Osteoporosis is a systemic bone disease. Due to osteopenia and deterioration of bone microstructure, the risk of fragility fractures increases[10]. The results of modern clinical medical research show that multiple factors cause the pathogenesis of osteoporosis, and the imbalance of bone remodeling is considered to be the main mechanism leading to osteoporosis. Growth hormone activity, musculoskeletal function, daily dietary calcium and vitamin D intake, genetics, and environment can affect bone remodeling and break the bone formation-resorption balance[11]. The myostatin signaling pathway increases bone resorption by activating the RANKL signaling pathway [12].
The results showed that the main active ingredients of RA combined with PRR in treating osteoporosis were mistletoe, kaempferol, isorhamnetin, verbascoside, diosgenin, and catechins. Kaempferol is a flavonol compound with biological effects such as anti-oxidation, anti-tumor, and immune regulation. It can promote the proliferation and differentiation of osteoblasts and increase osteoblast alkaline phosphatase's activity to promote bone formation[13]. Studies have shown that high-dose kanamycin can treat osteoporosis by regulating the balance of Ca2+ metabolism, promoting collagen production, and reducing bone loss[14]. Mistletoe is a kind of polyhydroxyflavonoids, which can effectively promote osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW264.7 cells[15]. At the same time, mistletoe can also promote the proliferation and bone differentiation of bone marrow stem cells[16].
PPI network topology analysis results showed that STAT3, Jun, SRC, AKT1, Mapk14, mapk1, TNF, IL6, and other proteins were the main targets of RA combined with PRR in the treatment of osteoporosis. JUN protein is a family of protein kinases in the mitogen-activated protein kinase (MAPK) signal transduction cascade, and c-Jun is also an essential substance for activating transcriptional activator protein 1 (AP-1)[17]. Studies have shown that the increase of JUN protein level can initiate more AP-1, AP-1 can affect the apoptotic process of cells, promote apoptosis, and accelerate the differentiation of osteoclast precursor cells into osteoclasts by regulating the expression of related genes[18]. In addition, it can also increase the activity of metalloproteinases to promote the degradation of collagen in human skin[19–20]. Src proteins are a class of non-receptor protein tyrosine kinases activated by various extracellular signaling molecules[21]. Many studies have shown that the high activation of Src protein is closely related to the occurrence and development of osteoporosis. Its inhibitors are expected to be a critical method for treating osteoporosis drugs[22]. AKT1 is a member of the protein kinase (B) family and is necessary for cell proliferation, cell growth, and differentiation in the body. The researchers found that mice with the AKT1 gene knocked out had skeletal muscle atrophy and impaired bone development. In recent years, TNF and IL6 factors have been considered the main factors regulating bone resorption under pathological conditions, and they have a close relationship with estrogen levels. Many experiments in vitro and in vivo have confirmed that IL-6 can elevate the formation of osteoclasts and increase bone resorption[23–24]. TNF indirectly activates mature osteoclasts by stimulating osteoblasts and inhibits the apoptosis of osteoclasts[25].
KEGG pathway enrichment analysis results showed that RA combined with PRR treatment of osteoporosis mainly involves PI3K-Akt, MAPK, TNF, Rap1, Toll-like receptors, HIF-1, Ras signaling pathways. PI3K/Akt pathway is closely related to osteogenesis and osteoclast pathways. The PI3K/Akt pathway can regulate the differentiation and apoptosis of osteoblasts and osteoclasts to maintain the balance of bone resorption and bone formation[26]. Decreasing the activity of caspases-9 through the PI3K-Akt pathway can weaken the absorptive capacity of osteoclasts and reduce the apoptosis caused by cell damage, thereby delaying the process of osteoporosis[27]. The MAPK pathway is directly involved in the regulation of bone metabolism. It has a complementary relationship with the PI3K/Akt pathway[28] and plays a vital role in promoting osteoblast growth and differentiation. MAPK signaling pathways mainly include extracellular regulated protein kinase (ERK) transduction pathway, Jun N-terminal kinase (JNK), ERK5/macrofilament mitin-activated protein kinase transduction pathway, and p38 signaling pathway. The JNK pathway mainly affects bone formation[29]. It has been shown that fucoidan promotes the osteogenesis of human bone marrow stem cells by increasing phosphorylation-induced BMP2 expression and stimulating the activation of ERK, JNK, and p38 signaling pathways[30]. The p38 pathway and ERK signaling pathway can promote the expression of osteoprotegerin, which is the key to maintaining the balance of bone metabolism[31]. Tumor necrosis factor (TNF) is the main cytokine regulating osteoclast activity and bone resorption[32]. Researchers isolated the glycoprotein osteocalcin (OPG) from TNF factors through fetal mouse gene sequencing experiments, which can be used as a soluble factor to regulate bone mass[33].
In addition, this study established a rat model of osteoporosis by removing the rat’s ovaries. The mechanism of RA combined with PRR in the treatment of osteoporosis was further explored through experimental methods such as dual-energy X-ray absorption assay, enzyme-linked immunosorbent assay, and HE staining method. Experimental results showed that RA combined with PRR could promote bone formation and increase BMD. The experimental results showed that the bone formation marker BGP was significantly increased compared with the model group, and the levels of β-CTX, MSTN, and PIIINP were significantly decreased. It showed that RA combined with PRR could increase bone formation markers to promote bone formation and have a therapeutic effect on osteoporosis. Pathological examination results showed that the bone tissue of the model group was disordered, and the bone mass was reduced. RA combined with PRR could significantly improve the microstructure of bone tissue in osteoporotic rats.