Induction of PI3K/Akt-Mediated Apoptosis in Osteoclasts Is a Key Approach for Buxue Tongluo Pills to Treat Osteonecrosis of the Femoral Head

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.

Programmed cell death of periodontal ligament cells

“…Studies have demonstrated that BTPs upregulated the expression of caspase‐3 and bax in osteoclasts, reduced the expression of bcl‐2, and downregulated the levels of p‐Akt and p‐PI3K in a dose‐dependent manner. These findings indicate that BTPs induce PI3K/Akt‐mediated osteoclast apoptosis and preserve bone mass to a certain extent (Wang, Liu, et al, 2021).…”

Cycloastragenol prevents bone loss via inhibiting osteoclast activity in glucocorticoid-induced osteonecrosis of the femoral head: An in vivo study

“…As a supplement or alternative, many TCM herbal formulations are prescribed for GIONFH patients in Chinese clinics or wards to alleviate symptoms and promote bone repair [ [27] , [28] , [29] ]. In recent years, osteoclast inhibition has attracted high interest as a potential preventive or therapeutic strategy for GIONFH [ 15 , 30 , 31 ]. In the present study, the protective effect of CAG on MPS-induced GIONFH in rats was identified, in which suppression of osteoclast hyperactivity may play a critical part.…”

“…What's more, enhanced osteoclast activity was proved to be positively correlated with the early progression of GIONFH, with evidence that the increased protein expression of RANKL in necrotic bone is correlated with the progression stage of GIONFH [ 13 ]. Recently, the efficacy of natural osteoclast inhibitors in the prophylaxis and treatment of GIONFH has been evaluated [ [14] , [15] , [16] ], indicative of their positive therapeutic potential.…”

Osteogenesis-Related Long Noncoding RNA GAS5 as a Novel Biomarker for Osteonecrosis of Femoral Head

“…HIF 1α is found to be upregulated in the ischemic side of the femoral head, and it can act as a positive regulator of Sox9 activity in femoral head osteonecrosis ( Zhang et al, 2011 ). In addition, the PI3K/AKT signaling pathway may play a significant role in the pathogenesis of ONFH by regulating the proliferation and apoptosis of osteoblasts and osteoclasts ( Deng et al, 2019 ; Wang et al, 2021 ). However, analysis of differences in lncRNA GAS5 expression between the osteogenic induction and non-osteogenic induction groups at the same time point would make this more reasonable.…”