The emerging field of network pharmacology can apply systematic research on the multitarget and multipathway characteristics of the compounds or monomers of Chinese herbal medicines and reveal them at the molecular level. Therefore, network pharmacology, a systems biology-based approach, provides great convenience and saves valuable time for researchers in conducting drug screening and validating disease-signaling pathways.
Previous studies reported that vaccarin has a wide range of biological effects, such as the induction of FGF2 expression and the promotion of angiogenesis . In terms of clinical translational applications, wound dressings containing vaccarin can inhibit inflammatory cell infiltration  and effectively promote endothelial cell migration and wound healing . However, the issue of whether vaccarin can prevent and treat osteoporosis by influencing these biological processes is not yet clear.
Six target proteins, namely, IL-6, TNF, VEGFA, HSP90AA1, CREB1, and IL-2, found in the PPI network analysis were considered to be the most critical targets for pharmacological intervention in osteoporosis. The occurrence of osteoporosis is a very complex pathological process that may involve several key target proteins. The monomers of Chinese herbal medicines, unlike small-molecule inhibition, often exert their therapeutic effects by affecting multiple targets. The biggest advantage of conducting a PPI network analysis is that the proteins with the greatest effect on gene interactions at the intersecting targets can be rapidly calculated, which in turn provides clear targets for drug intervention. Among the aforementioned key target proteins, IL-6, TNF, and IL-2 are important members of the inflammatory factor family, and they have been found to play critical roles in the differentiation of osteoclast precursor cells to mature osteoclasts. VEGFA is a member of the vascular endothelial growth factor family, HSP90AA1 is an important member of the heat shock protein family , and CREB1 belongs to the important family of leucine zippers of DNA-binding proteins.
GO annotation analysis suggested that vaccarin might influence the pathogenesis of osteoporosis through 23 different biological processes, including regulation of apoptosis, angiogenesis, endothelial cell migration, lipolysis metabolism, insulin secretion, and cellular response to hydrogen peroxide. Several studies have proved that vaccarin plays a role in the treatment of other diseases by influencing the aforementioned biological processes. For instance, high glucose-induced endothelial cell injury can be reversed by downregulating the expression level of the apoptotic protein caspase 3. Fibroblast growth factor receptor 1 activation can promote angiogenesis . Disorders of glycolipid metabolism can be corrected by improving insulin resistance . Increasing the activity of superoxide dismutase, catalase, and glutathione peroxidase in vivo can reduce the damage caused by H2O2 and oxidative stress to endothelial cells.
Notably, GO annotation analysis revealed positive regulation of osteoclast differentiation, which was inconsistent with the findings of previous studies that vaccarin negatively regulates osteoclast differentiation. This inconsistency may be attributed to the fact that network pharmacology can only predict the biological process in which the drug is involved, and the positive or negative regulation by the drug must be experimentally confirmed. Moreover, other biological processes in which vaccarin may be involved were identified, but extensive basic experiments are warranted to confirm them. Research on the potential signaling pathways of vaccarin in the treatment of osteoporosis mainly focused on the following four different pathological processes.
Effects on osteogenic cell differentiation and apoptosis pathways
Osteoblast apoptosis plays a crucial role in the pathogenesis of postmenopausal osteoporosis. Both osteoblasts and osteoclasts are engaged in the process of bone reconstruction, and any influence on either side may break the balance inside the bone. PI3K/Akt is a signaling pathway closely related to cell proliferation and apoptosis, and PI3K/Akt signaling activation can reportedly promote osteoblast apoptosis . Rap1 and Ras, which belong to the small GTPase family, are upstream signaling molecules of the MAPK signal and can be activated by various extracellular signals, such as growth factors, receptor tyrosine kinases, and Ca2+. Experimental studies have proved that Rap1 can promote osteoblast proliferation and differentiation by influencing the MAPK signaling pathway, whereas Ras activation can increase cyclinDl expression to promote osteoblast proliferation . HIF-1α is also involved in osteoclast formation and can effectively prevent the occurrence of osteoporosis by inhibiting its expression .
Internal secretion metabolic disorder
Postmenopausal osteoporosis is a common endocrine metabolism disorder. As the ovaries stop producing estrogen and serum estrogen levels drop precipitously after menopause, the postmenopausal female experiences a state of high inflammation and oxidative stress. This situation leads to osteoclast formation promotion, osteoblast proliferation inhibition, and steady state of bone rebuilding disruption.
Numerous inflammatory factors and inflammation-related signaling pathways are involved in the pathogenesis of osteoporosis. NOD-like receptors have been found to activate cysteine proteases, upregulate the secretion of the inflammatory factor IL-1B, and exacerbate inflammation-induced pathological bone loss.
Pathways associated with other diseases
The present KEGG pathway enrichment analysis revealed that rheumatoid arthritis, prostate cancer, tuberculosis, and amoebiasis may interact with osteoporosis. Overactive osteoclasts in advanced rheumatoid arthritis cause bone resorption, resulting in severe destruction of cartilage and subchondral bone. Bone metastasis, which is different from general malignant tumor metastasis, is most common in advanced prostate cancer. Bone tuberculosis often occurs in bones or joints that bear heavy weights and are thus prone to damage, with the greatest damage being joint deformity and nerve compression due to bone destruction. Both of these disease processes are closely related to osteoclast-induced bone resorption. Thus, signaling pathways associated with rheumatoid arthritis, prostate cancer, tuberculosis, and other diseases may also be targets for vaccarin in the treatment of osteoporosis.