Analysis results table by MMVEC.

TFRD has been widely used in China to treat osteoporosis (OP). However, the specific molecular mechanism of TFRD against OP has not been fully clarified. Our previous studies have also proved that TFRD could attenuate OP and the clinical equivalent dose of 67.5mg/kg/d is the effective dose for TFRD treating OP. Therefore, this study used 67.5mg/kg as the dosage of TFRD in combination with multi omics to investigate the mechanism of action of TFRD in the treatment of OP. The aim of this study was to further elucidate molecular mechanism of TFRD for treating OP based on metagenomic and metabolomic analyses. In this study, hematoxylin-eosin (H&E) staining, micro computed tomography (micro-CT) and bone mineral density (BMD) analysis were used to observe pharmacological effects of TFRD against ovariectomized (OVX)-induced OP. Subsequently, multiomics analysis including metagenomics, untargeted and short chain fatty acids (SCFAs) metabolomics were carried out to identify whether the anti-osteoporosis mechanism of TFRD correlated with gut microbiota and related metabolites. Our results indicate that TFRD could improve the microstructure and density of trabecular bone in OVX rats. 17 differential species, which mainly from Akkermansia, Bacteroides, and Phascolarctobacterium genus, 14 related differential metabolites and acetic acid in SCFAs were significantly altered by OVX and reversed by TFRD. Furthermore, according to results of untargeted metabolomics analysis, it was found that several metabolic pathways such as phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis and so on might play an important role in TFRD against OP. In order to further study the relationship between gut microbiota and related metabolites, spearman correlation analysis was used, and showed that gut microbiota such as Akkermansia muciniphila might be closely related to several metabolites and metabolic pathways. These findings suggest that TFRD treatment could reduce the effects of OVX-induced OP by altering community composition and abundance of gut microbiota, regulating metabolites and SCFAs. It was speculated that the gut microbiota especially Akkermansia muciniphila and related metabolites might play an important role in TFRD against OP, and deserve further study by follow-up experiment. This conclusion provides new theoretical support for mechanism research of TFRD against OP.

TFRD已在我国被广泛应用于骨质疏松症（osteoporosis, OP）的临床治疗，但TFRD对抗骨质疏松症的具体分子机制尚未完全阐明。我们前期研究已证实，TFRD可缓解骨质疏松症，其临床等效剂量67.5mg/kg/d为TFRD治疗骨质疏松症的有效给药剂量。据此，本研究以67.5mg/kg作为TFRD的给药剂量，联合多组学（multi omics）技术探究TFRD治疗骨质疏松症的作用机制，旨在基于宏基因组学（metagenomics）与代谢组学（metabolomics）分析，进一步阐明TFRD治疗骨质疏松症的分子机制。本研究采用苏木精-伊红（H&E）染色、显微计算机断层扫描（micro-CT）及骨密度（BMD）检测，观察TFRD对卵巢切除（OVX）诱导的骨质疏松症模型大鼠的药理效应。随后开展包含宏基因组学、非靶向代谢组学及短链脂肪酸（SCFAs）代谢组学在内的多组学分析，以明确TFRD抗骨质疏松症的作用机制是否与肠道菌群及相关代谢产物存在关联。研究结果显示，TFRD可改善卵巢切除大鼠的骨小梁微观结构与骨密度。卵巢切除造模可显著改变17种差异菌种（主要隶属于阿克曼菌属、拟杆菌属及考拉杆菌属）、14种差异代谢产物及短链脂肪酸组分中的乙酸水平，而TFRD干预可逆转上述异常变化。此外，非靶向代谢组学分析结果表明，苯丙氨酸代谢、苯丙氨酸-酪氨酸-色氨酸生物合成等多条代谢通路可能在TFRD抗骨质疏松症过程中发挥重要调控作用。为进一步解析肠道菌群与相关代谢产物的相互关系，本研究采用斯皮尔曼相关分析，结果显示嗜黏蛋白阿克曼菌等肠道菌群可能与多种代谢产物及代谢通路密切相关。上述研究结果表明，TFRD可通过重塑肠道菌群的群落组成与丰度、调节代谢产物及短链脂肪酸水平，减轻卵巢切除诱导的骨质疏松症损伤。我们推测，肠道菌群（尤其是嗜黏蛋白阿克曼菌）及其相关代谢产物可能在TFRD抗骨质疏松症过程中扮演关键角色，值得后续实验进一步验证。本研究结论为TFRD抗骨质疏松症的机制研究提供了全新的理论支撑。