PART 1 Cistanche Deserticola Polysaccharide Inhibits OVX-induced Bone Loss in Mice And RANKL-induced Osteoclastogenesis

1. Introduction

Osteoporosis—a disease characterized by decreased bone mass, abnormal bone tissue microstructure, increased bone fragility, and fracture (De Martinis, Di Benedetto, Mengoli, & Ginaldi, 2006)— currently affects more than 200 million people worldwide and presents a substantial medical and socioeconomic burden on modern society (Strom et al., 2011). The clinical treatment of osteoporosis mainly focuses on hormone replacement, bisphosphonate, or denosumab therapy. These methods are effective but pose long-term side effects such as the potential risk of breast cancer and atypical femur fractures (Black, Bauer, Schwartz, Cummings, & Rosen, 2012; Rachner, Khosla, & Hof- Bauer, 2011). Hence, there is an urgent need to explore drugs that can not only inhibit osteoporosis but also possess fewer undesirable side effects.

Cistanche deserticola (CD), a tonic and medicinal food widely used in China, known as “desert ginseng”, is the dried succulent stem with scale leaf of C. deserticola YC Ma (Gu, Yang, & Huang, 2016) and has diverse and effective pharmacological activities, such as immune regulation, anti-oxidation, and anti-osteoporosis properties (Hu et al., 2020; Li et al., 2012; Zhang et al., 2014). Previous studies on the active substances of CD on osteoporosis treatment mainly focused on phenylethanoid glycosides (Li, Jiang, & Gu, 2018; Xu, Zhang, Wang, Yao, & Ma, 2017). However, the CD also contains other effective components such as iridoids, lignans, polysaccharides (Wang, Zhang, & Xie, 2012). The clinical application of traditional Chinese medicine (TCM) is mainly water decocting. Polysaccharides are water-soluble components and are most likely to be the main pharmacodynamic components of TCM. Polysaccharides extracted from TCM were wildly reported to have an anti-osteoporosis effect and a few undesirable side effects, which have been generally used in clinics (e.g., polysaccharides extracted from Epimedium brevicornum (Zheng, He, Wu, Cai, & Wei, 2020), Achyranthes bidentata (Zhang, Zhang, Zhang, Wang, & Yan, 2018), and Morinda officinalis (Yan et al., 2019)). Therefore, we hypothesized that Cistanche deserticola polysaccharide (CDP) extracted from CD may be a potentially safe drug for the treatment of osteoporosis.

Typically, bone resorption and bone formation maintain a dynamic balance during bone remodeling in coordination with several types of cells, including osteoclasts, osteoblasts, bone lining cells, and osteocytes (Kular, Tickner, Chim, & Xu, 2012). A break in this balance can result in a variety of bone metabolic diseases, such as osteoporosis (Zhu et al., 2018) and osteosclerosis (Ihde et al., 2011). Osteoclasts, as end- differentiated cells derived from the mononuclear/macrophage lineage, are the only cells with bone resorption function (Teitelbaum, 2000). There are two key cytokines that participate in the osteoclast differentiation and maturation (Koga et al., 2004): macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). M-CSF mediates the differentiation of hematopoietic stem cells into osteoclast progenitors and promotes the differentiation of osteoclasts by upregulating the expression of the RANK receptor (Takayanagi, 2007). The binding of RANKL and RANK on the osteoclast cell surface results in the following: (1) recruitment of signaling adapter molecules such as TNF receptor-associated factor 6 (TRAF6); (2) activation of multiple downstream targets, including NF- κB and mitogen-activated protein kinases (MAPKs); and (3) upregulation of the expression levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) (Liu et al., 2019; Yamashita et al., 2007). The activation of these signaling pathways directly regulates the expression of osteoclast genes, including acid phosphatase 5 (Acp5) [encoding tartrate-resistant acid phosphatase (TRAcP)], matrix metalloproteinase 9 (MMP9), and cathepsin K (CTSK) (Boyle, Simonet, & Lacey, 2003). Therefore, the inhibition of RANKL-induced osteoclast differentiation-related signaling pathways is a potential therapeutic method for osteoporosis.

In this study, we aimed to determine the effects of CDP treatment on the ovariectomized (OVX)-induced osteoporosis mouse model in vivo and on RANKL-induced osteoclast activity in vitro, with a focus on the expression of osteoclast-specific genes and the activation of NFATc1 and the NF-κB and MAPKs signaling pathways. Our findings may provide new insights into the potential of CDP as a safe and effective drug for treating osteoporosis.

For more information please contact: Joanna.jia@wecistanche.com

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2. Materials and methods

2.1. Chemicals and sample collection

CD (no. 180801) was purchased from Anhui Jishun Traditional Chinese Medicine Co., Ltd. (Anhui, China) and identified by Professor Haibo Huang from the School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China. Estradiol valerate tablets were purchased from Bayer (Leverkusen, North-Rhine- Westphalia, Germany). Alpha-modified minimal essential medium

(α-MEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin/streptomycin and the TRAcP staining kit were acquired from Solarbio (Beijing, China). Recombinant mouse M-CSF and recombinant mouse RANKL were pro- cured of R&D Systems (Minneapolis, MN, USA). Hydroxyapatite-coated plates were purchased from Corning Life Sciences (St. Lowell, MA, USA). The primary antibodies for NFATc1 and CTSK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); IκB-α, p65, P-p65, p38, P-p38, ERK1/2, P-ERK1/2, JNK, and P-JNK (Cell Signaling Technology, Dan- vers, MA, USA); and β-actin (CWBIO, Beijing, China) was also obtained. The other reagents used in this study were of analytical grade, and the water was purified by the Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.2. CDP extraction

The CD was ground to a fine powder and mixed with petroleum ether thrice to defeat it. The solid residue was collected by filtration and then dried at room temperature. The polysaccharides were extracted from the pre-treated samples thrice with water for 2 h, concentrated, precipitated by the addition of anhydrous ethanol to a final concentration of 80% (v/ v), and stored at 4 ◦C for 24 h. The precipitates were collected by centrifugation at 3000 rpm for 20 min, dissolved in distilled water, and purified (removal of free proteins) using the Sevag method (Zhao et al., 2019). The recovered polysaccharides were dialyzed, dried, and stored until further use. Furthermore, the chemical composition results showed that the total sugar, uronic acid, sulfate, and protein contents of CDP were 67.63 0.98%, 21.05 0.49%, 1.90 0.24%, and 8.81 0.36%.

The monosaccharide composition and Fourier transform infrared analysis of CDP were shown in Supplementary Fig. 1 and 2.

2.3. OVX-induced osteoporosis mouse model

All animal experiments were approved by the Animal Ethics Committee of the Guangzhou University of Chinese Medicine (approved SYXK 2019-0202). Thirty 6-week-old female C57BL/6 mice were supplied by the Experiment Animal Center of the Guangzhou University of Chinese Medicine. After acclimatization for 1 week, one group of mice was given a sham operation (Sham group), while the remaining mice were subjected to an ovariectomy operation (OVX group). After surgery, the mice were allowed to recover for 1-week. Then, mice successfully modeled were randomly divided into 5 groups, with 6 mice per group:

(1) Sham group: treated with distilled water, (2) OVX group: treated with distilled water, (3) OVX E2 group (E2): treated with 0.13 mg/kg estradiol valerate tablets, (4) OVX CDP low dose group (CDP-L): treated with 300 mg/kg of CDP, (5) OVX CDP high dose group (CDP- H): treated with 600 mg/kg of CDP. Distilled water, E2, or CDP was administered by gavage once daily. All mice were sacrificed after the 12- week treatment period (Fig. 1A). The uteruses were collected and weighed, and the left tibias were collected for subsequent examinations. Whole blood samples were collected and centrifuged at 5000 rpm for

15 min at 4 ◦C. The serum supernatants were aspirated and stored at

—80 ◦C until further analysis.

2.4. Micro-computed tomography (micro-CT) analysis and bone histomorphometry

The left tibias were fixed with 4% paraformaldehyde for 48 h and subsequently placed in centrifuge tubes containing normal saline. The fixed samples were scanned with Skyscan 1172 micro-CT instrument (Bruker micro-CT, Skyscan, Kontich, Belgium) using the following settings: voltage, 80 kV; source current, 100 μA; Al, 0.5 mm filter; pixel

size, 9.76 μm; and rotation step, 0.6◦. Several parameters of the

trabecular bone, including bone mineral density (BMD), bone volume fraction (BV/TV), bone surface per total volume (BS/TV), trabecular number (Tb. N), and trabecular spacing (Tb. Sp) were measured using CT- Analyser® software (Bruker micro-CT, Skyscan). Three-dimensional images were generated using CTVol software (Bruker micro-CT. Skyscan).

Following micro-CT analysis, the left tibias were decalcified in 14% EDTA solution and embedded into paraffin for sectioning. The samples were sliced into 5 µm sections using a microtome prior to the hematoxylin and eosin (H&E) and TRAcP staining experiments. The stained sections were examined and documented using Olympus CX 31 micro- scope (Olympus Optical Co., Ltd, Tokyo, Japan).

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2.5. Serum analysis

The calcium (Ca) and phosphorus (P) concentrations were determined according to the kit instructions designed by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRAcP-5b and RANKL levels in the serum were analyzed using TRAcP-5b ELISA kit (CUSABIO, Wuhan, China) and RANKL ELISA kit (Cloud-CloneCorp., Wuhan, China), respectively.

2.6. In vitro osteoclastogenesis assay

BMMs were isolated from the tibia and femur of 6-week-old female C57BL/6 mice. The isolated cells were cultured in α-MEM medium containing 25 ng/mL M-CSF, 10% FBS, and 1% penicillin/streptomycin. Upon reaching confluence, the BMMs (1 104 cells/well) were seeded in 96-well plates and treated with CDP in the presence of 50 ng/mL RANKL. The medium and CDP were replaced every 2 days. After 7 days, the cells were fixed with 4% paraformaldehyde and stained for the presence of TRAcP. The number of osteoclasts, defined as TRAcP- positive cells with three or more nuclei, was counted and documented using a light microscope.

2.7. Cell proliferation assay

The BMMs (1 × 104 cells/well) were seeded in a 96-well plate and incubated overnight. Following this, the cells were treated with different concentrations of CDP (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µg/mL) for 48 h. The cell confluence was detected and analyzed using IncuCyte ZOOM® (Essen BioScience, Ann Arbor, MI, USA), a long-term, real-time dynamic live cell imaging system.

2.8. Hydroxyapatite resorption assay

The BMMs (1 104 cells/well) were seeded in a hydroxyapatite-coated plate, treated with CDP at the indicated concentrations (0, 5, and 10 µg/mL), and cultured in complete α-MEM containing 25 ng/mL M-CSF and 50 ng/mL RANKL. After 7 days, the cells were washed with a 10% bleach solution to remove the cell components. The images of hydroxyapatite resorption areas were captured using IncuCyte ZOOM® and analyzed using ImageJ software (NIH, Bethesda, MD, USA) (Abramoff, Magelhaes, & Ram, 2003).

2.9. RNA isolation and real-time reverse transcription-quantitative PCR (RT-qPCR) analysis

The BMMs (1 105 cells/well) were seeded in a 6-well plate and stimulated with RANKL and M-CSF in the presence of CDP at different concentrations (0, 5, and 10 µg/mL) for 5 days. Total RNA was isolated from the cells or bone tissue of mice using TRIzol reagent (Sigma- Aldrich). Complementary DNA was synthesized from 2 µg total RNA using a reverse transcriptase kit (TransGen Biotech, Beijing, China). The RT-qPCR reactions were prepared using PerfectStart™ Green qPCR SuperMix (TransGen Biotech) and detected by ABI 7500 system (Applied

Biosystems, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cycling parameters for PCR were set as follows: 95 ◦C for 5 min, followed by 40 cycles of 95 ◦C for 15 s, and 60 ◦C for 30 s. The specific primers used are shown in Table 1, and the quantity of each target gene was normalized to GAPDH (internal control).

2.10. Western blot analysis

The BMMs (5 105 cells/well) were seeded in a 6-well plate. For short time-course experiments, the cells were pre-incubated with the different concentrations of CDP (0, 5, and 10 µg/mL) for 1 h, and then treated with 50 ng/mL RANKL for 30 min. For a long time course, the cells were stimulated with 50 ng/mL RANKL on days 3, 5, and 7 in the presence of CDP (0, 5, and 10 µg/mL). Total proteins were extracted using the RIPA lysis buffer (CWBIO). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, incubated with primary

antibodies overnight at 4 ◦C, and then with the corresponding secondary

antibodies for 1.5 h. The membranes were developed using ECL reagents (Millipore Corp., Billerica, MA, USA), and images were taken using Tanon 5200 Chemiluminescence Imaging System (Tanon Science and Technology, Shanghai, China).

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2.11. Detection of NFATc1 translocation using immunofluorescence test

The BMMs were seeded onto 12-well coverslips, stimulated with RANKL and M-CSF, and treated with 10 µg/mL CDP for 5 days. The cells were fixed with 4% paraformaldehyde for 15 min, washed thrice with PBS, and permeabilized with 0.1% Triton X-100 for 10 min. The cells were mixed with 10% goat serum (CWBIO) and incubated for 2 h. Subsequently, the cells were incubated with the primary antibody overnight at 4 ◦C and then with Alexa Fluor 488 goat anti-mouse secondary antibody (Abcam, Cambridge, MA, USA) in the dark for 1 h. The coverslips were washed with PBS, mounted in Prolong Gold Antifade Reagent with 4′, 6-diamidino-2-phenyl in-dole (DAPI) (Solar), and

inspected using Zeiss LSM 800 with Airyscan confocal microscope (Carl Zeiss, Oberkochen, Germany).

2.12. Statistical analysis

All experimental data were analyzed using SPSS 25.0 statistical software (IBM Corporation, Armonk, NY, USA). The data for animal and cell studies are expressed as means ± SEM and means ± SD, respectively. Results are using one-way analysis of variance or Student’s t-test. Values of p < 0.05 were considered statistically significant.

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