Abstract
The Buxue Tongluo pill (BTP) is a self-made pill with the functions of nourishing blood, promoting blood circulation, dredging collaterals, and relieving pain. It consists of Angelica sinensis (Oliv.) Diels, Pheretima aspergillum (E.Perrier), Panax notoginseng (Burk.) F. H. Chen, Astragalus membranaceus (Fisch.) Bge, and Glycyrrhiza uralensis Fisch. Various clinical practices have confirmed the therapeutic effect of BTP on osteonecrosis of the femoral head (ONFH), but little attention has been paid to the study of its bioactive ingredients and related mechanisms of action. In this study, UPLC/MS-MS combined with GEO data mining was used to construct a bioactive ingredient library of BTP and a differentially expressed gene (DEG) library for ONFH. Subsequently, Cytoscape (3.7.2) software was used to analyze the protein–protein interaction between BTP and DEGs of ONFH to screen the key targets, and functional annotation analysis and pathway enrichment analysis were carried out. Finally, 34 bioactive compounds were screened, which acted on 1,232 targets. A total of 178 DEGs were collected, and 17 key genes were obtained after two screenings. By bioinformatics annotation on these key genes, a total of 354 gene ontology (GO) functional annotation analyses and 42 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were obtained. The present study found that GO and KEGG enrichment were mainly related to apoptosis, suggesting that BTP may exert an anti-ONFH effect by promoting osteoclast apoptosis. Experiments in vitro demonstrated that BTP could increase the mitochondrial membrane potential (MMP) and induce remarkable apoptosis in osteoclasts. Furthermore, we determined the apoptosis marker of cleaved(C)-caspase-3, bcl-2, and bax and found that BTP could upregulate the C-caspase-3 and bax expression in osteoclasts and decrease the expression of bcl-2, p-Akt, and p-PI3K in a dose-dependent manner, indicating that BTP could induce PI3K/Akt-mediated apoptosis in osteoclasts to treat ONFH. This study explored the pharmacodynamic basis and mechanism of BTP against ONFH from the perspective of systemic pharmacology, laying a foundation for further elucidating the therapeutic effects of BTP against ONFH.
Highlights
Osteonecrosis of the femoral head (ONFH) refers to a disease in which the blood supply of the femoral head is damaged or interrupted, leading to the death of bone marrow components and bone cells and the structural change and collapse of the femoral head (Pouya and Kerachian, 2015)
A total of 34 components were identified from Buxue Tongluo pill (BTP), of which six compounds belonged to Angelica sinensis (Oliv.) Diels, seven to Astragalus membranaceus (Fisch.) Bge, six to Glycyrrhiza uralensis Fisch., six to Panax notoginseng (Burk.) F
The results showed that the targets of TP53, EGFR, and CDK2 and the pathways of the PI3K-AKT signaling pathway and cell cycle were more critical in the entire network
Summary
Osteonecrosis of the femoral head (ONFH) refers to a disease in which the blood supply of the femoral head is damaged or interrupted, leading to the death of bone marrow components and bone cells and the structural change and collapse of the femoral head (Pouya and Kerachian, 2015). It has been considered a debilitating disease of multifactorial genesis, predominately affecting young people aged 20–40 years with the destruction of hip joints in their third, fourth, or fifth decade of life (Sun and Li, 2013). A growing amount of TCM compounds and monomers, such as Bushen Huoxue decoction (Sun and Li, 2013), Huangqi Shenggu decoction (Ping, 2021), Taohong Siwu decoction (Zhao, 2020), Gastrodin (Zheng et al, 2014), and luteolin (Yan et al, 2020), have been reported to exert a significant therapeutic effect on ONFH
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