.015）。（4）药物干预后骨折端骨组织形态。药物干预7 d后,模型对照组、接骨七厘胶囊组、接骨续筋丸组骨折端骨组织中可见少量成骨细胞聚集;药物干预14 d后成骨细胞显著增多,且接骨续筋丸组及接骨七厘胶囊组的成骨细胞数量较模型对照组明显增多;药物干预21 d后成骨细胞开始减少;3组破骨细胞在药物干预21 d后出现增长趋势。（5）药物干预后骨折端骨组织中BMP-2的表达量。药物干预7 d后,4组大鼠骨折端骨组织中BMP-2表达量比较,差异有统计学意义（0.10±0.01,0.14±0.02,0.23±0.03,0.27±0.03,F=59.960,P=0.000）;组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.018,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组低于接骨续筋丸组（P=0.015）。药物干预14 d后,4组大鼠骨折端骨组织中BMP-2表达量比较,差异有统计学意义（0.12±0.02,0.15±0.02,0.24±0.03,0.28±0.03,F=47.588,P=0.000）;组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.047,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组低于接骨续筋丸组（P=0.006）。药物干预21 d后,4组大鼠骨折端骨组织中BMP-2表达量比较,差异有统计学意义（0.11±0.02,0.15±0.02,0.24±0.03,0.28±0.02,F=80.017,P=0.000）;组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.004,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组低于接骨续筋丸组（P=0.004）。（6）药物干预后骨折端骨组织中VEGF的表达量。药物干预7 d后,4组大鼠骨折端骨组织中VEGF表达量比较,差异有统计学意义（0.11±0.02,0.14±0.02,0.26±0.01,0.27±0.04,F=69.567,P=0.000）;组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.015,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组与接骨续筋丸组的差异无统计学意义（P=0.905）。药物干预14 d后,4组大鼠骨折端骨组织中VEGF表达量比较,差异有统计学意义（0.11±0.02,0.15±0.02,0.25±0.03,0.28±0.02,F=72.334,P=0.000）;组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.026,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组与接骨续筋丸组的差异无统计学意义（P=0.071）。药物干预21 d后,4组大鼠骨折端骨组织中VEGF表达量比较,差异有统计学意义（0.11±0.02,0.15±0.02,0.24±0.02,0.27±0.05,F=39.739,P=0.000）;组间两两比较,假手术组低于模型对照组、接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000,P=0.000）,模型对照组低于接骨七厘胶囊组、接骨续筋丸组（P=0.000,P=0.000）,接骨七厘胶囊组低于接骨续筋丸组（P=0.029）。结论:接骨续筋丸促进大鼠骨折愈合的作用机制可能是通过提高大鼠血清P、ALP的含量,促进其骨组织中BMP-2及VEGF的表达,从而促进成骨细胞增殖,但其具体作用机制有待进一步深入研究。更多还原

【Abstract】 Objective: To explore the mechanism of action of Jiegu Xujin Wan( 接骨续筋丸,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( 接骨七厘胶囊,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 2 nd 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 bonebetween 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.更多还原