淫羊藿总黄酮联合骨营养剂抗骨质疏松作用探讨

Studies on the anti-osteoporosis effect of total flavonoids of epimedium combined with bone nutrients

目的探索淫羊藿总黄酮联合骨营养剂治疗骨质疏松的协同作用,讨论中西药联用的潜在作用机制。方法通过去除卵巢建立绝经后骨质疏松大鼠模型,设置假手术组(Sham)、模型组(OVX)、戊酸雌二醇组(EV)、淫羊藿总黄酮提取物组(T)、淫羊藿总黄酮提取物配伍骨营养剂组(TG),每组7只。给药干预8周后,通过酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清骨代谢标志物和炎症因子的变化,利用Micro-CT和生物力学分析测定骨密度和显微结构、采用苏木精-伊红染色(hematoxylin-eosin staining,H&E)和抗酒石酸酸性磷酸酶染色(tartrate resistant acid phosphatase,TRAP)观察骨病理损伤及破骨细胞数量,使用免疫组化和实时荧光定量聚合酶链式反应(qRT-PCR)测量大鼠股骨中TLR4、PI3K和AKT的基因转录和表达。结果与OVX组相比,EV组、T组和TG组的骨小梁数目增多,排列整齐,结构较为完整,血清骨代谢指标、血清炎症因子、骨小梁参数、骨生物力学参数和破骨细胞数量显著改善(P<0.05),PI3K、AKT的阳性区域及mRNA表达明显升高(P<0.05),TLR4的阳性区域及mRNA表达明显降低(P<0.05)。与T组相比,TG组的药效作用更显著(P<0.05),TLR4 mRNA表达明显降低(P<0.05),PI3K mRNA表达明显升高(P<0.05)。结论淫羊藿总黄酮联合骨营养剂可在TLR4/PI3K/AKT信号通路中协同发挥药效,缓解大鼠骨质疏松的症状。

Objective To explore the synergistic effect of total flavonoids of epimedium in combination with bone nutrients on the treatment of osteoporosis and to discuss the potential mechanism of combination therapy.Methods A postmenopausal osteoporosis rat model was established by removing the ovaries.Seven rats in each group were set up in the sham-operated group(Sham),model group(OVX),estradiol valerate group(EV),total flavonoid extract of epimedium extract group(T),and total flavonoid extract of epimedium extract with bone nutrients group(TG).After 8 weeks of the treatment,serum bone metabolism markers and inflammatory factors were measured using enzyme-linked immunosorbent assay(ELISA).Micro-CT and biomechanical analysis were used to determine bone density and microstructure.Hematoxylin-eosin staining(H&E)and tartrate resistant acid phosphatase staining(TRAP)were used to determine bone pathological damage and osteoclastic bone resorption.Immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)were used to determine gene transcriptions and expressions of TLR4,PI3K,and AKT in the bone.Results Compared with the OVX group,the EV,T,and TG groups showed an increased number of bone trabeculae,neat arrangement and more complete structure,significantly improved serum bone metabolic indexes,serum inflammatory factors,bone trabecular parameters,bone biomechanical parameters and the number of osteoclasts(P<0.05),and significantly higher positive regions and mRNA expressions of PI3K and AKT(P<0.05).In addition,the expressions of TLR4 and mRNA were significantly lower(P<0.05).Compared to those in the T group,the pharmacodynamic effects were more significant in the TG group(P<0.05),with significantly lower TLR4 mRNA expression(P<0.05)and significantly higher PI3K mRNA expression(P<0.05).Conclusion Total flavonoids of epimedium combined with bone nutrients synergistically exerts pharmacological effects on the TLR4/PI3K/AKT signaling pathway to alleviate the symptoms of osteoporosis in OVX rats.