股骨头坏死愈胶囊联合重组人骨形态发生蛋白-2治疗激素性股骨头坏死的作用机制

Effect and Mechanism of Femoral Head Necrosis Healing Capsule Combined with H-BMP-2 on the Treatment of Steroid-Induced Femoral Head Necrosis

目的:探究股骨头坏死愈胶囊联合重组人骨形态发生蛋白-2(hBMP-2)治疗激素性股骨头坏死的机制。方法:将30只3周龄雌性SD大鼠采用随机数字表法分为空白组、模型组和骨愈组,每组10只,模型组和骨愈组采用臀肌注射甲基强的松龙40 mg/kg,阿莫西林溶液10 mg/kg,每48 h注射1次,连续注射8周。对照组注射等体积的生理盐水。造模8周后,骨愈组给予股骨头坏死愈胶囊水溶液0.4 g/kg灌胃,1次/d,连续8周,模型组大鼠给予体积生理盐水灌胃。MRI观察髋关节影像学改变和组织病理学观察。获取正常大鼠和造模大鼠骨髓间充质干细胞(BMSCs),分组培养:对照组、巨噬细胞系统(MPS)组、骨愈组、hBMP-2组和骨愈+hBMP-2组。碱性磷酸酶(ALP)和茜素红染色测定成骨分化情况,油红染色测定成脂分化情况。通过RT-qPCR和Western Blot法评估成骨相关基因的表达水平。结果:体内实验中,MRI扫描和组织学分析显示,造模成功且股骨头坏死愈胶囊可防止大鼠股骨头坏死模型的骨丢失。体外实验显示,股骨头坏死愈胶囊联合重组人骨形态发生蛋白-2共处理的骨髓间充质干细胞增殖、成骨分化能力明显优于MPS组,与对照组相比,差异无统计学意义(P>0.05)。股骨头坏死愈胶囊联合hBMP-2可使骨髓间充质干细胞成骨分化过程中碱性磷酸酶的表达和钙结节的形成提高,其成脂分化被抑制。Western Blot法结果显示,与MPS组相比,骨愈组和骨愈+hBMP-2组表达更高水平的BMP-2、Runx2和ALP,差异有统计学意义(P<0.05),表达更低水平的过氧化物酶增殖物激活受体α(PPARα),差异有统计学意义(P<0.05)。结论:中药股骨头坏死愈胶囊联合重组人骨形态发生蛋白-2,可显著改善骨髓间充质干细胞成脂和成骨之间平衡能力,促进成骨分化,可成为预防或延缓骨转移相关股骨头坏死进展的有效组合。

Objective:To investigate the mechanism of femoral head necrosis capsule combined with human bone morphogenetic protein-2(hBMP-2)in treating steroid-induced femoral head necrosis.Methods:303-week-old female SD rats were randomly divided into blank group,model group and femoral head necrosis group,with 10 rats in each group.The model group and femoral head necrosis group were injected with methylprednisolone(40 mg/kg)and amoxicillin solution(10 mg/kg)by intramuscular injection once every 48 h for 8 weeks.The control group was injected with physiological saline of the same volume.After modeling for 8 weeks,the rats in the femoral head necrosis group were given femoral head necrosis capsule solution(0.4 g/kg)by gavage once a day for 8 weeks,and the rats in the model group were given physiological saline by gavage.MRI was used to observe joint imaging changes,and histopathological observations were performed.Normal rat and model rat bone marrow mesenchymal stem cells(BMSCs)were obtained and divided into control group,MPS group,femoral head necrosis group,hBMP-2 group,and femoral head necrosis+hBMP-2 group for culture.Alkaline phosphatase and eosin red staining were used to determine osteogenic differentiation,and oil red staining was used to determine adipogenic differentiation.The expression levels of osteogenesis-related genes were evaluated by RT-qPCR and Western Blot.Results:In the in-vivo experiment,MRI scans and histological analysis showed that the model was successful and that femoral head necrosis capsule prevented bone loss in the rat femoral head necrosis model.In vitro experiment showed that compared with the MPS group,femoral head necrosis capsule combined with hBMP-2 significantly improved the proliferation and osteogenic differentiation ability of BMSCs,and there was no statistical difference compared with the control group.Femoral head necrosis capsule combined with hBMP-2 can enhance the expression of ALP and calcium nodules in BMSCs during osteogenic differentiation,and inhibit adipogenic differentiation.Western Blot results showed that femoral head necrosis group and femoral head necrosis+hBMP-2 group had higher levels of BMP-2,Runx2,and ALP expression(P<0.05),and lower PPARαexpression(P<0.05)compared with the MPS group.Conclusion:The combination of femoral head necrosis capsule and hBMP-2 can significantly improve the balance between adipogenic and osteogenic abilities of BMSCs,promote osteogenic differentiation,and can be an effective combination to prevent or delay the progression of bone metastasis related femoral head necrosis.