补肾活血方通过DLL4/Notch通路促进激素性股骨头坏死模型的修复作用及机制研究

Study on the Repairing Effect and Mechanism of Bushen Huoxue Recipe on Promoting Steroid-induced Osteonecrosis of the Femoral Head by DLL4/Notch Pathway

目的:探讨补肾活血方通过调控Delta样配体(DLL)4/Notch通路对激素性股骨头坏死(SIONFH)模型的修复作用。方法:Wistar大鼠随机分为正常对照组、模型组、阳性对照组(阿仑膦酸钠片,8.6 mg·kg^(-1))、补肾活血方组(644.47 mg·kg^(-1))和补肾活血方+Jagged1/Fc组(644.47 mg·kg^(-1)+13μg·kg^(-1)),每组15只。除正常对照组外的各组大鼠均采用肌注甲基泼尼松龙琥珀酸钠的方法建立SIONFH模型。苏木精伊红(HE)染色观察股骨头病理变化;微型计算机断层(Micro-CT)扫描观察骨体积分数(BV/TV)、骨小梁数量(Tb·N)、骨小梁模式因子(Tb·Pf)、骨小梁厚度(Tb·Th)、骨小梁分离度(Tb·Sp)、骨密度(BMD)和血管化情况;酶联免疫吸附法检测血清碱性磷酸酶(AKP)、骨钙素(OST)的活性及总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)含量;Western blot检测股骨头组织中Notch配体(Jagged1)、DLL4、Notch1、Notch细胞内片段(NICD)、发状分裂相关增强子1(HES1)蛋白表达水平。结果:与模型组相比,补肾活血方组大鼠股骨头坏死症状明显减轻,血管生成明显增加,空腔率、空腔面积、脂肪组织面积显著下降,骨小梁面积及BV/TV、Tb·N、Tb·Pf、Tb·Th和BMD显著升高,Tb·Sp显著降低,ALP、OST活性显著升高,TC、TG、LDL/HDL比值及Jagged1、DLL4、Notch1、NICD、HES1蛋白表达显著降低(P<0.05),阳性对照组的上述指标表达趋势与补肾活血方组一致,且两组之间差异无统计学意义(P>0.05)。添加Jagged1/Fc上调股骨头组织中Jagged1、DLL4、Notch1、NICD、HES1蛋白表达后,补肾活血方对SIONFH大鼠模型的修复作用被抑制,各项指标水平被逆转(P<0.05)。结论:补肾活血方可通过抑制DLL4/Notch通路减轻SIONFH模型的骨退化,进而对SIONFH模型产生修复作用。

Objective:To investigate the repairing effect of Bushen Huoxue Recipe on steroid-induced osteonecrosis of the femoral head(SIONFH)model by regulating DLL4/Notch pathway.Methods:Wistar rats were randomly divided into normal control group,model group,positive control group(groupalendronate sodium tablets,8.6 mg·kg^(-1)),Bushen Huoxue Recipe group(644.47 mg·kg^(-1))and Bushen Huoxue Recipe+Jagged1/Fc group(644.47 mg·kg^(-1)+13μg·kg^(-1))with 15 rats in each group.SIONFH model was established by intramuscular injection of methylprednisolone sodium succinate except the normal control group.Hematoxylin and eosin(HE)staining was performed to observe the pathological changes of the femoral head;micro-computed tomography(Micro-CT)scanning was performed to observe bone volume fraction(BV/TV),trabecular bone number(Tb·N),trabecular bone pattern factor(Tb·Pf),trabecular bone thickness(Tb·Th),trabecular bone separation(Tb·Sp),bone mineral density(BMD)and vascularization;ELISA was performed to detect serum alkaline phosphatase(ALP)and osteocalcin(OST)activities,and total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL)and high density lipoprotein(HDL)contents;Western blot was performed to detect the protein expression levels of Notch ligand(Jagged1),DLL4,Notch1,Notch intracellular fragment(NICD)and hair-mitation-related enhancer 1(HES1)in femoral head tissue.Results:Compared with those of the model group,the symptoms of osteonecrosis of the femoral head in Bushen Huoxue Recipe group were obviously relieved,the angiogenesis was obviously increased,and the cavity rate,cavity area and adipose tissue area were obviously decreased,the trabecular bone area and BV/TV,Tb·N,Tb·Pf,Tb·Th and BMD were obviously increased,Tb·Sp was obviously decreased,ALP and OST activities were obviously increased,the ratio of TC,TG,LDL/HDL and the protein expression of Jagged1,DLL4,Notch1,NICD and HES1 were obviously decreased(P<0.05),the expression trends of the above indexes in alendronate sodium tablets group were consistent with those in Bushen Huoxue Recipe group,and there were no significant differences between the two groups(P>0.05).After adding Jagged1/Fc to up-regulate the protein expression of Jagged1,DLL4,NOTCH1,NICD and HES1 in the femoral head tissue,the repairing effects of Bushen Huoxue Recipe on the SIONFH rat model were inhibited,and the level of each index was reversed(P<0.05).Conclusion:Bushen Huoxue Recipe can reduce the bone degeneration of SIONFH model by inhibiting DLL4/NOTCH pathway,and then producing a repairing effect on the SIONFH model.