PART 1 Anti-Osteoporotic Activity Of An Edible Traditional Chinese Medicine Cistanche Deserticola On Bone Metabolism Of Ovariectomized Rats Through RANKL/RANK/TRAF6- Mediated Signaling Pathways

Bo Zhang 1†, Ling-Ling Yang 1†, Shu-Qin Ding 1†, Jing-Jing Liu 1, Yan-Hong Dong 1,

Yan-Ting Li 1, Nan Li 1, Xiao-Jun Zhao 1, Chang-Ling Hu 2, Yiping Jiang 3* and Xue-Qin Ma 1*

1 Key Laboratory of Hui Ethnic Medicine Modernization, Department of Pharmaceutical Analysis, Ministry of Education, School of Pharmacy, Ningxia Medical University, Yinchuan, China, 2 Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest Technologies, North Carolina Research Campus, North Caroline A&T State University, Greensboro, NC, United States, 3 Department of Pharmacognosy, The Second Military Medical University, Shanghai, China

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Given the limitations of existing therapeutic agents for the treatment of postmenopausal osteoporosis, there still remains a need for more options with both efficacy and fewer adverse effects. Cistanche deserticola Y. C. Ma is known as a popular tonic herb traditionally used to treat deficiency of kidney energy including muscle weakness in minority areas of Asian counties. Based on the theory of “kidney dominate bone,” an ovariectomized (OVX) rat model of postmenopausal osteoporosis was used to evaluate the therapeutic effect of C. deserticola extract (CDE) on bone loss. Forty-eight female Sprague-Dawley rats, aged about 12 weeks, were randomly assigned into six groups including the sham group orally administrated with 0.5% carboxymethyl cellulose sodium (CMC-Na) (sham), the positive group treated with 1 mg/kg of estradiol valerate (EV), low, moderate, and high dosage groups orally administrated with 200, 400, and 800 mg/ kg/day of CDE, respectively. After 3 months of continuous intervention, CDE exhibited significant anti-osteoporotic activity evidenced by the enhanced total bone mineral density, ameliorated bone microarchitecture; increased alkaline phosphatase activity; decreased deoxypyridinoline, cathepsin K, tartrate-resistant acid phosphatase, and malondialdehyde levels; whereas the body, uterus, and vagina weights in OVX rats were not influenced by CDE intervention. In addition, a seemingly contradictory phenomenon on levels of calcium and phosphorus between OVX and sham rats was observed and elucidated. Mechanistically, CDE significantly down-regulated the levels of TRAF6, RANKL, RANK, NF-κB, IKKβ, NFAT2, and up-regulated the phosphatidylinositol 3-kinase (PI3K), AKT, osteoprotegerin, and c-Fos expressions, which implied CDE could suppress RANKL/ RANK-induced activation of downstream NF-κB and PI3K/AKT pathways, and ultimately, preventing the activity of the key osteoclastogenic proteins NFAT2 and c-Fos. All of the data suggested CDE possessed potential anti-osteoporotic activity and this effect was, at least in part, involved in modulation of RANKL/RANK/TRAF6-mediated NF-κB and PI3K/AKT signaling as well as c-Fos and NFAT2 levels. Therefore, CDE may represent a useful promising remedy candidate for the treatment of postmenopausal osteoporosis.

Keywords: Cistanche deserticola, antiosteoporotic, TRAF6, RANKL, RANK

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

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INTRODUCTION

Since osteoporosis has not been regarded as part of normal aging, extensive advances in the osteoporosis field have been obtained. Today, osteoporosis has become a major health hazard afflicting more than 200 million populations all over the world with the attendant 10 billion dollars were spent every year on these individuals (Ma et al., 2015). Osteoporosis, including postmenopausal and senile osteoporosis, was characterized by micro-architectural deterioration and low bone mass density (BMD) as a result of an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, respectively (Kanis et al., 2015). In consideration of the limitations of current therapeutic options and some unwanted adverse effects of synthetic agents (Ye et al., 2015) for the treatment of osteoporosis disease, it is necessary for alternatives with both efficacy and minimal side effects (Tan et al., 2017). As a remarkable complementary and alternative therapy way for the treatment of various ailments including postmenopausal osteoporosis, the natural medicinal herb especially traditional Chinese medicines (TCM) were gradually recognized and highly warranted (Czerwinska and Melzig, 2018; Ma et al., 2018). Two thousand years ago, Cistanche deserticola Y. C. Ma was found beneficial for human health and recorded in the Chinese classical medicinal book named Shen Nong Herbal (Anonymous, 1981). Now, it is an important TCM officially recorded in the Chinese pharmacopeia (Chinese and Pharmacopoeia, 2015) and also as a famous tonic herb that has been used in Asian counties including China and Japan for centuries of years (Wong et al., 2006). C. deserticola was found to possess a favorable safety profile (Liao et al., 2018) and broad medicinal functions: in folk, it was widely used to cook with kinds of sorts of herbal cuisine for the treatment of kidney deficiency, muscle debility, and lumbar weakness; nowadays, increasing attentions have been paid for its various pharmacological functions including anti-inflammatory, anti-fatigue, antitumor, antioxidant activity, enhancing immunity, and so on (Gu et al., 2016; Fu et al., 2018). It is also well known as “ginseng of the desert” in China due to its excellent clinical medical curative effect. Both the traditional and modern application of C. deserticola made this herb popular in both medicine and the health food industry, thus having been developed into medicinal and nutritional liquid approved by the State Food and Drug Administration. Given C. deserticola was usually employed to deal with kidney deficiency in Chinese folk medicine, which implied this edible medicinal herb can be regarded as a promising alternative agent to intervene osteoporosis based on the theory of “kidney dominate bone” (Wang et al., 2016). Published data had proved the anti-osteoporotic effect of C. deserticola extracts both in vivo and in vitro (Liang et al., 2011; Li et al., 2012; Liang et al., 2013; Xu et al., 2017; Song et al., 2018), and several isolated compounds including echinacoside (Li et al., 2013), acteoside (Lee et al., 2013), and cistanoside A (Xu et al., 2017) which also had been reported processing anti-osteoporotic activities; and other compounds like 2’-acetylacteoside was confirmed possessed antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, immune-enhancing (Li et al., 2016), and anti-aging potentials (Peng et al., 2016). To date, dozens of bioactive phenylethanoid glycosides have been identified from the Cistanche herb (Wang et al., 2012), echinacoside, and acteoside are the main compounds existing in most of the Cistanche species with the contents were 1.83–41.49 and 0.27–8.28 mg/ kg, respectively; whereas the other phenylethanoid glycosides including 2’-acetylacteoside, 6’-acetylacteoside, cistanoside A, cistanoside C, and isoacteoside was 1.56–3.16 mg/g, 0.49–1.66 mg/kg, 1.41–10.11 mg/kg, 0.33–2.24 mg/kg, and 0.08–5.00 mg/ kg, respectively (Dong et al., 2018). Based on the above published data, we found that the underlying anti-osteoporotic molecular mechanisms of C. deserticola especially the upstream signaling was remain unclear; and the compound itself can not represent the effect of the herb which contained the synergy property contributed by different types of components; furthermore, the specific targets of signaling pathways were totally different between different components. The current work aimed at investigating the potential protective effect of C. deserticola against ovariectomized (OVX) rats in vivo with emphasis on the regulation of tumor necrosis factor receptor-associated factor 6 (TRAF6), receptor activator of nuclear factor kappa B ligand (RANKL), RANK expressions and RANKL/RANK/TRAF6- induced nuclear factor kappa B (NF-κB), and phosphoinositide 3-kinase (PI3K)/protein kinase (AKT) signalings. Bone remodeling, including reconstruction and repairment, is an intricate process governed by the balance between osteoblastic bone formation and osteoclastic bone resorption, and increased bone resorption mainly caused by enhanced osteoclastogenesis usually led to osteoporosis even bone fracture. Most of the cell signals, hormones, and growth factors that were essential for osteoclast differentiation and function were controlled by RANK and its two ligands, RANKL and osteoprotegerin (OPG) (Lacey et al., 1998). And recent findings further discovered TRAF6 was a key regulatory factor in RANKL/RANK triggered signaling cascades (Tan et al., 2017). By recruitment of TRAF6, RANKL bound to its receptor RANK, and then the downstream signaling cascades including NF-κB and PI3K/AKT were triggered, then the key osteoclastogenic proteins of c-Fos and nuclear factor of activated T cells c2 (NFAT2) (Takayanagi, 2007) were up-regulated, and finally, the differentiation of osteoclast was initiated. In the present study, the underlying anti-osteoporotic mechanism of CDE on RANKL/RANK/TRAF6-mediated bone resorption is investigated and discussed.

MATERIALS AND METHODS

Animals, Cells, Antibodies, and Reagents

Female adult Sprague-Dawley rats (Ningxia Medical University, Yinchuan, China); RAW264.7 cells (Zhong Qiao Xin Zhou Biotechnology Co., Ltd., Shanghai, China); macrophage colonystimulating factor (M-CSF) and RANKL (PerroTech, Inc. USA); estradiol valerate [estradiol valerate (EV), Delpharm Lille SAS, Paris, France]; cathepsin K (CK) ELISA kit (BioVision, American); deoxypyridinoline (DPD) and bone gla-protein (BGP) cross-links ELISA kits (Xinyu Biological Engineering Co., Ltd, Shanghai, China); malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) reagent kits (Jianchen Biological Engineering, Nanjing, China); penicillin and streptomycin (Gibco, USA); polyvinylidene fluoride (PVDF) membrane (Millipore Life Sciences, USA); primary antibodies including GAPDH (18AF0401), β-actin (17AV0411), IκB kinase β (IKKβ) (AD01134589), TRAF6 (2), RANKL (GR3193842-5), RANK (AA02113656), NFκBIA (AH04138226), PI3K (AC09021266), OPG (AG06292776), c-Fos (AG12059411), AKT (AF05173234), NFAT2 (AO11015648), and secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G, 134658) were offered by ZSGB-BIO (Beijing, China); Dulbecco’s modified eagle’s medium (DMEM), bicinchoninic acid protein quantization assay kit, fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (PBS) were purchased from Hylcone (Logan, UT, USA); echinacoside [highperformance liquid chromatography (HPLC)≥98%, 11167-200503, National Institutes for Food and Drug Control, China]; acteoside (HPLC≥98%, AB0497, ALFA, China); isoacteoside (HPLC≥98%, AB17021202, ALFA, China); 2’-acetyl acteoside (HPLC≥98%, PRF7081243, Chengdu Pu Rui Fa Technology, Co., Ltd. China); cistanoside A, cistanoside C, and 6’-acetyl acteoside (HPLC≥95%, isolated and purified in our lab) (Xu et al., 2017).

Plant Materials and Preparation

Dried stems of C. deserticola Y. C. Ma were purchased from Yongning County, Ningxia province in September 2015, the specific coordinates of plant picking were (106.026597, 38.262816). The herb was identified by Prof. Ling Dong (department of pharmacognosy, Ningxia Medical University), with a voucher specimen (#20150901) was available in the herbarium of pharmaceutical analysis. A total of 10.0 kg dried and powdered C. deserticola was extracted by using the heat reflux method, the detailed procedure was as follows: the solvent was 75% ethanol, the ratio of C. deserticola material to solvent was 1:8, the reflux time has lasted 2 h, and the materials were reflux for three times; then all of the filtrates were mixed together and concentrated under reduced pressure condition to afford 6.5 kg C. deserticola ethanol extracts (CDE). For the animal experiment, 0.5% carboxymethyl cellulose sodium (CMC-Na) was used to suspend CDE, and the orally administered dosage was set as 200, 400, and 800 mg/kg/day, with a volume of 1 ml/100 g body weight of each animal; for western blot analysis, dimethyl sulfoxide was employed to dissolve CDE and then diluted with DMEM to acquire the final concentration of 0.01, 0.1 1 mg/ml; for HPLC analysis, methanol was applied to dissolve CDE, followed by filtered and diluted with initial mobile phase to get the proper concentration.

High-Performance Liquid Chromatography Analysis of Cistanche deserticola Extract

The major chemical constituents of CDE were identified by using HPLC with the corresponding reference substances. The chromatography conditions were set as follows: Agilent 1220 RP-HPLC instrument with C18 column (4.6 i.d. × 250 mm; 5 μm), a gradient elution mobile phase mainly contained water and acetonitrile was used to obtain well-separated peaks: solvent A (water containing 0.5% acetic acid) and B (acetonitrile): 0–4 min, 85–83% A; 4–10 min, 83–80% A; 10–30 min, 80–75% A; 30–40 min, 75–70% A. The sample injection volume was 5 μl, the temperature was controlled at 30°C, and UV detection was performed at 333 nm (Dong et al., 2018). Reference substances including echinacoside, acteoside, isoacteoside, cistanoside A, cistanoside C, 2’-acetyl acteoside, and 6’-acetyl acteoside were used to identify the corresponding peak.

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Animal Experiments

For the animal experiments, all procedures were performed in strict accordance with the protocol approved by the institutional animal care and use committee guide of Ningxia Medical University (NXMU-20130311). Forty-eight adult female Sprague-Dawley rats weighing about 237 ± 25 g were obtained (Ningxia Medical University, Yinchuan, China) and maintained for 1 week with a standard pellet diet and tap water under an environmentally controlled condition. Then, each rat underwent either anesthesia (chloral hydrate, 100 mg/kg, i.p.) only, sham ovariectomized (sham), or two ovaries were both removed and then randomly divided into five groups: model group (OVX) was orally administrated with 0.5% CMC-Na; positive group (EV) with 1 mg/kg/day of estradiol valerate; low (CDEL), moderate (CDEM), and high (CDEH) dosage groups with 200, 400, and 800 mg/kg/ day of CDE, respectively. The experiments have lasted for 12 weeks; the bodyweight of each rat was measured biweekly with the administration dose adjusted accordingly. After the last day of the intervention, 24 h urine, serum, femora, tibia, and uterus were collected and stored at −80°C for further different analysis.

Biochemical Parameters

The levels of serum SOD, GSH, MDA, cathepsin K, BGP, and urinary DPD were determined by the corresponding reagent kits, while the activity of tartrate-resistant acid phosphatase (TRAP) was evaluated according to a reference (Jiao et al., 2009). The activity of alkaline phosphatase (ALP) and the levels of Ca and P were estimated by employing an automatic analyzer machine (Ciba-Corning 550, USA).

Bone Mineral Density and Micro-Computed Tomography Analysis

A dual-energy X-ray absorptiometry machine (Lunar, USA) was employed to estimate the bone mineral density (BMD, g/ cm2) of the right femur of each rat with the scan mode was set as a small animal. Then, the same femur was used to evaluate the 3D image of trabecular bone microarchitecture by applying a micro-CT scanner apparatus (GE, American), the region of interest (ROI) was chosen by setting the same coordinates in the growth plate of the femur of each sample, the microarchitecture parameters including bone mineral content (BMC), tissue mineral content (TMC), tissue mineral density (TMD), trabecular separation (Tb. Sp), trabecular number (Tb. N), and trabecular thickness (Tb. Th) were obtained by analyzing the ROI.

Western Blot Determination

Osteoclasts were induced by using RAW 264.7 cells added with MCSF (25 ng/ml) and RANKL (20 ng/ml) (Yang et al., 2019). After 6 days of lasted stimulated, the matured osteoclast cells which were identified for TRAP activity were treated with or without CDE (0.01, 0.1, and 1 mg/ml, respectively) for 48 h, then the cells were lysed by lysis buffer, and the supernatants were centrifuged and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membrane was blocked with 5% non-fat milk at ambient temperature for 1 h and then incubated with primaries antibodies against TRAF6 (1:400), RANKL (1:400), RANK (1:400), IKKβ (1:400), NFκBIA (1:400), OPG (1:400), PI3K (1:400), AKT (1:400), c-Fos (1:400), NFAT2 (1:400), β-actin, and GAPDH (1:1,000). The same membranes were stripped and probed again with corresponding antibodies, detected by the Image Lab Software at the end. Each of the experiments was repeated three times, with β-actin or GAPDH as an internal control.

Statistical Analysis

The data of our experiments, described as the mean ± SD, were analyzed by using a one-way analysis of variance followed by Dennett’s test (SPSS 22.0 software, SPSS, USA), p < 0.05 was considered as statistically significant.

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RESULTS Main Chemical Constituents of Cistanches deserticola

Extract By using corresponding reference substances, seven mainly phenylethanoid glycosides compounds including echinacoside, cistanoside A, acteoside, isoacteoside, cistanoside C, 2’-acetyl acteoside, and 6’-acetyl acteoside were identified existing in this extract, their corresponding chromatography peaks and structures were shown in Figure 1.

Effects of Cistanches deserticola Extract on the Body, Uterine, and Vagina Weights

There was no significant difference in the initial mean body weight before surgery. However, 3 months after the ovariectomized operation, the body weights of the rats in the OVX model group were significantly increased by 35.7%, whereas the uterine and vagina wet weights were sharply declined by 91.2 and 61.8%, respectively, as compared to the sham rats (p < 0.001). CDE, with the dosage of 200–800 mg/kg/day, showed no influence on these noticeably increased body weights and decreased uterine and vagina wet weights, as Figure 2 showed. However, totally different to CDE, 1 mg/kg/day of EV exhibited a significant estrogenic effect, of which the gained body weight and decreased uterine and vagina weights in OVX rats were significantly reversed by EV supplemented.

Effects of Cistanches deserticola Extract on Bone Mineral Density and Bone Microarchitecture

An intuitively declining tendency was observed in the OVX group with BMD was 0.158 ± 0.016 g/cm2 as compared to the sham rats with the BMD was 0.180 ± 0.010 g/cm2 (p < 0.001), which implied the BMD of the rats in OVX model group decreased about 12.2% after 12 weeks of the ovariectomized surgery as compared to the sham rats. As Figure 3 showed, all CDE treated rats showed an increased BMD by 15.1% (p < 0.001), 8.1% (p < 0.05), and 9.2% (p < 0.05), respectively, as compared to OVX model group. The 3D image and the corresponding quantitative results of the trabecular bone microarchitecture of rats were shown in Figure 4. Consistent with the results of BMD, an obvious reduction of the trabecular area was obtained in the OVX model group as compared with the sham rats. However, the deterioration of trabecular bone was partly improved by CDE intervention, except TMD and Tb. Th, the other trabecular bone parameters include BMC, TMC, and Tb. N were significantly increased and Tb. Sp was notably decreased after CDE treatment.

Effects of Cistanches deserticola Extract on Urine and Serum Biochemical Parameters

Twelve weeks after the ovariectomized operation, a declining but non-statistically trend of urinary excretion level of Ca was observed in the OVX model group, as Figure 5 showed, whereas the level of urine P in rats of the OVX model group was declined about three times less than the sham rats (p < 0.001). After treatment with CDE (200–400 mg/kg/day) for 12 weeks, the reduction of serum Ca and P were significantly prevented (p < 0.05), and the decreased urinary excretion level of P was also inhibited in all the CDE treated groups (p < 0.01)

Effects of Cistanches deserticola Extract on Bone Formation and Resorption Markers

Concerning the bone formation markers of ALP and BGP (Figure 6), the activity of ALP, not BGP, was significantly improved in all three doses of CDE (200–800 mg/kg/day) treated groups as compared to the sham rats. Concerning the bone resorption markers of TRAP, DPD, and cathepsin K (Figure 7), the activities of all the three bone resorption

markers were significantly increased about 20.9~74.8% in the rats of the OVX model group as compared to the sham rats; and expectedly, CDE exhibited great potential in suppressing all of these indices, of which the activity of cathepsin K was decreased by 49.9~66.7% (p < 0.001), the level of DPD was declined by 22.9~39.3% (p < 0.01), and the property of TRAP was prevented by 20.1~27.6% (p < 0.01), respectively, as compared with the rats of OVX model group.

Effects of Cistanches deserticola Extract on Activities of Glutathione and Superoxide Dismutase as Well as Malondialdehyde Content

There were no significant differences in the activities of SOD and GSH between the OVX and sham groups after 3 months of ovariectomized operation, as Figure 8 showed, whereas the content of MDA was obviously enhanced in rats of OVX

model group by 68.2% in comparison to the sham rats, and these increased MDA levels were significantly suppressed by low, moderate, and high dosage of CDE treated groups with the inhibition percentages were 66.1, 44.0, and 62.8%, respectively, as compared with the rats of OVX model group.

Effects of Cistanches deserticola Extract on Protein Expression Levels of TRAF6, RANKL, RANK, NFκBIA, IKKβ, PI3K, AKT, OPG, c-Fos, and NFAT2

As Figure 9 showed, the expressions of TRAF6, RANKL, RANK, IKKβ, NFκBIA, and NFAT2 were significantly down-regulated whereas the levels of PI3K, AKT, OPG, and c-Fos were obviously up-regulated after treatment with CDE as compared to the control. Hypothesized mechanism (Figure 10) by which CDE could down-regulate the levels of RANKL, RANK, and TRAF6 and up-regulate the expression of OPG, thus the binding quantities of RANKL with RANK were reduced; consequently, the downstream pathways including NF-κB signal were suppressed and PI3K/AKT was stimulated, and these signaling cascade together led the activation of c-Fos was promoted and NFAT2 was inhibited, and finally the osteoclastic bone resorption was prevented. DISCUSSION