背景:临床研究表明,绝经后骨质疏松症(PMOP)常伴有铁过载的危险因素,这加剧了骨代谢紊乱和加速PMOP。先前的研究发现,女贞子(FLL)或酒蒸FLL(WFLL)中的多组分同时作用于铁过载和PMOP的共同目标,这表明FLL和WFLL可能双重调节铁/骨代谢。此外,根据几千年来的中医理论,WFLL具有更优越的疗效。
目的:揭示“高级多组分结构(SMCS)”及其在WFLL平行下调铁过载和挽救骨代谢中的分子机制。
方法:建立HPLC指纹图谱以比较FLL和WFLL的化学特征;然后,通过UPLC-Orbitrap-MS/MS与OPLS-DA联用分析了FLL和WFLL的化学成分和质量标志物;通过HPLC-DAD同时测定了不同蒸酒时间(WST)下质量标志物的动态含量和多组分结构。同时,通过系统的斑马鱼模型研究了FLL在不同WST下的动态功效。随后,从网络药理学(NP)和分子对接(MD)获得了WFLL治疗伴有铁过载的PMOP的潜在机制。最后,进行斑马鱼和去卵巢大鼠模型以验证这种潜在的机制。
结果:15批FLL和WFLL的HPLC指纹图谱相似性在0.9-1.0之间。确定了126种成分,包括58个环烯醚萜类化合物,25萜烯,30苯乙醇,7类黄酮和其他6。揭示了20个与WFLL相关的质量标记,通过对11种质量标记物的定量计算,在蒸(0-24h)过程中,苯乙醇:环烯醇:三萜的比例(P/I/T)从1:15:4.5转换为1:0.8:0.9;斑马鱼幼虫的骨矿化和运动性能表明,WFLL在12h时的最佳功效(p<0.05),其中P/I/T的SMCS转换为1:1.8。NP发现BMP-Smad通路是FLL抗PMOP的潜在作用机制之一,可同时调节骨形成和铁过载。MD显示17个活性成分和10个核心靶基因可以以适当的亲和力自发结合。大鼠模型证实FLL和WFLL明显逆转PMOP,基于骨形成指标的改善(ALP,OPG,OGN),铁代谢指标(铁调素,铁蛋白),骨微结构(BMD,BV/TV,TB。Th,TB。N);此外,与FLL相比,WFLL在抗PMOP中的逆转作用显着增强(p<0.05)。FLL和WFLL增加了基因和蛋白质的表达(Hep,BMP-6,p-Smad1/5,Smad4)与模型组比较,WFLL优于FLL(p<0.05)。
结论:通过葡萄酒蒸汽优化了FLL的SMCS,WFLL在下调铁过载和促进骨形成方面表现出双重作用,BMP-Smad通路是潜在的分子机制之一。
BACKGROUND: Clinical studies indicated that postmenopausal osteoporosis (PMOP) often accompanied by iron overload risk factor, which exacerbated bone metabolism disorders and accelerated PMOP. Previous research found that multicomponent in Ligustri Lucidi Fructus (FLL) or wine-steamed FLL (WFLL) acted on the common targets of iron overload and PMOP simultaneously, which indicated that FLL and WFLL probably regulated iron/bone metabolism dually. Additionally, WFLL had more superior effect according to the theory of Chinese medicine for thousands of years.
OBJECTIVE: To reveal the \"superior multi-component structure (SMCS)\" and its molecular mechanisms in parallelly down-regulating iron overload and rescuing bone metabolism by WFLL.
METHODS: HPLC fingerprinting was established to compare the chemical profiles of FLL and WFLL; Then, the chemical compositions and quality markers of FLL and WFLL were analyzed by UPLC-Orbitrap-MS/MS coupled with OPLS-DA; the dynamic contents of quality markers and the multi-component structure at different wine steaming times (WST) were simultaneously determined by HPLC-DAD. Meanwhile, the dynamic efficacy of FLL at different WST were hunt by systematic zebrafish model. Subsequently, potential mechanism of WFLL in treating PMOP accompanied with iron overload was obtained from network pharmacology (NP) and molecular docking (MD). Finally, zebrafish and ovariectomy rat model were carried out to validate this potential mechanism.
RESULTS: HPLC fingerprints similarity of 15 batches in FLL and WFLL were among 0.9-1.0. 126 compositions were identified, including 58 iridoids, 25 terpenes, 30 phenylethanoids, 7 flavonoids and 6 others. 20 quality markers associated with WFLL was revealed, and the ratio of phenylethanols: Iridoids: Triterpenes (P/I/T) was converted from 1: 15: 4.5 to 1: 0.8: 0.9 during steaming (0 - 24 h) calculated by the quantification of 11 quality markers; the bone mineralization and motor performance of zebrafish larvae indicated that the optimum efficacy of WFLL at 12 h (p < 0.05) in which the SMCS of P/I/T was converted to 1: 4: 1.8. NP discovered that BMP-Smad pathway is one of the potential mechanisms of FLL in anti PMOP and then regulated bone formation and iron overload simultaneously. MD revealed that 17 active ingredients and 10 core targets genes could spontaneously bind with appropriate affinity. Rats model verified that FLL and WFLL significantly reversed PMOP, based on the improvement in bone formation indexes (ALP, OPG, OGN), iron metabolism indicators (hepcidin, ferritin), bone microstructure (BMD, BV/TV, Tb. Th, Tb. N); Moreover, WFLL significant enhanced reversal effect in anti-PMOP compared to FLL (p < 0.05). FLL and WFLL increased genes and proteins expression (Hep, BMP-6, p-Smad1/5, Smad4) related to BMP-Smad pathway compared with model group, and WFLL was more superior than FLL (p< 0.05).
CONCLUSIONS: The SMCS of FLL was optimized by wine-steam, WFLL represented a dual effect in downregulating iron overload and promoting bone formation, and the BMP-Smad pathway is one of the potential molecular mechanisms.