柴胡桂枝汤加减通过调节Wnt/β-catenin信号通路对骨质疏松大鼠的改善作用及机制研究
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1.河南省中医院;2.郑州中医骨伤病医院

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.2021年河南省中医药拔尖人才培养项目资助【豫卫中医函(2021)15号】 2.河南省中医药科学研究专项课题(2021JDZX2121)


The improvement effect and mechanism of modified Chaihu Guizhi Decoction on osteoporosis rats by regulating Wnt/β-catenin signaling pathway
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1.Henan Hospital of Traditional Chinese medicine;2.Zhengzhou Hospital of Traditional Chinese medicine

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    摘要:

    的:探讨柴胡桂枝汤加减(CGD)调节Wnt/β-连环蛋白(β-catenin)信号通路对骨质疏松大鼠的改善作用。方法:将SD大鼠分为CK组、Model组、低剂量CGD组(CGD-L组,5 g/kg)、高剂量CGD组(CGD-H组,20 g/kg)、戊酸雌二醇组(EV组,9 mg/kg)、DKK-1(Wnt/β-catenin通路抑制剂)组(100 mg/kg)、CGD-H DKK-1组(20 g/kg 100 mg/kg),每组12只。除CK组外,其它组大鼠均通过灌胃70 mg/kg维甲酸的方式构建OP大鼠模型,CK组大鼠灌胃等量的生理盐水。造模4周开始给予相应的药物干预4周。ELISA法检测大鼠血清中Ⅰ型胶原C端肽( CTX-Ⅰ)、骨钙素(BGP)水平;观察股骨微观结构相关指标骨体积分数、骨小梁厚度、骨密度、骨小梁数量的变化;HE染色检测大鼠股骨病理变化;碱性磷酸酶(ALP)钙-钴染色检测大鼠股骨中成骨细胞活性;抗酒石酸酸性磷酸酶(TRAP)染色检测大鼠股骨组织中破骨细胞活性;western blot检测各组大鼠股骨组织中Wnt、β-catenin蛋白。结果:与CK组比较,Model组大鼠股骨组织病理损伤严重,CTX-Ⅰ水平、破骨细胞活性升高,BGP水平、骨体积分数、骨小梁厚度、骨密度、骨小梁数量、成骨细胞活性、Wnt、β-catenin蛋白表达降低(P<0.05);与Model组比较,CGD-L组、CGD-H组、EV组大鼠股骨组织病理损伤减轻,CTX-Ⅰ水平、破骨细胞活性降低,BGP水平、骨体积分数、骨小梁厚度、骨密度、骨小梁数量、成骨细胞活性、Wnt、β-catenin蛋白表达升高,而DKK-1组对应指标变化趋势与上述相反(P<0.05);与CGD-H组比较,CGD-H DKK-1组大鼠股骨组织病理损伤严重,CTX-Ⅰ水平、破骨细胞活性升高,BGP水平、骨体积分数、骨小梁厚度、骨密度、骨小梁数量、成骨细胞活性、Wnt、β-catenin蛋白表达降低(P<0.05)。结论:CGD可能通过激活Wnt/β-Catenin通路改善大鼠OP。

    Abstract:

    Objective: To investigate the improvement effect of modified Chaihu Guizhi Decoction (CGD) on osteoporosis rats by regulating the Wnt/β-catenin signaling pathway. Methods: SD rats were separated into CK group, Model group, low-dose CGD group (CGD-L group, 5 g/kg), high-dose CGD group (CGD-H group, 20 g/kg), and estradiol valerate group (EV group, 9 mg/kg), DKK-1 (Wnt/β-catenin pathway inhibitor) group (100 mg/kg), and CGD-H DKK-1 group (20 g/kg 100 mg/kg), 12 per group. Except for the CK group, the rats in other groups were given 70 mg/kg retinoic acid to build the OP rat model, and the rats in the CK group were given the same amount of normal saline. From the 4th week of modeling, the corresponding drug intervention was given for 4 weeks. ELISA was applied to detect the levels of collagen type I C-terminal peptide (CTX-Ⅰ) and osteocalcin (BGP) in serum of rats; the changes of bone volume fraction, bone trabecular thickness, bone mineral density and bone trabecular number were observed; HE staining was applied to detect the pathological changes of rat femur; Alkaline phosphatase (ALP) calcium-cobalt staining was applied to detect osteoblast activity in rat femur; the activity of osteoclasts in rat femur tissue was detected by tartrate-resistant acid phosphatase (TRAP) staining; Western blot was applied to detect Wnt and β-catenin proteins in femoral tissue of rats in each group. Results: Compared with the CK group, the femoral tissue of the Model group had severe pathological damage, CTX-I level and osteoclast activity increased, BGP level, bone volume fraction, bone trabecular thickness, bone mineral density, bone trabecular number, osteoblast activity, Wnt, β-catenin protein expression decreased (P<0.05); compared with the Model group, the pathological damage of the femur in the CGD-L group, the CGD-H group and the EV group was alleviated, CTX-I level and osteoclast activity decreased, BGP level, bone volume fraction, trabecular thickness, bone mineral density, bone trabecular number, osteoblast activity, Wnt, β-catenin protein expression increased, the trends of the corresponding indicators in the DKK-1 group were opposite to the above (P<0.05); compared with the CGD-H group, the femoral tissue of the CGD-H DKK-1 group was severely damaged, CTX-I level and osteoclast activity increased, BGP level, bone volume fraction, bone trabecular thickness, bone mineral density, bone trabecular number, osteoblast activity, Wnt, β-catenin protein expression decreased (P<0.05). Conclusion: CGD may improve OP of rats by activating the Wnt/β-Catenin pathway.

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  • 收稿日期:2022-11-17
  • 最后修改日期:2023-08-12
  • 录用日期:2023-10-09
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