瑞香素调节缺氧诱导因子-1α/血管内皮生长因子信号通路对胫骨骨折大鼠骨痂形成和血管生成的影响实验研究

Effect of daphnetin regulating HIF-1α/VEGF signaling pathway on callus formation and angiogenesis in tibial fracture rats

目的:探究瑞香素对胫骨骨折大鼠骨痂形成、血管生成及缺氧诱导因子-1α(HIF-1α)/血管内皮生长因子(VEGF)信号通路的影响。方法:构建胫骨骨折大鼠模型,将120只胫骨骨折大鼠随机分成模型组、瑞香素低剂量组(30 mg/kg)、瑞香素中剂量组(60 mg/kg)、瑞香素高剂量组(90 mg/kg)剂量组、瑞香素高剂量+HIF-1α抑制剂YC-1组(90 mg/kg瑞香素+10 mg/kg YC-1),每组24只。另取24只正常大鼠仅暴露胫骨,穿入克氏针,不切断胫骨,设为假手术组。各组大鼠进行相应给药干预8周。X射线观察骨痂形成情况,并计算骨痂体积。微计算机断层扫描(Micro-CT)分析骨折折断处骨痂组织和假手术组相同部位骨组织骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)和骨密度(BMD)。HE染色观察胫骨骨折处骨组织及假手术组大鼠相同部位骨组织形态学变化,并计算新生血管数量及血管面积。酶联免疫吸附法(ELISA)检测血清中碱性磷酸酶(ALP)、骨钙素(OCN)水平。蛋白印迹实验检测胫骨骨折处骨痂组织及假手术组骨膜组织HIF-1α和VEGF蛋白表达。结果:与假手术组比较,模型组胫骨骨组织破坏严重,骨小梁稀疏紊乱,成骨细胞数量明显减少,BV/TV、Tb.N、Tb.Th、BMD、新生血管数量及血管面积、HIF-1α和VEGF蛋白表达水平降低,ALP、OCN水平升高(均P<0.05)。与模型组比较,瑞香素低、中、高剂量组骨组织中骨痂依次增多,骨小梁生成增加且排列有序,毛细血管逐渐丰富,聚集的成骨细胞数量依次增加,骨组织破坏减轻,骨痂体积、BV/TV、Tb.N、Tb.Th、BMD、新生血管数量及血管面积、ALP、OCN、HIF-1α和VEGF蛋白表达水平依次升高(均P<0.05)。与瑞香素高剂量组比较,瑞香素高剂量+YC-1组骨痂、骨小梁生成、毛细血管、成骨细胞数量减少,骨组织破坏加重,骨痂体积、BV/TV、Tb.N、Tb.Th、BMD、新生血管数量及血管面积、ALP、OCN、HIF-1α和VEGF蛋白表达水平降低(均P<0.05)。结论:瑞香素可能通过激活HIF-1α/VEGF信号通路促进胫骨骨折大鼠骨痂形成、血管生成,从而加速骨愈合。

Objective:To investigate the effects of daphnetin on callus formation,angiogenesis and hypoxia inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)signaling pathway in rats with tibial fracture.Methods:A rat model of tibial fracture was constructed,120 rats with tibial fracture were randomly divided into model group,daphnetin low(30 mg/kg),medium(60 mg/kg),high(90 mg/kg)dose group,and daphnetin high dose+HIF-1αinhibitor YC-1 group(90 mg/kg daphnetin+10 mg/kg YC-1),with 24 animals in each group.Another 24 normal rats were selected as sham operation group with only tibia exposed and the kirschner wire inserted without cutting the tibia.The rats in each group were given corresponding drug intervention for 8 weeks.The formation of bone callus was observed by X-ray,and the volume of bone callus was calculated.Bone volume fraction(BV/TV),bone trabecular number(Tb.N),bone trabecular thickness(Tb.Th)and bone mineral density(BMD)were analyzed by Micro-CT.HE staining was used to observe the morphological changes of bone tissue in tibial fracture and bone tissue in the same part of rats in sham operation group,and the number of new vessels and the area of blood vessels were calculated.The serum levels of alkaline phosphatase(ALP)and osteocalcin(OCN)were detected by ELISA.The expressions of HIF-1αand VEGF protein in bone callus tissue at the site of tibial fracture and periosteal tissue in the sham operation group were detected by Western blot.Results:Compared with sham operation group,tibial bone tissue was seriously damaged,bone trabeculae were sparse and disregulated,the number of osteoblasts was significantly reduced in model group,the BV/TV,Tb.N,Tb.Th,BMD,number of new vessels and vascular area,HIF-1αand VEGF protein expression levels were significantly decreased,and the levels of ALP and OCN were significantly increased(all P<0.05).Compared with model group,the bone callus in daphnetin low,medium and high dose groups increased successively,the formation of bone trabeculae increased and arranged in an orderly manner,the capillaries gradually enriched,the number of osteoblasts increased significantly successively,and the bone tissue destruction was reduced,volume of bone callus,BV/TV,Tb.N,Tb.Th,BMD,number of new vessels and vascular area,ALP,OCN,HIF-1αand VEGF protein expression levels were increased successively(all P<0.05).Compared with daphnetin high dose group,the number of bone callus,bone trabecular formation,capillaries and osteoblasts in the daphnetin high dose+YC-1 group decreased significantly,and the bone tissue destruction increased,volume of bone callus,BV/TV,Tb.N,Tb.Th,BMD,number of new vessels and vascular area,ALP,OCN,HIF-1αand VEGF protein expression levels were significantly decreased(all P<0.05).Conclusion:Daphnetin may promote callus formation and angiogenesis in rats with tibial fractures by activating HIF-1α/VEGF signaling pathway,thus accelerating bone healing.