At the molecular level, multiple factors participate in the development of PMOP, interacting with each other and playing a role in regulating bone metabolism in a complex regulatory network. Among them, the low level of estrogen is the main cause of PMOP, which leads to the disorder of bone metabolism. Bone resorption is greater than bone formation, and eventually osteoporosis is caused . Compared with hormone replacement therapy (HRT), traditional Chinese medicine, as a viable alternative therapy, has the advantages of simplicity and low side effects. Modern research has found that traditional Chinese medicine contains a variety of biologically active ingredients, which can act on multiple targets through multiple pathways, reflecting the overall concept of traditional Chinese medicine and the theory of syndrome differentiation and treatment . Compared with the “single component-single target-single pathway” research model of traditional pharmacology, network pharmacology combines the concepts of bioinformatics and multi-directional pharmacology to analyze the relationship between biological systems, medicines, and diseases from a network perspective. It has opened a new research model of traditional Chinese medicine from empirical medicine to evidence-based medicine .
Key active ingredient of ZYTLF for anti-PMOP
According to the network analysis results of the "herb-active ingredient-overlapping target gene", the key active ingredients of ZYTLF are mainly quercetin, kaempferol, luteolin, rhubarbin, formononetin and other flavonoid compounds. At the same time, they are also plant-like estrogen, which has two-way regulating effects. It plays the role of estrogen when the level of estrogen is low, and plays the role of anti-estrogen when the level of estrogen increases. It can improve a series of symptoms caused by the postmenopausal low estrogen level and avoid the occurrence of tumors of the reproductive system caused by estrogen replacement therapy . Quercetin with the highest degree value in the network is a typical flavonoid compound. It has multiple pharmacological effects, including free radical scavenging, anti-cancer, anti-infection, and cardiovascular protection [26-29]. Quercetin has also been proven to be an effective component against osteoporosis, with the dual effects of inhibiting osteoclastogenesis and osteoblast differentiation. Studies such as Li Zhixing’s found that quercetin can relieve osteoporosis symptoms in ovariectomized rats, possibly by up-regulating ALP gene expression and inhibiting JNK, ERK, and p38 MAPK signaling pathways . Quercetin can also directly or indirectly down-regulate the expression of RANKL, inhibit osteoclast differentiation, reduce bone resorption, and stimulate osteoblast activity through estrogen signaling pathway and ERK signaling pathway . Kaempferol has also been shown to have bone protective effects on ovariectomized rats , possibly through estrogen receptor, MAPK, NF-κB and other signaling pathways . Luteolin can prevent bone loss after osteoporosis by inhibiting osteoclast differentiation.
Key Genes of ZYTLF for Preventing PMOP
Combined with the analysis of PPI network and key gene network, it was found that among the key genes of ZYTLF for anti-PMOP, JUN, MAPK8, AKT1, IL-6, MMP9, PTGS2, TNF, MAPK1, CASP3, etc. were all related to the inflammatory response process. Studies have found that the reduction of estrogen levels in postmenopausal women can stimulate the immune system to produce a large number of osteoclastogenic factors, including TNF-α, RANKL, IL-17A, etc., which in turn activates related signaling pathways, further aggravating bone loss . Li Zha and other scholars have found that postmenopausal women with osteoporosis have significantly increased levels of TNF-α. In-vitro experiments have found that TNF-α and RANKL synergistically enhance bone resorption of osteoclasts through NF-κB and PI3K/Akt signaling pathways . TNF-α and IL6 play an important role in the immune response and bone metabolism, mainly affecting the differentiation and proliferation of osteoclasts by regulating complex mechanisms, and they are important pathogenic factors for immune-mediated bone diseases . Studies have shown that increased levels of inflammatory factor TNF-α in the serum of postmenopausal women may be one of the important causes of osteoporosis. TNF-α can activate the RANK/RANKL signaling pathway and induce the formation of osteoclasts . The author speculates that elevated TNF-α level in the serum of postmenopausal women with osteoporosis may be related to estrogen deficiency.
The signal pathways ZYTLF in preventing PMOP
The enrichment analysis of KEGG pathways for the key genes of ZYTLF in preventing PMOP showed that 15 signaling pathways were related to the occurrence and development of PMOP, including hormone pathways and inflammation-related pathways. Hormones in postmenopausal women, including estrogen , parathyroid hormone , and prolactin, if not normally secreted could affect bone metabolism . Estrogen bound with the estrogen receptor in osteoblasts and osteoclasts to act on the OPG/RANK/RANKL signaling pathway, which further promoted OPG secretion, down-regulated the expression of RANKL, and inhibited the formation of osteoclasts . After the combination of estrogen and estrogen receptor, it could also regulate the expression of various target genes through the estrogen signaling pathway, thereby activating downstream PI3K/Akt, MAPK, WNT and other signaling pathways to promote the differentiation and proliferation of osteoblasts [42-43]. The TNF signaling pathway had the largest number of enriched genes, which might be the key signaling pathway for ZYTLF to prevent PMOP. The TNF signaling pathway was mainly opened by TNF-α and interacted with multiple signaling pathways to synergistically inhibit osteoclast differentiation and bone resorption function .