Total Glycosides And Polysaccharides Of Cistanche Deserticola Prevent Osteoporosis

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Fujiang Wang 1 , Pengfei Tu, Kewu Zeng * , Yong Jiang **

ABSTRACT

Ethnopharmacological relevance: Traditional Chinese medicine Cistanche deserticola Y. C. Ma has the effect of "tonifying kidney and strengthening bone". However, the specific active extracts of C. deserticola and mechanisms for the treatment of osteoporotic are not clear.

Aim of the study: We wanted to identify the effective component extracts of C. deserticola for the treatment of osteoporosis and the potential mechanisms.

Materials and methods: Our group researched the extracts of C. deserticola with anti-osteoporotic activity, including total glycosides (TGs), polysaccharides (PSs), and oligosaccharides (OSs) in senescence-accelerated mouse prone 6 (SAMP6) mice. The Goldner’s Trichrome, Van Gieson’s (VG), Safranin O-Fast Green staining, and von Kossa staining were performed to investigate the bone structure formation and calcium deposits. Serum was collected for detecting biochemical markers. Bone micro-architecture was detected by micro-CT. Expressions of bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), osteoprotegerin (OPG), receptor activator of nuclear factor-κ B ligand (RANKL), p-glycogen synthetase kinase-3β (p-GSK-3β), and p-β-catenin were analyzed by western blotting and immunohistochemistry.

Results: TGs and PSs ameliorated bone histopathological damages, promoted the formation of new bone, collagenous fiber, and chondrocytes, and accelerated the calcium deposits. Moreover, they remarkably altered the biomarkers of bone turnover and effectively ameliorated bone microarchitecture. The further mechanisms study showed that TGs and PSs significantly decreased the expressions of RANKL, p-β-catenin, as well as up-regulated the expression of BMP-2, OCN, OPG, and p-GSK-3β (Ser9).

Conclusion: The findings of this study suggest that TGs and PSs can promote osteoclastogenic bone formation and improve bone microstructure damage in SAMP6 mice, and their therapeutic effect on osteoporosis is via activating the Wnt/β-catenin signaling pathway.

Cistanche can promote osteoclastogenic bone formation and improve bone microstructure damage.

1. Introduction

Osteoporosis is a common disease in elderly individuals that seriously endangers the health of mankind (Ye et al., 2020). Osteoporosis patients may be completely asymptomatic before fracture, thus effective prevention and treatment of osteoporosis are paramount (Tella and Gallagher, 2014).

Research shows that increasing bone resorption and decreasing bone formation give rise to an imbalance in bone remodeling, leading to osteoporosis (Sims and Gooi, 2008). Osteoblasts and osteoclasts are two cell types that are critical for bone formation and resorption. The wnt/β-catenin pathway is essential for the growth, development, and maintenance of bone tissue (Cadigan and Nusse, 1997), and also plays a key role in regulating beagle dog bone marrow stromal cells (BMSCs) differentiation (Jing et al., 2018). GSK-3β prevents the degradation of β-catenin. β-Catenin subsequently enters into the nucleus, and then it can associate with the T-cell factor/lymphoid-enhancer binding factor and regulates the expression of Wnt target genes. Meantime, it is found that Wnt/β-catenin signaling decreases osteoclast differentiation by stimulating the production and secretion of OPG (Glass et al., 2005), which is a natural antagonist of the RANKL (Lacey et al., 1998). OPG plays an important regulatory role in bone formation and bone resorption. In any case, deletion of β-catenin in osteoclasts increases osteoclast number and bone resorption and decreases bone mass (Wei et al., 2011). Bone resorption inhibitors and bone formation promoters are mainly used in the treatment of osteoporosis. Considering that the adverse reactions of the presently used drugs in clinical with accurate efficacy are also obvious, it is urgent to look for the drugs with fewer side effects.

Cistanche deserticola Y. C. Ma (C. deserticola) is one of the source plants of the widely used tonic traditional Chinese medicinal herb Cistanches Herba, Roucongrong in Chinese, for the treatment of several diseases, such as kidney deficiency, female infertility, and senile constipation for more than 1000 years in China (National Pharmacopoeia Committee, 2020). According to the theories of traditional Chinese medicine (TCM), “kidney dominating bones” and “tonifying kidney-strengthening bone”, C. deserticola was used to treat osteoporosis. Studies indicated that C. deserticola can improve the serum alkaline phosphatase (ALP), osteocalcin, and calcium ion levels, promote the expression of BMP-2 in osteoblasts of rats (Gang et al., 2018). In addition, studies have shown that C. deserticola exerted protective effects against RANKL induced osteoclastogenesis (Zhang et al., 2019). Although C. deserticola has a therapeutic effect on osteoporosis, its specific active components are not very clear. It is of great significance to explore the effective component types of C. deserticola for the treatment of osteoporosis and related mechanisms.

Therefore, we conducted this study to determine if there were some beneficial effects of the extracts containing different types of chemical constituents from C. deserticola on SAMP6 mice. The finding results may give accurate guides for clinical application of C. deserticola as well as reveal the material basis of the C. deserticola for the treatment of osteoporosis.

2. Materials and methods

2.1. Chemicals and reagents

Cistanche deserticola Y. C. Ma was purchased from Mandela Biotechnology Co., Ltd (Alashan, Inner Mongolia, China), and then one person from the authors identified them (P. F. Tu). TGs, PSs, and OSs were prepared as the method previously mentioned (Gao et al., 2015). Constituent analysis of each extract was accomplished via using HPLC according to the report. (Li et al., 2019; Wang et al., 2020). H&E, Goldner’s trichrome staining, Van Gieson’s (VG) staining, and Safranin O-Fast Green kits were purchased from Boster (Hubei, China). Rabbit anti-mouse BMP-2 (ab14933), OCN (ab93876), OPG (ab183910), and RANKL (ab216484) were purchased from Abcam (Cambridge, Britain). Cell Signaling Technology was the source of rabbit anti-mouse p-GSK-3β (Ser9) (#5558), GSK3β (#12456), β-catenin (#13727), and p-β-catenin (#4176). Secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China).

High-quality cistanche extract from Chengdu Wecistanche Bio-Tech Co., Ltd

2.2. Animals

5-month-old female senescence-accelerated mouse/resistant 1 (SAMR1) and SAMP6 mice were obtained from Vital River Laboratory Animal Technology (Beijing, China). All animal manipulations were performed in accordance with the guidelines issued by the Institutional Animal Care and Use Committee of Peking University Health Science Center.

2.3. Drug administration

The mice were randomly separated into the following five groups: SAMR1 group (normal saline, n = 10); SAMP6 group (normal saline, n = 10); TGs group (400 mg/kg, n = 10); PSs group (400 mg/kg, n = 10), and OSs group (400 mg/kg, n = 10) (Gao et al., 2015). All drugs wereadministrated ig and daily for 12 weeks.

2.4. Goldner’s trichrome staining, H&E staining, SafraninO-Fast Green staining, and Van Gieson’s (VG) staining

After 12 weeks, the femurs were rapidly removed and fixed using 10% ethylene diamine tetraacetic acid (EDTA) for 7 d at 4 ◦C. Next, the femurs were sectioned into a slice (5 μm) and stained with Goldner Trichrome, Van Gieson (VG), SafraninO-Fast Green, and H&E staining following the manufacturer’s instructions. The images were observed with a light microscope (Leica, Solms, Germany).

2.5. Measurement of the levels of BGP, BALP, P1NP, PICP, ALP, S-CTX, TRACP, U-CTX, U-NTX, D-Pyr, and Pyr

The concentrations of bone gla protein (BGP), bone-specific alkaline phosphatase (BALP), procollagen type 1 N-terminal propeptide (P1NP), procollagen type I C-terminal propeptide (PICP), alkaline phosphatase (ALP), S–C-telopeptide of type-I-collagen (S-CTX), tatrate-resistant acid phosphatase (TRACP), U–C-telopeptide of type-I-collagen (U-CTX), U–N-telopeptide of type-I-collagen (U-NTX), D-pyridinoline (D-Pyr), and pyridinoline (Pyr) were determined by enzyme-linked immunosorbent assay (Jiangsu Meimian Industrial Co., Ltd, Jiangsu, China).

2.6. Von Kossa staining

The femur slices were immersed in 1% silver nitrate for 30 min under an intense sunbeam and were then washed three times with deionized water. Subsequently, 5% sodium thiosulfate was added for 5 min to remove un-reacted silver. The calcium phosphate salts were visualized as black staining. To quantitatively analyze femur calcium content, Image-Pro Plus software version 6.0 and Adobe Photoshop were applied.

2.7. Micro-computed tomography analysis

All femur samples were scanned at 9 μm resolution by a micro-CT scanner (PerkinElmer, MA, USA). Further analysis was performed using Analyze12.0 software to calculate the bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), and tissue mineral density (TMD). Three-dimensional images were reconstructed using CTVox software (PerkinElmer, MA, USA).

2.8. Tetracycline and calcein accumulation

Each animal was intraperitoneally given 25 mg/kg tetracycline and 5 mg/kg calcein on the 13th day and 3rd day before euthanasia, respectively. Tetracycline and calcein accumulation were probed using Vectra® Polaris™ Automated Quantitative Pathology Imaging System (PerkinElmer, MA, USA). The distance between tetracycline and calcein can be observed by Image-Pro Plus software version 6.0.

2.9. Western blotting analysis

Femur tissues were homogenized and lysed in RIPA lysis buffer. The protein concentration was determined using a bicinchoninic acid (BCA) Protein assay reagent kit (Beijing TransGen Biotech, Beijing, China). Total proteins were loaded on 10% or 12% SDS-PAGE gels and then transferred onto a nitrocellulose membrane. The membrane was blocked and then incubated overnight with primary antibodies and GAPDH (Los Angeles, USA) at 4 ◦C followed by incubation with secondary antibody. The bands of proteins analysis were analyzed using Tanon 5200 Multi (Shanghai, China).

2.10. Immunohistochemical analysis

Femur tissues sections were incubated with the primary antibodies at 4 ◦C. Polyclonal antibodies against BMP-2, OCN, OPG, and RANKL were diluted to 1:200 and 1:100, respectively. Secondary antibody mouse anti-rabbit IgG (1:200) at 37 ◦C for 1 h. Using Vectra® Polaris™ Automated Quantitative Pathology Imaging System (PerkinElmer, MA, USA). To quantitatively analyze the expression of the protein, Image-Pro Plus software version 6.0 and Adobe Photoshop were applied.

2.11. Statistical analysis

Results are expressed as mean ± standard deviation. One-way ANOVA was performed when comparing the different groups. SPSS software version 22.0 was used for statistical analysis, and P < 0.05 was considered statistically significant.

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3. Results

3.1. TGs and PSs ameliorate bone histopathological damages and promote collagenous fiber and chondrocytes formation in SAMP6 mice

The femur pathological damage can be observed by H&E staining. The histomorphological structures of bone in the SAMR1 group are arranged regularly. However, the structures of bone mentioned above were damaged in the SAMP6 group. The morphology changes in TGs and PSs groups were less than those in the SAMP6 group. However, the OSs treatment group showed no significant amelioration for the morphology changes (Fig. 1A). The Goldner’s Trichrome, Van Gieson’s (VG), and Safranin OFast Green staining were done to reveal the bone structure formation. The results illustrated that the new bone, collagenous fiber, and chondrocytes in TGs and PSs groups were ameliorated compared with the SAMP6 group. However, the OSs treatment group showed no significant amelioration for bone structure changes (Fig. 1B–D).

Fig. 1. TGs and PSs ameliorate histopathological damage in SAMP6 mice.

3.2. TGs and PSs alter biomarkers of bone turnover in SAMP6 mice

When osteoporosis occurred, the levels of bone formation biomarkers will be significantly decreased, such as serum BGP and PICP. In contrast, the biomarkers levels associated with bone resorption were remarkably increased, including serum TRACP and S-CTX (Fig. 2). Encouragingly, TGs and PSs groups could reverse the levels of BGP, BALP, P1NP, PICP, ALP, S-CTX, TRACP, U-CTX, U-NTX, D-Pyr, and Pyr, but OSs could not.

Fig. 2. Effects of TGs and PSs on biomarkers of bone turnover in SAMP6 mice.

3.3. TGs and PSs promote new bone formation and calcium deposit in SAMP6 mice

To test whether bone formation and deposition of phosphate minerals might be promoted by TGs, PSs, and OSs treatment, tetracyclinecalcein labeling, and Von Kossa staining were performed. The results showed that new bone formation in SAMP6 mice was significantly lower than that in SAMR1 mice, while TGs and PSs significantly promoted the formation of new bone (Fig. 3A). The Von Kossa staining revealed that a lot of calcium was deposited in the TGs and PSs treatment groups (Fig. 3B).

Fig. 3. TGs and PSs promote new bone formation and mineral deposition in SAMP6 mice.

3.4. TGs and PSs ameliorate bone microarchitecture in SAMP6 mice

The bone micro-architecture was detected by micro-CT. Compared to SAMR1 mice, SAMP6 mice had more deteriorated micro-architecture, while the bone condition in mice treated with TGs and PSs for 12 weeks was improved (Fig. 4). We also found that the indexes of BMD, BV/TV, Tb.N, and Tb. Th were reduced and the indexes of Tb. Sp and TMD were increased in SAMP6 mice, compared with those of SAMR1 mice. TGs and PSs significantly increased BMD, BV/TV, Tb. N, Tb. Th and decreased Tb. Sp and TMD compared with SAMP6 mice. However, no significant changes were observed in the OSs group.

3.5. TGs and PSs alter BMP-2, OCN, OPG, and RANKL expressions in SAMP6 mice

We examined the protein expressions of BMP-2, OCN, OPG, and RANKL. TGs and PSs induced the remarkable upregulation of BMP-2, OCN, and OPG, while the expression of RANKL was downregulated (Fig. 5). However, there was no significant difference in the OSs therapy group.

Fig. 4. Effects of TGs and PSs on bone mineral density and bone microarchitecture.

3.6. TGs and PSs alter p-GSK-3β (Ser9) and p-β-catenin expressions in SAMP6 mice

To understand the mechanisms of TGs and PSs promoting osteoblastogenesis, the expressions of p-GSK-3β (Ser9) and p-β-catenin in SAMP6 mice bone tissues were measured using Western blotting (Fig. 6). Results showed that TGs and PSs treatments prominently improved the expression of p-GSK-3β (Ser9) and reduced the expression of p-β-catenin in the femur compared with the SAMP6 group. However, the OSs treatment group showed no significant change.

4. Discussion

In the current work using SAMP6 and SAMR1 mice to find the effective components from C. deserticola against osteoporosis. Three extracts from C. deserticola were used to evaluate the therapeutic effects, as well as the possible mechanisms. In addition, the expressions of RANKL, OPG, OCN, and BMP-2 as well as other bone resorption regulators were also analyzed. Compared with the SAMP6 group, TGs and PSs can improve the histopathological damage of bone, as well as promote the formation of new bone, collagen fiber, chondrocyte, and calcium deposition. Meantime, both can change the biomarkers of bone turnover, and effectively improve the bone microstructure. However, no protective effects were observed for OSs treatment.

Fig. 5. TGs and PSs promote the expressions of BMP-2, OCN, and OPG and reduce the expression of RANKL.

TCMs have been extensively used to relieve symptoms of many diseases such as osteoporosis. alleviate various symptoms of diseases including osteoporosis. Numerous anti-osteoporotic bioactive compounds have been identified from dozens of natural Chinese medicinal herbs that are usually used to tonify kidneys as well as preserve kidney essence (Xu et al., 2017; Liu et al., 2018). C. deserticola possesses a relatively high safety and a wide range of therapeutic functions for the treatment of kidney deficiency. Many research studies have discovered the therapeutic effects of the extracts of C. deserticola on osteoporosis (Li et al., 2012; Liang et al., 2013; Song et al., 2018).

Bone-forming osteoblasts and bone-resorbing osteoclasts, which were from the differentiation of multipotential mesenchymal stem cells (MSCs), are terminally differentiated cells with short lives (Teitelbaum and Ross, 2003). They both need to be continuously replaced with new ones originating from stem cells (Long, 2011). Wnt/β-catenin signaling pathway, which plays a vital role in the differentiation of bone tissues, stimulates osteoblast production by promoting the orientation and differentiation of multipotential MSCs into osteoblasts (Rodda and McMahon, 2006). In addition, Wnts prevent the apoptosis of mature osteoblasts and thereby prolong their lifespan by both β-catenin-dependent and independent pathways (Almeida et al., 2005). Therefore, the Wnt/β-catenin signaling pathway plays an important role in clarifying the pathogenesis of osteoporosis. In the present experiment, SAMP6 mice were used to examine the effectiveness of anti-osteoporotic from different C. deserticola extracts. TGs and PSs significantly down-regulated the levels of RANKL and p-β-catenin and up-regulated the expressions BMP-2, OCN, OPG, and p-GSK-3β. In summary, the therapeutic effect of TGs and PSs on SAMP6 mice was mainly through activating the Wnt/β-catenin signaling pathway.

As excessive absorption of osteoclasts is an important cause of osteoporosis, the factors linked to the activation and differentiation of osteoclasts may be considered as important aims to prevent bone loss (Takatsuna et al., 2005). In our study, the expression of RANKL was prominently down-regulated, at the same time the level of OPG could be up-regulated by TGs and PSs. It has been well documented that the process of bone formation and remodeling of osteoblast cell differentiation is characterized mainly by increased expressions of BMP-2 and OPN (Canalis, 2009). In this study, we found that TGs and PSs of C. deserticola increased BMP-2 and OPG expressions and enhanced bone mineralization. Therefore, TGs and PSs mediate bone formation by upregulating BMP-2 and OPN and downgulating RANKL.

Bone formation markers reflect the activity of bone-building cells, the same bone resorption markers reflect the activity of osteoclasts. The microarchitecture deteriorates as a result of altered bone turnover markers. In our study, bone resorption markers (S-CTX, TRACP, U-CTX, D-Pyr, U-NTX, Pyr) were significantly reduced in the TGs and PSs groups, in the reverse, the bone formation markers (BGP, BALP, P1NP, PICP, ALP) remarkably increase. Hence one can see that TGs and PSs of C. deserticola promote the reconstruction of osteoporotic bone.

Wnt/β-catenin pathway stimulates the expression of osteoblast differentiation markers and mineralization, at the same time it activates the expression of the master osteogenic factor BMP-2 in osteoblasts (Zhang et al., 2013). Additionally, β-catenin increases the expression of OPG in the osteoblast, which indirectly represses osteoclast differentiation by inhibiting bone resorption (Baron and Kneissel, 2013). Our present study shows that TGs and PSs significantly up-regulate p-GSK-3β and down-regulate the level of p-β-catenin. These results support the conclusion that the function of TGs and PSs on anti-osteoporotic is regulated through activating the Wnt/β-catenin signaling pathway.

Our previous HPLC analysis showed that five phenylethanoid glycosides including echinacoside, cistanoside A, acteoside, isoacteoside, and 2′ -acetylacteoside were the major components in TGs (Li et al., 2019; Shi et al., 2019). The structures of the above five phenylethanoid glycosides were all rich in phenolic hydroxyl groups, which are responsible for the antioxidant property of C. deserticola (Chen et al., 2016). It has been reported that improving the antioxidant system can prevent bone loss, thus these phenylethanoid glycosides could be the potential active components of C. deserticola responsible for the anti-osteoporotic activity. There are reports that echinacoside and acteoside can ameliorate the typical pathological features of osteoporosis, such as, improving bone quality and total femur BMD, promoting bone formation, and inhibition of bone resorption (Chen et al., 2020). Additionally, echinacoside has a striking anti-osteoporotic effect (Li et al., 2013). Previous studies showed that cistanoside A could promote bone formation and prevent bone resorption through inhibiting NF-κB and activating PI3K/Akt pathways (Xu et al., 2017). The anti-osteoporotic effect of C. deserticola polysaccharides has not been reported. However, other studies have demonstrated that astragalus polysaccharides could suppress the expression of RANKL, increase serum OPG level and finally block osteoclast differentiation (Huo and Sun, 2016; Hwang et al., 2018). OSs of C. deserticola is mainly composed of mannitol, betaine, fructose, glucose, and sucrose (Shi et al., 2019), which have no report for the anti-osteoporotic activity, therefore, OSs has no therapeutic effect on osteoporosis. In summary, TGs and PSs are the active components in C. deserticola for the effect of anti-osteoporosis.

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5. Conclusions

In conclusion, TGs and PSs from C. deserticola can enhance bone formation in SAMP6 mice through regulating the Wnt/β-catenin signaling pathway but not OSs. In the future, TGs and PSs may become promising bone-protective therapeutic agents for osteoporosis.