摘要
目的:探究补骨膏对绝经后骨质疏松症(PMOP)大鼠的骨保护作用及其对骨保护素(OPG)-核因子κB受体激活蛋白(RANK)-核因子κB受体激活蛋白配体(RANKL)信号通路的调控作用。方法:将36只大鼠随机分为假手术组(9只)和手术组(27只),手术组采用双侧卵巢切除建立PMOP大鼠模型。造模成功后,将24只PMOP大鼠随机分为模型组、戊酸雌二醇组、补骨膏低剂量组、补骨膏高剂量组,然后予相应药物灌胃8周。骨密度仪检测股骨近端骨密度;Micro-CT三维重建分析股骨微结构;苏木精-伊红(HE)染色观察股骨组织病理形态;酶联免疫吸附试验(ELISA)法检测血清中骨碱性磷酸酶(BALP)、骨钙素(BGP)、OPG水平,测定盒检测血清中磷、钙水平;蛋白质印迹法(Western blotting)检测股骨组织中OPG、RANK、RANKL蛋白表达水平;RT-qPCR法检测股骨组织肿瘤坏死因子-α(TNF-α)mRNA、干扰素-γ(IFN-γ)mRNA、精氨酸酶-1(Arg-1)mRNA、转化生长因子-β1(TGF-β1)mRNA、基质金属蛋白酶-9(MMP-9)mRNA、OPG mRNA、RANK mRNA、RANKL mRNA表达水平。结果:假手术组大鼠股骨结构连续完整,骨小梁数目较多,形态较厚,结构致密;模型组大鼠股骨近端骨密度明显降低;补骨膏低剂量组、补骨膏高剂量组和戊酸雌二醇组大鼠股骨近端骨小梁数量、骨组织形态结构均得到不同程度改善。模型组大鼠骨密度及血清中钙、BALP、BGP、OPG水平均低于假手术组(P<0.01),血清磷水平高于假手术组(P<0.01);补骨膏低剂量组、补骨膏高剂量组及戊酸雌二醇组大鼠骨密度及血清中钙、BALP、BGP、OPG水平均高于模型组(P<0.05或P<0.01),血清磷低于模型组(P<0.01)。模型组大鼠股骨组织OPG蛋白相对表达量低于假手术组(P<0.01),RANK、RANKL蛋白相对表达量均高于假手术组(P<0.01);补骨膏低剂量组、补骨膏高剂量组及戊酸雌二醇组大鼠股骨组织中OPG蛋白相对表达量高于模型组(P<0.05)或(P<0.01),RANK、RANKL蛋白相对表达量均低于模型组(P<0.01)。模型组大鼠股骨组织TNF-α mRNA、IFN-γ mRNA、MMP-9 m RNA、RANK m RNA、RANKL mRNA对表达量均高于假手术组(P<0.01),Arg-1 m RNA、TGF-β1 mRNA、OPG mRNA对表达量均低于假手术组(P<0.01);补骨膏高剂量组及戊酸雌二醇组大鼠股骨组织TNF-α mRNA、IFN-γ m RNA、MMP-9mRNA、RANK mRNA、RANKL mRNA相对表达量均低于模型组(P<0.01),Arg-1 mRNA、TGF-β1 mRNA、OPG m RNA相对表达量均高于模型组(P<0.01)。结论:补骨膏可能通过调控OPG-RANK-RANKL信号通路,抑制免疫炎症反应,调节骨基质胶原合成与降解,从而维持骨代谢平衡,改善PMOP大鼠骨密度及骨微结构病理损伤。
Objective:To investigate the osteoprotective effects of Bugugao on rats with postmenopausal osteoporosis(PMOP)and its regulatory role in the osteoprotegerin(OPG)-receptor activator of nuclear factor-κB(RANK)-RANK ligand(RANKL)signaling pathway.Methods:A total of 36 rats were randomly assigned to a sham-operated group(n=9)and an ovariectomized group(n=27).PMOP models were established by bilateral ovariectomy.After successful modeling,24 PMOP rats were randomly divided into a model group,an estradiol valerate group,a low-dose Bugugao group,and a high-dose Bugugao group,followed by 8 weeks of intragastric administration of corresponding treatments.Bone mineral density(BMD)of the proximal femur was measured using a bone densitometer;femoral microstructure was analyzed by Micro-CT three-dimensional reconstruction;histopathological morphology of the femur was observed via hematoxylin-eosin(HE)staining;serum levels of bone alkaline phosphatase(BALP),osteocalcin(BGP),and OPG were detected by enzyme-linked immunosorbent assay(ELISA),while serum phosphorus and calcium levels were measured using assay kits;protein expression levels of OPG,RANK,and RANKL in femoral tissue were determined by Western blotting;expression levels of tumor necrosis factor-α(TNF-α)mRNA,interferon-γ(IFN-γ)mRNA,arginase-1(Arg-1)mRNA,transforming growth factor-β1(TGF-β1)mRNA,matrix metalloproteinase-9(MMP-9)mRNA,OPG mRNA,RANK mRNA,and RANKL mRNA in femoral tissue were detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR).Results:The sham-operated group exhibited continuous and intact femoral structure with numerous,thick,and densely arranged trabeculae;the model group showed significantly reduced BMD in the proximal femur;the low-dose Bugugao group,high-dose Bugugao group,and estradiol valerate group all displayed varying degrees of improvement in trabecular number and bone histomorphology in the proximal femur.Compared with the sham-operated group,the model group showed lower BMD and serum levels of calcium,BALP,BGP,and OPG(P<0.01),but higher serum phosphorus levels(P<0.01).Compared with the model group,the low-dose Bugugao group,high-dose Bugugao group,and estradiol valerate group showed higher BMD and serum levels of calcium,BALP,BGP,and OPG(P<0.05 or P<0.01),and lower serum phosphorus levels(P<0.01).The model group showed lower relative protein expression of OPG in femoral tissue than sham-operated group(P<0.01),while higher expression levels of RANK and RANKL proteins than sham-operated group(P<0.01).Compared with the model group,the expression of OPG protein was increased in the low-dose Bugugao group,high-dose Bugugao group,and estradiol valerate group(P<0.05 or P<0.01),while the expression of RANK and RANKL proteins was decreased in the low-dose Bugugao group,high-dose Bugugao group,and estradiol valerate group(P<0.01).The expression levels of TNF-αmRNA,IFN-γmRNA,MMP-9 mRNA,RANK mRNA,and RANKL mRNA in the model group were higher than that of the sham-operated group(P<0.01),whereas the expression levels of Arg-1 mRNA,TGF-β1 mRNA,and OPG mRNA were lower than that of the sham-operated group(P<0.01).In the high-dose Bugugao group and estradiol valerate group,the expression of TNF-αmRNA,IFN-γmRNA,MMP-9 mRNA,RANK mRNA,and RANKL mRNA was lower(P<0.01),while the expression of Arg-1 mRNA,TGF-β1 mRNA,and OPG mRNA was higher(P<0.01)compared with the model group.Conclusion:Bugugao may maintain bone metabolic balance and improve BMD and pathological damage to bone microstructure in PMOP rats by regulating the OPG-RANK-RANKL signaling pathway,inhibiting immune-inflammatory responses,and modulating the synthesis and degradation of bone matrix collagen.
作者
罗娟
曾朝辉
袁尚锋
朱莹莹
罗杰
彭真灵
龙厚任
LUO Juan;ZENG Zhaohui;YUAN Shangfeng;ZHU Yingying;LUO Jie;PENG Zhenling;LONG Houren(Hunan University of Chinese Medicine,Changsha Hunan 410208,China;Zhuzhou Hospital of Traditional Chinese Medicine Orthopedics and Traumatology,Zhuzhou Hunan 412007,China;Hunan Traditional Chinese Medical College,Zhuzhou Hunan 412006,China)
出处
《中医药导报》
2026年第1期7-13,共7页
Guiding Journal of Traditional Chinese Medicine and Pharmacy
基金
国家中医药管理局2022年全国名老中医专家传承工作室建设项目(国中医药人教函[2022]75号)
湖南省中医药管理局中医药科研计划项目(B2023153)
湖南省中医药管理局-局市(州)联合攻关科研项目(E2023016,E2023017)。
