Analysis of the mechanism of action of Liuwei Dihuang Pill in treating osteoporosis based on network pharmacology and molecular docking technology
Osteoporosis (OP) is a systemic bone disease characterized by low bone mass and damage to the microstructure of bone tissue. With the aging of the population in China, the incidence of osteoporosis and fractures is also increasing. Epidemiological data shows that there are currently over 200 million OP patients worldwide. In China, about 112 million people are affected by this disease, mainly concentrated in the population over 40 years old. Osteoporosis has become a serious public health problem. At present, the main treatment methods in modern medicine are supplementing calcium supplements and vitamin D. Based on this, anti OP treatment is carried out from two aspects: bone resorption and bone formation, through inhibition and promotion. Although the therapeutic effect is significant, the toxic side effects and poor long-term compliance of drug treatment have not been effectively addressed. Traditional Chinese medicine has greater advantages in preventing and treating osteoporosis. Both traditional Chinese medicine formulas and single drugs have multi-target and multi system regulatory effects, but the specific molecular mechanisms are still unclear.
OP belongs to the categories of “bone weakness”, “bone obstruction”, “back and waist pain” in traditional Chinese medicine, and its occurrence is closely related to kidney deficiency. The book “Su Wen: On Impotence” records: “If the kidney qi is hot, the waist and spine will not be lifted, the bones will wither and the marrow will decrease, resulting in bone impotence. The “Ling Shu · Jing Mai” records: “If there is a lack of Yin Qi in the feet, the bones will wither. During the Song Dynasty, Qian Yi recorded the widely circulated Liu Wei Di Huang Wan in his book “Direct Formula for Preschool Medicine Diagnosis”, which has the effects of nourishing yin and tonifying kidney, as well as seeking yin in yang. Liuwei Dihuang Wan, as a recommended medication for the treatment of OP, has a good therapeutic effect on OP. Lu et al. found through bioinformatics analysis that Liuwei Dihuang Pill can promote estrogen secretion by downregulating the expression of XRN1 gene and ZNF family gene, upregulate the expression of MAP3K10 gene, and downregulate the expression of XPO1 gene and NUP54 gene to balance immune response and promote bone tissue formation; Wu et al. found that Liuwei Dihuang Wan can increase bone density and peripheral serum osteocalcin, thereby increasing bone mass in OP patients; Yu et al.’s research shows that Liuwei Dihuang Pill significantly improves the clinical efficacy and effectiveness of OP patients on the basis of conventional calcium supplementation. The therapeutic effect of Liuwei Dihuang Pill on OP has been validated in clinical practice, therefore, the study of its specific mechanism of action is of great practical significance. Network pharmacology can reveal the mechanism of multi-component, multi-target, and multi pathway synergistic effects of traditional Chinese medicine and compound formulas. Therefore, this article uses network pharmacology methods to analyze the chemical components, target proteins, targets, signaling pathways, and biological processes involved in Liuwei Dihuang Wan, explore the mechanism of drug treatment for diseases, and provide new research ideas and data references for further treatment of OP.

This study found that the main active ingredients that exert therapeutic effects in Liuwei Dihuang Wan include quercetin, stigmasterol, kaempferol, diosgenin, β – sitosterol, etc. Among them, quercetin, sitosterol, and kaempferol play a key role in bone metabolism processes. Gu et al. confirmed that quercetin can effectively inhibit the pre maturation aging of osteoblast cell line MC3T3-E1, and through this pathway, it can effectively reduce bone loss in castrated osteoporotic mouse models, exerting estrogen like effects. A study has found that kaempferol can activate estrogen receptor activity, enhance the proliferation and differentiation ability of osteoblast cell line MG-63, and promote osteoblast mineralization. Osteoblasts treated with diosgenin can significantly upregulate the ratio of osteoprotegerin/nuclear factor kappa B receptor activator ligand system in cells, thereby reducing bone resorption and inhibiting osteoclast formation. Zeng et al. found that β – sitosterol promotes and enhances osteogenesis by increasing the ratio of osteoprotegerin/osteoclast differentiation factor (OPG/ODF) and stimulating the differentiation of ovarian granulosa cells into estradiol. In summary, some potential molecular active ingredients in Liuwei Dihuang Wan have potential therapeutic effects on osteoporosis by inhibiting the generation of osteoclasts, reducing bone loss, and promoting osteogenesis.
GO pathway enrichment analysis showed that the action of Liuwei Dihuang Wan on diseases mainly involves various biological processes such as oxidative stress response, lipopolysaccharide response, active regulation of cell migration, reactive oxygen species response, etc. Multiple biological processes involve nuclear and cardiac targets such as AKT1, IL6, TP53, VEGFA, TNF, etc. Among them, AKT1 can control the differentiation of osteoblasts (OB) and the growth of osteoclasts (OC) by activating downstream effector proteins mTORC1/S6K1. Studies have confirmed that AKT1 gene knockout mice exhibit delayed ossification, severely disrupting bone growth and development. Recent studies have found that the occurrence and development of osteoporosis are directly related to immune system dysfunction leading to inflammatory reactions. Inflammatory cytokines such as IL6 and TNF, which play a key role in the inflammatory response, disrupt the balance of bone turnover by increasing bone resorption by osteoclasts or decreasing bone formation by osteoblasts, and participate in the occurrence and development of osteoporosis. VEGFA can promote endothelial cell proliferation and neovascularization, accelerate bone formation and remodeling processes, directly promote bone marrow mesenchymal stem cells and increase osteogenic activity of bone marrow (OB), reduce OC osteolytic activity to promote bone formation and increase bone density, playing an important role in the occurrence and development of osteoporosis.
The pathways analyzed by KEGG mainly involve TNF signaling pathway, MAPK pathway, HIF-1 signaling pathway, PI3K/Akt pathway, Toll like receptor signaling pathway, etc., indicating that the chemical components of Liuwei Dihuang Pill can intervene in certain pathways to achieve the goal of treating OP. The chronic inflammation related bone remodeling process mediated by the tumor necrosis factor signaling pathway (TNF) is an important pathway for the treatment of osteoporosis. Studies have found that TNF – α can lead to the occurrence of postmenopausal osteoporosis, and its mechanism of action is to promote the formation of osteoclasts induced by RANKL. The core genes TNF and IL-6 regulate the balance between bone resorption and bone remodeling by communicating the signaling pathways of TNF and RANK/RANKL/OPG. The MAPK pathway can affect the progression of OP by regulating the differentiation of OC. There is research confirming that MAPK directly participates in the differentiation of OC precursor cells into mature OC after activation, and participates in the differentiation of OC through autophagy. The main cause of OP is the overactivation of osteoclasts, an important therapeutic target. The PI3K/Akt pathway is closely related to the osteogenic and osteoclast pathways, regulating the survival and differentiation processes of osteoblasts and osteoclasts to maintain the balance of bone mass and bone turnover. Activation of the PI3K/Akt signaling pathway can promote the expression of osteogenic differentiation markers such as alkaline phosphatase (ALP) and BMP-2, and promote the proliferation and differentiation of osteoblasts. The presence of the PI3K/Akt signaling pathway plays a crucial role in the survival of osteoclasts. Toll like receptors are a type of non-specific immune receptor, and the Toll like signaling pathway plays an important role in bone metabolism. The Toll like receptor 4 (TLR4) signaling pathway can interact with other osteogenic pathways by activating ERK, p38, JNK, and NF – κ B pathways, and affect osteoblast differentiation, proliferation, mineralization, and apoptosis. The dynamic balance between bone resorption and bone remodeling is formed on the basis of osteoblast osteoclast coupling, which is crucial for the occurrence and treatment of osteoporosis. The hypoxia inducible factor-1 signaling pathway (HIF-1) is closely related to the proliferation, differentiation, and apoptosis of osteoblasts and osteoclasts. HIF-1 has the function of activating osteoclasts, and the lack of estrogen in patients with osteoporosis can reduce the inhibitory effect of HIF-1 on osteoclasts, exacerbating the development of osteoporosis. Therefore, it is speculated that Liuwei Dihuang Pill regulates key targets such as AKT1, IL6, TP53, VEGFA, and TNF in the treatment of OP through signaling pathways such as TNF signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, PI3K/Akt pathway, and Toll like receptor signaling pathway. Molecular docking was conducted between the potential active ingredients of Liuwei Dihuang Wan and key targets, and it was found that compounds such as quercetin, stigmasterol, and kaempferol had binding energies of less than -5.0 kcal/mol with corresponding targets such as TP53, AKT1, and IL6. This suggests that the potential active ingredients in Liuwei Dihuang Wan have good binding activity with key targets, indicating that they may be potential active ingredients for treating OP. The prediction of the potential active ingredients and key targets of Liuwei Dihuang Pill for treating OP through network pharmacology and molecular docking methods has certain scientific basis and theoretical support.
This article suggests through network pharmacology and molecular docking verification that Liuwei Dihuang Pill can prevent and treat osteoporosis by regulating bone metabolism, inflammatory response, cell proliferation and differentiation, and has the characteristics of multi-component, multi-target, and multi pathway effects. At the same time, providing a multidimensional perspective on the specific mechanism of action of traditional Chinese medicine in the prevention and treatment of osteoporosis is more conducive to the inheritance, innovation, and development of traditional Chinese medicine, as well as the deepening of subsequent experimental research and the expansion of clinical thinking. It also provides basic theoretical support for future drug research and development. However, due to the fact that this study mainly relies on database information and considers the complexity of traditional Chinese medicine decoction and possible changes in active ingredients in vivo, further experiments are needed to confirm the specific mechanism of action of the drug.