桃红四物汤联合二甲双胍对2型糖尿病合并骨质疏松大鼠骨折愈合的影响

Effect of Taohong Siwu Decoction combined with metformin on fracture healing in rats model of type 2 diabetes mellitus complicated with osteoporosis

关键词:  桃红四物汤  二甲双胍  骨质疏松  骨折  2型糖尿病

Key words:  Taohong Siwu Decoction  metformin  osteoporosis  fracture  type 2 diabetes mellitus

摘要:

[目的] 研究桃红四物汤联合二甲双胍对2型糖尿病（T2DM）合并骨质疏松（OP）大鼠骨折愈合的影响。[方法] 选取40只SPF级雌性大鼠随机分为正常组、模型组、桃红四物汤组和联合组，每组10只，除正常组外，其余3组大鼠均建立T2DM合并OP模型，4组大鼠均建立股骨骨折模型。正常组和模型组每日灌胃10 mL/kg生理盐水，桃红四物汤组每日灌胃6 g/kg桃红四物汤药液，联合组在此基础上灌胃100 mg/kg二甲双胍生理盐水混悬液。X射线检查各组大鼠骨折愈合情况；双能X线全身骨密度测量仪检测股骨骨密度（BMD）值；苏木精-伊红（HE）染色观察骨折组织形态；显微电子计算机断层扫描（microCT）扫描股骨标本检测各组大鼠骨痂相关指标[相对骨体积（BV）、骨小梁数（Tb.N）、骨小梁厚度（Tb.Th）、骨小梁分离度（Tb.Sp）、连接密度（Conn.D）、骨体积分数（BV/TV）和骨表面积体积比（BS/BV）]；采用力学实验仪检测股骨的生物力学性能；每周定时检测各组大鼠空腹血糖（FBG）；分离血清，采用酶联免疫吸附（ELISA）法检测各组大鼠血清中血管内皮生长因子（VEGF）和纤维蛋白原（FIB）的表达水平，采用速率法检测血清中碱性磷酸酶（ALP）和D-二聚体的表达水平。[结果] X射线可见桃红四物汤组和联合组骨折线不清晰，骨性骨痂连接于骨折端。与模型组相比，桃红四物汤组和联合组的BMD值提高（P<0.05）；与桃红四物汤组相比，联合组BMD值更高（P<0.05）。HE染色可见桃红四物汤组和联合组的骨性骨痂明显增多，软骨和纤维组织减少，编织骨和板层骨开始融合。桃红四物汤组和联合组的Tb.N、Tb.Th、Conn.D、BV/TV均高于模型组（P<0.05），Tb.Sp、BS/BV低于模型组（P<0.05）；与桃红四物汤组相比，联合组的Tb.N、Tb.Th、Conn.D、BV/TV均较高，BS/BV较低（P<0.05）。桃红四物汤组和联合组的最大载荷和最大载荷恢复率均高于模型组（P<0.05）；与桃红四物汤组相比，联合组最大荷载和最大载荷恢复率较高（P<0.05）。实验期间桃红四物汤组和联合组大鼠FBG平稳下降并接近正常组水平。与模型组相比，桃红四物汤组和联合组VEGF、FIB、ALP、D-二聚体的表达水平均降低（P<0.05）；与桃红四物汤组相比，联合组VEGF、FIB、D-二聚体的表达水平更低，ALP表达水平较高（P<0.05）。[结论] 桃红四物汤联合二甲双胍可有效降低血糖水平，提高BMD值和生物力学性能，改善骨痂的形成，降低VEGF、FIB、ALP和D-二聚体的表达水平，从而有效促进T2DM合并OP大鼠骨折的愈合。

Abstract:

[Objective] To investigate the effect of Taohong Siwu Decoction combined with metformin on fracture healing in rats model of type 2 diabetes mellitus (T2DM) complicated with osteoporosis (OP).[Methods] The 40 SPF female rats were randomly divided into normal group, control group, Taohong Siwu Decoction group and combination group, with 10 rats in each group. Model of T2DM complicated with OP fracture were established in all rats except for the normal group, the fracture model were established in all four groups. The normal group and the control group were given 10 ml/kg of normal saline everyday, the Taohong Siwu Decoction group was given 6 g/kg of Taohong Siwu Decoction everyday, and the combination group was given 100 mg/kg of metformin saline suspension on this basis. After the treatments, fracture healing states of all rats were inspected by X-ray;bone mineral density (BMD) of each rat was detected by dual energy X-ray absorptiometry (DEXA);bone histomorphologies were observed by HE staining;bone volume (BV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular resolution (Tb.Sp), junction density (Conn.D), bone volume fraction (BV/TV) and bone surface volume ratio (BS/BV) were measured by microCT;biomechanical properties of femoral were detected by mechanical tester;fasting blood glucose (FBG) of each rat was detected at the same time of every week during the experiment;serum vascular endothelial growth factor (VEGF), fibrinogen (FIB), alkaline phosphatase (ALP) and D-dimer were detected.[Results] The X-ray showed that the fracture lines of Taohong Siwu Decoction group and combination group wereblurry, bone callus connected to fracture end. As compared with control group, BMD of Taohong Siwu Decoction group and combination group were significantly increased (P<0.05), and combination group was higher than Taohong Siwu Decoction group (P<0.05). HE staining showed that osseous callus of Taohong Siwu Decoction group and combination group were increased, braided bone and lamellar bone began to fuse. Tb.N, Tb.Th, Conn.D and BV/TV of Taohong Siwu Decoction group and combination group were higher than control group (P<0.05), Tb.Sp and BS/BV were lower than control group (P<0.05);as compared with Taohong Siwu Decoction group, Tb.N, Tb.Th, Conn.D and BV/TV of combination group were higher and BS/BV was lower (P<0.05). Max loads of Taohong Siwu Decoction group and combination group were greater than control group (P<0.05);and max load of combination group was greater than Taohong Siwu Decoction group (P<0.05). FBG of Taohong Siwu Decoction group and combination group were decreased to close to normal group after the treatment. Compared with the control group, the expression levels of VEGF, FIB, ALP and D-dimer in the Taohong Siwu Decoction group and the combination group were significantly reduced(P<0.05). Compared with the Taohong Siwu Decoction group, the expression levels of VEGF, FIB, ALP and D-dimer in the combination group were significantly reduced (P<0.05). The expression level of FIB and D-dimer were lower, and the expression level of ALP were higher (P<0.05).[Conclusion] Taohong Siwu Decoction combined with metformin could effectively decrease the blood glucose level, increase the BMD and biomechanical property osteoporosis rats, improve the formation of callus, decrease the expression levels of serum VEGF, FIB, ALP and D-dimer, which promotes the fracture healing ofrats model of T2DM complicated with OP.