Curculigoside is a Promising Osteoprotective Agent for Osteoporosis: Review

Introduction

Osteoporosis (OP) is a chronic metabolic bone disease characterized by decreased bone mineral density and impaired bone tissue microarchitecture.¹ The pathogenesis of OP is mainly due to abnormal changes in bone homeostasis between bone formation and bone resorption, eg, oxidative stress of bone-associated cells, chronic inflammation, and other factors can disrupt this dynamic equilibrium and lead to OP.^2,3 With the increasing aging of the population, OP has become an important public health problem facing the world. It has been reported that more than 200 million people suffer from OP worldwide.⁴ About half of women and 20% of men over the age of 50 are at risk for osteoporotic fracture.⁵ Currently, conventional pharmacologic regimens for OP include calcium supplements, hormone replacement therapy, and bisphosphonates, which can, to some extent, mitigate the progression of OP. However, long-term use presents risks of damaging the gastrointestinal tract, increasing blood clots, and seriously affecting patient adherence.⁶ Therefore, there is an urgent need for a safe and effective treatment.

Herbal medicine has a long history of preventing and treating OP and has achieved certain results. Chinese herbal medicines have multi-target and multi-pathway regulatory effects, showing unique advantages in OP treatment. In addition, Compared with chemically synthesized drugs, herbal medicines are characterized by fewer adverse reactions, lower cost and more obvious effects in long-term use, which have broad application prospects. Curculigoside is a naturally occurring polyphenol compound that has a wide range of pharmacological effects and is used in the treatment of various diseases such as ulcerative colitis (UC),⁷ Ischemic brain injury (IBI),⁸ Alzheimer’s disease (AD)⁹ and rheumatoid arthritis (RA),¹⁰ etc. OP as a common age-related skeletal disorder, is on the rise with the aging of society. However, effective therapeutic countermeasures are still lacking. As the main active ingredient of the traditional Chinese herb centella asiatica, curculigoside may be a potential option for the treatment of OP. Over the past decade. Curculigoside has shown significant osteoprotective effects in the OP field. Its molecular or cellular mechanisms include regulating the activity and differentiation of mesenchymal stem cells, osteoblasts and osteoclasts, reducing oxidative stress and inflammatory responses, etc, which helps to promote bone health, promises to be a safe and effective therapeutic drug. However, curculigoside still needs to be further developed in studies on the prevention and treatment of OP. Based on this, this article reviews the in vivo and in vitro experimental evidence that curculigoside is effective in ameliorating OP, aiming to emphasize its potential role in the prevention and treatment of osteoporosis, with a view to informing further studies on the prevention and treatment of OP.

Origin and Basic Characteristics of Curculigoside

Curculigo orchioides Gaertn is a perennial herb, one of the important Chinese herbs in the Chinese medicine system, widely distributed in Zhejiang, Guangxi, Sichuan, Yunnan, Guizhou, Taiwan and other provinces and regions of China, also produced in Japan and Southeast Asia and other regions.¹¹ For thousands of years, Curculigo orchioides Gaertn has been regarded as a commonly used traditional Chinese medicine in China and India, with the effect of nourishing the essence, tonifying the liver and kidneys, and has been used as an aphrodisiac and tonic, as well as for the treatment of impotence, jaundice, asthma, and bone-related diseases.¹² Modern research has shown that Curculigo orchioides Gaertn is rich in chemical constituents such as phenols, phenolic glycosides, lignans, etc.^13,14 Among them, curculigoside was identified as the main anti-OP active substance.¹⁵ Curculigoside is a benzoic acid ester derivative, molecular formula is C₂₂H₂₆O₁₁, relative molecular mass is 466.44. Curculigoside monomer standard is white crystal, soluble in warm water, methanol, ethanol, n-butanol, acetone, etc, insoluble in petroleum ether, hashish, aether, and so on.¹⁶

Protective Effect of Curculigoside on Bone: An in vitro Test

Normal bone homeostasis is the result of the interaction between bone marrow mesenchymal stem cells (MSCs), osteoblasts and osteoclasts. As an important “executive” of bone metabolism in the human body, they are the key target cells in bone repair and bone reconstruction. We found that curculigoside, as an exogenous factor, has regulatory effects on mesenchymal stem cells, osteoblasts and osteoclasts of different origins. This includes regulating the balance between osteogenesis and lipogenesis, promoting angiogenesis, antagonizing oxidative stress and inhibiting inflammatory responses. Here, we summarize the data on the availability of curculigoside for the treatment of OP in in vitro studies (Table 1).

Table 1 Effects of Curculigoside in Regulating Different Cells

Regulatory Effects of Curculigoside on Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal, multidirectional differentiation potential and immunomodulatory functions.²⁷ MSCs are present in almost all organs and tissues, such as bone marrow, adipose tissue, liver and cord blood.²⁸ MSCs have been shown to have good therapeutic potential in a variety of diseases such as diabetes mellitus complicated by severe limb ischemia,²⁹ skin defects,³⁰ fibrotic diseases,³¹ and tumors.³² In bone metabolic diseases, osteoblasts differentiated from MSCs play an important role in bone formation as well as remodeling processes.³³

Curculigoside Regulates Bone Marrow Tissue-Derived Mesenchymal Stem Cells

Currently, BMSCs are the most commonly used MSCs, and BMSCs have potential for osteogenic and lipogenic differentiation. The balance between osteogenic and lipogenic differentiation is one of the important factors in regulating as well as maintaining bone homeostasis, and an imbalance of osteogenic/lipogenic differentiation may occur if the number, proliferative capacity, and differentiation potential of BMSCs decline due to aging or specific physiological conditions. Curculigoside, as a potential anti-OP natural active product, is able to exert osteoprotective effects by modulating the differentiation pathway of BMSCs. Protective effects.

Shen et al¹⁷ set three groups of different concentrations of curculigoside to intervene on BMSCs, and found that cells cultured in curculigoside grew faster than those cultured in the control group, which had a pro-proliferative effect on BMSCs. After 7 days of culture, the alkaline phosphatase (ALP) activity in BMSCs cultured in the three concentrations of curculigoside was approximately twice as high as that in the control group. Meanwhile, curculigoside treatment significantly enhanced the expression of collagen type 1 (Col1), osteocalcin (OCN), osteoprotegerin (OPG), and Runt-related transcription factor 2 (RUNX2) in BMSCs, especially at a dose of 100 μm. In addition, this study showed that curculigoside could directly stimulate osteoprotegerin (OPG) production by BMSCs, suggesting that curculigoside may inhibit osteoclastogenesis by regulating the OPG system, but the study has not yet elucidated the underlying mechanism. Transcriptional co-activator (TAZ) with a PDZ-binding motif has been shown to be a nuclear transcription factor that plays a key role in stem cell differentiation.³⁴ TAZ binding to RUNX2 promotes osteogenesis and interacts with peroxisome proliferator-activated receptorγ (PPARγ) to inhibit adipogenesis.³⁵ Yu et al³⁶ reported that PGC-1α-targeted TAZ could alter the bone-fat balance during skeletal aging. Previously, Wang et al³⁷ indicated that the MEK-ERK pathway was associated with TAZ during osteogenic differentiation of BMSCs. It was subsequently found that appropriate concentrations of curculigoside could up-regulate the p-ERK/ERK ratio, significantly increase the expression of TAZ, RUNX2, and OCN in BMSCs, and down-regulate the protein and mRNA levels of PPARγ, a marker of adipogenesis, during osteogenesis, thus stimulating the osteogenic ability and inhibiting the adipogenic ability of BMSCs, and reversing the osteogenic/adipogenic Imbalance of osteogenic/lipogenic differentiation.¹⁸ In contrast, TAZ knockdown or use of MEK-ERK pathway inhibitors reduced this regulatory effect of curculigoside. Notably, the optimal concentration of curculigoside to induce differentiation of BMSCs up to was 100 μm, which is consistent with the results of Shen et al.¹⁷

Curculigoside Regulates Adipose Tissue-Derived Mesenchymal Stem Cells

Adipose-derived stem cells (ADSCs) are a class of pluripotent stem cells with multidirectional differentiation potential isolated from adipose tissue, which are mesenchymal stem cells. In recent years, it has been found that compared with BMSCs, ADSCs are easier to isolate, more abundant and more productive.³⁸ Since the proliferation and differentiation of ADSCs are less affected by age and multiple passages, making them a potential source of cell-based therapies.³⁹ Similarly, ADSCs have a high potential to differentiate into multilineage cells, including osteoblasts, chondrocytes, and adipocytes.^40–42

You and Xu¹⁹ treated ADSCs with different concentrations of curculigoside, and found that curculigoside enhanced ALP activity and calcium deposition, and significantly up-regulated the expression of RUNX2, ALP and Osterix. In addition, the PI3K/AKT signaling pathway has been shown to be critical in all stages of osteoblast differentiation and maturation, bone development and growth.⁴³ In this study, curculigoside demonstrated the ability to significantly upregulate the phosphorylation of AKT to P-AKT to promote osteogenic differentiation in ADSCs, these promotional effects were reversed by the PI3K/AKT signaling pathway inhibitor LY294002. However, which receptors or proteins are associated with the role of curculigoside in ADSCs is not clear, and thus more studies are needed. Furthermore, there is an inverse relationship between osteogenic and lipogenic differentiation in ADSCs,⁴⁴ inhibition of adipogenesis and specificity in promoting osteogenesis provide a possible novel therapeutic approach for the treatment of osteoporosis.⁴⁵

Curculigoside Regulates Human Amniotic Fluid-Derived Mesenchymal Stem Cells

Amniotic fluid-derived stem cells (AFSC) have a strong potential to differentiate into osteoblasts and to become a novel cell source for the treatment of bone diseases.^46,47 AFSC have a MSC phenotype with the ability to migrate and engraft into a wide range of musculoskeletal tissues (especially injury sites) and undergo site-specific osteogenic differentiation.⁴⁸ Amniocentesis is a widely accepted procedure for prenatal testing with low risk to both mother and fetus, and AFSC can be easily obtained.⁴⁹ In addition, AFSC has no ethical implications for clinical use and has no tumorigenic risk.⁵⁰ Thus, AFSC has emerged as an ideal candidate for cell-based therapies with the promise of improving bone formation in patients suffering from a variety of metabolic and genetic bone diseases, including osteoporosis.

The results of Liu et al²⁰ showed that curculigoside dose-dependently promoted ALP activity and calcium deposition during osteogenic differentiation of human amniotic fluid-derived mesenchymal stem cells (hAFSCs) within a certain concentration range. As analyzed by RT-PCR, curculigoside upregulated Col1 and osteoblast protein (OPN). Meanwhile, the ratio of OPG to RANKL was increased, suggesting that curculigoside inhibited osteoclastic differentiation of hAFSCs. The classical Wnt signaling pathway is involved in the osteogenic differentiation of MSCs.⁵¹ Wnt signaling promotes osteoblast differentiation through β-catenin (β-catenin) activation,⁵² and knockdown of the β-catenin gene at early stages of development leads to aberrant osteoblast differentiation.⁵³ In a study by Liu et al,²⁰ curculigoside upregulated the mRNA expression of β-catenin and cell cycle proteins and promoted osteogenic differentiation of hAFSCs. And DKK-1 (a specific inhibitor of β-catenin signaling) significantly inhibited the above effects. Thus, it is clear that curculigoside can promote hAFSC osteogenic differentiation and has potential application in the treatment of bone diseases.

Regulation of Osteoblasts by Curculigoside

During normal bone metabolism, osteoblasts mediate bone formation and osteoclasts mediate bone resorption, and both maintain a dynamic balance. Osteoblasts are the cell type that plays a direct role in bone formation in the osteogenic spectrum of cells, and are able to secrete bone matrix proteins including COLI, OPN and OCN, etc.⁵⁴ In addition, RANKL secreted by osteoblasts promotes osteoclast recruitment, differentiation, activation, and survival by binding to RANK, a specific receptor on the surface of osteoclasts. Osteoblasts also secrete OPG, a soluble receptor for RANKL, which inhibits osteoclast differentiation and activity by preventing RANK/RANKL binding.⁵⁵ Based on the above properties, osteoblasts are essential for the balance of bone metabolism, bone development, fracture repair, and bone remodeling. Curculigoside is a potential osteoblast promoter that promotes the proliferation and differentiation of osteoblasts, as well as the formation of bone nodules.⁵⁶

Ma et al²¹ found that curculigoside significantly enhanced the proliferation of mouse osteoblast precursor MC3T3-E1 cells and stimulated the production of vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase-1 (Flt-1) and bone morphogenetic protein-2 (BMP-2). VEGF, as an important angiogenic factor, not only mediates bone angiogenesis but also influences the differentiation of progenitor cells into osteoblasts to stimulate bone repair.⁵⁷ Flt-1, also known as VEGF receptor 1, is another factor that plays an important role in vascular maintenance, endothelial precursor recruitment.⁵⁸ Mice deficient in Flt-1 exhibit lower bone conversion rates at an early age, suggesting that Flt-1 signaling is important not only for osteogenic cell differentiation but also for osteoblast activity during early stages of growth.⁵⁹ In conclusion, the results of these studies and the data from the current study suggest that the role of curculigoside for bone protection is related to the VEGF/Flt-1 system. Oxidative stress due to high ROS levels is considered to be a major cause of various degenerative diseases including osteoporosis.⁶⁰ H₂O₂ is one of the major reactive oxygen species, and H₂O₂ stimulates ERK-dependent NF-κB activation, leading to impaired osteoblast differentiation.⁶¹ Curculigoside can increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities by regulating the phosphorylation of p38MAPK, ERK, and NF-kB pathways, and can significantly restore ALP activity, calcium deposition, and RUNX2 levels in osteoblasts under H₂O₂-induced oxidative stress, as well as increase the expression of COLI.²² It was shown that curculigoside promotes the differentiation of osteoblasts under oxidative stress and contributes to the amelioration and prevention of OP. In addition, the body can regulate the transcription level of forkhead transcription factor 1 (FoxO1) under hyperoxia to increase the activity of SOD and catalase (CAT) to counteract the damage of oxidative stress on bone.⁶² Curculigoside enhances the capacity of osteoblasts to resist oxidative damage by regulating FoxO1a expression and nuclear translocation to increase proliferation, differentiation and bone mineralization nodule formation in H₂O₂-injured osteoblasts.²³ Zhang et al²⁴ found that curculigoside up-regulated the levels of FoxO1 and nuclear factor E2-related factor 2 (Nrf2), down-regulated the levels of p53 and the levels of FoxO1 phosphorylation, and promoted FoxO1 nuclear translocation by inhibiting the IGFR/AKT signaling pathway, which in turn improved cellular autophagy and reduced the MC3T3-E1 cell apoptosis triggered by hyperoxia. Zhu et al²⁵ found that curculigoside not only increased the mitochondrial membrane potential (MMP) level and decreased ROS generation in osteoblasts under dexamethasone (DEX)-induced osteoclasts, but also inhibited the release of inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and COX-2, to protect osteoblasts injured by DEX. These results provide new insights for further investigation of the osteoblast protective mechanism of curculigoside.

Regulation of Osteoclasts by Curculigoside

Osteoclasts are specialized multinucleated cells differentiated from hematopoietic stem cells that are primarily responsible for bone resorption and maintain bone metabolic homeostasis in conjunction with osteoblast-mediated bone formation.⁶³ However, excessive bone resorption usually leads to an imbalance in bone homeostasis, resulting in net bone loss and ultimately OP.As mentioned previously, oxidative stress affects the process of osteoblast differentiation.⁶¹ Similarly, there is growing evidence that oxidative stress is involved in osteoclastogenesis.⁶⁴ Excessive ROS production activates NF-κB and MAPK pathways and induces osteoclastogenesis.⁶⁴ Activated NF-κB activates T-cell cytoplasmic nuclear factor 1 (NFATc1), which is responsible for the regulation of genes encoding tartrate-resistant acid phosphatase (TRAP) and histone K (Ctsk), and subsequently increases osteoclast formation and bone resorption.⁶⁵ Nrf2 is a major transcription factor that controls the gene expression of many cytoprotective enzymes, such as heme oxygenase-1 (HO-1), quinone reductase (NQO-1), and γ-glutamylcysteine synthetase (GCS), with antagonistic effects on oxidative stress.⁶⁶ Therefore, drugs with antioxidant activity and modulation of Nrf2, NF-κB signaling pathways may be able to inhibit osteoclast formation and differentiation to prevent and treat OP.

In a study by Liu et al,²⁶ they found that curculigoside inhibited TRAP activity induced by RANKL and H₂O₂ in osteoclasts, and decreased the release of bone fragment degradation products and the expression of matrix metallopeptidase 9 (MMP9). In addition, curculigoside inhibited ROS levels in osteoclasts. What’s more, curculigoside enhanced Nrf2 expression and nuclear translocation and decreased NF-κB expression and p65 phosphorylation and nuclear translocation in osteoblasts. And Nrf2 inhibitor ML385 and NF-κB inhibitor Bay11-7082 antagonized the above effects of curculigoside. Therefore, it is suggested that attenuating oxidative stress and osteoclastogenesis by activating Nrf2 and inhibiting the NF-κB pathway is a potential mechanism for curculigoside treatment of OP.

Protective Effect of Curculigoside on Bone: In vivo Experiments

The construction and selection of appropriate animal models is not only an important foundation for the in-depth study of the pathogenesis of OP, but also an effective means for clinical screening and comprehensive evaluation of drugs against OP. Researchers have established various animal models to evaluate the therapeutic value of curculigoside according to the pathogenesis of OP. These models include the animal model of OP caused by ovariectomy, ^19,67 Glucocorticoids cause OP in animal models ^68,69 and the OP animal model of natural aging.¹⁸ The osteoprotective effect of curculigoside in animal models of gene-induced Alzheimer’s disease (AD) has also been explored,⁷⁰ In addition, Zhu et al ⁷¹ explored the interventional effects of curculigoside in a mouse model of cranial capitellar osteolysis, exemplifying the promising application of curculigoside in bone tissue engineering. Here, we summarize the data collected from curculigoside studies on the availability of several of these animal models (Table 2).

Table 2 Interventional Effects of Curculigoside on Different Animal Models

Bone Protection by Curculigoside in a Postmenopausal OP Model

Ovariectomized animal model is the most commonly used classical model to simulate postmenopausal osteoporosis. You et al¹⁹ established an OVX mouse osteoporosis model by removing bilateral ovaries. The results showed that the bone trabeculae in the OVX model group were sparse and reduced compared with those in the sham-operated group, the expression of RUNX2 was significantly down-regulated, and the expression of p-AKT was lower in osteoblasts. However, treatment with curculigoside significantly reversed these conditions. Similarly, Wang et al⁶⁷ used bilateral ovariectomy to establish a rat model of osteoporosis, and treatment with curculigoside was able to significantly increase the elevated serum OCN and ALP levels, and decrease the levels of TNF-α and IL-6 in rats.Micro-CT showed that curculigoside contributed to increased bone mineral density, trabecular bone thickness, number of trabeculae, and bone volume fraction were elevated. It has been reported, Long-stranded noncoding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) expression levels were significantly reduced in tissue samples from OP patients,⁷² Upregulation of miR-214-5p expression in bone marrow mononuclear cells from OP patients may promote osteoclast differentiation and exacerbate bone loss,⁷³ And bioinformatics showed that lncRNA KCNQ1OT1 had some targeting relationship with miR-214-5p. When curculigoside acted on OP rats, lncRNA KCNQ1OT1 expression was up-regulated and miR-214-5p expression was down-regulated in rat femur tissues, however, the KCNQ1OT1 inhibitor reversed this effect. These results suggest that the lncRNA KCNQ1OT1/miR-214-5p axis may be a potential mechanism by which curculigoside promotes osteogenesis and improves OP symptoms.

Bone Protection by Curculigoside in the Glucocorticoid OP Model

Glucocorticoids are widely used to treat a variety of inflammatory and autoimmune diseases, with glucocorticoidogenic OP being one of their toxic side effects. Wang et al⁶⁸ used intramuscular dexamethasone sodium phosphate to induce the establishment of an OP model in SD male rats, and the results of treatment with curculigoside showed that the morphological evaluation of tibial bone in rats improved significantly compared with that of the model group, and it was further found that the expression of LC3II/I, Beclin1, miR-181a-5p increased, and the lncRNA MEG3 expression was decreased, suggesting that curculigoside may promote autophagy activity of rat osteoblasts by regulating the LncRNA MEG3/miR-181a-5p signaling axis, thus exerting an osteoprotective effect.^74,75 Similarly, Han et al⁶⁹ found that curculigoside intervention significantly reduced SOD content in serum, increased SOD and CAT content, and attenuated oxidative stress levels, which had a modulating and ameliorating effect on osteoporosis symptoms in mice compared to dexamethasone-induced mice in the model group. It was further found that curculigoside could promote the expression of osteogenic differentiation-related proteins. In conclusion, the above results preliminarily demonstrated that curculigoside had good effects in the glucocorticoid-induced osteoporosis treatment group.

Bone Protection by Curculigoside in a Geriatric Model of OP

The aging mouse model can well simulate the process of age-related OP bone loss. Compared with 3-month-old mice, 18-month-old mice showed a significant decrease in femoral trabecular volume fraction, trabecular thickness and trabecular number, and a significant increase in trabecular spacing. Also, significant accumulation of fat in bone marrow with age was confirmed by more osmium tetroxide (OsO4)-stained lipid droplets in bone marrow.¹⁸ It was found that oral administration of curculigoside to 18-month-old mice significantly increased bone mass and decreased the number of adipocytes in the bone marrow in senescent mice. It was further found that curculigoside regulated TAZ expression through the MEK-ERK pathway to promote osteogenesis at the expense of reducing adipogenesis in BMSC and ameliorate bone loss in senescent mice. This provides a rationale for the clinical application of curculigoside in the treatment of senile osteoporosis.¹⁸

Bone Protection by Curculigoside in a Model of Alzheimer’s Disease Combined with OP

Alzheimer’s disease (AD) and OP are both common chronic degenerative diseases in the elderly population, and epidemiology shows a very high co-morbidity between the two diseases.⁷⁶ In addition, there may be common pathogenic factors between AD and OP, among which amyloid-β (Aβ) is one of the most widely investigated ones.Aβ is neurotoxic, leading to impaired synaptic plasticity and neuronal apoptosis, accelerating the rate of cognitive decline; at the same time, Aβ deposition in the brain also exacerbates the deposition of Aβ through a series of signaling, thus creating a vicious circle.⁷⁷ Previous studies have observed that Aβ42, one of the isoforms of Aβ, is expressed at higher levels than controls in both osteoporotic human and animal bone tissues, and is negatively correlated with bone mineral density levels, suggesting that Aβ42 may enhance osteoclast differentiation and activation.⁷⁸ APP/PS1 mutant transgenic mice are one of the common AD models. Compared with wild-type mice, APP/PS1 mice showed significantly increased levels of Aβ40 and Aβ42 in the brain and femur, and in addition, the elevated levels of Aβ led to increased levels of the bone resorption marker, histone K, and decreased levels of the biochemical marker of bone formation, osteocalcin. Serum levels of markers of osteoclast activity (TRACP5b), IL-6, and TNF-α were significantly higher in APP/PS1 mice than in wild-type mice. IL-6 and TNF-α levels were significantly higher than those of wild-type mice, and IL-6 and TNF-α not only directly stimulated osteoclastogenesis and bone resorption, but also synergistically stimulated RANKL production in osteoblasts.⁷⁹ Zhao et al⁷⁰ intervened APP/PS1 mice with curculigoside, which was able to reverse the above alterations, significantly improve the academic performance of APP/PS1 mutant transgenic mice, and improve bone loss. It suggests that curculigoside is highly likely to be a potential therapeutic drug for patients with AD combined with OP.

Osteoprotection by Curculigoside in a Model of Cranial Capitellar Osteolysis

Total Joint Arthroplasty (TJA) offers significant benefits in terms of joint pain relief and joint function restoration in patients with bone and joint disorders.⁸⁰ However, TJA failure can occur due to infection, prosthesis fracture or loosening and wear. Of these, aseptic prosthetic loosening accounts for 38% of cases, with the majority of aseptic loosening being caused by particle abrasion,^81,82 This induces osteolysis, which has become a major problem limiting the longevity of artificial joints. In view of the advantages that curculigoside embodies in bone protection, Zhu et al⁷¹ chose titanium particles (Ti) to simulate periprosthetic osteolysis in vivo and then investigated the effect of curculigoside on osteolysis. They first prepared a model of Ti induced osteolysis in the mouse skull by surgical means, and gave mice intraperitoneal injection of curculigoside for intervention on the 2nd day, and then took cranial bone tissues for further experiments after executing each group of mice after 14 days. The results showed that titanium injection induced osteolysis and bone loss in the cranial bones of the mice, and the cranial bones of the mice exhibited osteolysis characteristics. In addition, significant changes in osteolysis-related parameters, including BMD, BV/TV, and Tb.Th, occurred in the Ti group compared with the sham-operated group. However, in the curculigoside-treated group, the extent of Ti-induced osteolysis was attenuated and also reversed osteolysis-related parameters. In addition, Ti injection increased the expression of IL-1β, IL-6, RANKL, and TNF-α and suppressed the expression of OPG in mouse cranium, which increased the production of pro-inflammatory cytokines and the RANKL/OPG ratio, leading to bone destruction. In contrast, curculigoside significantly reversed these changes and promoted bone formation. In summary, curculigoside can effectively alleviate attenuate titanium-induced osteolysis, which is expected to provide curculigoside as a potential bone protective agent for the prevention and treatment of bone defects.

Pathway Related to the Effect of Curculigoside on Bone

As the study of OP molecular pathology network continues to deepen, its intrinsic cellular signaling mechanisms have been gradually and systematically elucidated. It is worth noting that curculigoside has the characteristics of multi-pathway regulation, and it can intervene in key signaling axes such as OPG/RANKL/RANK, PI3K/Akt, Wnt/β-catenin, MAPKs, Nrf2, and NF-κB, etc (Figure 1). Curculigoside has the advantage of multi-targeting in bone metabolism homeostasis. Advantage lays an important theoretical foundation for its precise therapeutic strategy and new drug development based on signaling pathways.

Figure 1 Pathway related to the effect of curculigoside on bone.

The OPG/RANKL/RANK signaling pathway has been found to regulate osteoclast differentiation, induction, activation and maintenance and is recognized as a potential target for the treatment of OP.⁸³ In this pathway, RANKL binds to RANK on the surface of osteoclasts and will form a RANKL-RANK complex and stimulate osteoclast activation, formation, and differentiation.⁸⁴ OPG, a decoy receptor produced primarily by osteoblast-profiling cells, inhibits osteoclast formation.⁸⁵ Specifically, OPG regulates osteoclasts to inhibit bone resorption and delay bone loss by competitively binding to RANKL, thereby preventing RANKL-RANK complex formation. Normally, under normal conditions, OPG and RANKL are in relative equilibrium, and if the level of RANKL/OPG is elevated, it directly affects osteoclasts.⁸⁶ The experimental study¹⁷ found that curculigoside synergistically promoted bone formation and inhibited bone resorption by up-regulating Runx2 and OPG expression and inhibiting osteoclast activity through regulating OPG/RANKL balance. Similarly, consistent results were obtained by Liu et al.²⁰

Phosphoinositide 3-kinase (PI3K) is a lipid kinase found in the cytoplasm, and serine/threonine kinase (AKT) is an essential signaling target downstream of PI3K.⁸⁷ Activation of PI3K results in the phosphorylation of phosphatidylinositol 2-phosphate (PIP2) to PIP3, which causes a conformational change and translocation of Akt to the cell membrane and exposure of the activation site, which is then activated by PI3K, leading to complete phosphorylation of Akt.⁸⁸ Activated AKT affects downstream signaling molecules such as mammalian target of rapamycin palladium protein (mTOR) and participates in the regulation of cellular life activities. Several studies have demonstrated that the PI3K/AKT signaling pathway, once activated, significantly promotes the proliferation and differentiation of osteoblasts and plays an important role in regulating and maintaining the homeostasis of bone tissue.^89,90 You et al¹⁹ combined in vivo as well as in vitro experiments to demonstrate that curculigoside significantly increased the phosphorylation levels of PI3K and AKT (p-PI3K and p-AKT) and promoted bone remodeling. In contrast, this osteoprotective effect of curculigoside was partially reversed using the PI3K inhibitor LY294002.

The Wnt/β-catenin pathway is recognized as a classical pathway in the Wnt signaling pathway. The key to the activation of this pathway lies in the binding of Wnt ligands to specific curculigoside and co-receptors, which triggers a series of intracellular events that stabilize β-catenin and promotes the transcription and expression of related target genes.⁹¹ The Wnt/β-catenin pathway plays a key role in osteoblast differentiation, bone remodeling and skeletal homeostasis. Studies have shown that curculigoside activates the Wnt/β-catenin pathway to enhance the osteogenic differentiation of hAFSCs by up-regulating the expression of β-catenin and Cyclin D1, providing a molecular basis for its stem cell-based bone regeneration therapy.²⁰

The mitogen-activated protein kinase (MAPK) signaling pathway consists of three core members, namely, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38MAPK. MAPK signaling is one of the key pathways in the eukaryotic signaling network, which mediates cellular responses to a variety of stimuli and is closely related to inflammation, apoptosis, growth and differentiation.⁹² The MEK-ERK pathway was first recognized in the early 1990s as a member of the MAPK family.⁹³ Activation of p-ERK was previously shown to be critical for RUNX2 upregulation and transcriptional activity.⁹⁴ Wang et al¹⁸ found that curculigoside mediates osteogenic differentiation of BMSCs by upregulating TAZ signaling through the MEK-ERK pathway.

There is growing evidence that ROS accumulate in bone with age and estrogen deficiency, and that excess ROS can activate the NF-κB pathway and induce osteoclastogenesis.⁶⁴ Specifically, NF-κB activation induces nuclear factor activation in activated NFATc1, which is responsible for the regulation of genes encoding TRAP and Ctsk, and subsequently increases osteoclastogenesis and bone resorption.⁶⁵ Nrf2 is a major endogenous antioxidant defense regulator that controls the gene expression of many cytoprotective enzymes, such as HO-1 and gamma-glutamylcysteine synthetase (GCS), to counteract oxidative stress.⁶⁶ Therefore, drugs with antioxidant activity and the ability to modulate Nrf2 and NF-κB signaling pathways may be able to inhibit osteoclast formation and differentiation. The study²⁶ has shown that Curculigoside effectively inhibits osteoclastogenesis and bone resorption function and alleviates oxidative stress injury by activating the Nrf2/Keap1 pathway and inhibiting the NF-κB pathway, providing a novel candidate molecule for OP therapy.

Pharmacokinetics and Modified Applications of Curculigoside

Given the wide range of pharmacological activities of curculigoside and its applications, it is important to understand the pharmacokinetics and in vivo behavior of curculigoside, which is rapidly distributed to multiple tissues after oral administration, including the heart, lungs, spleen, intestines, stomach, kidneys, liver, brain, and bone marrow; however, the amount absorbed into the systemic circulation is relatively small.⁹⁵ The absolute bioavailability of oral curculigoside was 0.38%, 0.22%, and 0.27% at 100, 200, and 400 mg/kg, respectively, and the distribution and clearance of curculigoside in vivo following intravenous administration was essentially the same as for oral administration.⁹⁵ This result is similar to a previous report where Zhao et al⁹⁶ reported that the absolute bioavailability of curculigoside was also only 1.27% after oral administration of 32 mg/kg dose of curculigoside to rats. Therefore, it can also be concluded that curculigoside may have a low absolute bioavailability. This will greatly hinder its pharmacological action and clinical application, therefore, improving the bioavailability of curculigoside is an important research topic in its development.

Studies have shown that co-administration with verapamil enhances the absorption of some natural compounds with poor oral bioavailability, as verapamil, by inhibiting the activity of P-gp and some CYP enzymes, increases the absorption of the drug in the intestine and slows down the rate of its elimination in the liver.⁹⁷ Wang et al⁹⁸ administered verapamil (10 mg/kg) pretreated and unpretreated rat curculigoside (20 mg/kg) orally, respectively; verapamil increased the peak plasma concentration of curculigoside from 60.17 ng/mL to 93.66 ng/mL and inhibited the exocytosis of curculigoside and increased the uptake of curculigoside in Caco-2 cells. Rat liver microsomal incubation experiments demonstrated that verapamil significantly reduced the intrinsic clearance of curculigoside (from 38.8 to 23.6 μL/min/mg protein) and effectively increased bioavailability. Recent studies have discussed the protective effects of delivering senecioside to modulate bone reconstruction through three-dimensional (3D) printing of bionic hydroxyapatite composites. Weng et al⁹⁹ developed a bionic composite scaffold (HGSC) loaded with senecioside by utilizing 3D printing technology. Eight weeks after implantation of HGSC in cranial defects in rats, micro-computed tomography and histological observations showed significant angiogenesis and new bone growth in the area treated with HGSC composite scaffold. It is noteworthy that the composite stent simultaneously achieved sustained drug release for up to 12 days, which significantly improved the drug utilization of senecioside. This is an exciting news, which provides a solid theoretical foundation for Xianmaooside in clinical bone repair.

Conclusion

Over the years, natural products have shown great potential in the prevention and treatment of osteoporosis, leading to the emergence of more acceptable complementary alternatives that are less costly, have fewer side effects, and are more widely used in the long term.^100–102 According to our findings, curculigoside acts on osteoblasts, osteoclasts, and mesenchymal stem cells through a variety of mechanisms, which can reverse bone loss and protect bone homeostasis. In addition, evidence from various animal models confirms the therapeutic effects of curculigoside on bone destruction, which will likely provide a basis for the therapeutic use of curculigoside to be transferred to clinical practice. However, this therapeutic approach is still in the early stages of clinical translation, and there are still many obstacles between its experimental results and clinical application. For example, the absorption and metabolic stability of curculigoside is not ideal, and the drug delivery route as well as bioavailability still need to be optimized, which is an urgent issue. Overall, as a readily available and inexpensive natural compound, curculigoside has a favorable osteoprotective effect. Therefore, we hypothesize that curculigoside is expected to be a potential alternative therapy for the treatment of OP, and the application of curculigoside in bone tissue engineering is also expected.