Abstract

Objective:
To investigate the effects of acteoside on osteoblast and osteoclast activity in ovariectomized (OVX) mice.

Methods:
A postmenopausal osteoporosis mouse model was established by ovariectomy (OVX). Fifty C57BL/6 mice were randomly divided into five groups: sham-operated group, model group, estrogen group, low-dose acteoside group, and high-dose acteoside group, with 10 mice in each group.

Body weight was recorded. Bone histomorphometric analysis was performed using micro-computed tomography (micro-CT). Serum levels of estradiol (E2), CTX-1, PINP, and ALP were measured using corresponding assay kits. Uterine weight was recorded. TRAP staining was used to observe the number of osteoclasts. Immunohistochemistry was used to detect Runx2 protein expression. Real-time quantitative RT-PCR (qRT-PCR) and Western blot analysis were used to evaluate the expression of markers related to bone formation and bone resorption.

Results:
Compared with the sham-operated group, mice in the model group showed significantly increased body weight, significantly decreased uterine weight and serum E2, PINP, and ALP levels, and significantly increased serum CTX-1 levels. Bone parameters including BMD, BV/TV, Tb.N, Tb.Th, PINP, ALP, Runx2, OCN, and Col1α were significantly reduced, while Tb.Sp was significantly increased. In femoral tissue, ALP, OCN, and Col1α mRNA expression was significantly decreased, whereas NFATc1 and c-fos mRNA expression, as well as Runx2, p-IκBα, and p-Akt protein expression, were significantly increased (P<0.05P < 0.05P<0.05). The number of osteoclasts was also increased.

Compared with the model group, all treatment groups showed significantly reduced body weight, significantly increased serum PINP and ALP levels, and significantly decreased serum CTX-1 levels. BMD, BV/TV, Tb.N, Tb.Th, PINP, ALP, Runx2, OCN, and Col1α were significantly increased, while Tb.Sp was significantly decreased. In femoral tissue, ALP, OCN, and Col1α mRNA expression was significantly increased, whereas NFATc1 and c-fos mRNA expression, as well as Runx2, p-IκBα, and p-Akt protein expression, were significantly decreased (P<0.05P < 0.05P<0.05). The number of osteoclasts was reduced.

Conclusion:
Acteoside exerts a dual regulatory effect on bone remodeling in OVX mice. It may improve OVX-induced bone loss by inhibiting osteoclast-mediated bone resorption through suppression of the NF-κB/Akt/NFATc1/c-fos pathway, while promoting osteoblast-driven bone formation through activation of the Runx2/OCN/Col1α axis.

Keywords:
Acteoside; postmenopausal osteoporosis; osteoblasts; osteoclasts

What Is Osteoporosis?

Osteoporosis (OP) is a common and often silent skeletal disease characterized by reduced bone mineral density, deterioration of bone microstructure, increased bone fragility, and a higher risk of fractures. Globally, approximately 18.3% of the population is affected, with postmenopausal women being especially vulnerable. This condition is known as postmenopausal osteoporosis (PMOP) [1].

Estrogen plays a key regulatory role in bone remodeling through multiple mechanisms. During bone remodeling, osteoclasts and osteoblasts maintain a dynamic balance between bone resorption and bone formation. When osteoblast activity declines and/or osteoclast activity becomes excessive, this balance is disrupted, leading to serious bone matrix loss [2].

Therefore, regulating the function and formation of osteoblasts and osteoclasts may be an effective strategy for treating estrogen-deficiency-related osteoporosis.

Why Look at Herbal Compounds for Osteoporosis?

Current mainstream medications for osteoporosis include bisphosphonates, teriparatide, raloxifene, and selective estrogen receptor modulators. These drugs can be effective, but they may also cause adverse effects such as gastrointestinal discomfort and, in rare cases, osteonecrosis of the jaw. These concerns can limit long-term use.

In recent years, increasing attention has been directed toward traditional herbal medicine for supporting bone health, especially in postmenopausal osteoporosis.

Rehmannia root, a traditional Chinese herb, is known for nourishing the kidney and promoting blood circulation. It is often used in herbal formulas for osteoporosis [3]. Several clinical studies have reported that Chinese herbal formulas containing Rehmannia may help improve bone mineral density, relieve pain, and improve clinical symptoms in patients with osteoporosis [4].

Acteoside is a water-soluble phenylethanoid glycoside found in Rehmannia, Cistanche, Plantago seed, and other medicinal plants. It has been reported to possess antioxidant, anti-inflammatory, antidiabetic, and bone formation-promoting activities [5].

A study based on systematic review and data mining of multiple databases suggested that acteoside may be one of the core active components in Chinese herbal formulas for postmenopausal osteoporosis [6]. Network pharmacology studies have also indicated that acteoside may be a key active ingredient in herbal formulas such as Qigu Capsule and Zuogui/Yougui Pills for treating postmenopausal osteoporosis [7,8].

These findings suggest that acteoside may be a promising natural compound for bone health. However, its precise mechanism of action has not yet been fully clarified.

This study was designed to investigate the effects of acteoside on osteoblast and osteoclast activity in an ovariectomized mouse model, with the aim of providing a scientific basis for further development of acteoside in osteoporosis-related research.

 

1. Materials and Instruments

1.1 Experimental Animals

Fifty SPF-grade 6-week-old female C57BL/6 mice were purchased from the Hebei Experimental Animal Center, license number SCXK (Hebei) 2022-001. The mice weighed 20–23 g.

All mice were maintained under a 12-hour light/dark cycle, with room temperature controlled at 22±3∘C22 \pm 3^\circ C22±3∘C, humidity at 55±5%55 \pm 5\%55±5%, and adequate food and water provided. After one week of adaptive feeding, the experiment was conducted.

1.2 Drugs and Reagents

Acteoside, with a purity of 98% and batch number CFN97048, was purchased from Wuhan ChemFaces.

Estradiol valerate tablets, specification 1 mg × 21 tablets, were purchased from Bayer Healthcare Co., Ltd., Guangzhou.

Mouse serum estradiol (E2), alkaline phosphatase (ALP), type I collagen cross-linked C-terminal telopeptide (CTX-1), and procollagen type I N-terminal propeptide (PINP) assay kits were purchased from Beyotime Biotechnology, Shanghai.

RIPA lysis buffer was obtained from Sigma, USA. The Bio-Rad DC Protein Assay Kit was obtained from Bio-Rad Laboratories, USA. Anti-phospho-Akt, anti-Akt, anti-phospho-IκB, and anti-IκB antibodies were purchased from Cell Signaling Technology, USA. Anti-Runt-related transcription factor 2 (Runx2) antibody was purchased from Santa Cruz Biotechnology, USA. TRAP staining kit was purchased from Sigma, USA. TRIzol reagent and RevertAid First Strand cDNA Synthesis Kit were purchased from Thermo Fisher Scientific, USA.

1.3 Main Instruments

The main instruments used included:

SKYSCAN 1276 micro-computed tomography scanner, Bruker Technology, Beijing

SpectraMax M microplate reader, Molecular Devices, Shanghai

FA2004N electronic analytical balance, Sailun Instruments, Beijing

WMP-6105 optical microscope, Wumo Optical Instruments, Shanghai

VeritiPro real-time quantitative PCR system and PowerEase electrophoresis apparatus, Thermo Fisher Scientific, Shanghai

 

2. Methods

2.1 Model Establishment and Grouping

C57BL/6 mice were randomly divided into five groups:

Sham-operated group

Model group

Estrogen group, 1 mg/kg1 \text{ mg/kg}1 mg/kg

Low-dose acteoside group, 50 mg/kg50 \text{ mg/kg}50 mg/kg

High-dose acteoside group, 150 mg/kg150 \text{ mg/kg}150 mg/kg

Each group contained 10 mice.

All animals were anesthetized with 2% sodium pentobarbital. A small incision was made to expose the ovaries. The ovaries were gently located and removed to establish the OVX model. In the sham-operated group, only the fat tissue near the ovaries was removed.

After surgery, all mice received intramuscular penicillin at 50,000 U/day for 3 consecutive days.

After surgery:

The estrogen group received estradiol valerate at 1 mg/kg1 \text{ mg/kg}1 mg/kg by gavage once daily.

The low- and high-dose acteoside groups received acteoside by gavage once daily.

The sham-operated and model groups received purified water at 10 mg/kg10 \text{ mg/kg}10 mg/kg by gavage.

Mice were sacrificed 4 weeks after surgery. Femoral tissue and blood samples were collected. Femurs were fixed in 4% paraformaldehyde for 48 hours and evaluated using micro-CT.

2.2 Micro-CT Detection

The left femur of each mouse was scanned using high-resolution micro-CT. The scanning parameters were:

Voltage: 50 kV

Current: 55 μA

Isotropic resolution: 6 μm

Femoral scanning was performed at 2 mm proximal to the growth plate, with 720 sections scanned each time.

Micro-CT data were analyzed using INFINITT-Xelis software. The evaluated structural parameters included:

Bone mineral density (BMD)

Bone volume/total volume (BV/TV)

Trabecular thickness (Tb.Th)

Trabecular separation (Tb.Sp)

Trabecular number (Tb.N)

2.3 Blood Biochemical Indicators

Blood samples were collected and centrifuged at 1,300 r/min for 15 minutes at 4∘C4^\circ C4∘C. ELISA kits were used to measure serum levels of:

CTX-1, a bone resorption marker

PINP, a bone formation marker

ALP, a bone formation marker

2.4 TRAP Staining

After fixation, mouse femurs were decalcified in EDTA for 10 days. They were then dehydrated, embedded in paraffin, and prepared into 6 μm sections.

The sections were subjected to xylene dewaxing, ethanol rehydration, and TRAP staining. Subsequently, the sections were dehydrated, cleared, and mounted.

2.5 Immunohistochemistry (IHC) Staining

For Runx2 immunohistochemistry, 5 μm sections were cut using a microtome, then dewaxed and hydrated.

Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in PBS for 30 minutes.

To reduce nonspecific staining, sections were incubated for 1 hour in PBS containing 10% normal horse serum and 0.1% Triton X-100.

Sections were incubated overnight at 4∘C4^\circ C4∘C with anti-Runx2 primary antibody at a dilution of 1:1,000.

Next, sections were washed with PBS and incubated with biotinylated rabbit anti-goat IgG at a dilution of 1:200 for 2 hours, followed by incubation with avidin-biotin-peroxidase complex for 1 hour.

Antigen color development was performed using 3,3′-diaminobenzidine tetrahydrochloride solution containing 0.003% hydrogen peroxide. Hematoxylin solution was used for counterstaining.

2.6 Real-Time Quantitative RT-PCR (qRT-PCR)

RNA was extracted using TRIzol reagent. The amount of RNA in each sample was normalized to 1 μg for cDNA synthesis.

cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit.

qRT-PCR was performed using the Exicycler-96 real-time quantitative thermal cycler.

The amplification parameters were:

Initial denaturation at 95∘C95^\circ C95∘C for 5 minutes

40 cycles of three-step PCR:

Denaturation at 95∘C95^\circ C95∘C for 1 minute

Annealing at 60∘C60^\circ C60∘C for 30 seconds

Final extension at 72∘C72^\circ C72∘C for 1 minute

Data were normalized using GAPDH and evaluated as average fold changes. Relative gene expression was calculated using the 2−ΔΔCt2^{-\Delta\Delta Ct}2−ΔΔCt method.

2.7 Western Blot Analysis

Cells were lysed using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors I and II.

Protein concentration was determined using the Bio-Rad DC Protein Assay Kit.

Equal amounts of protein, 20μg20 \mu g20μg per lane, were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to Immobilon-P polyvinylidene fluoride membranes.

Membranes were blocked with 5% skim milk for 1 hour and then incubated overnight at 4∘C4^\circ C4∘C with primary antibodies.

After washing three times with Tris-buffered saline containing 0.1% Tween-20 (TBST), membranes were incubated with anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody at a dilution of 1:3,000 for 1 hour.

Proteins were visualized by chemiluminescence using an ECL kit.

2.8 Statistical Analysis

All experimental tests were performed at least three times.

Comparisons among multiple groups were performed using one-way analysis of variance. Pairwise comparisons between groups were conducted using Tukey's multiple comparison test.

SPSS 14.0 software was used for statistical analysis. A value of P<0.05P < 0.05P<0.05 was considered statistically significant.

 

3. Results

3.1 Effects of Acteoside on Serum E2 Level, Uterine Weight, Body Weight, and Uterine Index in OVX Osteoporotic Mice

As shown in Figure 1, compared with the sham-operated group, mice in the model group had significantly lower serum E2 levels, uterine weight, and uterine index, while body weight was significantly increased (P<0.05P < 0.05P<0.05).

Compared with the model group, mice in all treatment groups showed significantly reduced body weight (P<0.05P < 0.05P<0.05).

Figure 1. Effects of acteoside on serum E₂ level, uterine weight, body weight, and uterine index in OVX osteoporotic mice

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Note:
∗P<0.05*P < 0.05∗P<0.05 vs. sham-operated group;
#P < 0.05 vs. model group;
△P<0.05\triangle P < 0.05△P<0.05 vs. estrogen group;
▲P<0.05\blacktriangle P < 0.05▲P<0.05 vs. low-dose acteoside group.

 

3.2 Effects of Acteoside on Bone Histomorphometric Parameters in OVX Osteoporotic Mice

As shown in Figure 2, compared with the sham-operated group, mice in the model group showed significantly decreased BMD, BV/TV, Tb.N, and Tb.Th, while Tb.Sp was significantly increased (P<0.05P < 0.05P<0.05).

Compared with the model group, all treatment groups showed significantly increased BMD, BV/TV, Tb.N, and Tb.Th, while Tb.Sp was significantly decreased (P<0.05P < 0.05P<0.05).

Figure 2. Effects of acteoside on bone histomorphometric parameters in OVX osteoporotic mice

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Note:
∗P<0.05*P < 0.05∗P<0.05 vs. sham-operated group;
#P < 0.05 vs. model group;
△P<0.05\triangle P < 0.05△P<0.05 vs. estrogen group;
▲P<0.05\blacktriangle P < 0.05▲P<0.05 vs. low-dose acteoside group.

 

3.3 Effects of Acteoside on Serum Bone Metabolism-Related Indicators in OVX Osteoporotic Mice

As shown in Figure 3, compared with the sham-operated group, mice in the model group showed significantly decreased serum PINP and ALP levels, while CTX-1 levels were significantly increased (P<0.05P < 0.05P<0.05).

Compared with the model group, all treatment groups showed significantly increased serum PINP and ALP levels, while CTX-1 levels were significantly decreased (P<0.05P < 0.05P<0.05).

Figure 3. Effects of acteoside on serum bone metabolism-related indicators in OVX osteoporotic mice

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Note:
∗P<0.05*P < 0.05∗P<0.05 vs. sham-operated group;
#P < 0.05 vs. model group;
△P<0.05\triangle P < 0.05△P<0.05 vs. estrogen group;
▲P<0.05\blacktriangle P < 0.05▲P<0.05 vs. low-dose acteoside group.

Figure 4. Effects of acteoside on the number of osteoclasts in the femur of OVX osteoporotic mice (TRAP staining)

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Figure 5. Effects of acteoside on Runx2 protein expression in femoral tissue of OVX osteoporotic mice (IHC, ×400)

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Figure 6. Effects of acteoside on ALP, OCN, Col1α, NFATc1, and c-fos mRNA expression in femoral tissue of OVX osteoporotic mice

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Note:
∗P<0.05*P < 0.05∗P<0.05 vs. sham-operated group;
#P < 0.05 vs. model group;
△P<0.05\triangle P < 0.05△P<0.05 vs. estrogen group;
▲P<0.05\blacktriangle P < 0.05▲P<0.05 vs. low-dose acteoside group.

3.4 Effects of Acteoside on the Number of Osteoclasts in Femoral Tissue of OVX Osteoporotic Mice

As shown in Figure 4, compared with the sham-operated group, the number of TRAP-positive osteoclasts in the model group was increased.

Compared with the model group, the number of TRAP-positive osteoclasts was decreased in all treatment groups.

3.5 Effects of Acteoside on Runx2 Protein Expression in Femoral Tissue of OVX Osteoporotic Mice

As shown in Figure 5, compared with the sham-operated group, Runx2 protein-positive expression in femoral tissue was reduced in the model group.

Compared with the model group, Runx2 protein-positive expression in femoral tissue was increased in all treatment groups.

3.6 Effects of Acteoside on ALP, OCN, Col1α, NFATc1, and c-fos mRNA Expression in Femoral Tissue of OVX Osteoporotic Mice

As shown in Figure 6, compared with the sham-operated group, femoral ALP, OCN, and Col1α mRNA expression was significantly decreased in the model group, while NFATc1 and c-fos mRNA expression was significantly increased (P<0.05P < 0.05P<0.05).

Compared with the model group, all treatment groups showed significantly increased ALP, OCN, and Col1α mRNA expression, while NFATc1 and c-fos mRNA expression was significantly decreased (P<0.05P < 0.05P<0.05).

 

Figure 7. Effects of acteoside on p-IκBα and p-Akt protein expression in femoral tissue of OVX osteoporotic mice

A. Sham-operated group
B. Model group
C. Estrogen group
D. Low-dose acteoside group
E. High-dose acteoside group

Note:
∗P<0.05*P < 0.05∗P<0.05 vs. sham-operated group;
#P < 0.05 vs. model group;
△P<0.05\triangle P < 0.05△P<0.05 vs. estrogen group;
▲P<0.05\blacktriangle P < 0.05▲P<0.05 vs. low-dose acteoside group.

 

4. Discussion

Osteoporosis is a common metabolic bone disease characterized by reduced bone mineral density, destruction of bone microstructure, increased bone fragility, and elevated fracture risk.

Approximately 200 million people worldwide are affected by osteoporosis. With population aging, its high incidence and mortality place a heavy burden on society.

Postmenopausal osteoporosis is mainly caused by estrogen deficiency and is the most common type of osteoporosis. Its typical features include declining estrogen levels and continuous calcium loss with aging.

The pathological mechanism of osteoporosis is characterized by reduced bone mass and destruction of trabecular microstructure. This directly results from an imbalance between osteoclast formation and osteoblast formation [9].

Given the important influence of estrogen deficiency on osteoporosis, this study used an ovariectomy-induced mouse model to evaluate the anti-osteoporotic effects of acteoside.

First, this study confirmed that OVX caused estrogen deficiency, uterine atrophy, and bone loss in mice, while exogenous E2 supplementation improved uterine atrophy and bone loss in OVX mice.

In addition, this study found that acteoside did not alter serum E2 levels or uterine weight in OVX mice, but it significantly improved bone mineral density and bone microstructure, thereby improving bone loss.

This was reflected by increased femoral BMD, BV/TV, Tb.N, and Tb.Th, as well as decreased Tb.Sp.

Among these parameters:

BMD effectively evaluates the degree of bone loss.

BV/TV reflects cancellous bone mass.

Tb.N, Tb.Sp, and Tb.Th are mainly used to evaluate the spatial morphology of trabecular bone [10].

 

How Acteoside May Support Bone Remodeling

An imbalance between bone formation and bone resorption is considered a major mechanism of osteoporosis.

Current osteoporosis therapies mainly include:

Anti-resorptive drugs, such as bisphosphonates, which inhibit osteoclast activity

Anabolic agents, such as parathyroid hormone, which promote osteoblast function

Although these drugs have clear benefits for bone health, they may also be associated with adverse reactions, including nausea, vomiting, stomach pain, and increased risks related to breast cancer and endometrial cancer [11].

An ideal osteoporosis strategy would be a compound with dual regulatory activity and fewer side effects: one that can inhibit osteoclast differentiation while enhancing osteoblast activity.

This study found that acteoside reduced serum CTX-1 levels and increased serum PINP and ALP levels.

Serum ALP and PINP are markers of bone formation, while CTX-I is a marker of bone resorption [12].

These results suggest that acteoside may regulate bone metabolism in OVX mice by promoting bone formation and inhibiting bone resorption, showing a potential dual regulatory effect on bone turnover.

 

Acteoside May Help Reduce Osteoclast-Mediated Bone Resorption

Histological analysis showed that acteoside treatment reduced the number of TRAP-positive cells, namely osteoclasts, in the trabecular bone near the femoral growth plate of OVX mice.

This suggests that acteoside may inhibit OVX-induced osteoclast activation.

In the bone microenvironment, osteoclasts are specialized multinucleated macrophages formed by the differentiation of monocytes induced by RANKL and macrophage colony-stimulating factor.

The RANKL-induced signaling cascade plays a core role in regulating osteoclast activity. After RANKL binds to its receptor RANK, it recruits TNF receptor-associated factor 6, which further activates the downstream NF-κB pathway.

In the classical NF-κB pathway, IκBα binds to NF-κB and prevents its nuclear translocation. During osteoclast differentiation, IκBα is phosphorylated and degraded, resulting in activation of the NF-κB pathway.

In addition, Akt plays an important role in osteoclast differentiation [13]. RANKL and M-CSF stimulation can activate Akt, which promotes NFATc1 nuclear localization through phosphorylation of glycogen synthase kinase 3β, thereby inducing osteoclast formation.

Activation of NF-κB and Akt triggers expression of the key osteoclast differentiation factors c-fos and NFATc1. These two core regulators influence the differentiation of osteoclast precursors into mature osteoclasts by regulating the expression of osteoclast-specific genes.

This study found that acteoside treatment inhibited IκBα degradation in femoral tissue of OVX mice, thereby blocking NF-κB pathway activation. It also specifically reduced Akt phosphorylation and inhibited c-fos and NFATc1 protein expression.

These results indicate that acteoside may inhibit osteoclast differentiation and prevent OVX-induced bone resorption by blocking the NF-κB and Akt pathways and downregulating NFATc1 and c-fos expression.

 

Acteoside May Promote Osteoblast-Driven Bone Formation

Runx2 is a key transcription factor in bone formation. It is expressed only in osteoblasts and mineralized tissues and plays an important role in osteoblast differentiation by inducing key osteoblast-specific marker genes, including ALP, OCN, and Col1α [14].

Among these:

ALP is an early phenotypic marker of osteoblast differentiation. Its activity reflects the initial stage of osteoblast differentiation and plays an important role in bone mineralization by initiating and/or promoting hydroxyapatite crystal formation in osteoblast matrix vesicles.

OCN and Col1α are expressed in the later stages of osteoblast differentiation, accompanying the initiation of extracellular matrix mineralization [15].

Through immunohistochemical analysis, this study found that acteoside increased the protein expression of the osteogenic marker Runx2 in femoral tissue, suggesting that it may promote bone formation in OVX mice.

At the same time, downregulation of osteoblast-specific marker genes ALP, OCN, and Col1α was observed in the femoral tissue of OVX mice, while acteoside treatment partly reversed OVX-induced suppression of osteoblast-specific gene transcription.

These results suggest that acteoside may promote osteogenesis by enhancing both the early and late stages of osteoblast differentiation, thereby supporting osteoblast differentiation and mineral deposition.

 

What This Means for People Interested in Herbal Support for Osteoporosis

For people who are looking for herbal or natural approaches to support bone health, this study offers a meaningful scientific direction.

Acteoside is not simply a "calcium supplement." Instead, this research suggests that it may work more deeply by helping regulate the balance between two important types of bone cells:

Osteoblasts, which build bone

Osteoclasts, which break down bone

Healthy bones depend on the balance between these two processes. In postmenopausal osteoporosis, bone resorption often becomes stronger than bone formation, leading to gradual bone loss.

According to this mouse study, acteoside may help restore this balance by:

Supporting osteoblast-related bone formation

Reducing osteoclast-related bone resorption

Improving bone mineral density and trabecular bone structure

Regulating key molecular pathways related to bone remodeling

For customers interested in herbal bone health products, this provides a promising research basis for acteoside-rich botanicals such as Rehmannia and Cistanche.

However, it is important to be responsible and realistic: this study was performed in mice, not humans. Therefore, acteoside should not be presented as a proven cure for human osteoporosis. People diagnosed with osteoporosis should consult a qualified healthcare professional, especially if they are already using prescription medication or hormone-related therapy.

Still, for those seeking a natural bone-support strategy, acteoside is a compound worth paying attention to because it is supported by emerging mechanistic research.

 

Conclusion

In summary, acteoside appears to exert a dual regulatory effect on bone remodeling in OVX mice.

It may inhibit osteoclast-mediated bone resorption by suppressing activation of the NF-κB/Akt/NFATc1/c-fos pathway.

At the same time, it may promote osteoblast-driven bone formation by activating the Runx2/OCN/Col1α axis.

Through these mechanisms, acteoside improved OVX-induced increases in bone resorption and reductions in bone formation, ultimately helping reduce bone loss in mice.

However, this research was conducted only in an animal model. Future studies should establish corresponding cell models to further explore how acteoside regulates osteoblast and osteoclast function in vitro. More human clinical studies are also needed before acteoside can be recommended as a therapeutic option for osteoporosis.

For now, acteoside represents a promising natural compound for further development in the field of bone health and osteoporosis-related research.

---

References

[1] Brown JP. Long-term treatment of postmenopausal osteoporosis. Endocrinol Metab (Seoul), 2021, 36(3): 544–552.

[2] Fischer V, Haffner-Luntzer M. Interaction between bone and immune cells: Implications for postmenopausal osteoporosis. Semin Cell Dev Biol, 2022, 123: 14–21.

[3] He Yuchi, Liu Ya. Exploration of the molecular mechanism of Liuwei Dihuang Pill in the treatment of diabetes-related osteoporosis based on network pharmacology and molecular docking. Traditional Chinese Medicine, 2025, 14(7): 6.

[4] Tan Xiaoyan, Li Xinhua, Chen Guanghua, et al. Clinical efficacy of Liuwei Dihuang Pill combined with zoledronic acid in the treatment of postmenopausal osteoporosis. China Journal of Pharmaceutical Economics, 2024, 19(10): 47–51.

[5] Xiao Y, Ren Q, Wu L. The pharmacokinetic property and pharmacological activity of acteoside: A review. Biomed Pharmacother, 2022, 153: 113296.

[6] Zhou Yu. Systematic evaluation and medication rule exploration of traditional Chinese medicine compound prescriptions for postmenopausal osteoporosis and the mechanism of Gujindan intervention therapy. Dissertation. Changchun: Changchun University of Chinese Medicine, 2023.

[7] Wu Tingyu, Yu Pei, Liu Zhiqiang, et al. Mechanism of Qigu Capsule against osteoporosis based on network pharmacology. Journal of Xinyu University, 2021, 26(5): 29–37.

[8] Sun Qianhui. Study on the effects of Zuogui Pill and Yougui Pill on osteogenesis, adipogenesis, and apoptosis of bone marrow mesenchymal stem cells in ovariectomized rats. Dissertation. Shenyang: Liaoning University of Traditional Chinese Medicine, 2017.

[9] Zhang QY, Gong HB, Jiang MY, et al. Regulation of enzymatic lipid peroxidation in osteoblasts protects against postmenopausal osteoporosis. Nat Commun, 2025, 16(1): 758.

[10] Wang Dongdong, Ren Weimin, Chen Kai, et al. Predictive value of micro-CT parameters for recurrence after osteoporotic vertebral compression fracture surgery. Imaging Science and Photochemistry, 2022, 40(1): 22–27.

[11] Brown JP. Long-term treatment of postmenopausal osteoporosis. Endocrinol Metab (Seoul), 2021, 36(3): 544–552.

[12] Migliorini F, Maffulli N, Spiezia F, et al. Biomarkers as therapy monitoring for postmenopausal osteoporosis: A systematic review. J Orthop Surg Res, 2021, 16(1): 318.

[13] Udagawa N, Koide M, Nakamura M, et al. Osteoclast differentiation by RANKL and OPG signaling pathways. J Bone Miner Metab, 2021, 39(1): 19–26.

[14] Gomathi K, Akshaya N, Srinaath N, et al. Regulation of Runx2 by post-translational modifications in osteoblast differentiation. Life Sci, 2020, 245: 117389.

[15] Sharma AR, Lee YH, Gankhuyag B, et al. Effect of alumina particles on the osteogenic ability of osteoblasts. J Funct Biomater, 2022, 13(3): 105.