基于VEGF/VEGFR-2信号通路研究淫羊藿苷促进大鼠胫骨干骨折愈合的作用机制

[目的]基于血管内皮生长因子/血管内皮生长因子受体-2(vascular endothelial growth factor/vascular endothelial growth factor receptor-2,VEGF/VEGFR-2)信号通路研究淫羊藿苷(icariin,ICA)对大鼠胫骨干骨折愈合的影响。[方法]将41只胫骨干骨折模型SD大鼠随机分为对照组,ICA低、高剂量组,每组各10只,另设阳性药物组,大鼠11只。ICA低、高剂量组以40、80mg/(kg·d)的ICA腹腔注射,阳性药物组以0.4mL复方骨肽注射液腹腔注射。对照组以等量0.9%氯化钠溶液腹腔注射,各组均为1次/d,连续21d。干预第7、14、21天进行微计算机断层扫描技术(micro-computed tomography,Mirco-CT)活体扫描,计算样本骨体积分数(bone volume/total volume,BV/TV),骨小梁数量(trabecular number,Tb.N)。脱颈处死大鼠,进行下肢生物力学测试,采用苏木精-伊红(hematoxylin-eosin,HE)染色观察骨痂组织血管新生情况,实时荧光定量聚合酶链式反应(quantitative Real-time polymerase chain reaction,QRT-PCR)检测骨痂组织VEGF、VEGFR-2 mRNA相对表达量,Western blot检测骨痂组织VEGF、VEGFR-2蛋白相对表达量。[结果]与对照组比较,ICA低、高剂量组、阳性药物组BV/TV更高,Tb.N更多,最大载荷更大,新生血管数量增多,面积也更大,VEGF、VEGFR-2 mRNA及蛋白相对表达量较高(P<0.05);与ICA低剂量组比较,ICA高剂量组、阳性药物组BV/TV更高,Tb.N更多,最大载荷更大,新生血管数量增多,面积也更大,VEGF、VEGFR-2 mRNA及蛋白相对表达量较高(P<0.05);与ICA高剂量组比较,阳性药物组BV/TV更高,Tb.N更多,最大载荷更大,新生血管数量增多,面积也更大,VEGF、VEGFR-2 m RNA及蛋白相对表达量较高(P<0.05)。[结论]ICA可加快胫骨干骨折大鼠骨量新生,促进血管形成,增强抵抗外力冲击的能力,且该效应呈剂量依赖性,推测可能与上调VEGF、VEGFR-2表达有关。

Mechanism of Icariin in Healing of Tibial Shaft Fracture in Rats Based on VEGF/VEGFR-2 Signaling Pathway

[Objective] To study the effect of icariin(ICA) on the healing of tibial shaft fractures in rats based on the vascular endothelial growth factor/vascular endothelial growth factor receptor-2(VEGF/VEGFR-2) signaling pathway. [Methods]Forty-one tibial shaft fracture model SD rats were randomly divided into control group, ICA low-dose and high-dose groups, with 10 in each, and 11 in positive drug group. ICA low and high-dose groups were injected intraperitoneally with 40, 80 mg/(kg·d) ICA, and positive drug group was injected intraperitoneally with 0.4 mL of compound Osteopeptide injection. Control group was injected intraperitoneally with the same amount of 0.9% sodium chloride solution, 1 time/d, continuous for 21 days. On the7 th, 14 th, and 21 st day of the intervention, micro-computed tomography(Mirco-CT) biopsy was performed, the sample bone volume/total volume(BV/TV) and trabecular number(Tb.N) was calculated. After the last Mirco-CT in vivo scan, the rats were sacrificed by cervical dislocation and biomechanical testing of lower limbs was carried out, the angiogenesis of callus tissue was observed by hematoxylin-eosin(HE) staining. The relative expression of VEGF and VEGFR-2 mRNA of callus tissue was detected by quantitative Real-time polymerase chain reaction(QRT-PCR), and the relative expression of VEGF and VEGFR-2 protein of callus tissue was detected by Western blot. [Results] Compared with control group, ICA low, high-dose group and positive drug group had higher BV/TV, Tb.N, maximum load, number of new blood vessels, larger area of new blood vessels and higher relative expression of VEGF and VEGFR-2 mRNA and protein(P<0.05). Compared with ICA low-dose group, ICA high-dose group and positive drug group had higher BV/TV, Tb.N, maximum load, number of new blood vessels, larger area of new blood vessels, and higher relative expression of VEGF and VEGFR-2 mRNA and protein(P<0.05). Compared with ICA high-dose group, positive drug group had higher BV/TV, Tb.N, maximum load, number of new blood vessels, larger area of new blood vessels, and higher relative expression of VEGF and VEGFR-2 m RNA and protein(P<0.05). [Conclusion]ICA can accelerate the bone regeneration of tibial shaft fracture rats, promote the formation of blood vessels, enhance the ability to resist external force shock, and the effect is dose-dependent. It is speculated that it may be related to the up-regulation of VEGF and VEGFR-2 expression.