Molecular Mechanisms and Therapeutic Potential of Puerariae Lobatae Radix in Bone Metabolism: A Comprehensive Review

Abstract

Dysregulated bone metabolism is the core pathological trigger of osteoporosis (OP) and other orthopedic disorders. Puerariae Lobatae Radix (PLR; kudzu) is a classic traditional Chinese medicinal herb, and its bioactive constituents including isoflavonoids and triterpenoid saponins exert well-documented bone-modulating effects. However, the specific mechanisms underlying PLR’s regulation of bone metabolism have not been systematically elucidated, due to the complexity of bone metabolic signaling networks. Based on 127 relevant literature retrieved from Web of Science, Scopus and PubMed, this review systematically summarizes the molecular mechanisms of PLR and its core active components in regulating bone metabolism. We focus on PLR’s regulation of pathways implicated in metabolic bone diseases (MBDs), including NF-κB, RANKL/RANK/OPG, PI3K/AKT, and MAPK, via multiple mechanisms including anti-inflammation, anti-oxidative stress, modulation of ferroptotic signaling, ie, regulation of iron-dependent lipid peroxidation-related cell death, autophagy modulation, and osteoimmune regulation, ie, modulation of interactions between immune responses and bone cells. This review suggests that PLR regulates bone homeostasis through multi-component and multi-pathway mechanisms, with potential effects on inhibiting osteoclast activity, promoting osteoblast function and maintaining bone microenvironment stability. It also provides a theoretical reference for further research on PLR, and highlights that future studies should focus on high-quality clinical trials and multi-omics exploration of signaling crosstalk to promote its clinical translation in MBDs.

Introduction

Metabolic bone diseases (MBDs) refer to a group of systemic metabolic disorders characterized by an imbalance in bone metabolism, alongside structural or mineralization abnormalities. The clinical hallmark of MBDs is dysregulated bone turnover, which may manifest as generalized bone pain, pathological fractures, and skeletal deformities.1 Mechanistically, the pathogenesis is driven by a fundamental disruption of bone homeostasis, where the rate of osteoclastic bone resorption consistently exceeds that of osteoblastic bone formation, ultimately culminating in decreased bone mass and skeletal destruction.2 MBDs primarily encompasse osteoporosis, osteomalacia, Paget’s disease, and renal osteodystrophy, with osteoporosis representing the most prevalent metabolic bone disorder to date.3 Among the Chinese population aged 50 and older, the prevalence of osteoporosis is 6.46% in men and significantly higher at 29.13% in women. Currently, approximately 60.2 million individuals over the age of 50 in China suffer from osteoporosis, comprising 10.9 million men and 49.3 million women. As the aging of the population accelerates, the number of osteoporosis patients in China is projected to exceed 120 million by 2050.4 Osteoporosis not only inflicts profound suffering upon individual patients but also imposes a substantial economic burden on society. In an Italian survey of patients with osteoporotic hip fractures, the average treatment cost per patient was 4963 euros. Across Europe, the total expenditure for managing osteoporosis exceeded 56.9 billion euros in 2019.5

According to the latest guidelines from the International Osteoporosis Foundation (IOF),6 current therapeutic strategies for MBDs are primarily categorized into pharmacological and non-pharmacological interventions. Non-pharmacological interventions, serving as a foundational approach throughout the entire course of management, are strongly recommended by clinical guidelines. These primarily include dietary supplementation of calcium, vitamin D, and protein, alongside exercise interventions and lifestyle modifications. Pharmacological therapies include baseline agents such as calcium and vitamin D, anti-resorptive medications like bisphosphonates and RANKL inhibitors, and bone-forming anabolic agents including abaloparatide and romosozumab.7 Currently, each existing therapeutic modality has its own limitations. In patients receiving non-pharmacological treatment, clinical efficacy may be constrained by poor adherence and significant inter-individual variability.8 Regarding pharmacological options, adverse side effects often preclude effective treatment for some patients. For instance, calcium supplementation may lead to adverse outcomes such as nephrolithiasis and gastrointestinal complications.9 As a first-line therapeutic option, bisphosphonates are associated with severe adverse effects, such as osteonecrosis of the jaw.10–12 Furthermore, biologics such as denosumab entail substantial medication costs, rendering effective treatment inaccessible for patients who cannot afford the financial burden.5 As a cornerstone of traditional Chinese medicine (TCM), this therapeutic modality offers distinct historical advantages, such as profound clinical efficacy and relatively favorable tolerability in traditional use. Its high patient compliance and cost-effectiveness have further contributed to its significant success in managing orthopedic disorders.13 A variety of traditional herbs, including Rhizoma Drynariae, Fructus Psoraleae, and Fructus Ligustri Lucidi, have been empirically proven to play a pivotal role in modulating bone metabolism.14–17

Puerariae Lobatae Radix (PLR; kudzu), derived mainly from Pueraria lobata, is a classical traditional Chinese medicinal herb. PLR has a long history as both a traditional medicine and an edible plant in China. It was first recorded in Shen Nong Ben Cao Jing and has been continuously described in successive Materia Medica texts. Traditionally, PLR has been used to relieve fever, generate body fluid, alleviate thirst, stop diarrhea, promote rash eruption, and detoxify alcohol. Its long-standing medicinal and dietary use provides an important historical basis for its modern pharmacological investigation.18 It is an important component of Gegen Decoction and Bushen Qianggu Decoction.19 These prescriptions have been used in clinical practice for disorders related to musculoskeletal function and bone health. Recent preclinical studies suggest that PLR and its major constituents may regulate bone metabolism by promoting osteoblast function, suppressing osteoclast differentiation, and modulating the bone microenvironment.20

However, due to the myriad of signaling pathways involved in bone turnover and the inherent complexity of these regulatory networks,21 the specific molecular mechanisms by which PLR regulates bone metabolism have yet to be systematically elucidated. This review aims to summarize the regulatory effects of PLR and its bioactive constituents on bone metabolism. It focuses on key signaling pathways, bone-related cell types, disease models, and translational challenges. It also integrates current evidence from domestic and international studies. This review may provide a clearer framework for future research on PLR and support further evaluation of PLR in metabolic bone diseases.

Literature Search Strategy and Evidence Scope

This review was designed as a comprehensive narrative review to summarize the current evidence regarding the regulatory effects of PLR and its major active constituents on bone metabolism. A literature search was conducted in three electronic databases, including PubMed, Scopus, and Web of Science, from database inception to September 30, 2025. The search strategy combined terms related to bone metabolism and bone metabolic disorders with terms related to PLR and its botanical or phytochemical synonyms. Taking PubMed as an example, the following search terms were used: (“Osteoclast” OR “Odontoclasts” OR “Cementoclasts” OR “Cementoclast” OR “Odontoclast” OR “Osteoblast” OR “Osteocyte” OR “Osteoporosis” OR “Osteopenia” OR “Osteonecrosis” OR “Osteophyte” OR “Osteomalacia” OR “Osteogenesis Imperfecta” OR “Bone Metabolism” OR “Bone Remodeling”) AND (“pueraria” OR “puerarin” OR “kudzu” OR “Pueraria lobata” OR “Pueraria tuberosa” OR “tuberosa” OR “Pueraria montana” OR “montana, Pueraria” OR “Pachyrhizus thunbergianus”). The search strategy was adapted as appropriate for Scopus and Web of Science. The initial search yielded 674 records, including 219 records from Web of Science, 331 from Scopus, and 124 from PubMed. After duplicate records were removed, 507 records remained for title and abstract screening. Studies were considered potentially eligible if they investigated PLR, puerarin, or other PLR-related constituents/extracts in relation to bone metabolism, bone remodeling, osteoblasts, osteoclasts, osteocytes, bone marrow mesenchymal stem cells, metabolic bone diseases, or bone-related signaling pathways. After screening titles and abstracts, 127 studies were retained and used as the main evidence base for this review. The study selection process is shown in Figure 1.

Figure 1.

PRISMA flowchart of the literature screening.

The included evidence consisted predominantly of preclinical studies, including in vitro experiments using osteoblasts, osteoclast precursors, osteoclasts, osteocytes, bone marrow mesenchymal stem cells, and related cell models, as well as in vivo studies using animal models of osteoporosis, inflammatory bone loss, alveolar bone loss, glucocorticoid-induced bone impairment, and bone defect repair. Clinical evidence was limited, and available human studies were not sufficient to establish definitive clinical efficacy or long-term safety. Therefore, this review primarily summarizes mechanistic and translationally relevant preclinical evidence, while clinical implications are discussed cautiously.

The Chemical and Pharmacological Basis of PLR and Its Active Monomers

Puerariae Lobatae Radix (PLR; kudzu) is the dried root of the perennial deciduous vine Pueraria lobata (Willd.) Ohwi or Pueraria thomsonii Benth., belonging to the Fabaceae family.22 In clinical TCM, PLR is widely utilized for managing various ailments. It has demonstrated significant therapeutic efficacy across a diverse range of pharmacological activities, including anti-tumor, immunomodulatory, hypoglycemic, and hypolipidemic effects.23 The primary bioactive constituents of PLR are isoflavonoids, which include puerarin, daidzin, daidzein, genistein, formononetin, and biochanin A. These isoflavones exhibit a diverse range of biological activities—including anti-inflammatory, anti-diabetic, anti-osteoporotic, and neuroprotective effects—underpinning their broad pharmacological profile.24 Furthermore, PLR is enriched with triterpenoids, such as soyasapogenol B and kudzusaponins A1 and A2, as well as coumarins, including scopolin and coumarin-7-O-rhamnoside. Among these, the triterpenoids have demonstrated significant potential in anti-tumor, anti-inflammatory, and anti-oxidative stress pathways, while the coumarins similarly possess robust antioxidant, anti-inflammatory, and anti-viral properties.25 To provide a clearer overview of the pharmacological material basis of PLR in bone metabolism, the major chemical categories and representative bioactive monomers are summarized in Table 1. Furthermore, PLR possesses a high starch content of up to 449.85 mg/g, with resistant starch accounting for 23.14%, making it a high-quality source of dietary fiber capable of modulating the composition of gut microbiota. It is also enriched with water-soluble polysaccharides, a diverse profile of amino acids, and essential micronutrients, which collectively enhance immune function and support systemic energy metabolism, neurotransmission, and skeletal mineralization.26 Accumulating preclinical evidence suggests that PLR and its primary bioactive constituents may have potential benefits in the regulation of osteoporosis-related bone metabolism.27 In summary, PLR and its primary constituents exhibit multifaceted pharmacological effects in the treatment of osteoporosis, with mechanisms spanning bone metabolism regulation, anti-inflammation, and antioxidant pathways. The core biological activities underlying the bone-protective effects of PLR are systematically summarized in Figure 2. A variety of bioactive compounds within PLR have been demonstrated in vitro to modulate the activity of intraosseous cells via specific signaling cascades. By synthesizing these signaling pathways, this review offers novel perspectives and therapeutic strategies for managing osteoporosis. These findings further establish a theoretical framework for the development of PLR as a candidate anti-osteoporotic agent.

Table 1.

Major Chemical Constituents and Representative Bioactive Monomers of PLR with Relevance to Bone Metabolism

Category	Representative Compounds	Reported Content in PLR	Major Pharmacological Relevance	Chemical Formula	Structural Formula	Reference
Isoflavones	Puerarin	4.28–76.10 μg/g	Anti-inflammatory; antioxidant; anti-apoptotic; autophagy regulation; anti-ferroptotic effects	C21H20O9		[28–30]
	Daidzein	0.36–16.48 μg/g	Anti-osteoporotic; osteogenic; anti-resorptive; antioxidant; estrogen-like effect	C15H10O4		[31–33]
	Genistin	7.63–51.43 μg/g	Anti-osteoporotic; osteogenic; anti-resorptive; antioxidant; anti-inflammatory	C21H20O10		[34–36]
	Formononetin	Trace level	Anti-osteoporotic; osteogenic; anti-resorptive; anti-inflammatory; antioxidant	C16H12O4		[37–39]
Coumarins	Coumestrol	Trace level	Anti-osteoporotic; bone-protective; estrogen-like effect; antioxidant; anti-inflammatory	C15H8O5		[40–42]
Triterpenoids and saponins	Soyasapogenol B	Trace level	Anti-inflammatory; anti-osteoporotic; bone-protective; antioxidant	C30H50O3		[43]

Figure 2.

PLR exerts anti-osteoporotic effects through five key complementary actions: anti-inflammatory; antioxidant; anti-apoptotic; autophagy regulation and anti-ferroptotic effects, which collectively regulate bone metabolism and maintain bone homeostasis.

Key Molecular Mechanisms of PLR in Regulating Bone Metabolism

Core Signaling Pathways

The bone-protective effects of PLR and its bioactive constituents are primarily mediated by the precise modulation of multiple core signaling pathways closely associated with bone homeostasis, including MAPK, RANKL/RANK/OPG, ER, NF-κB, PI3K/Akt, and Wnt cascades. These pathways synergistically regulate the proliferation, differentiation, and functional activity of osteoblasts and osteoclasts, thereby maintaining the dynamic balance of bone remodeling. The key preclinical evidence regarding the regulatory effects of PLR and its active components on these core pathways is systematically summarized in Table 2.

Table 2.

Effects of PLR on Core Signaling Pathways in Bone Metabolism

Pueraria Constituent	Study Type	Intervention	Subjects	Optimal Dose	Dose Range	Outcomes	References
Puerarin	In vitro	N/A	Rat BMSCs	20 μM	20, 50, 100, 200 μM	Runx2, Osx, OCN, Col-I, Bcl-2/Bax↑; TRAP, NFATc1, c-Fos↓	[44]
	In vivo	Intragastric administration	OVX rats	4 mg/kg/d	4 mg/kg/d	Bone mass, bone microstructure↑
Puerarin	In vitro	N/A	MC3T3-E1 cells	1 μM	0.1, 1, 10, 100, 1000 μM	Cell viability, Col I, p-ERK1/2↑	[45]
Puerarin	In vitro	N/A	MC3T3-E1 cells	1 μM	0.1 μM, 1 μM, 10 μM	Calcium, BMP-2, p-ERK1/2, p-p38↑	[46]
Puerarin	In vitro	N/A	HOBs	0.01 μM	0.001, 0.01, 0.1, 1 μM	Bcl-2, p-ERK↑; Bax↓	[47]
Puerarin	In vitro	N/A	ROBs	0.1 μM	0.01, 0.03, 0.1 μM	Mineralization, Col I↑, p-p38↑, β-catenin↑	[48]
	In vivo	Intragastric administration	OVX rats	20 mg/kg/d	20 mg/kg/d	BMD, Tb.N, BMC, Tb.Th, Tb.Sp↑
PLR	In vitro	N/A	Mice BMMs	25 μg/mL	1, 5, 25 μg/mL	TRAP, c-Fos, NFATc1, p-p38, p-JNK↓	[49]
HX112	In vitro	N/A	RAW264.7 cells	200 μg/mL	10, 50, 100, 200 μg/mL	TRAP, c-FOS, MMP9, p-SRC, p-PI3K, p-AKT↓	[50]
	In vivo	Oral gavage	OVX mice	300 mg/kg/d	100, 300 mg/kg/d	BMD, BV/TV, Tb.Th↑; Tb.Sp↓
Puerarin	In vivo	Oral gavage	OVX rats	18 mg/kg/d	18 mg/kg/d	BV/TV, Tb.N, OPG/RANKL, E2↑; Tb.Sp, CTX, TRACP l↓	[51]
Puerarin	In vivo	Oral gavage	Periodontitis rats	200 mg/kg/d	100, 200, 400 mg/kg/d	CEJ-ABC, IL-1L, TNF-α, MMP-2↓; BV/TV↑	[52]
PLR	In vitro	N/A	RAW264.7 cells	100 μg/mL	0.01, 0.1, 1, 10, 100 μg/mL	OPG/RANKL, OCN, Runx2↑; TRAP, p-NF-κB↓	[53]
	In vivo	Oral gavage	OVX mice	100 mg/kg	100 mg/kg	BV/TV, Tb.N, OPG↑; Tb.Sp↓
Puerarin	In vivo	Oral gavage	OVX rats	50 mg/kg/d	50 mg/kg/d	BV/TV, Tb.Th, zinc, calcium, OCN, OPG↑; Tb.Sp, ALP, CTX, RANKL↓	[54]
Puerarin	In vivo	Oral gavage	SD rats	4 mg/kg/2d	4 mg/kg/2d	BMD, BV/TV, Tb.N, biomechanical properties, OPG, OPN↑, calcium↑; Tb.Sp↓; RANKL↓, TRAP↓	[55]
PXY	In vitro	N/A	ROBs	20 μM	5, 10, 20 μM	OPG↑, OPG/RANKL↑, RANKL↓	[56]
	In vivo	Intraperitoneal injection	OVX mice	60 mg/kg/d	20, 40, 60 mg/kg/d	Uterineindex, calcium, ALP↑; femoral pathological injury ameliorated
Puerarin	In vivo	Subcutaneous injection	IUGR Rats	50 mg/kg/d	50 mg/kg/d	BALP, IGF-1, OPG, OCN, OPG/RANKL↑	[57]
FRAC	In vivo	Oral administration	OVX rats	62.5 μg/mL	31.25, 62.5, 125, 250, 500 μg/mL	Body/uterine weight↓; femoral biomechanical parameters↑	[58]
PLR	In vivo	Oral administration	OVX rats	1600 mg/kg/d	25, 100, 400, 1600 mg/kg/d	Calcium, E2, ER-α, OC, CTX-1, DPD, PYD; ER-α↑	[59]
Puerarin	In vitro	N/A	MG-63 cells	0.01 μM	0.01, 0.1, 1 μM	IL-6, MG63 cell proliferation↓	[60]
Puerarin	In vitro	N/A	ROBs	1 μM	0.01, 0.1, 1 μM	Cell proliferation, ALP, ERα/ERβ↑	[61]
DG	In vitro	N/A	RAW264.7 cells	50 μg/mL	25, 50 μg/mL	TRAP, RANK, c-FOS, NFATc1, p-p65, autophagy, ROS↓	[62]
	In vivo	Oral administration	OVX rats	200 mg/kg/d	50, 200 mg/kg/d	BMD, BV/TV↑; RANKL, CR, BUN↓
Puerarin	In vitro	N/A	RAW264.7 cells	100 μM	10, 50, 100, 500 μM	ROS↓; NFATc1, MMP9↓, p-p65↓	[63]
	In vivo	Intraperitoneal injection	OVX mice	100 mg/kg/2d	100 mg/kg/2d	Femoral BMD, BV/TV↑; osteoclast number, ROS, NOX, HO-1↓
Puerarin	In vitro	N/A	RAW264.7 cells	100 μM	1, 10, 50, 100, 500 μM	Bone resorption pits, F-actin ring formation↓; IκBα, p65, c-Fos, NFATc1, MMP-9↓	[64]
	In vivo	Intraperitoneal injection	Osteolysi Rats	30.8 mg/kg/d	15.4, 30.8 mg/kg/d	BMD↑, BV/TV↑; TNF-α↓, IL-6↓
Puerarin	In vitro	N/A	RAW264.7 cells	10 μM	10 μM	TRAP, MMP-9, p-AKT, p-FoxO1↓; FoxO1, catalase↑	[65]
	In vivo	Subcutaneous injection	OVX rats	100 mg/kg/d	100 mg/kg/d	BMD, BV/TV, OPG/RANKL, GSH-Px↑; ROS↓
Puerarin	In vitro	N/A	MG-63 cells	0.1 μM	0.01, 0.1, 1, 10 μM	Cyclin D1/B1, ALP, COL I, Bcl-xL, p-ERK, p-Akt↑; apoptosis↓	[66]
Puerarin	In vivo	Culture medium	ROBs	10 μM	2.5, 5, 10, 25, 50, 100 μM	Cell viability, ALP, p-Akt(Ser473)↑	[67]
Puerarin	In vitro	N/A	Rat BMSCs	1 μM	0.1, 1, 10, 100 μM	Cell proliferation, ALP, mineralized nodule formation, Col I, β-catenin↑	[68]
	In vivo	Local injection	RME Rats	15 mg/kg/d	15 mg/kg/d	Bone microstructure, ALP, BMP2↑
Puerarin	In vitro	N/A	Rat BMSCs	10 μM	0.01, 0.1, 1, 10, 100 μM	ALP↑, OCN↑, Wnt5b↑	[69]

Notes: ↑ indicates increase/upregulation; ↓ indicates decrease/downregulation; N/A indicates not applicable.

MAPK Signaling Pathway

Mitogen-activated protein kinases (MAPKs) serve as essential transducers that relay signals from the cell surface to the nucleus. They play a pivotal role in diverse cellular processes, including proliferation, differentiation, development, inflammatory responses, and programmed cell death.70 Mammals express at least four distinct MAPK subgroups, namely ERK1/2, JNK, p38 MAPK, and ERK5. Current research indicates that PLR and its active constituents significantly modulate bone metabolism via the p38 MAPK, ERK1/2, and JNK signaling pathways. While each pathway functions independently, they also exhibit intricate crosstalk and synergistic interactions.71 For instance, Li et al72 demonstrated that osteogenesis requires the coordinated regulation of both the JNK and p38 signaling pathways.

ERK Signaling Pathway

The ERK signaling pathway is a canonical member of the MAPK family. It is activated via the Ras/Raf/MEK tertiary phosphorylation cascade, mediating the transduction of extracellular signals into the intracellular compartment to regulate fundamental cellular processes such as proliferation and differentiation. This pathway typically exerts a stimulatory effect on osteoblasts, promoting the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts and enhancing bone matrix mineralization by upregulating key osteogenic transcription factors such as Runx2 and OSX.73 In contrast, it exhibits a bidirectional regulatory effect on osteoclasts, modulating their differentiation and activity through the regulation of NFATc1. Research indicates that PLR can activate the ERK1/2 signaling pathway to regulate bone metabolism, thereby exerting a bone-protective effect.74

Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells originating from the bone marrow. Under specific induction conditions, they can differentiate into mesenchymal lineages—including osteoblasts, chondrocytes, and adipocytes—as well as myoblasts, fibroblasts, and neuron-like cells, making them pivotal for maintaining skeletal homeostasis. Research has demonstrated that activation of the ERK pathway promotes the proliferation and differentiation of BMSCs.75 Yang et al44 demonstrated that puerarin concentrations exceeding 200 μM inhibit the proliferation of BMSCs and induce apoptosis in a dose-dependent manner. Conversely, at dosages of 20μM and 50μM, puerarin significantly facilitates BMSC differentiation, an effect that is markedly suppressed upon the addition of an ERK1/2 inhibitor. Zheng et al45 reported that 1 μM puerarin significantly promotes the proliferation and differentiation of the osteoblast precursor cell line MC3T3-E1 within 48 hours, elevating alkaline phosphatase (ALP) levels. However, this effect is attenuated by ERK1/2 inhibition. Similarly, Zhong et al46 observed that 1μM puerarin intervention for seven days maximizes the proliferation of MC3T3-E1 cells, while ERK1/2 pathway blockers effectively negate these osteogenic effects. Focusing on human osteoblasts (hOBs), Liu et al47 identified an optimal dosage of 0.01 μM puerarin, which significantly inhibits hOB apoptosis and increases the phosphorylation levels of the ERK signaling pathway. These protective effects were diminished by both ERK pathway inhibitors and estrogen receptor (ER) antagonists. Consequently, it can be inferred that the puerarin-mediated inhibition of hOB apoptosis via the ERK pathway is dependent on the presence of ER, positioning the ERK pathway as a downstream signaling cascade mediated by estrogen receptors.

P38 MAPK Signaling Pathway

Under steady-state conditions, p38 MAPK remains stable in a dephosphorylated form. However, upon exposure to extracellular or intracellular stimuli—such as BMP/TGF-β, hormonal fluctuations, or mechanical loading—the pathway is activated via a MKK3/6-mediated tertiary phosphorylation cascade. This activation subsequently triggers downstream biological responses, including inflammatory cascades and apoptosis.76 In the context of bone metabolism, the p38 MAPK pathway plays a central role in promoting osteoblast proliferation, inhibiting osteoclast activation, and driving the lineage commitment and maturation of mesenchymal stem cells into osteoblasts.77

Both ERK1/2 and the p38 MAPK pathway belong to the MAPK family and play regulatory roles in bone metabolism, yet their interaction mechanisms and relative dominance remain unclear. In studies by Yang et al44 and Zhong et al,46 the osteogenic effects of puerarin were attenuated following the application of p38 MAPK inhibitors. However, Yang observed that this attenuation was less pronounced than that caused by ERK1/2 inhibition, suggesting that ERK1/2 may play a relatively dominant role. In contrast, the findings reported by Zhong et al indicated the opposite, with p38 MAPK inhibition exerting a greater impact. These discrepancies may reflect differences in the targeting specificity of puerarin-regulated pathways during osteogenic differentiation. Specifically, Yang et al utilized BMSCs and covered the entire osteogenic cycle, whereas Zhong et al employed MC3T3-E1 pre-osteoblasts, focusing primarily on the initial differentiation stage. Consequently, it can be hypothesized that puerarin’s regulatory function via the ERK1/2 pathway is more significant throughout the full cycle of bone metabolism, while the p38 MAPK pathway plays a more critical role during the early stages of osteoblast differentiation. Research has shown48 that puerarin at concentrations of 0.03–0.1 μM promotes osteoblast differentiation and significantly increases p38 phosphorylation levels. However, when an ER antagonist was applied to these cells, the puerarin-induced phosphorylation of p38 was significantly inhibited. This suggests that the activation of the p38 pathway may be ER-dependent. Therefore, it can be inferred that in the regulation of bone metabolism, the p38 pathway serves as one of the downstream pathways mediated by the ER receptor.

JNK Signaling Pathway

JNK represents a canonical member of the MAPK family. It is encoded by three distinct genes including JNK1, JNK2, and JNK3. Upon activation by upstream signaling cascades, JNK translocates to the nucleus to phosphorylate transcription factors, subsequently activating c-Jun and enhancing its transcriptional activity. Its core function involves transducing extracellular stimuli to regulate cellular differentiation, proliferation, and stress responses.78 Interestingly, its role in bone metabolism remains somewhat controversial. Some studies suggest that JNK pathway activation facilitates osteogenic differentiation and exhibits synergistic effects with the p38 pathway.79 Conversely, other evidence indicates that the JNK signaling cascade may accelerate SMAD1 degradation via osteoclast activation, thereby inhibiting bone formation.74 Baek et al49 demonstrated that PLR suppresses the formation of Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, by markedly inhibiting JNK phosphorylation and downregulating the expression of osteoclast-related genes. This regulatory effect ultimately leads to a reduction in bone resorptive activity. Similarly, Song et al50 found that HX112, a combination extract of PLR and Platycodi Radix, downregulates osteoclastogenesis-related genes by inhibiting the JNK/p38 pathways. In contrast, the experimental findings of Yang et al44 indicated that JNK phosphorylation levels remained unchanged following puerarin treatment. This discrepancy may be attributed to cell-type specificity. Specifically, PLR extracts exert anti-osteoporotic effects by inhibiting the high JNK pathway activity that promotes RANKL-induced osteoclast differentiation and bone resorption. However, during the osteogenic differentiation of BMSCs, puerarin primarily initiates osteogenic gene transcription through the specific activation of the ERK1/2 and p38 MAPK pathways, while the JNK pathway may not be involved in this particular osteogenic regulatory network.

RANK/RANKL/OPG Signaling Pathway

The RANKL/RANK/OPG signaling pathway is a member of the tumor necrosis factor (TNF) superfamily, and it is composed of three core components. These three components are Receptor Activator of Nuclear Factor-κB Ligand (RANKL), Receptor Activator of Nuclear Factor-κB (RANK), and Osteoprotegerin (OPG). Surface RANK on osteoclast lineages binds with RANKL to promote cellular activation and maturation. This interaction concurrently triggers intracellular signaling cascades to drive further differentiation. In contrast, OPG functions as a decoy receptor for RANKL, competitively binding to it to prevent its interaction with RANK, thereby inhibiting osteoclast development and ultimately suppressing bone resorption.80 Consequently, the OPG/RANKL expression ratio plays a pivotal role in modulating bone repair. Furthermore, OPG effectively blocks the RANKL-RANK interaction, thus arresting osteoclastogenesis.81 Research indicates that pathological bone loss is frequently associated with excessive RANKL expression resulting from an imbalanced immune response. Therefore, targeting RANKL-related pathways may mitigate immune-mediated skeletal damage.82 In summary, the RANKL/RANK/OPG signaling axis is of fundamental significance in regulating osteoblast proliferation, differentiation, and mineralization, as well as the bone-resorptive activities of osteoclasts.83

Huang et al51 demonstrated that puerarin intervention improved body weight and bone microstructure in rats with postmenopausal osteoporosis, elevated estrogen levels, reduced osteoclast metabolic markers, and concurrently upregulated the OPG/RANKL ratio. Similarly, Yang et al52 found that puerarin improved bone microstructure in male Sprague-Dawley (SD) rats with ligation-induced periodontitis, while increasing the OPG/RANKL ratio, reducing osteoclast counts, and modulating key inflammatory and bone metabolic mediators, including IL-1β, TNF-α, and MMP-2/9. Kim et al53 reported that PLR and its extract fermented with Lactobacillus paracasei JS1 (FPE) significantly promoted osteoblast proliferation and enhanced the OPG/RANKL ratio. As an essential trace element, zinc is indispensable for bone growth and stimulation. Evidence suggests that decreased serum zinc levels are associated with osteoporosis, likely due to zinc’s role in modulating the anabolic effects of estrogen on bone and inhibiting osteoclastogenesis.84 Wang and Lin54 observed that puerarin, especially when combined with zinc supplementation, significantly improved bone microstructure and serum levels of zinc, calcium, and phosphorus in female SD rats compared to control groups. Regarding bone turnover markers, this combination led to a decrease in the Osteocalcin/C-terminal telopeptide of type I collagen (OC/CTX) ratio and an increase in the OPG/RANKL ratio, indicating a synergistic therapeutic effect. In a related study, Liu et al55 found that the combination of puerarin and zinc significantly enhanced mandibular bone mineral density (BMD), microstructure, and biomechanical properties in female SD rats, while increasing the OPG/RANKL ratio and decreasing TRAP levels. The authors attributed this mechanism partly to improved gastrointestinal calcium absorption facilitated by zinc. Notably, another study by Liu85 demonstrated that the combination of puerarin and zinc did not increase uterine weight in rats, thereby avoiding estrogenic side effects and underscoring its safety profile. Furthermore, Li et al56 discovered that PXY, a major isoflavone monomer from PLR, significantly improved body weight and uterine indices in ovariectomized (OVX) mice, elevated serum calcium and phosphorus, and repaired femoral pathology. PXY was also found to promote osteoblast proliferation and enhance the OPG/RANKL ratio in a dose-dependent manner. Maternal malnutrition during pregnancy has a profound impact on the skeletal development of children, and intrauterine growth restriction (IUGR) induced by maternal nutritional deficiencies directly affects the neonatal skeletal status.86 Chen et al57 found that puerarin significantly reversed the negative effects of malnutrition in IUGR rats, markedly increasing body weight and the OPG/RANKL ratio without altering serum calcium and phosphorus levels compared to controls.

Estrogen Receptor (ER) Signaling Pathway

The estrogen receptor (ER) signaling pathway serves as a critical nexus linking endocrine regulation with skeletal remodeling and is a cornerstone in maintaining systemic bone homeostasis. Estrogen (E2), acting as a primary extracellular messenger, traverses the cell membrane to bind with cytoplasmic estrogen receptors ERα and ERβ, inducing conformational changes, dimerization, and subsequent nuclear translocation. Within the nucleus, these receptor complexes function as transcription factors, binding specifically to estrogen response elements (EREs) in the promoter regions of target genes to initiate transcriptional programs. Concurrently, membrane-bound receptors such as GPER1 mediate rapid non-genomic effects by triggering intracellular kinase cascades. Activation of this pathway significantly promotes osteoblast proliferation and differentiation while inducing osteoclast apoptosis.87 Furthermore, research has demonstrated that this pathway supports the metabolic environment essential for skeletal health by maintaining gut microbiota homeostasis and intestinal barrier function, thereby blocking systemic inflammation triggered by endotoxemia.88 Within the osteoimmune microenvironment, ER pathway activation inhibits the differentiation of pro-inflammatory Th17 cells and promotes the expansion of regulatory T cells (Tregs), fostering an osteogenic microenvironment by downregulating TNF-α and RANKL levels.89 Additionally, studies have found that ER signaling upregulation enhances antioxidant enzyme systems and preserves mitochondrial function, thereby delaying the senescence of osteoblasts and osteocytes by mitigating oxidative stress-induced damage.

Satpathy et al58 demonstrated that the flavonoid-rich antioxidant fraction (FRAC) from PLR tubers significantly improved skeletal biomechanical parameters and bone microstructure in OVX rats, while concurrently reducing body weight and increasing uterine weight. Molecular docking analysis revealed a high binding affinity of this extract for both ERα and ERβ, suggesting that its bone-protective effects are mediated through phytoestrogenic activities. Lee et al59 found that PLR extract dose-dependently decreased bone turnover markers, elevated serum calcium and estradiol levels, and upregulated ER-α expression in OVX rats, accompanied by increases in uterine weight and endometrial thickness. Notably, even at a chronic high dosage of 1600 mg/kg, no significant alterations in AST or ALT levels were observed, indicating a low potential for hepatotoxicity. Luo et al60 identified that puerarin significantly inhibited the mRNA and protein expression of IL-6 in MG-63 cells—an effect that was markedly attenuated by ER antagonists. In the study by Wang et al (2012),48 both in vivo and in vitro experiments reported that puerarin significantly enhanced the phosphorylation levels of the ERK and Wnt signaling pathways. These effects were reversed by ER receptor antagonists, thereby establishing the ER pathway as a critical upstream mediator of the p38 MAPK and Wnt cascades in puerarin-induced bone metabolism regulation. Furthermore, Wang et al61 reported that puerarin significantly stimulated the proliferation of rat primary osteoblasts and increased both intracellular and extracellular ALP activity, while upregulating the expression of ERα and ERβ in a concentration-dependent manner.

NF-κB Signaling Pathway

Nuclear Factor-kappa B (NF-κB) is a generic term for a family of transcription factors that function as dimers, primarily regulating genes involved in immunity, inflammation, and cell survival.90 Its activation occurs via two pathways, among which the canonical pathway is most intimately linked to bone metabolism. Triggered by upstream factors, the canonical pathway involves IKK-mediated phosphorylation and subsequent degradation of IκBα at Ser32/36, allowing the p50/p65 heterodimer to translocate into the nucleus and activate transcription. This process directly regulates the transcription of target genes associated with osteoclast differentiation and activation, driving the transformation of osteoclast precursors into mature osteoclasts and enhancing bone resorptive function. Furthermore, inflammatory cytokines such as TNF-α can activate p65/p50, which interferes with the binding of SMAD complexes to DNA, thereby inhibiting key osteogenic transcription factors like Runx2 and Osx and arresting osteoblast differentiation.91 Extensive in vitro and in vivo evidence suggests that NF-κB, by simultaneously modulating both inflammatory responses and bone remodeling processes, represents a critical therapeutic target for inflammation-related bone diseases.92 Consequently, the activation of the NF-κB signaling pathway is an indispensable requirement for osteoclastogenesis and maturation. Blocking this cascade serves as a potential strategy for preventing inflammatory osteolysis.

Qin et al62 utilized network pharmacology to predict the mechanisms of the “Salvia miltiorrhiza-Puerariae” herb pair and subsequently suggested that it significantly inhibits the differentiation of RAW264.7 cells into osteoclasts by suppressing RANKL-induced p65 phosphorylation. Similarly, Xiao et al63 found that puerarin markedly inhibits the formation of multinucleated osteoclasts and reduces intracellular reactive oxygen species (ROS) levels. It downregulates the mRNA and protein expression of NFATc1 and MMP-9 in a concentration-dependent manner, indicating the suppression of NF-κB signaling transduction. Tang et al64 further demonstrated that puerarin significantly decreases RANKL-induced TRAF6 protein expression, leading to a marked reduction in the phosphorylation levels of IκBα and p65, as well as the protein expression of NFATc1 and MMP-9. Notably, experiments showed that puerarin exerts these inhibitory effects on osteoclastogenesis without significant toxicity to precursor cells. Yang et al52 reported that puerarin significantly lowered the levels of p-NF-κB, IL-1β, TNF-α, and MMP-2/9 in rat gingival tissues, effectively inhibiting NF-κB activation. In a ligation-induced periodontitis rat model, puerarin reduced the cemento-enamel junction to alveolar bone crest (CEJ-ABC) distance, decreased the RANKL/OPG ratio, and diminished the number of osteoclasts.

PI3K/Akt Signaling Pathway

The PI3K/Akt signaling pathway serves as a critical nexus linking extracellular signals to intracellular effectors and is a cornerstone in the regulation of bone metabolic homeostasis. PI3K, a key lipid kinase, is stimulated by extracellular primary messengers such as growth factors or cytokines to catalyze the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) into phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3). As a vital intracellular second messenger, PIP3binds to the PH domain of Akt, inducing its full activation through phosphorylation at the Thr308 and Ser473 sites. Activated Akt subsequently phosphorylates downstream target proteins to extensively regulate cellular proliferation, differentiation, and survival.93 As a major downstream branch of insulin signaling, this pathway provides essential bioenergetic support for anabolic processes by promoting glucose uptake and utilization in osteoblasts.94 Furthermore, within the osteoimmune microenvironment, activation of this pathway facilitates the polarization of macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory/pro-reparative M2 phenotype. This process fosters a bone microenvironment conducive to osteogenesis, ultimately achieving multidimensional regulation of bone metabolism. Research has also revealed that PI3K/Akt activation phosphorylates and activates the downstream regulator mTOR, which mitigates oxidative stress-induced damage to osteocytes through the fine-tuning of autophagy.95

FoxO1 is a member of the forkhead box (FOX) transcription factor family. As a key molecular regulator of oxidative stress and bone metabolism, it plays a central role in osteoclast differentiation and antioxidant homeostasis, primarily by binding to the promoter regions of target genes to modulate their transcriptional activity.96 Feng et al65 demonstrated that puerarin significantly inhibits the formation of TRAP-positive osteoclasts and mitigates H₂O₂-induced intracellular ROS accumulation. This effect was accompanied by a reduction in the phosphorylation levels of MMP-9, AKT, and FoxO1, a profile mirrored by the application of PI3K/AKT inhibitors. Wang et al66 reported that puerarin markedly stimulates osteoblast proliferation and enhances ALP activity and Type I collagen (Col-I) expression, concurrently elevating p-ERK and p-Akt levels. Through shRNA-mediated knockdown, they suggested that puerarin’s regulatory effects on osteoblasts are contingent upon the presence and functional integrity of the ER, thereby establishing that puerarin improves bone metabolism via the ER-mediated PI3K/Akt signaling axis. Furthermore, Song et al50 identified that the PLR extract HX112 significantly suppresses osteoclastogenesis and bone resorption by downregulating the mRNA levels of NFATc1, c-Fos, and MMP-9. Mechanistically, it inhibits RANKL-induced Src phosphorylation, subsequently intercepting the Src-dependent PI3K/Akt signaling cascade. Zhang et al67 also found that puerarin significantly enhances osteoblast viability and increases AKT phosphorylation at the Ser473 site, an effect that was effectively abolished by PI3K inhibitors.

Wnt Signaling Pathway

The Wnt signaling pathway is a fundamental regulator of organ development and adult tissue homeostasis, governing critical cellular processes such as proliferation, differentiation, migration, polarity, and gene expression. Based on its dependence on β-catenin, this system is categorized into canonical and non-canonical Wnt signaling pathways.97 The canonical Wnt pathway, the β-catenin-dependent cascade, is initiated upon the binding of Wnt ligands to Frizzled receptors and low-density lipoprotein receptor-related protein 5/6 (LRP5/6), leading to the intracellular accumulation and nuclear translocation of β-catenin. Subsequently, nuclear β-catenin interacts with T-cell factor/lymphoid enhancer-binding factor to activate the transcription of downstream target genes. This pathway is further modulated by endogenous inhibitors, including secreted frizzled-related proteins, Dickkopf (DKK), and Sclerostin (SOST). Extensive in vivo and in vitro evidence underscores the indispensable role of canonical Wnt signaling in osteoblast proliferation, differentiation, and functional activity, thereby regulating bone formation and maintenance. In contrast, non-canonical Wnt signaling activates β-catenin-independent cascades and, in certain contexts, can antagonize the canonical pathway. Non-canonical signaling is further subdivided into the Planar Cell Polarity and Wnt/Ca²⁺ pathways. Emerging research increasingly highlights the significant contribution of non-canonical Wnt signaling to bone formation. Most studies have indicated that PLR modulates bone metabolism primarily through the canonical Wnt pathway, exerting a dual regulatory effect that not only promotes the differentiation of mesenchymal stem cells (MSCs) into osteoblasts but also suppresses osteoclastogenesis and bone resorption by increasing the OPG/RANKL ratio.98

Yang et al68 demonstrated that puerarin significantly stimulates the proliferation of rat BMSCs, enhancing ALP activity, mineralized nodule formation, and upregulating osteogenic genes alongside proteins such as Collagen I and β-catenin. In a rat model of rapid maxillary expansion (RME), puerarin markedly increased BMP2 expression at the midpalatal suture, elevated microvessel density, and promoted chondrocyte hypertrophy, thereby accelerating bone regeneration by improving blood supply. Zhang and Luo69 found that 10μM puerarin significantly enhanced ALP activity and osteocalcin (OCN) content in BMSCs by elevating Wnt5b levels. However, this effect was concentration-dependent. Furthermore, Wang et al48 identified that puerarin signaling is initially triggered by the ER, which subsequently activates both the p38 MAPK and Wnt/β-catenin pathways to improve bone microstructure and metabolism. Their study suggested that these two cascades are independent downstream branches mediated by the ER, rather than having a hierarchical relationship.

Regulated Cell Death

Regulated cell death, including autophagy and ferroptosis, has been identified as a critical regulatory mechanism in the maintenance of bone homeostasis, with aberrant activation or inhibition of these processes closely linked to the pathogenesis of metabolic bone diseases. Accumulating evidence has demonstrated that PLR and its active components can modulate bone metabolism via the precise regulation of autophagy and ferroptosis in bone-related cells, with the key research findings summarized in Table 3.

Table 3.

Effects of PLR on Regulated Cell Death in Bone Metabolism

Pueraria Constituent	Study Type	Intervention	Subjects	Optimal Dose	Dose Range	Outcomes	References
Puerarin	In vitro	N/A	MC3T3-E1 cells	1 μM	0.1, 1, 10 μM	LC3B-II/LC3B-I, Beclin1, Autophagosomes↑; miR-204↓	[99]
Puerarin	In vitro	N/A	Rat BMSCs	50 μM	10, 25, 50 μM	Atg5, Atg7, Beclin1, LC3II/I, TRAP, CTSK, MMP-9↓	[100]
Puerarin	In vitro	N/A	RAW264.7 cells	40 μM	10, 20, 40, 80 μM	IL-6, TNF-α, NO↓, GSSG↑	[101]

Notes: ↑ indicates increase/upregulation; ↓ indicates decrease/downregulation; N/A indicates not applicable.

Autophagy

Autophagy is a highly conserved, lysosome-dependent degradation pathway in eukaryotic cells. It maintains intracellular energy homeostasis and quality control by forming double-membraned autophagosomes that sequester damaged organelles, misfolded proteins, or invading pathogens, subsequently delivering them to lysosomes for degradation and recycling.102 In bone metabolism, autophagy plays a pivotal role in maintaining skeletal homeostasis through the precise regulation of various bone cell types. In osteoblasts, basal autophagic flux is indispensable for supporting survival, differentiation, and mineralization. Under conditions of oxidative stress, autophagy prevents osteoblast apoptosis by mitophagy. In osteoclasts, moderate autophagy contributes to the formation of the ruffled border and the secretion of lysosomal enzymes. However, either excessive autophagy or impaired autophagic flux can disrupt the balance of bone resorptive function. Consequently, the targeted modulation of autophagy has emerged as a significant strategy for the prevention and treatment of metabolic bone diseases, such as osteoporosis.103

Feng et al99 demonstrated that puerarin enhances autophagy by downregulating the expression of miR-204 in MC3T3-E1 cells, thereby relieving the post-transcriptional inhibition of LC3B. This autophagic activation subsequently upregulates the expression of Runx2, OCN, and ALP, significantly bolstering osteoblast proliferation and mineralized nodule formation. Conversely, autophagic activation is generally considered an indispensable step in the RANKL-driven maturation of bone marrow-derived macrophages (BMMs) into osteoclasts.104 Zhang et al100 reported that puerarin inhibits RANKL-induced osteoclast autophagy by reducing LC3-II conversion and promoting p62 accumulation. The rescue of autophagy through the overexpression of Atg5, Atg7, or Becn1 reversed these effects, indicating that puerarin disrupts osteoclast differentiation and reduces bone resorption specifically by suppressing autophagic flux.

Ferroptosis

Ferroptosis, first defined by Dixon et al105 is an iron-dependent form of regulated cell death characterized by intracellular iron overload and the lethal accumulation of lipid peroxides. Mechanistically, it is initiated by the functional impairment of the System X_c transporter, leading to the depletion of glutathione (GSH) and decreased expression of glutathione peroxidase 4 (GPX4). Subsequently, when intracellular iron levels become excessive, the Fenton reaction catalyzes the peroxidation of polyunsaturated fatty acids within the cell membrane. This process results in the rapid generation and accumulation of lethal lipid reactive oxygen species, which overwhelm the cellular antioxidant defense systems. These accumulated peroxides directly compromise membrane integrity and mitochondrial function, ultimately culminating in cell death.106 Recent studies have reported that ferroptosis disrupts the equilibrium between osteoblasts and osteoclasts, leading to the dysregulation of bone homeostasis. On one hand, intracellular iron overload and mitochondrial dysfunction directly inhibit the Wnt/β-catenin signaling pathway and downregulate the key osteogenic transcription factor Runx2. This effect ultimately arrests osteogenic differentiation and matrix mineralization. On the other hand, ROS accumulation activates the NF-κB and MAPK signaling cascades, and synergistically upregulates transferrin receptor 1 (TFR1). These changes significantly enhance RANKL-induced osteoclast differentiation and bone resorptive activity. Consequently, ferroptosis has emerged as a novel therapeutic target for osteoporosis. Current research indicates that PLR has also shown promising therapeutic potential in bone metabolic disorders via targeted regulation of ferroptosis.

Deng et al107 identified through network pharmacology screening that puerarin acts on four core targets, namely PTGS2, IL6, VEGFA, and PLIN2, to modulate the VEGF, IL-17, and TNF signaling pathways. Their study demonstrated that puerarin binds tightly to PTGS2, effectively intercepting IL-1β-induced ferroptosis, lipid peroxidation, and the inflammatory cascade, thereby mitigating the progression of osteoarthritis. Furthermore, Zeng et al101 discovered that puerarin can reverse the hallmarks of lipopolysaccharide (LPS)-induced ferroptosis in macrophages by reducing intracellular ROS and iron levels. The underlying mechanism involves the regulation of arachidonic acid and glutathione metabolism, specifically upregulating the antioxidant enzyme GPX4 while inhibiting Acyl-CoA synthetase long-chain family member 4 (ACSL4) activity, which collectively thwarts lipid peroxidation and modulates ferroptosis.108

Epigenetic Regulation

Epigenetic regulation, including post-transcriptional modulation by microRNAs (miRNAs) and chromatin modification by histone deacetylases (HDACs), plays a pivotal role in the orchestration of bone remodeling, and has emerged as a novel therapeutic target for metabolic bone diseases. Recent studies have revealed that PLR exerts osteogenic effects via epigenetic modulation, with the core evidence from preclinical studies systematically collated in Table 4.

Table 4.

Effects of PLR on Epigenetic Regulation in Bone Metabolism

Pueraria Constituent	Study Type	Intervention	Subjects	Optimal Dose	Dose Range	Outcomes	References
Puerarin	In vitro	N/A	Rabbit osteoblasts	0.1 μM	0.1, 1, 5, 10 μM	Cell viability, mineralized nodule formation↓; RUNX2, Col-I, ALP↑	[109]
	In vivo	Oral gavage	SONFH rabbit	0.5 g/kg/d	0.5 g/kg/d	miR-34a↑; empty bone lacunae, bone marrow necrosis↓
Puerarin	In vitro	N/A	MC3T3-E1 cells	0.1 μM	0.1, 1, 10 μM	TRPM3, miR-204, Runx2↓	[110]
Puerarin	In vitro	N/A	SaoS-2 cells	20 μM	10, 20, 40 μM	p-NF-κB, TNF-α, IL-1β, COX2, MMP-14, HDAC1, HDAC3↓	[111]

Notes: ↑ indicates increase/upregulation; ↓ indicates decrease/downregulation; N/A indicates not applicable.

MicroRNAs

MicroRNAs (miRNAs) are a class of endogenous, evolutionarily highly conserved non-coding single-stranded RNA molecules, typically 18–25 nucleotides in length. As pivotal post-transcriptional regulators of gene expression, miRNAs do not encode proteins but instead bind specifically to the 3′-untranslated regions (3′-UTRs) of target mRNAs. This interaction induces mRNA degradation or translational inhibition, thereby silencing target gene expression at the post-transcriptional level.112 In recent years, miRNAs have been identified as key orchestrators of bone remodeling, maintaining a dynamic equilibrium between osteoblast-mediated bone formation and osteoclast-mediated bone resorption.113

MiR-34a is a canonical tumor-suppressive miRNA activated by p53, primarily responsible for inducing cell cycle arrest and apoptosis.114 In physiological bone metabolism, overexpression of miR-34a is generally regarded as a negative regulator of bone formation and is closely associated with skeletal aging.115 However, Jiang et al109 discovered that in glucocorticoid-induced osteonecrosis models, miR-34a expression is paradoxically suppressed, leading to impaired osteogenic function. Puerarin restores the expression of the key osteogenic transcription factor Runx2, Col-I, and ALP by specifically upregulating miR-34a. Conversely, miR-204 is widely recognized as a major negative regulator of osteogenesis.116 The aberrant overexpression of miR-204 significantly hinders osteoblast differentiation and mineralization, representing a critical pathological mechanism in the development of osteoporosis. Zeng et al110 found that puerarin downregulates miR-204 expression by inhibiting TRPM3 transcription, thereby relieving the suppression of Runx2. This subsequently leads to the significant upregulation of ALP and OCN expression, synergistically promoting osteoblast proliferation, differentiation, and mineralization.

HDACs

Histone deacetylases (HDACs) play a pivotal role in cellular epigenetic regulation. Their core function involves catalyzing the deacetylation of amino acid residues on histone tails. This modification increases the positive charge of histones, thereby tightening their binding to negatively charged DNA, which induces chromatin condensation and subsequent transcriptional repression.117 In the context of bone homeostasis, HDAC-mediated chromatin compaction negatively regulates the expression of key osteogenic factors, such as Runx2, thereby inhibiting osteoblast differentiation. Concurrently, specific HDACs participate in RANKL-induced signal transduction to promote osteoclastogenesis. Consequently, HDACs have emerged as crucial epigenetic targets for balancing bone remodeling and treating pathological bone loss.

Specifically, HDAC1 and HDAC3, both belonging to Class I HDACs, exert critical regulatory effects on the NF-κB signaling pathway, osteoclast differentiation, and cartilage matrix metabolism by modulating chromatin structure and transcription factor activity.118 Guo et al111 discovered that puerarin inhibits the upregulation of HDAC1 and HDAC3 induced by streptozotocin (STZ) in SaoS-2 cells both in vivo and in vitro. This was accompanied by a significant reduction in NF-κB phosphorylation and protein expression, thereby ameliorating the inflammatory state.

Gut Microbiota

The gut microbiota influences bone metabolism through diverse mechanisms, including nutrient absorption, immune modulation, and the gut-brain-bone axis.119 Short-chain fatty acids (SCFAs) produced by microbial fermentation activate G protein-coupled receptors to stimulate the secretion of insulin-like growth factor-1 (IGF-1) and inhibit HDAC activity, thereby enhancing osteogenic differentiation. Under physiological conditions, a healthy gut microbiota maintains bone homeostasis by regulating the Treg/Th17 cell balance, which reduces systemic inflammation and suppresses LPS-induced bone resorption.120

Li et al121 discovered that PLR can remodel the dysbiotic gut microbiota in OVX rats, significantly increasing the abundance of beneficial bacteria and elevating intestinal SCFA levels. Furthermore, PLR upregulated the expression of ZO-1 and Occludin, demonstrating its reparative effect on the intestinal mucosal metabolism. Yang et al122 discovered that puerarin can remodel the dysbiotic gut microbiota in type 2 diabetic mice, significantly increasing the abundance of beneficial bacteria such as Alloprevotella and elevating systemic α-linolenic acid downregulated the abundance of the pro-inflammatory genus Alistipes and reduced bone marrow adiposity. The key findings regarding the regulatory effects of PLR on bone metabolism via the gut-bone axis are summarized in Table 5.

Table 5.

Effects of PLR on the Gut-Bone Axis in Bone Metabolism

Pueraria Constituent	Study Type	Intervention	Subjects	Optimal Dose	Dose Range	Outcomes	References
Puerarin	In vivo	Oral administration	OVX rats	100 mg/kg/d	100 mg/kg/d	BMD, Tb.N, BMC, Tb.Th, Tb.Sp, microbiota diversity, SCFAs, ZO-1↑, CTX-1, BALP↓	[121]
Puerarin	In vivo	Intragastric administration	Type 2 diabetic mice	50 mg/kg/d	25, 50 mg/kg/d	Tb.Th, Tb.Sp, Tb.N, Alloprevotella↑; Alistipes, Rodentibacter↓	[122]
	In vitro	N/A	MC3T3-E1 cells	10 μM	1, 10 μM	Col1a1, Alp, Runx2↑

Notes: ↑ indicates increase/upregulation; ↓ indicates decrease/downregulation; N/A indicates not applicable.

Regulatory Mechanisms of PLR in Different Bone Metabolic Disease Models

Bone Metabolism Regulation of PLR in Postmenopausal Osteoporosis Models

Postmenopausal osteoporosis is the most studied disease model for PLR. Most studies used ovariectomized animals to mimic estrogen deficiency-induced bone loss. PLR extract, puerarin, PXY, and related preparations have been tested in these models. These interventions improved bone mineral density and bone microstructure in OVX animals. Several studies also showed increased trabecular bone volume, trabecular thickness, and trabecular number. In contrast, trabecular separation and osteoclast activity were reduced.

Zhao et al123 demonstrated that puerarin alleviates bone loss in ovariectomized rats. Puerarin increased femoral and lumbar BMD. It also improved trabecular microstructure. Mechanistically, puerarin reduced the activation of the JAK2/STAT3 signaling pathway in femoral tissue. Tanaka et al124 found that long-term dietary intake of low-dose kudzu vine ethanol extract (PVEE) prevented bone loss in ovariectomized mice. Clinical studies have shown that kudzu and related preparations may have potential in regulating postmenopausal bone metabolism. Current evidence mainly suggests reductions in bone resorption-related markers and possible improvement of menopausal symptoms. However, most studies used bone turnover markers as primary outcomes, and sufficient evidence on BMD, fracture risk, and long-term safety is still lacking. Bihlet et al125 conducted a four-week exploratory clinical trial in postmenopausal women with menopausal symptoms. Kudzu root extract reduced CTX-I and CTX-II levels, indicating decreased bone resorption and cartilage degradation. The effect was more evident with three-times-daily dosing. The extract also improved menopausal symptom scores and showed good short-term safety. Kim et al126 reported that a kudzu flower–mandarin peel mixture reduced CTx levels and improved hot flash symptoms in menopausal-transition women. Manonai et al127 found that Pueraria mirifica decreased bone-specific alkaline phosphatase (BAP) levels in healthy postmenopausal women, suggesting a potential regulatory effect on bone turnover. However, Pueraria mirifica is slightly different from PLR in botanical origin and phytochemical composition.

In postmenopausal osteoporosis models, PLR mainly restores the balance between bone formation and bone resorption. Its effects are closely related to estrogen receptor-associated signaling, RANKL/RANK/OPG regulation, MAPK, PI3K/Akt, Wnt/β-catenin, and JAK2/STAT3 pathways. PLR may also regulate oxidative stress, autophagy, inflammation, and gut microbiota-related metabolism.

Bone Metabolism Regulation of PLR in Alveolar Bone Loss Models

Periodontitis-related alveolar bone loss is another important model. This model is usually induced by ligation or inflammatory stimulation. Puerarin has been reported to reduce alveolar bone loss in periodontitis models. It also decreased osteoclast number and inflammatory cytokine levels.

Xiang et al128 demonstrated that puerarin relieved periapical inflammation and bone destruction in periodontitis mice. Mechanistically, this study linked the protective effect to mitochondrial autophagy activation through mitochondrial Mitofusin 2, together with improved osteogenic differentiation in periapical tissues. Thus, puerarin was not limited to anti-inflammatory action, but also promoted a regenerative response in the periodontal bone microenvironment. Li and Peng129 found that puerarin promoted the osteogenic differentiation of human periodontal ligament stem cells. Puerarin increased intracellular ALP activity and mineralized nodule formation in a concentration-dependent manner, and upregulated osteogenic genes including COL-I, OPN, Runx2, and OCN. Cao et al130 further showed that puerarin promoted osteogenic differentiation of rat dental follicle cells by activating the nitric oxide pathway.

In alveolar bone loss models, PLR mainly acts through anti-inflammatory and anti-resorptive mechanisms. It may inhibit NF-κB activation, regulate the RANKL/RANK/OPG axis, and reduce osteoclast-mediated bone resorption. PLR may also protect periodontal tissue by reducing matrix degradation and improving mitochondrial homeostasis. In addition, it may promote local osteogenic differentiation of periodontal-related cells.

Bone Metabolism Regulation of PLR in Glucocorticoid-Induced Bone Impairment Models

Glucocorticoids can impair bone formation. They can also promote bone loss. Some studies suggest that puerarin may improve glucocorticoid-related bone impairment. The main target cells are BMSCs and osteoblast-lineage cells.

Jiang et al109 demonstrated that puerarin facilitated osteogenesis in steroid-induced necrosis of the rabbit femoral head and in dexamethasone-treated rabbit osteoblasts. Puerarin attenuated methylprednisolone-induced histopathological abnormalities, reduced empty bone lacunae and bone marrow necrosis, and restored the expression of ALP, RUNX2, COL1A1, and miR-34a. Research by Qi et al131 revealed that puerarin activates the Wnt/β-catenin pathway by upregulating Wnt10b and β-catenin expression while inhibiting the negative regulator GSK3β to reduce protein degradation. Notably, under glucocorticoid-induced osteoporosis (GIOP) conditions, puerarin can downregulate the key adipogenic factor PPARγ, thereby inhibiting the transdifferentiation of BMSCs into adipocytes and instead promoting their lineage commitment toward osteoblasts.

In glucocorticoid-induced bone impairment models, PLR mainly protects osteoblast-lineage cells and regulates the differentiation direction of BMSCs. Its effects are associated with Wnt/β-catenin signaling, miRNA-mediated regulation, NF-κB inhibition, and HDAC-related epigenetic modulation. PLR may promote osteogenic differentiation and suppress adipogenic shift under glucocorticoid stress.

Bone Metabolism Regulation of PLR in Bone Regeneration Models

PLR has also been studied in developmental and regenerative bone models. These include intrauterine growth restriction models and rapid maxillary expansion models. Puerarin improved bone-related markers in these settings. It also promoted bone remodeling and new bone formation.

Mao et al132 reported that puerarin promoted tibial shaft fracture healing in rats in a dose-dependent manner. The inclusion of BMP-2 and Noggin groups suggests that activation of BMP-Smad signaling is a key mediator of puerarin-induced fracture repair. Cao et al133 developed a puerarin-loaded PLGA/TCP porous scaffold for a rat calvarial critical-size defect model. Eight weeks after implantation, the scaffold induced new bone formation within the macropores and inside the scaffold. Mechanistically, puerarin-loaded scaffolds stimulated VEGF and BMP-2 expression, promoted vascular infiltration, recruited repair cells, and thereby coordinated angiogenesis and osteogenesis.

In bone regeneration models, PLR mainly promotes bone repair through osteogenesis-angiogenesis coupling. Its effects are associated with Wnt/β-catenin, BMP-Smad and VEGF-mediated angiogenesis. Local delivery systems may further enhance these effects by maintaining sustained release at the defect site. To further clarify the disease-specific regulatory patterns of PLR in bone metabolism, the current evidence from different experimental disease models is summarized in Table 6. Representative histological, imaging, and biochemical findings from different bone metabolic disease models are presented in Figure 3, illustrating the protective effects of PLR and its major active constituents under various pathological conditions.

Table 6.

Regulatory Mechanisms of PLR in Different Bone Metabolic Diseases

Disease Model	Experimental Model	Intervention	Main Outcomes	Main Mechanisms	Reference.
Postmenopausal osteoporosis	OVX rats/mice; postmenopausal women	Puerarin; PLR extract; PXY; HX112; DG; FRAC;	BMD, BV/TV, OPG/RANKL, OCN, Runx2, ALP↑; Tb.Sp, CTX/CTX-I, RANKL, NFATc1, MMP-9, ROS, IL-6, TNF-α↓	JAK2/STAT3; ER; RANKL/RANK/OPG; MAPK; PI3K/Akt; WNT; Apoptosis; Autophagy; Gut microbiota	[36–39,42,48,51–53,56,58–60,64,71,72,77–79,83,85,86,93–95,108]
Alveolar bone loss	Periodontitis rats; periodontitis mice	Puerarin	Mineralized nodules, COL-I, OPN, OCN, NO, Mfn2↑; CEJ-ABC, sulcus bleeding index, ROS↓	Autophagy; NO; RANKL/RANK/OPG; NF-κB	[46,109,115,116]
Glucocorticoid-induced bone impairment	SONFH rabbit model	Puerarin;	Col-I/COL1A1, miR-34a, Wnt10b, β-catenin↑; GSK3β, PPARγ, miR-204, IL-1β, HDAC1/3↓	miR-34a; WNT; GSK3β/β-catenin; PPARγ; TRPM3/miR-204; NF-κB	[62,102–104,106,110]
Bone regeneration	IUGR rats; RME rats	Puerarin; PTP	BALP, IGF-1, OCN, ALP, β-catenin, BMP2/BMP-2, BMD, VEGF, Smad1/5↑;	IGF-1; OPG/RANKL; WNT; BMP-Smad; VEGF	[50,91,117,118]

Notes: ↑ indicates increase/upregulation; ↓ indicates decrease/downregulation.

Figure 3.

This composite figure systematically illustrates the effects of PLR and its constituents on bone metabolism in different animal disease models. (A). Micro-CT 3D images of periodontitis mice treated with puerarin (M1: Maxillary first molar; M2: Maxillary second molar; Closed white dashed line: region of interest for alveolar bone quantification);128 (B). Hematoxylin-eosin staining results of Steroid-induced necrosis of the femoral head rabbits treated with puerarin;109 (C). TRAcP staining showing the effects of pioglitazone combined with puerarin on osteoporosis in type 2 diabetic mice;122 (D). Puerarin ameliorates bone loss in osteoporotic mice, as verified by TRAcP staining and immunohistochemical analysis of bone resorption markers MMP9 and NFATc1 (TB, trabecular bone; BM, bone marrow. Red arrows in TRAcP-stained images indicate TRAcP-positive osteoclasts, while red arrows in IHC images mark MMP9- and NFATc1-positive cells);63 (E). Bone mineral density measurement and serum osteocalcin detection in osteoporotic rats treated with Kyung-Ok-Ko combined with Pueraria lobata Ohwi (ns = not significant, **P < 0.01 and ***P < 0.001).134

Delivery Strategies for PLR in Bone Metabolic Diseases

Oral Formulation Optimization

PLR contains multiple bioactive constituents. Among them, puerarin is the most abundant and well-characterized compound. However, puerarin is classified as a Biopharmaceutics Classification System class IV compound. It has low solubility and limited intestinal permeability. Its oral bioavailability is usually less than 7%. These properties markedly restrict the clinical application of PLR and its active constituents. Therefore, current oral formulation optimization mainly aims to improve gastrointestinal dissolution, enhance intestinal absorption, and increase the systemic bioavailability of PLR-derived active compounds.

Wang et al135 investigated AEROPERL^® 300 Pharma, a mesoporous silica carrier, as a solid dispersion system for puerarin. This formulation improved the dispersion state of puerarin and converted most of the drug into an amorphous form. It also significantly enhanced drug dissolution and oral bioavailability. Zhang et al136 prepared sustained-release oral tablets of a puerarin derivative using succinylated whey protein isolate as a functional excipient. This formulation showed sustained-release behavior in vitro and improved pharmacokinetic performance in beagle dogs. In addition, cyclodextrin inclusion complexes, self-emulsifying drug delivery systems (SEDDS), and nanocrystal formulations have also shown potential for improving oral bioavailability in experimental studies.137–140 However, these strategies have rarely been evaluated in bone metabolic disease models. Further studies are needed to determine whether oral formulation optimization can improve the therapeutic efficacy of PLR in bone metabolism-related diseases.

Nanocarrier Technologies for Enhanced Drug Delivery

Nano-drug delivery systems (NDDS) utilize nanoparticles as carriers to deliver specific therapeutic agents to targeted cells or tissues. Due to the surface effect of nanoparticles, a decrease in particle diameter leads to a significant increase in the specific surface area, which substantially enhances drug loading efficiency compared to conventional carriers.141 Researchers have leveraged this high specific surface area to develop highly efficient drug delivery vehicles for biomedical applications. Nanomaterials offer distinct advantages in enhancing the bioavailability of TCM, achieving targeted drug delivery, and enabling controlled release.142

Zhang et al143 encapsulated puerarin within exosomes derived from BMSCs, which enhanced the cellular uptake of puerarin by chondrocytes and subsequently inhibited the activation of the pro-inflammatory NF-κB signaling pathway within the cartilage matrix. Furthermore, Wang et al144 demonstrated that TPGS-modified long-circulating liposomes loaded with puerarin could effectively suppress oxidative stress and inflammatory responses by activating the Wnt/β-catenin signaling pathway. In an OVX rat model, these nano-formulations exhibited superior efficacy in improving bone microstructure compared to free puerarin.

Bone Repair Scaffold Technology

For the repair of bone defects, an ideal bioactive scaffold should facilitate bone regeneration by locally releasing bioactive substances into the defect site. These scaffolds aim to modulate the osteogenic microenvironment to achieve superior osseointegration, ultimately promoting comprehensive bone repair and regeneration.145 However, traditional titanium implants often suffer from insufficient surface bioactivity. Current research focuses on constructing biomimetic coatings on titanium surfaces to serve as carriers for osteogenic drugs or biological nanovesicles, thereby endowing the implants with the capacity to induce bone regeneration and enhance osseointegration.146

Wang et al147 utilized layer-by-layer self-assembly technology to construct a coating loaded with puerarin and exosomes on titanium surfaces. This modification significantly improved the surface hydrophilicity and synergistically promoted osteoblast proliferation and differentiation by activating the p38 MAPK/ATF-2 signaling pathway. Additionally, Cao et al133 developed a puerarin-loaded scaffold using poly(lactic-co-glycolic acid) (PLGA) as the carrier. This scaffold was shown to significantly improve bone microstructure by enhancing local blood circulation at the site of the defect.

Other Emerging Platforms

In addition to oral formulations, nanocarriers, and scaffold-based delivery systems, other emerging platforms may provide new opportunities to improve the translational potential of PLR in bone metabolism. These platforms mainly include hydrogel systems and 3D-printed biomaterials.147,148 Compared with conventional formulations, these systems may offer better local retention, sustained release, and microenvironment-responsive delivery. However, direct evidence supporting their application in PLR-related bone metabolic research remains limited.

Overall, emerging delivery platforms may help shift PLR-based interventions from simple systemic administration toward precision local therapy. Future studies should focus on disease-specific delivery design, long-term release control, local pharmacodynamics, and biosafety evaluation. These efforts may help determine whether advanced delivery systems can enhance the therapeutic value of PLR in bone metabolism-related diseases.

Challenges in Clinical Translation

Limited Clinical Evidence

PLR shows potential value in the field of bone metabolism. However, current evidence is mainly derived from cell and animal studies. Direct clinical evidence evaluating PLR in bone metabolic diseases remains limited. Available clinical studies mainly focus on postmenopausal women with menopausal symptoms. Human studies on other bone metabolic diseases are still lacking. Most existing studies have small sample sizes and short intervention periods. They also mainly use bone turnover markers as outcome measures. Differences also exist in botanical source, medicinal part, extract composition, dose, and dosing frequency across studies. Therefore, current evidence is not sufficient to confirm a definitive therapeutic effect of PLR on osteoporosis or other bone metabolic diseases.

To address the lack of clinical evidence, future studies should prioritize staged and disease-specific clinical validation. Postmenopausal osteoporosis may be the most suitable initial clinical target. Future trials should include patients with osteopenia or early postmenopausal osteoporosis, rather than only healthy women with menopausal symptoms. Outcome measures should also extend beyond bone turnover markers to BMD, bone microarchitecture, fall risk, fracture incidence, and quality of life.

Heterogeneity of Preparations and Doses

The heterogeneity of PLR preparations and doses is an important barrier to clinical translation. Current studies have used various PLR-related interventions, including crude extracts, purified puerarin, total isoflavones, fermented extracts, compound formulas, plant-derived exosome-like nanovesicles, and scaffold-based delivery systems. These preparations are not equivalent. Purified puerarin is useful for clarifying specific molecular mechanisms. However, it cannot fully represent the multi-component effects of PLR. In contrast, crude extracts and compound formulas may exert broader regulatory effects, but their chemical composition and effective dose are often less clearly defined. Dose selection also shows certain patterns. In vitro studies often show concentration-dependent effects, but the effective range is relatively narrow. Low to moderate concentrations of puerarin usually promote osteogenic differentiation, whereas excessive concentrations may reduce cell viability or induce cytotoxicity. In animal studies, effective doses vary greatly across disease models, administration routes, and treatment durations. This dose heterogeneity reduces comparability among studies and limits the design of future clinical dosing strategies.

Therefore, future studies should not only report the dose of PLR-related interventions. They should also clearly report the botanical source, medicinal part, extraction method, puerarin content, purity, administration route, and treatment duration. More importantly, the preparation type should be matched with the research purpose. Purified puerarin may be more suitable for pathway validation. Standardized PLR extracts may be more suitable for systemic bone metabolism studies. Local delivery systems may be more suitable for bone defect repair. This preparation-specific research strategy may improve reproducibility and provide a clearer basis for clinical translation.

Bioavailability and Delivery Barriers

Bioavailability remains a key barrier to the clinical translation of PLR-related interventions. Puerarin is the most extensively studied active monomer of PLR. However, puerarin has poor solubility and limited intestinal permeability. Therefore, it is classified as a Biopharmaceutics Classification System class IV drug. Its oral bioavailability is usually below 7%.148 These properties limit systemic exposure after oral administration. Therefore, improving the absorption and effective exposure of puerarin is particularly important for systemic bone metabolic diseases, such as postmenopausal osteoporosis.

Current evidence suggests that delivery requirements differ across disease models. For systemic bone loss, oral or injectable formulations need to maintain sufficient circulating exposure. For bone defect repair and local bone regeneration, local delivery may be more suitable. Puerarin-loaded scaffolds, nanoparticles, liposomes, micelles, and other carrier systems can provide sustained release at the defect site. This may increase local drug concentration and reduce unnecessary systemic exposure. In this setting, the goal of delivery systems is not only to improve bioavailability, but also to promote osteogenesis, angiogenesis, and local bone repair. Therefore, conventional pharmacokinetic parameters may not fully explain all biological effects of PLR preparations. Future studies should evaluate both systemic exposure and local mechanisms. They should also further optimize disease-specific administration routes and delivery strategies. This may help improve the clinical translational value of PLR.

Safety and Model-to-Clinic Gaps

Safety evaluation is essential for the clinical translation of PLR. PLR has long been used as both food and medicine in traditional practice. This provides a certain basis for its safety. Existing animal studies suggest that PLR and puerarin are generally well tolerated in experimental models. Limited clinical evidence also indicates that kudzu-related preparations may affect bone turnover markers without obvious short-term endometrial or hormone-related adverse effects. Clinical studies in other disease fields also support the short-term tolerability of puerarin. Li et al149 evaluated puerarin at 150 mg/day as an adjunctive treatment for polycystic ovary syndrome. During the 3-month intervention, no puerarin-related adverse events were reported in patients receiving puerarin. Kwok et al150 conducted a randomized, double-blind, placebo-controlled crossover trial involving 217 Chinese men. Puerarin supplementation at 90.2 mg/day for 12 weeks did not significantly affect liver function, renal function, coagulation, inflammatory markers, or testosterone levels. These findings suggest that short-term oral puerarin is generally well tolerated.

However, bone metabolic diseases usually require long-term intervention. Most available clinical studies used intervention periods of no more than 3 months. Therefore, current short-term safety data cannot fully reflect the risks of chronic exposure. Future studies should not only evaluate BMD and histological changes, but also assess dose equivalence, systemic exposure, potential toxicity, and herb-drug interaction risks. Clinical studies should further evaluate BMD, fracture risk, pain, functional improvement, and long-term safety. Special attention should be given to elderly patients, patients receiving anti-osteoporotic drugs, and patients with liver, kidney, cardiovascular, or endocrine diseases. This model-to-clinic safety evaluation strategy may help define the real therapeutic value and safety boundary of PLR.

Conclusions and Perspectives

PLR and its major active constituents show potential regulatory effects on bone metabolism. Current evidence suggests that PLR may promote osteoblast function, inhibit osteoclast differentiation, and improve the bone microenvironment. These effects are mainly associated with MAPK, RANKL/RANK/OPG, ER, NF-κB, PI3K/Akt, Wnt/β-catenin, JAK2/STAT3, oxidative stress, autophagy, ferroptosis-related signaling, and osteoimmune regulation. The comprehensive molecular mechanisms underlying the bone-protective effects of PLR are systematically illustrated in Figure 4. This figure was created using BioGDP.151 Disease-model studies further suggest that PLR-related interventions may act differently in postmenopausal osteoporosis, alveolar bone loss, glucocorticoid-induced bone impairment, and bone regeneration. Overall, PLR appears to regulate bone homeostasis through multi-component and multi-pathway mechanisms. However, most evidence remains preclinical, and direct clinical evidence is still limited.

Figure 4.

PLR and its bioactive constituents exert comprehensive bone-protective effects by modulating core signaling pathways including MAPK, NF-κB, PI3K/AKT, Wnt/β-catenin, and RANKL/RANK/OPG. These effects are mediated through multiple biological processes, such as anti-inflammation, anti-oxidative stress, autophagy regulation, ferroptosis inhibition, epigenetic modulation, and gut microbiota remodeling, which collectively promote osteoblast differentiation, inhibit osteoclastogenesis, and maintain bone homeostasis.

Future studies should standardize PLR extracts and active monomers in terms of botanical source, chemical composition, purity, dose, and route of administration. Pharmacokinetic research is also needed to clarify absorption, distribution, metabolism, bioavailability, and effective exposure. Further mechanistic studies should define the crosstalk among signaling pathways in different bone-related cells and disease models. Multi-omics approaches may help clarify these complex regulatory networks. Well-designed clinical trials are required to evaluate efficacy, safety, optimal dose, and long-term outcomes. Herb-drug interactions and population-specific safety also deserve more attention. These studies will help determine whether PLR can be translated from experimental bone metabolism research into clinically useful interventions for metabolic bone diseases.

Funding Statement

This work was supported by the Key Project of Tianjin Major Special Projects for Public Health and Biomedicine Science and Technology (No. 24ZXGZSY00200).

Author Contributions

All authors made a significant contribution to the work reported, including the conception and design of the review, literature collection, data analysis and interpretation, drafting, revising, and critically reviewing the article. All authors approved the final version to be submitted, agreed on the journal to which the article has been submitted, and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.