补肾益气化瘀冲剂促进骨质疏松大鼠骨折术后骨痂血管形成的作用机制

Mechanism by which Bushen Yiqi Huayu granule improves callus angiogenesis in osteoporotic rats after fracture operation

背景:补肾益气化瘀冲剂具有益气化瘀、补肾通络的功效,常用于治疗骨质疏松,目前关于补肾益气化瘀冲剂对骨质疏松性骨折骨痂血管形成的影响研究较少。目的:探究补肾益气化瘀冲剂上调细胞外调节蛋白激酶/丝裂原活化蛋白激酶(extracellular signal-regulated kinase/mitogen-activated protein kinases,ERK/MAPK)信号通路改善骨质疏松大鼠骨折术后骨痂血管形成的具体机制。方法:①收集骨质疏松SD大鼠骨髓间充质干细胞、C57BL/6小鼠骨髓单核细胞,MTT法检测不同剂量补肾益气化瘀冲剂对骨髓间充质干细胞的毒性。分别使用添加0,1.5 mg/mL补肾益气化瘀冲剂的培养基培养骨髓间充质干细胞、骨髓单核细胞,进行体外成骨分化、破骨分化实验。②144只SD大鼠随机分为假手术组、模型组、冲剂组、冲剂+PD98059组,每组36只,后3组切除双侧卵巢建立骨质疏松模型,假手术组仅切除卵巢附近部分脂肪组织。8周后所有大鼠接受左侧胫骨横断截骨,冲剂+PD98059组给予5 g/kg补肾益气化瘀冲剂灌胃,尾静脉注射0.3 mg/kg PD98059(ERK1/2抑制剂);冲剂组给予5 g/kg补肾益气化瘀冲剂灌胃,尾静脉注射等体积生理盐水;假手术组、模型组给予等体积生理盐水,尾静脉注射等体积生理盐水;每日给药1次,持续8周。X射线、微型计算机断层扫描、微血管灌注造影观察骨折愈合与骨痂血管生成情况,检测骨痂骨密度与机械强度,苏木精-伊红、Masson染色观察胫骨组织形态,Western blotting、免疫组化检测ERK/MAPK信号通路相关分子表达情况。结果与结论:①与0 mg/mL补肾益气化瘀冲剂组相比,1.5 mg/mL补肾益气化瘀冲剂组成骨细胞碱性磷酸酶活性、矿化水平、p-ERK1/2、p-p38 MAPK表达水平升高(P<0.05),破骨细胞密度、体积减小(P<0.05);②与假手术组相比,冲剂组大鼠第4,8周骨折愈合程度相近,Lane-Sandhu评分、愈伤组织总体积、矿化愈伤组织体积、矿化愈伤组织体积/愈伤组织总体积、骨小梁厚度、血管数量、间距、厚度、血管体积/总体积、骨密度、骨痂极限荷载、刚度、p-ERK1/2、p-p38 MAPK、p-IκB-α、p-p65表达水平差异无显著性意义(P>0.05),模型组、冲剂+PD98059组大鼠骨折愈合缓慢,Lane-Sandhu评分、愈伤组织总体积、矿化愈伤组织体积、矿化愈伤组织体积/愈伤组织总体积、骨小梁厚度、血管数量、厚度、血管体积/总体积、骨密度、骨痂极限荷载、刚度、p-ERK1/2、p-p38 MAPK、p-IκB-α、p-p65表达水平降低(P<0.05),血管间距增加(P<0.05);③提示补肾益气化瘀冲剂能够通过增强ERK/MAPK信号通路相关分子表达改善骨质疏松大鼠骨折愈合情况,促进骨痂血管形成,减轻骨质疏松症状。

BACKGROUND:Bushen Yiqi Huayu granule has the effect of replenishing qi and eliminating blood stasis,tonifying kidney and smoothing collages,and is often used in the treatment of osteoporosis.At present,there are few studies on the effect of Bushen Yiqi Huayu granule on the callus angiogenesis of osteoporotic fractures.OBJECTIVE:To explore the mechanism of Bushen Yiqi Huayu granule by up-regulating extracellular signal-regulated kinase/mitogen-activated protein kinases(ERK/MAPK)signaling pathway to improve callus angiogenesis after fracturing in osteoporotic rats.METHODS:(1)Bone marrow mesenchymal stem cells from osteoporotic SD rats and bone marrow monocytes from C57BL/6 mice were collected.MTT assay was used to detect different doses of Bushen Yiqi Huayu granule on bone mesenchymal stem cell toxicity.Bone mesenchymal stem cells and bone marrow monocytes were cultured in the medium supplemented with 0 and 1.5 mg/mL Bushen Yiqi Huayu granules,respectively,and osteogenic differentiation and osteoclast differentiation were carried out in vitro.(2)144 SD rats were randomly divided into sham operation group,model group,granule group and granule+PD98059 group with 36 rats in each group.The osteoporotic model was established in the model group,granule group and granule+PD98059 group,and only part of the adipose tissue near the ovary was removed in the sham operation group.8 weeks later,all rats received left tibial osteotomy.In the granule+PD98059 group,5 g/kg Bushen Yiqi Huayu granule was given intragastrically,and 0.3 mg/kg PD98059 was injected into the tail vein.The granule group was given 5 g/kg Bushen Yiqi Huayu granule,and the same volume of normal saline was injected into the tail vein.The sham operation group and model group were given an equal volume of normal saline,and the caudal vein was injected with an equal volume of normal saline.Drug administration was conducted once a day for 8 weeks.Fracture healing and callus angiogenesis were observed by X-ray,micro-computed tomography and microvascular perfusion angiography.Bone mineral density and mechanical strength of the callus were measured.The tibia was observed by hematoxylin-eosin staining and Masson staining.Western blot assay and immunohistochemistry were used to examine the expression of ERK/MAPK signaling pathway-related molecules.RESULTS AND CONCLUSION:(1)Compared with the 0 mg/mL group,the alkaline phosphatase activity,mineralization level,p-ERK1/2 and p-p38 MAPK expression levels of osteocytes increased(P<0.05),while the density and volume of osteoclasts decreased in the 1.5 mg/mL group(P<0.05).(2)Compared with the sham operation group,the fracture healing degree of the granule group was similar at 4 and 8 weeks,but there was no significant difference in Lane-Sandhu score,total callus volume,mineralized callus volume,mineralized callus volume/total callus volume,trabecular thickness,vascular number,spacing,thickness,vascular volume/total volume,bone mineral density,ultimate load of callus,stiffness,p-ERK1/2,p-p38 MAPK,p-IκB-α,and p-p65 expression levels(P>0.05).In the model group and granule+PD98059 group,fracture healing was slow,Lane-Sandhu score,total callus volume,mineralized callus volume,mineralized callus volume/total callus volume,trabecular thickness,vascular number,thickness,vascular volume/total volume,bone mineral density,ultimate load of callus,stiffness,p-ERK1/2,p-p38 MAPK,p-IκB-α,and p-p65 expression levels decreased(P<0.05),and vascular spacing increased(P<0.05),compared with the sham operation group.(3)It is indicated that Bushen Yiqi Huayu granule can improve fracture healing,promote callus angiogenesis and alleviate the symptoms of osteoporosis by enhancing the expression of the ERK/MAPK signaling pathway-related molecules in osteoporotic rats.