Beneficial Effects Of Total Phenylethanoid Glycoside Fraction Isolated From Cistanche Deserticola On Bone Microstructure in Ovariectomized Rats Ⅰ

The present study was designed to estimate the antiosteoporotic activity of total phenylethanoid glycoside fraction isolated from C. deserticola (CDP) on rats induced by ovariectomy (OVX) as well as the related mechanisms. After 3 months of oral administration, the decreased bone mineral density, serum Ca, and P in OVX rats were recovered and the deteriorated trabecular bone microarchitecture was partly improved by CDP (60, 120, and 240 mg/kg) intervention, the activities of bone resorption markers were downregulated, and the bioactive of the bone formation index was upregulated; meanwhile, the content of MDA was declined, and GSH was increased by CDP treatment. Compositionally, 8 phenylethanoid glycoside compounds were identified in CDP, with the total contents quantified as 50.3% using the HPLC method. Mechanistically, CDP declined the levels of TRAF6, RANKL, and RANK, thus suppressing RANKL/RANK/TRAF6-induced activation of downstream NF-κB and PI3K/AKT signaling pathways and ultimately preventing activities of the key osteoclastogenic proteins of NFAT2 and c-Fos. All of the above data implied that CDP exhibited beneficial effects on bone microstructure in ovariectomized rats, and these effects may be related to the NF-κB and PI3K/AKT signaling pathways which were triggered by the binding of RANKL, RANK, and TRAF6.

 NATURAL ORGANIC CISTANCHE FOR  BONE GROWTH

1. Introduction

Postmenopausal osteoporosis, where 1 in 3 women older than 50 years will suffer, is becoming a main health hazard afflicting more than 200 million women all over the world [1]. At menopause, the sharp decline of the estrogen level usually leads to an exceeded bone resorption caused by enhanced osteoclastogenesis; then, the balance between osteoblast-induced bone formation and osteoclast-induced bone resorption is disrupted, and the accelerated bone resorption finally caused osteoporosis and even hip or spine fracture [2]. It was believed that the differentiation of the osteoclast was triggered when the receptor activator of nuclear factor kappa B (RANK) bound to RANKL, the ligand of RANK. However, the combination of RANK to RANKL cannot be activated unless protein tumor necrosis factor receptor-associated factor 6 (TRAF6) is joined in it [3], followed by the stimulation of the downstream signaling pathways including PI3K/AKT and NF-κB. Finally, the expressions of the nuclear factor of activated T cells c2 (NFAT2) and c-Fos were regulated [4] to modulate the differentiation of the osteoclast as well as bone resorption. Thus, the factors and regulators that are directly or indirectly related to the activation and differentiation of osteoclast were believed as crucial targets for preventing bone loss.

There are indeed some clinical and synthetic hormone replacement therapy drugs like estradiol valerate which are effective in the treatment of postmenopausal osteoporosis. Unfortunately, some of these enhanced the risk of serious cancers including breast and endometrial cancers [5], which limited their clinical applications. Therefore, it is necessary to select other alternatives with both efficacy and minimal side effects. Traditional Chinese medicines (TCM), as well as the isolated bioactive compounds and fractions [6–9], were proven effective on various ailments including postmenopausal osteoporosis. Among these bioactive components and fractions, phenylethanoid glycoside (PhG) compounds with potential efficacy were believed as promising agents for the treatment of osteoporosis [10–12]. The structures of PhGs consist of cinnamic acid aglycone, a hydroxyl phenyl ethyl group which is combined with β-glucopyranose, apiose, galactose, rhamnose, or xylose via a glycosidic bond. They widely exist in medicinal species of the genus Cistanche [13]. Cistanche deserticola Y.C. Ma is an official TCM that is recorded in Chinese pharmacopeia, and besides being an important TCM [14], C deserticola is also an antiaging tonic herb with few side effects which has been developed into medicinal liquor and nutritional liquid-approved by the State Food and Drug Administration. Based on the record of Chinese pharmacopeia, C. deserticola had been traditionally used by natives to handle kidney essence deficiency problems like muscle debility and lumbar weakness, and phenylethanoid glycoside compounds including echinacoside and acetonide are the main bioactive constituents in this herb. According to the TCM theory of "kidney-govern-bone," the bone system is governed by kidney essence [15], and bone-related troubles like osteoporosis could be recovered by herbs or compounds possessing the activity of nourishing the kidney essence. Therefore, we hypothesized that the total phenylethanoid glycoside fraction isolated from C. deserticola, at least partly, was beneficial in the treatment of osteoporosis. The current experiment was therefore devised to validate our hypothesis by using an ovariectomized (OVX) rat model; besides the bone resorption and formation markers which must be estimated, the antioxidation index as well as RANKL/RANK/TRAF6- induced PI3K/AKT and NF-κB signaling pathways were also employed to investigate the main mechanisms of the antiosteoporotic bioactivity.

2. Materials and Method

2.1. Plant Materials and Preparation. 

A total of 30 kg stems of Cistanche deserticola Y.C. Ma were collected from Yongning County in September 2015 with the coordinates 106.026597 and 38.262816, Ningxia Province, China. The herb was identified by Dr. Lin Dong (Department of Pharmacognosy, Ningxia Medical University), and a corresponding specimen (#20150901) was preserved in the Department of Pharmaceutical Analysis. Firstly, 30.0 kg of powdered C. deserticola was extracted by using the reflux method with 70% ethanol as solvent; the ratio of material to solvent was set as 1: 10, and the reflux time was 2 h for 3 times. Then, all of the filtrates were combined and condensed under reduced pressure at 60° C. Secondly, AB-8 macroporous resin columns were used for the preliminary separation, and different ratios of ethanol in water (0%, 20%, 30%, 40%, 50%, and 60%, each 60 L) were employed for eluting. Thirdly, the 40% and 50% eluents were combined and further purified by using repeated AB-8 macroporous resin columns with the eluents of 0%, 20%, 30%, 40%, and 50% ethanol in water, and each eluent was 12 L. Finally, the 40% fraction was collected and condensed under reduced pressure to obtain 150 g pale yellow sediment phenylethanoid glycoside fraction of C. deserticola (CDP, the yield was 0.5%). For in vivo experiments, 0.5% CMC-Na solvent was employed to dissolve CDP; oral administration to animals was set as 1 mL/100 g of body weight; for in vitro Western blot analysis, CDP was dissolved with DMSO and then diluted with DMEM to obtain the final concentrations of 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL.

2.2. Chemicals and Solvents. 

Estradiol valerate (EV) was from Delpharm Lille S.A.S., France; alkaline phosphatase (ALP), bone gla-protein (BGP), tartrate-resistant acid phosphatase (TRAP), and deoxypyridinoline (DPD) crosslink ELISA kits from Xinyu Biological Engineering Co. Ltd., Shanghai, China, 201605; malondialdehyde (MDA, 20181221), superoxide dismutase (SOD, 20121218), and glutathione (GSH, 20181221) reagent kits from Institute of Nanjing Jiancheng Biological Engineering, Nanjing, China; lntact parathormone (l-PTH, NEWASHE7UZ), calcitonin (2L9ISN7AIU), and estrogen-related receptor alpha (ERRα, Y3AY8QEWB3) crosslink ELISA kits from Elabscience Biotechnology Co. Ltd., Wuhan, China; cathepsin K ELISA reagent kit from BioVision, America, 1l300141; primary antibodies of RANKL (GR3193842-5), RANK (AA02113656), TRAF6 (2), c-Fos (AG12059411), NFAT2 (AO11015648), NF-κBp65 (AH04138226), PI3 kinase p85 alpha (AC09021266), AKT 1 (AF05173234), β-actin (17AV0411), and secondary antibodies of horseradish peroxidase-conjugated goat antirabbit IgG from ZSGB-BIO, China, 136080; total BCA protein assay kit and the commercial kit for the detection of osteoclast formation and fetal bovine serum and Dulbecco's modified Eagle's medium (DMEM) from HyClone, Logan, UT, USA; polyvinylidene fluoride (PVDF) membrane from Millipore Life Sciences, Billerica, MA, USA; penicillin and streptomycin from Gibco, Rockville, MD, USA. All the other chemical agents used were of AR grade.

2.3. HPLC Quantification of CDP. 

An Agilent 1220 HPLC instrument was employed to identify and quantify the composition of CDP. The chromatography conditions were as follows: C18 column (TSK-GEL, 4 6 I d × 250 mm, 5 μm); gradient elution contained solvents A (acetonitrile) and B (water containing 0.5% acetic acid) (0-10 min: 17-20% A; 10-30 min: 20-25% A; and 30-40 min: 25-30% A); the detection wavelength was 333 nm; ambient temperature; flow rate was 1.0 mL/min; sample injection volume was 5 μL. Eight PhG compounds, namely, cistanoside F, echinacoside,6′-acetylacteoside, cistanoside C, cistanoside A, acteoside, 2′-acetylacteoside, and isoacteoside, were identified; by using the corresponding reference substances and an external standard method, the contents of the above 8 PhGs were quantified by HPLC analysis (Figure 1).

Figure 1: HPLC fingerprint of CDP. Eight phenylethanoid glycoside compounds were found in this fraction, and the total contents were quantified as 50.7%. The compounds and their contents were as follows: (1) acteoside F (3.6%), (2) echinacoside (8.8%), (3) cistanoside A (5.0%), (4) acteoside (13.3%), (5) isoacteoside (3.3%), (6) acteoside C (3.6%), (7) 2′-acetylacteoside (9.9%), and (8) 6′-acetylacteoside (3.2%).

2.4. Animal Experimental Protocol. 

A total of 60 female adult Sprague-Dawley rats aged 3 months were purposed from the center of animal testing of Ningxia Medical University, with an average initial body weight of about 234 ± 25 g. The rats were housed in a standard specific pathogen-free environment meant to acclimate for 1 week. Then, all of the rats were anesthetized (chloral hydrate, 100 mg/kg, i.p.) only or sham ovariectomized (SHAM), or two ovaries were both removed and then randomly divided into 5 subgroups: orally treated with vehicle (0.5% CMC-Na) was set as the model group (OVX), estradiol valerate (1 mg/kg/day) as the positive group (EV), and 60, 120, and 240 mg/kg/day of CDP as low (CDPL), moderate (CDPM), and high (CDPH) dosage groups, respectively. All the rats were orally administered daily and lasted for 3 months with the dosage adjusted every 2 weeks which depended on the change of the whole body weights. On the last day of the animal experiment, 24-hour urine was obtained by using metabolic cages; serum was collected from the femoral artery of anesthetic-tized rats; the right femora, tibia, and all the organs were dissected and stored at -80° C for further analysis. The animal experiments that we conducted were approved by the Institutional Animal Care and Use Committee of Ningxia Medical University.

2.5. Bone Mineral Density Determination and Micro-CT Analysis. 

Firstly, a dual-energy X-ray absorptiometry machine (Lunar, USA) was used to estimate the total bone mineral density of the right femur of each rat; secondly, the same femur was used to estimate the 3D image of trabecular bone microarchitecture by employing a micro-CT scanner apparatus (GE, America). The isotropic resolution was set as 14 μm to obtain an ideal 3D image; the region of interest (ROI) was chosen by setting the same coordinates in the growth plate of the femur of each sample; and the bone morphometric parameters including trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular thickness (Tb. Th), bone mineral content (BMC), tissue mineral density (TMD), and tissue mineral content (TMC) were obtained by analyzing the ROI.

2.6. Serum and Urine Biochemical Assay. 

The activities of serum cathepsin K, TRAP, SOD, and GSH, as well as the contents of serum PTH, calcitonin, ERRα, MDA, BGP, and urine DPD, were determined by employing corresponding reagent kits according to the manufacturer's instruction, and the level of alkaline phosphatase (ALP) and the contents of serum and urine calcium (Ca) and phosphorus (P) were estimated by employing an automatic machine (Ciba-Corning 550 Diagnostics Corp., Oberlin, OH, USA). 

2.7. Western Blot Analysis. 

Osteoclasts were induced by using RAW 264.7 cells added with macrophage colony-stimulating factor (MCSF) and RANKL. Briefly, 1 × 107 RAW 264.7 cells were cultured in a 6-well plate in the presence of 30 ng/mL of MCSF and 25 ng/mL of RANKL. After 6 days of induction, the matured osteoclast cells were identified by using the TRAP-stained method with the corresponding kit, then treated with CDP (0.1, 0.01, and 0.001 mg/mL, respectively) for 24 h; then, the cells were lysed with a lysis buffer containing 0.5 mmol phenylmethylsulfonyl fluoride, protease and phosphatase inhibitors. The lysate was then separated by using 10% SDS-PAGE and transferred to a PVDF membrane, which was probed with AKT1, NF-κB-p65, RANKL, PI3Kp85α, RANK, NFAT2, TRAF6, c-Fos, and β-actin (1 : 400) after blocking with 5% nonfat milk for 2 h. The same membranes were stripped and probed again with the above 9 corresponding antibodies, respectively, then were detected by the Image Lab software at the end. The experiments were repeated three times. 

2.8. Statistical Analysis. 

All of the data obtained from in vivo and in vitro experiments, described as the mean ± SD, were analyzed by using one-way ANOVA followed by Dunnett's test (SPSS 22.0 software, SPSS, USA); p < 0 05 was statistically significant.

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