PART 2: Cistanche Deserticola Polysaccharide Inhibits OVX-induced Bone Loss in Mice And RANKL-induced Osteoclastogenesis
Mar 02, 2022
Contact: joanna.jia@wecistanche.com
Osteoporosis is a serious bone disease affecting the aging population. Cistanche deserticola (CD), a tonic and medicinal food widely used in China, has been proved to provide effective treatment for osteoporosis. Cistanche deserticola polysaccharide (CDP), extracted from CD, possesses various pharmacological properties, but its role in osteoclastic formation and function, as well as osteoporosis, remains unknown, The purpose of this study was to extract and purify CDP(Cistanche deserticola polysaccharide) to further explore its potential mechanism of action on osteoporosis. Results showed that the CDP(Cistanche deserticola polysaccharide) treatment prevented OVX-induced osteoporosis and ameliorated bone loss by repressing osteoclast activity and function. Furthermore, CDP(Cistanche deserticola polysaccharide) treatment significantly inhibited RANKL-induced osteoclastogenesis. bone resorption, and osteoclast-specific gene expression. Mechanistically, CDP(Cistanche deserticola polysaccharide) inhibited RANKL-induced NF-xB and MAPKs signaling pathways activation and consequently affected the downstream NEATc1 activation. The findings in this study suggest that CDP(Cistanche deserticola polysaccharide) is a potentially safe drug for treating osteoporosis.
Treatment for osteoporosis: Cistanche Deserticola Polysaccharide
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3. Results
3.1. CDP(Cistanche deserticola polysaccharide) prevents OVX-induced bone loss in vivo
We used an OVX-induced osteoporosis mouse model to investigate the effects of CDP(Cistanche deserticola polysaccharide) treatment on osteoporosis in vivo. Compared with the Sham group, the bodyweight of OVX mice significantly increased whereas the uterine index decreased, indicating that the osteoporosis model was constructed successfully (Fig. 1B and D). micro-CT analysis of the tibias in the OVX group showed an extensive bone loss. Compared with the OVX group, the number of bone trabeculae increased in the CDP-L group, while the increase in the CDP-H and E2 groups were more significant, and the trabeculae in the latter two groups became coarser and the gap became smaller. Quantitative analysis confirmed that several bone histomorphometric parameters, including BMD, BV/TV, BS/TV, and Tb. N had significantly increased values, whereas Tb. Sp showed decreased values in the CDP-H group (Fig. 1E and F). Histopathological examination using H&E staining revealed that the number of bone trabeculae in the CDP-H and E2 groups was significantly higher than that in the OVX group while there was no significant improvement in the CDP-L group (Fig. 1G). Additionally, TRAcP staining showed that the number of osteoclasts markedly increased in the OVX group but decreased in the CDP-H and E2 groups (Fig. 1H). CDP-L, CDP-H, and E2 groups showed inhibited the OVX-induced increase of P content but increased the Ca content in the blood serum. The bone absorption markers, RANKL and TRAcP-5b showed a decreasing trend in the CDP-L.
3.2. CDP(Cistanche deserticola polysaccharide) suppresses RANKL-induced osteoclastogenesis in vitro
We monitored the cell proliferation of CDP-treated BMMs using a long-term real-time dynamic live cell imaging analyzer. Compared to the control group, cell confluence in the CDP-treated groups did not change at a dose <10 µg/mL, but was significantly reduced at the 20 and 40 µg/mL dosages (Fig. 2A). To investigate the effect of CDP(Cistanche deserticola polysaccharide) on RANKL-induced osteoclastogenesis, BMMs were stimulated with RANKL and M- CSF in the presence of CDP for 7 days. TRAcP, a characteristic enzyme of osteoclasts, is considered an indicator of osteoclast function (Minkin, 1982). TRAcP staining showed that the osteoclasts treated with>1.25 µg/mL CDP(Cistanche deserticola polysaccharide) showed significantly reduced size and number. These results showed that CDP(Cistanche deserticola polysaccharide) effectively inhibited the expected RANKL-induced osteoclastogenesis without affecting the cell viability.
3.3. CDP(Cistanche deserticola polysaccharide) inhibits RANKL-induced osteoclastic resorption activity and osteoclast-specific gene expression
We further determined the effects of CDP(Cistanche deserticola polysaccharide) treatment on the bone resorption activity of osteoclasts using hydroxyapatite-coated plates. Bone resorption is the main function and standard for measuring the activity and ability of osteoclasts (Novack & Faccio, 2011). The results showed that CDP(Cistanche deserticola polysaccharide) can significantly reduce the bone-resorptive area of osteoclasts in a dose-dependent manner, indicating that CDP(Cistanche deserticola polysaccharide) inhibited the bone resorption activity induced by RANKL (Fig. 3A and B). In particular, the osteoclastic bone-resorptive area decreased to approximately 6.3% after treatment with 10 µg/mL CDP(Cistanche deserticola polysaccharide). To better figure out the hindrance of CDP(Cistanche deserticola polysaccharide) on osteoclastogenesis, we measured the transcription levels of osteoclast-related genes. Without treatment with CDP(Cistanche deserticola polysaccharide), induction with RANKL promoted the expression of NFATc1, CTSK, Acp5, Atp6v0d2, and Mmp9 genes. On the contrary, the concomitant application of CDP and RANKL significantly down-regulated these five genes.
3.4. CDP(Cistanche deserticola polysaccharide) inhibits RANKL-induced NFATc1 activation
TRAcP staining revealed that CDP(Cistanche deserticola polysaccharide) treatment significantly reduced the expected increase in the number of mature osteoclasts after RANKL stimulation for 3–7 days (Fig. 4A and B). Furthermore, we investigated whether CDP treatment can effectively impair osteoclast differentiation by inhibiting NFATc1 expression. Results showed that NFATc1 was highly expressed after RANKL stimulation for 5 days, which was significantly inhibited after CDP(Cistanche deserticola polysaccharide) treatment (Fig. 4C and D). In addition, CDP treatment suppressed the expression levels of CTSK, a protein required for normal osteoclast formation and function (Bossard et al., 1996) (Fig. 4E and F). We selected a point-in-time representation of RANKL stimulation for 5 days to further investigate the effect of 10 µg/ mL CDP(Cistanche deserticola polysaccharide) treatment on NFATc1 translocation. As expected, CDP(Cistanche deserticola polysaccharide) treatment significantly inhibited the RANKL-induced NFATc1 nuclear translocation.
3.5. CDP(Cistanche deserticola polysaccharide) inhibits RANKL-induced activation of NF-κB and MAPKs signaling pathways
To elucidate the mechanisms involved in the CDP-induced inhibition of osteoclast differentiation, we examined the impact of CDP(Cistanche deserticola polysaccharide) treatment on the NF-κB and MAPKs pathways. Results revealed that CDP(Cistanche deserticola polysaccharide) treatment had suppressive effects on IκB-α degradation and p65 phosphorylation in a dose-dependent manner. Phosphorylation of three MAPKs family members, specifically extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were upregulated.
Cistanche Deserticola
4. Discussion
Osteoporosis is a common and degenerative bone disease. At present, there are many chemical synthesis drugs used in clinical treatments for osteoporosis, such as estrogen and bisphosphonate. However, the long-term use of these drugs will gradually weaken clinical efficacy and even cause many serious side-effects, including increased risk of ovarian, breast, and endometrial cancers and osteonecrosis of the jaw (Beral, 2003; Khan et al., 2009; Lacey et al., 2002). While in this study, we selected CDP(Cistanche deserticola polysaccharide), an effective component extracted from CD, as the therapeutic agent and demonstrated that CDP(Cistanche deserticola polysaccharide) had a protective effect against
bone loss in OVX mice and inhibited RANKL-induced osteoclast formation and function. Meanwhile, according to the “kidney governing bone” traditional Chinese medicine theory, bone loss is attributed to kidney deficiencies. As a kidney-tonifying medical food, the CD has been proved to provide effective treatment for osteoporosis (Fan et al., 2019; Jiang, Wang, Li, & Zhang, 2016). Thus, CDP(Cistanche deserticola polysaccharide) extracted from this natural herb is a better and safer drug option.
Currently, the OVX mouse model is a classic model of post-menopausal bone loss in women, investigating the causes of bone loss and conducting intervention (Wehrle et al., 2015). Accordingly, we performed bilateral oophorectomy on C57BL/6J female mice to construct an osteoporosis model. In previous studies, the CDP doses given to animals were 50–200 mg/kg (low dose) and 1800 mg/kg (high dose) (Guo et al., 2016; Zhang et al., 2018). Here, the high dose of CDP(Cistanche deserticola polysaccharide) in mice was 600 mg/kg, which was based on the high clinical dosage of 30 g CD in decoction; thus, it is similar to current clinical practice. In this study, treatment with CDP(Cistanche deserticola polysaccharide) significantly inhibited OVX-induced body weight gain, which is a common phenomenon observed in OVX mice (Davis et al., 2019). By using micro-CT scanning and 3D reconstruction, we found that OVX led to a noticeable reduction in BMD, BV/TV, BS/TV, and Tb. N, and an increase in Tb. Sp, while CDP treatment reversed all these alterations. In particular, compared with the positive drug, CDP could significantly improve the BMD of OVX mice, which is an important marker of bone quality and used to reflect the degree of osteoporosis (Fuggle et al., 2019), indicating that CDP has an advantage over the positive drug in improving BMD. Furthermore, the histological results indicated that CDP treatment could reduce the increased amount of TRAcP-positive osteoclasts induced by OVX. These results suggest that CDP administration effectively mitigated bone loss in OVX-induced osteoporosis, possibly by inhibiting osteoclast formation. Consistent with the above results, we found CDP dose-dependently inhibited RANKL-induced osteoclastic formation and function, while CDP had little effect on M-CSF-induced osteoclast precursor cell proliferation in our in vitro experiments.
The binding of RANKL and RANK can lead to the recruitment of TRAF6 and the subsequent activation of several downstream signaling pathways, such as NF-κB and MAPKs (Boyle et al., 2003; Huang et al., 2006). The NF-κB signaling pathway plays an important role in the regulation of osteoclast differentiation, and the silencing of genes coding for key proteins in this pathway can lead to abnormal bone development (Leibbrandt & Penninger, 2008). It is well established that NF-κB is present in the cytoplasm of unstimulated cells in a complex with IκB but rapidly enters into the nucleus after RANKL stimulation (Boyle et al., 2003). The released IκB is then rapidly degraded, whereas NF-κB activates specific gene transcription, consequently promoting osteoclast differentiation, maturation, and apoptosis (Abdelmagid et al., 2015). In this study, results showed that CDP(Cistanche deserticola polysaccharide) treatment suppressed RANKL- induced activation of the NF-κB signaling pathway, as demonstrated by inhibition of the degradation of IκBα and phosphorylation of p65 in a dose-dependent manner, which may involve in its anti-osteoclastogenic effect. In addition, the MAPKs signaling pathway, including ERK, JNK, and p38, are closely involved in RANKL-induced osteoclastogenesis (Li et al., 2002). Furthermore, the dominant inhibitors of p38 and JNK can prevent RANKL-induced osteoclastogenesis (Chang et al., 2008; Kim et al., 2019), whereas ERK plays a significant role in osteoclast survival (Miyazaki et al., 2000). It is well established that RANKL activates the MAPKs signaling pathway by increasing the phosphorylation of ERK, JNK, and p38 (Mizukami et al., 2002). In our study, we demonstrated that CDP suppressed RANKL-induced phosphorylation of key proteins in the MAPKs signaling pathway. Taken together, these results indicate that inhibiting the activation of the NF-κB and MAPKs signaling pathways is contributed to the inhibitory effect of CDP against osteoclastogenesis.
Activation of the NF-κB and MAPKs signaling pathways promotes the expression of several key transcription factors, such as cellular oncogene fos, activator protein 1, and NFATc1 (Huang et al., 2006; Wagner & Matsuo, 2003). NFATc1, an important member of the NFAT family, is involved in terminal osteoclast differentiation as a master transcriptional regulator (Asagiri et al., 2005). It has been reported that NFATc1- deficient embryonic stem cells can not differentiate into osteoclasts under the stimulation of RANKL and the ectopic expression of NFATc1 causes precursor cells to undergo differentiation bypassing RANKL signaling, which suggests that NFATc1 has a critical role in osteoclast differentiation (Takayanagi et al., 2002). The results of our study showed the suppressive effect of CDP(Cistanche deserticola polysaccharide) on NFATc1 expression and its following protein CTSK. Interestingly, we observed that the protein expression level of NFATc1 first increased and then decreased, reaching the highest level on the 5th day after RANKL stimulation. This was consistent with the results of Baek et al. (2014), who observed M-CSF- induced NFATc1 degradation during late-stage osteoclastogenesis through the Cbl-induced ubiquitination of NFATc1 in an Src kinase-dependent manner. Immunofluorescence assay results consistently supported the inhibitory effect of CDP(Cistanche deserticola polysaccharide) on NFATc1 transcriptional activation. Furthermore, osteoclast-specific genes, including CTSK, Acp5, Mmp9, and Atp6v0d2, are all regulated by NFATc1 directly (Y. Kim et al., 2005), were suppressed by CDP. These findings illustrate that the NFATc1 is a target of the inhibitory effect of CDP(Cistanche deserticola polysaccharide) on the NF-κB and MAPKs signaling pathways.
Cistanche deserticola polysaccharide
5. Conclusion
In summary, we extracted and purified the active polysaccharide from CD and demonstrated that CDP(Cistanche deserticola polysaccharide) exhibited a protective effect on OVX-induced osteoporosis and ameliorated bone loss by repressing osteoclast activity and function. Furthermore, CDP(Cistanche deserticola polysaccharide) can inhibit RANKL- induced osteoclast differentiation and function by interfering with the NF-κB and MAPKs signaling pathways and consequently affecting the downstream NFATc1 activation. In conclusion, our findings provide the foundation for the expansion and application of CDP in the treatment of osteoporosis.
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