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  

Improving effect and mechanism of modified Chaihu Guizhi Decoction on osteoporosis rats by regulating the Wnt/β⁃catenin signaling pathway

作　　者：郭中华[1] 史栋梁[1] 曹玉举 王云飞 杨少祥 秦合伟[3] 张仲博[1] 任博文[1] 周松林[4] 王俊杰[1] 王莉莎[1] 万小冠 GUO Zhonghua;SHI Dongliang;CAO Yuju;WANG Yunfei;YANG Shaoxiang;QIN Hewei;ZHANG Zhongbo;REN Bowen;ZHOU Songlin;WANG Junjie;WANG Lisha;WAN Xiaoguan(Department 1 of Bone Diseases,Henan Hospital of Traditional Chinese Medicine,Zhengzhou 450053;China.2.Department of Orthopaedics,Zhengzhou Traditional Chinese Medicine Orthopedics Hospital,Zhengzhou 450016;.3.Department of Rehabilitation,Henan Hospital of Traditional Chinese Medicine,Zhengzhou 450053;.4.Department of Pain,Henan Hospital of Traditional Chinese Medicine,Zhengzhou 450053;.5.Department of Orthopaedics,Zhengzhou Ninth People’s Hospital,Zhengzhou 450053)

机构地区：[1]河南省中医院骨病一科,郑州450053 [2]郑州中医骨伤病医院骨科,郑州450016 [3]河南省中医院康复科,郑州450053 [4]河南省中医院疼痛科,郑州450053 [5]郑州市第九人民医院骨科,郑州450053

出　　处：《中国比较医学杂志》2023年第10期15-22,共8页Chinese Journal of Comparative Medicine

基　　金：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);河南省中医药科学研究专项课题(2021JDZX2121)。

摘　　要：目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大鼠分组为Model组、CK组、高剂量CGD组(CGD-H组,20 g/kg)、低剂量CGD组(CGD-L组,5 g/kg)、戊酸雌二醇组(EV组,9 mg/kg)、DKK-1组(Wnt/β-catenin通路抑制剂,100 mg/kg)和CGD-H+DKK-1组(20 g/kg+100 mg/kg),每组12只。除CK组外,其他组大鼠均通过灌胃70 mg/kg维甲酸的方式构建骨质疏松症(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Wnt3a、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、Wnt3a、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、Wnt3a、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、Wnt3a、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。Objective To investigate the improving effect of modified Chaihu Guizhi Decoction(CGD)on osteoporosis rats by regulating the Wnt/β-catenin signaling pathway.Methods SD rats were assigned to Model,CK,high-dose CGD(CGD-H;20 g/kg),low-dose CGD(CGD-L;5 g/kg),and estradiol valerate(EV;9 mg/kg),Wnt/β-catenin pathway inhibitor(DKK-1,100 mg/kg),and CGD-H+DKK-1(20 g/kg+100 mg/kg)groups with 12 rats per group.Except for those in the CK group,rats were administered 70 mg/kg retinoic acid to establish the osteoporosis(OP)model,and rats in the CK group were administered the same amount of normal saline.From week 4 of modeling,the corresponding drug was administered for 4 weeks.ELISA were applied to measure serum levels of collagen type I C-terminal peptide(CTX-Ⅰ)and osteocalcin(BGP).Changes in the bone volume fraction,bone trabecular thickness,bone mineral density,and bone trabecular number were observed.HE staining was applied to assess pathological changes in the rat femur.Alkaline phosphatase(ALP)calcium-cobalt staining was applied to detect osteoblast activity in the rat femur.Osteoclast activity in rat femur tissue was detected by tartrate-resistant acid phosphatase(TRAP)staining.Western blot was applied to detect Wnt3a andβ-catenin proteins in femoral tissue.Results Compared with the CK group,femoral tissue of the Model group had severe pathological damage,the CTX-Ⅰlevel and osteoclast activity were increased,and the BGP level,bone volume fraction,bone trabecular thickness,bone mineral density,bone trabecular number,osteoblast activity,and Wnt3a andβ-catenin protein expression were decreased(P<005).Compared with the Model group,pathological damage of the femur in CGD-L,CGD-H,and EV groups was alleviated,the CTX-Ⅰlevel and osteoclast activity were decreased,the BGP level,bone volume fraction,trabecular thickness,bone mineral density,bone trabecular number,osteoblast activity,and Wnt3a andβ-catenin protein expression were increased,and the opposite trends of the corresponding indicators were observed in the DKK

关 键 词：柴胡桂枝汤加减 Wnt/β-连环蛋白信号通路 骨质疏松症 成骨细胞 破骨细胞 

分 类 号：R-33[医药卫生]