探讨藿骨牡蛎方调节绝经后骨质疏松症大鼠破骨-成骨平衡的作用及其机制

Mechanism of Huogumuli Prescription in regulating the osteoclast-osteogenesis balance in postmenopausal osteoporosis rats

目的基于破骨-成骨平衡探讨藿骨牡蛎方(淫羊藿、骨碎补、生牡蛎)对绝经后骨质疏松症(PMOP)大鼠的防治作用及其机制。方法将48只雌性SD大鼠按体质量分为假手术组、模型组、强骨胶囊组(0.054 g/kg)、碳酸钙组(1.670 g/kg)、藿骨牡蛎方低剂量组(0.188 g/kg)、藿骨牡蛎方高剂量组(0.375 g/kg),每组8只。适应性喂养后,除假手术组仅切除卵巢附近等体积脂肪,其余大鼠以双侧卵巢去势手术建立PMOP模型。造模成功后灌胃相应药物,连续90 d。酶联免疫吸附测定(ELISA)法检测血清雌二醇(E2)、雌激素受体α(ERα)、Ⅰ型前胶原氨基端原肽(PINP)、抗酒石酸酸性磷酸酶5b(TRACP-5b);比色法检测血清总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA);显微计算机断层成像检测大鼠股骨骨密度(BMD)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)、骨小梁分离度(Tb.Sp)和结构模型指数(SMI),并观察松质骨显微结构;蛋白质印迹法检测大鼠股骨组织骨保护素(OPG)、核因子κB受体活化因子配体(RANKL)、核因子κB受体激活因子(RANK)、叉头框蛋白O3α(FoxO3α)、Wnt2、β-连环蛋白(β-catenin)、过氧化物酶体增殖物激活受体γ(PPARγ)蛋白表达。结果(1)强骨胶囊组与藿骨牡蛎方低、高剂量组大鼠血清E2、ERα含量较模型组增加(P<0.05)。(2)与模型组比较,强骨胶囊组与藿骨牡蛎方高剂量组大鼠血清PINP、TRACP-5b含量下降(P<0.05),PINP/TRACP-5b升高(P<0.05)。(3)与模型组比较,强骨胶囊组、碳酸钙组及藿骨牡蛎方低、高剂量组T-AOC、SOD、CAT活性升高,MDA含量下降(P<0.05)。(4)与模型组比较,强骨胶囊组及藿骨牡蛎方低、高剂量组大鼠BMD、Tb.Th、Tb.N增加,Tb.Sp、SMI降低(P<0.05);碳酸钙组大鼠BMD增加,Tb.Sp降低(P<0.05)。(5)与模型组比较,强骨胶囊组及藿骨牡蛎方低、高剂量组大鼠RANKL、RANK、FoxO3α、PPARγ蛋白表达下降,OPG、Wnt2、β-catenin蛋白表达增加(P<0.05)。结论藿骨牡蛎方可明显提高PMOP大鼠雌激素水平,抑制破骨细胞分化,降低骨吸收水平;缓解氧化应激,促进成骨分化、抑制成脂分化,提高骨形成水平,恢复破骨细胞与成骨细胞稳态平衡,从而达到防治PMOP的作用。此作用可能是通过OPG/RANKL/RANK及FoxO3α/Wnt2/β-catenin/PPARγ通路调节的。

Objective We aimed to investigate(i)the preventive and therapeutic effects of Huogumuli Prescription(HGMLP),a Chinese medical compound consists of epimedii folium,drynariae rhizoma,and ostreae concha,on postmenopausal osteoporosis(PMOP)rats and(ii)whether it exerts its effects by regulating the osteoclast-osteogenesis balance.Methods Forty-eight female Sprague-Dawley rats were randomly divided into the following six groups:(i)the sham-operated group,(ii)the model group,(iii)the Qianggu Capusule group,(iv)the calcium carbonate group,and(v,vi)the HGMLP low-dose and high-dose groups(n=8 rats per group).After adaptive feeding,rats in all groups except the sham-operated group were treated with bilateral ovarian castration to establish the PMOP model.Each day,rats in the Qianggu Capsule group received 0.054 g/kg Qianggu Capsule suspension intragastrically,rats in the calcium carbonate group received 1.670 g/kg calcium carbonate suspension intragastrically,and rats in the HGMLP low-dose and high-dose groups received 0.188 g/kg and 0.375 g/kg HGMLP intragastrically.Rats in the sham operation group and the model group received an equal volume of normal saline intragastrically.After 90 consecutive days,serum estradiol(E2),estrogen receptorα(ERα),and procollagen typeⅠN propeptide(PINP),tartrate-resistant acid phosphatase 5b(TRACP-5b),were detected by ELISA.Total antioxidant capacity(T-AOC),superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)levels were measured by colorimetry.Bone mineral density(BMD),trabecular number(Tb.N),trabecular separation/spacing(Tb.Sp),trabecular thickness(Tb.Th),and structure model index(SMI)were measured by Micro-CT,and the microstructure of cancellous bone was observed.The expression of osteoprotegerin(OPG),receptor activator of nuclear factor-κB(RANK),RANK ligand(RANKL),phosphorylation of forkhead box O3(FoxO3α),Wnt2,β-catenin,and peroxisome proliferator-activated receptorγ(PPARγ)in rat femur tissue was detected by Western blotting.Results The serum levels of E2 and ERαincreased in the Qianggu Capsule group and HGMLP groups,compared with the model group(all P<0.05).(ⅱ)Compared with the model group,the serum levels of PINP,TRACP-5b decreased and PINP/TRACP-5b increased in both the Qianggu Capsule group and HGMLP high-dose group(all P<0.05).(ⅲ)The activities of T-AOC,AOD,and CAT in the Qianggu Capsule group and HGMLP groups were higher than those in the model group,while the content of MDA lower(all P<0.05).(ⅳ)Compared with the model group,the femoral BMD,Tb.Th,and Tb.N increased in the Qianggu Capsule group and HGMLP groups,while the femoral Tb.Sp and SMI decreased(all P<0.05);the femoral BMD increased and the Tb.Sp decreased in the calcium carbonate group(all P<0.05).(ⅴ)The protein expressions of RANKL,RANK,FoxO3α,and PPARγin the Qianggu Capsule group and HGMLP groups were lower than those in the model group,while the protein expressions of OPG,Wnt2,andβ-catenin were higher(all P<0.05).Conclusion HGMLP can significantly increase estrogen levels,inhibit osteoclast differentiation,and inhibit bone resorption in the PMOP rats.It also alleviates oxidative stress,promotes osteogenic differentiation,inhibits lipogenic differentiation,improves bone formation,and recovers the balance between osteoclasts and osteoblasts,thus achieving prevention and treatment of PMOP.The potential mechanism of HGMLP is regulated via the OPG/RANKL/RANK or FoxO3α/Wnt2/β-catenin/PPARγpathways.