What Stimulates New Bone Growth? Cistanche (CPhGs) Activates The SIRT2–C/EBPβ–AREG Pathway To Build Stronger Bones

Results

 

2.1 CPhGs alleviate ovariectomy-induced osteoporosis in mice

micro-CT findings (Figure 1A) showed that, compared with the Sham group, the OVX group exhibited severe destruction of trabecular bone microarchitecture in the distal femur, characterized by sparse and fractured trabeculae. In the OVX+CPhGs group, trabecular number and connectivity were significantly restored, approaching the Sham level. These results indicate that CPhGs can mitigate ovariectomy-induced bone loss. HE staining (Figure 1B) further confirmed that trabecular number was reduced in the OVX group versus the Sham group, with residual trabeculae becoming thinner and fractured; in contrast, the OVX+CPhGs group showed increased trabecular number compared with OVX and markedly improved structural integrity. Calcein double-labeling (Figure 1C) demonstrated blurred and narrowly spaced fluorescent bands in the OVX group, suggesting inhibited bone formation; in the OVX+CPhGs group, fluorescent bands were clear with significantly increased spacing, indicating that CPhGs promote bone matrix mineralization rate and markedly enhance new bone formation capacity.

 

 

A representative micro-computer tomography image of the trabecular bone in the femoral metaphysis of mice;B  hematoxylin and eosin staining of the femoral sections of mice; C calcein double-labeling images of mice in the OVX and OVX+CPhGs groups.

Fig.1  CPhGs alleviate ovariectomy-induced osteoporosis in mice

 

Cistanche Other Benefits

 

 

 

 

Cistanche Listing For Sale

 

 

2.2 Effects of CPhGs on osteogenic differentiation of MC3T3-E1 cells

In vitro, the CPhGs group showed significantly higher protein and mRNA expression levels of OCN, OPN, and RUNX2 than the NC group (P < 0.05). Alkaline phosphatase (ALP) activity on day 10 was also significantly higher in the CPhGs group than in NC (P < 0.05). These results indicate that CPhGs significantly promote osteoblast differentiation. See Table 1.

 

2.3 Effects of CPhGs on the SIRT2–C/EBPβ–AREG axis

Cell experiments (Table 2) showed that, compared with the NC group, the CPhGs group had significantly increased protein and mRNA expression of C/EBPβ and elevated AREG mRNA (P < 0.05), indicating activation of downstream C/EBPβ target genes. Relative to NC, SIRT2 mRNA was significantly upregulated in the CPhGs group (P < 0.05), with a concomitant increase at the protein level (P < 0.05). In contrast, mRNA levels of SIRT1, SIRT3, SIRT4, and SIRT5 showed no significant changes (P > 0.05), confirming pathway specificity for SIRT2.

Table 1. CPhGs Enhance the Osteogenic Differentiation Markers in MC3T3-E1 Cells

Item	NC Group	CPhGs Group
OCN protein	0.48 ± 0.14	1.24 ± 0.18¹
OCN mRNA	1.00 ± 0.11	1.61 ± 0.23¹
OPN protein	0.51 ± 0.15	1.05 ± 0.44¹
OPN mRNA	1.03 ± 0.12	2.11 ± 0.24¹
RUNX2 protein	0.77 ± 0.21	1.08 ± 0.25¹
RUNX2 mRNA	1.02 ± 0.14	1.58 ± 0.17¹
ALP activity at day 10	0.21 ± 0.02	0.44 ± 0.05¹

¹ P < 0.05 vs. NC group.

 

Table 2. Effects of CPhGs on the Expression of SIRT2, C/EBPβ, and AREG

Item	NC Group	CPhGs Group
C/EBPβ protein	0.51 ± 0.06	1.21 ± 0.11¹
C/EBPβ mRNA	1.00 ± 0.10	1.15 ± 0.14¹
AREG mRNA	1.01 ± 0.15	2.05 ± 0.24¹
SIRT1 mRNA	1.03 ± 0.12	1.01 ± 0.11
SIRT2 protein	0.26 ± 0.08	0.37 ± 0.07¹
SIRT2 mRNA	0.87 ± 0.08	1.88 ± 0.21¹
SIRT3 mRNA	1.01 ± 0.14	1.28 ± 0.17
SIRT4 mRNA	1.21 ± 0.12	1.04 ± 0.05
SIRT5 mRNA	1.06 ± 0.05	1.08 ± 0.03

¹ P < 0.05 vs. NC group.

 

2.4 Effects and mechanism of CPhGs on SIRT2 and C/EBPβ

Immunofluorescence (Figure 2A) revealed markedly enhanced expression and nuclear co-localization of SIRT2 and C/EBPβ in the CPhGs group versus NC. While SIRT2 and C/EBPβ signals were weak in NC, their fluorescence intensities were significantly increased by CPhGs, with clear nuclear co-localization signals, suggesting strengthened interaction. Co-immunoprecipitation further confirmed that, in C/EBPβ pull-down complexes, SIRT2 protein was significantly increased in the CPhGs group versus NC (Figure 2B). In C/EBPβ immunoprecipitates, the acetyl-lysine (Acetyl) signal decreased in the CPhGs group, whereas total C/EBPβ protein was unchanged (Figure 2C), indicating that CPhGs significantly enhance the SIRT2–C/EBPβ interaction and suppress endogenous C/EBPβ acetylation in MC3T3-E1 cells.

2.5 Effects of SIRT2 on C/EBPβ protein and mRNA expression

Compared with the Control group, SIRT2 knockdown (SIRT2 KD) significantly reduced C/EBPβ protein and mRNA levels in MC3T3-E1 cells. Relative to Control+CPhGs, the SIRT2 KD+CPhGs group also showed decreased C/EBPβ protein and mRNA, and CPhGs could not reverse the reduction caused by SIRT2 knockdown (Figures 3A, 3B). These findings indicate that loss of SIRT2 markedly diminishes C/EBPβ protein accumulation and C/EBPβ mRNA expression.

A,immunofluorescence analysis of SIRT2 (red) and C/EBPβ (green) in CPhGs-treated MC3T3-E1 cells (×200);B,co-immunoprecipitation (co-IP) analysis of SIRT2-C/EBPβ interaction;C,acetylation level of C/EBPβ detected by pan-acetyllysine antibody after IP purification.

Fig.2  Effect of CPhGs on SIRT2 and C/EBPβ and its mechanism

 

2.6 Effects of C/EBPβ deacetylation on osteogenic differentiation

C/EBPβ acetylation-site mutants (K102R and K211R) were constructed to mimic sustained deacetylation. We assessed ALP activity at the early differentiation stage (day 7) and mineralization at the terminal stage (day 14). Compared with WT, both K102R and K211R mutants displayed increased ALP-positive area, and Alizarin Red S (ARS) staining showed enhanced extracellular matrix mineralization. However, the EX group exhibited greater increases in ALP and ARS than either single mutant, suggesting that CPhGs cooperatively modulate both K102 and K211 sites to comprehensively promote bone formation (Figure 3C). These results indicate that the pro-osteogenic effect of CPhGs is associated with C/EBPβ deacetylation-mediated early differentiation and terminal mineralization, providing functional evidence that CPhGs influence osteogenic differentiation through the SIRT2/CEBPβ/AREG axis.

Discussion

 

Osteoporosis has become a global public health issue that seriously affects patients' quality of life. Traditional Chinese medicines such as Cistanche (Rou Cong Rong), Ligustrum lucidum, and Morinda officinalis play important roles in osteoporosis management and are attracting growing attention due to fewer adverse effects and stable efficacy [8–9]. Cistanche and its constituents can modulate bone formation and resorption and hold strong potential for osteoporosis prevention and treatment. One study [10] found that Cistanche extract promotes mesenchymal stem cell differentiation toward osteoblasts, effectively increasing bone mass while reducing bone resorption. Another study [11] showed that Cistanche polysaccharides inhibit osteoclastogenesis and hydroxyapatite resorption, in part by suppressing osteoclast marker genes (e.g., Ctsk, MMP9, and ACP5).

This study systematically investigated the effects and molecular mechanisms of CPhGs in osteoporosis therapy. Through in vivo and in vitro experiments, we found that CPhGs significantly promote osteogenic differentiation by modulating the SIRT2–C/EBPβ–AREG axis, providing a new theoretical basis for regulating bone metabolism with natural products.

We demonstrated that CPhGs improve the bone-loss phenotype in ovariectomized mice by activating the SIRT2–C/EBPβ–AREG axis, significantly increasing trabecular number and promoting bone formation. Our results show that CPhGs markedly upregulate osteogenic markers (OCN, OPN, RUNX2) and ALP activity. As a master regulator of osteogenic differentiation [12], RUNX2 upregulation may directly drive bone matrix mineralization, and enhanced ALP activity further supports maturation of osteoblast function. We reveal for the first time that CPhGs upregulate SIRT2 expression, strengthen its interaction with C/EBPβ, and induce C/EBPβ deacetylation. Deacetylated C/EBPβ likely undergoes conformational changes that enhance transcriptional activity and significantly upregulate AREG mRNA; as an EGFR ligand, AREG may activate the PI3K–AKT pathway to promote osteogenesis-consistent with ZHANG et al. [13], who reported that Cistanche extract suppresses osteoclast differentiation via PI3K/AKT. Using C/EBPβ acetylation-site mutants (K102R and K211R), we directly confirmed that C/EBPβ deacetylation promotes extracellular matrix calcification, while SIRT2 knockdown established the necessity of this pathway in CPhG action, indicating that SIRT2-mediated C/EBPβ deacetylation is central to the pro-osteogenic effects of CPhGs. These findings broaden the recognized diversity of Sirtuin family functions in bone homeostasis and suggest new strategies for treating osteoporosis by targeting post-translational modifications.

Osteoblasts play key roles in the complex process of bone remodeling. Imbalance between bone formation and resorption-largely regulated by osteoblasts-is a major driver of osteoporosis. Therefore, enhancing bone formation is a fundamental therapeutic strategy. Effective remodeling depends on coordinated interactions between osteoblasts and osteoclasts. AREG, a member of the EGF family, is critical in regulating cell growth, apoptosis, and migration across diverse cell types [14]. SIRT2 is an NAD+-dependent deacetylase expressed mainly in the cytoplasm but also found in mitochondria and the nucleus, making it a unique Sirtuin family member [15]. As a transcription factor, C/EBPβ undergoes deacetylation under SIRT2 regulation, increasing the transcriptional activity of various genes. During osteoblast differentiation, C/EBPβ binds multiple genes to modulate transcription. Our results show that CPhGs enhance C/EBPβ protein expression and its interaction with SIRT2, markedly suppress C/EBPβ acetylation, and in turn upregulate AREG mRNA, thereby promoting osteogenic differentiation. Although C/EBPβ expression correlates positively with AREG transcription, whether C/EBPβ directly serves as an AREG transcription factor warrants further validation.

In summary, CPhGs promote C/EBPβ deacetylation via SIRT2, thereby increasing AREG expression, enhancing osteoblast differentiation, and improving osteoporosis. CPhGs hold potential as therapeutic agents for osteoporosis.

 

Trusted Cistanche sourcing for osteoporotic bone health

If you are a nutraceutical brand, functional food manufacturer, or R&D buyer searching for reliable, high-quality Cistanche phenylethanoid glycosides (CPhGs) or standardized Cistanche extracts for bone health, it is essential to partner with a supplier that controls the full herb industrial chain and complies with international quality systems. Chengdu Wecistanche Bio-Tech Co., Ltd (WECISTANCHE) is the sales company of Hotan Dicheng (Hetian Dicheng) and our headquarters is located in Hotan, Xinjiang-China's premier Cistanche origin region. WECISTANCHE is a leading China Cistanche manufacturer with:

Hotan Dicheng (Hetian Dicheng)

The world's largest Cistanche processing capacity linked to a 200,000-acre base, including 85,000+ acres of cultivation and a 20,000-ton GMP factory for fresh Cistanche and other botanicals.

100,000-class GMP production workshop; advanced ultrafiltration/nanofiltration purification and concentration; and a 10,000-class microbiology lab.

 

Hotan Dicheng (Hetian Dicheng)
 

Certifications: China SC, HACCP, organic certifications (IOIA/International Organic Crop Improvement Association) and NOP (US Organic Program).

14 Cistanche-related invention patents, including membrane separation extraction technology.

Academic collaborations with top institutions (e.g., Peking University, Shanghai Jiao Tong University, Xinjiang Medical University, Kyoto Pharmaceutical University).

 

Business scope covers Cistanche extracts, health supplements, raw materials, and applications across TCM decoctions, functional foods, healthcare products, dietary supplements, pharmaceuticals, and skincare. During COVID-19, WECISTANCHE donated Cistanche products valued at over 1 billion RMB, reflecting strong social responsibility.

 

For buyers looking to wholesale or procure Cistanche extract for osteoporosis-focused formulas-targeting mechanisms like SIRT2–C/EBPβ–AREG modulation, osteoblast differentiation (RUNX2/ALP/OCN/OPN), and complementary anti-resorptive support-WECISTANCHE offers scalable, compliant supply aligned with US/EU standards. Learn more: About Us - WECISTANCHE (https://www.xjcistanche.com/about-us).

Email:wallence.suen@wecistanche.com
Address: No.1, Floor 26, Unit 1, Building 6, No.133 Sheng'an Street, High-tech District, Chengdu, Sichuan, China (Pilot Free Trade Zone).

 

References

[10] Li Y, Pei T, Zhu H, et al. Melatonin alleviates circadian rhythm disruption-induced enhanced luteinizing hormone pulse frequency and ovarian dysfunction. J Pineal Res. 2025;77(1):e70026. doi:10.1111/jpi.70026

[11] Liu J, Yang Y, He Y, et al. Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells. J Ethnopharmacol. 2023;304:116046. doi:10.1016/j.jep.2022.116046

[12] Yang ZN, Du X, Wang A, et al. Melatonin ameliorates Pb-induced mitochondrial homeostasis and ovarian damage through regulating the p38 signaling pathway. Ecotoxicol Environ Saf. 2025;292:117937. doi:10.1016/j.ecoenv.2025.117937

[13] Okoye CN, Koren SA, Wojtovich AP. Mitochondrial complex I ROS production and redox signaling in hypoxia. Redox Biol. 2023;67:102926. doi:10.1016/j.redox.2023.102926

[14] Keskin-Aktan A, Akbulut KG, Yazici-Mutlu Ç, et al. The effects of melatonin and curcumin on the expression of SIRT2, Bcl-2 and Bax in the hippocampus of adult rats. Brain Res Bull. 2018;137:306-310. doi:10.1016/j.brainresbull.2018.01.006

[15] Zhu T, Yan L, Deng S, et al. Mitochondria of porcine oocytes synthesize melatonin, which improves their in vitro maturation and embryonic development. Antioxidants. 2024;13(7):814. doi:10.3390/antiox13070814