This study aimed to investigate the efficacy and safety of echinacoside (ECH) using an osteopenia rat model. Forty-eight 6-month-old female Sprague-Dawley rats were randomly divided into one sham-operated group (SHAM) and five OVX (ovariectomized) subgroups: SHAM with vehicle 0.5% carboxymethylcellulose sodium (0.5% CMC-Na) and OVX with vehicle (OVX), OVX with 17
Osteoporosis is one kind of skeleton metabolic disorders characterized by reduction of bone mass and microarchitectural deterioration of bone tissue, which may result in skeletal fragility and fractures. It has already become one of the leading threats for the health of the aging population [
Estrogen depletion disrupts bone homeostasis, altering differentiation and activity of osteoblasts and osteoclasts and therefore played a very important role in the initiating and developing of osteoporosis and it has been shown to be a major risk factor for the development of postmenopausal osteoporosis in women [
The
Chemical structure of echinacoside (ECH).
Our previous study showed that ECH can stimulate bone regeneration through increasing OPG/RANKL ratio in MC3T3-E1 cells [
The fresh stems of the
Echinacoside (ECH) was separated and purified from an ethanol extract of
Forty-eight female Sprague-Dawley rats (Nantong University, China), aged 6 months with the body weight of
Rats were randomly divided into six groups (8 rats in each group): sham operated (SHAM), bilaterally ovariectomized (OVX), OVX and E2 treatment, and three other OVX and different doses of ECH treatment groups. All rats were anesthetized via intraperitoneal (i.p.) injection of 300 mg/kg chloral hydrate (Sinopharm, China) and then ovariectomized at week 0 and the SHAM group underwent a sham ovariectomy. The surgical procedure was performed under aseptic conditions following the University of China Pharmaceutical University Animal Care protocol. Rats were left untreated for 4 weeks to allow for rats to recover and develop osteopenia. After 4 weeks, rats in the OVX and E2 group received daily intragastric (i.g.) administrations of E2 (50
At necropsy, blood was collected from the carotid artery under general anesthesia in the early morning. The blood was allowed to clot and centrifuged at 3,000
Alkaline phosphatase (ALP) is known to be associated with bone metabolism and differentiation of osteoblasts and its activity is one of the most common indicators of osteoblast differentiation and osteogenic properties [
Bone mineral density (BMD) of the right total femora was measured by using Discovery W dual energy X-ray absorptiometry (DEXA, Hologic Inc., Boston MA, USA) equipped with appropriate software (edition 13.1.2) for bone density assessment in small laboratory animals. After scanning, BMD in right total femora was obtained for statistical analysis. The investigator performing the measurement was unaware of the treatments the rats had received.
The representative right distal femora were scanned to evaluate three-dimensional (3D) trabecular microarchitectures using microcomputed tomography (
The right femora were tested in three-point bending to evaluate the mechanical properties of the cortical bones at mid-diaphysis using a CSS-4420 material testing machine (Changchun Research Institute for Testing Machines Co. Ltd., China). The femur was placed on the lateral surface on a fixing supporter with two loading points. The distance between the loading points had a fixed length of 20 mm. A preload of 1 N was applied at the medial surface of the diaphysis by lowering a third rounded bar. A constant displacement rate of 1 mm/min was applied until breakage. The force and displacement data were automatically recorded into a computer which was interfaced to the material testing machine and the load-deformation curve was plotted simultaneously. The following mechanical parameters were directly determined from the load-deformation curve: (1) ultimate load (newtons, N), defined as the maximum load, (2) extrinsic stiffness (newtons per millimeter, N/mm), calculated as the slope in the linear region between 40% and 80% of the ultimate load, and (3) energy to ultimate load (millijoules, mJ), defined as the area under the load-deformation curve.
Selected femora for immunohistochemical evaluation of OPG and RANKL expression. The selected specimens (
Serum lipid parameters total cholesterol (TC) and triglycerides (TG) levels were determined by using commercial assay kits (Nanjing jiancheng Bioengineering Institute, Nanjing, China). Serum CEA and CA-125 levels were also measured with ELISA kits (R&D Systems Inc.). All of the measurements of ELISA kits were performed according to the protocols provided by the manufacturers.
At study end, uteri and mammary glands were fixed in neutral buffered formalin, trimmed, processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E) for microscopic examination. Additionally, the interested areas were assayed using a light microscope (Leica DM 100, Germany) equipped with Mini See 1.0.9.37 image analyzing system.
Immunostaining was performed using a 5030 kit (MaxVision, Fuzhou, China). Paraffin sections of 4
All results are presented as mean ± standard deviation (SD). Statistical differences among the sham control, OVX control, and different treatment groups were analyzed using one-way analysis of variance (ANOVA) followed by a post hoc multiple comparison using Fisher’s least significant difference (LSD)
Serum ALP levels and bone turnover markers TRACP-5b were assessed at the end of treatment. In Table
Serum parameters after 12-week administration of ECH.
Group | SHAM | OVX | E2 | ECH-L | ECH-M | ECH-H |
---|---|---|---|---|---|---|
ALP (U/100 mL) | 10.96 ± 1.36 | 14.76 ± 2.86## | 11.66 ± 1.88* | 12.20 ± 1.22* | 12.00 ± 1.56* | 10.95 ± 1.97** |
TRACP-5b (U/L) | 0.306 ± 0.026 | 0.367 ± 0.054# | 0.294 ± 0.054* | 0.277 ± 0.084* | 0.231 ± 0.088** | 0.227 ± 0.082** |
OPG (pg/mL) | 610.32 ± 85.29 | 516.36 ± 65.35# | 621.41 ± 121.78* | 631.77 ± 131.64* | 729.42 ± 122.59** | 775.29 ± 179.87** |
RANKL (pg/mL) | 6.44 ± 0.55 | 7.38 ± 0.83# | 7.19 ± 1.75 | 6.38 ± 0.58* | 6.15 ± 1.01* | 5.75 ± 1.21** |
OPG/RANKL ratio | 95.09 ± 13.44 | 70.32 ± 8.53## | 92.74 ± 35.72 | 100.75 ± 28.50* | 121.41 ± 29.23** | 138.98 ± 39.16** |
The data are expressed as mean ± SD,
At the end of the protocol, assays for serum OPG and RANKL levels were performed. All doses of ECH treatment groups had significantly higher levels of OPG and OPG/RANKL ratios, as well as a significantly lower RANKL levels than E2-treated or vehicle-treated OVX groups (Table
Results of the total femur BMD by DXA were presented in Table
BMD, Micro-CT properties of femoral trabeculae and biomechanical test of femur after 12-week administration of ECH.
Group | SHAM | OVX | E2 | ECH-L | ECH-M | ECH-H |
---|---|---|---|---|---|---|
BMD (g/cm2) | 0.28 ± 0.01 | 0.22 ± 0.01## | 0.26 ± 0.01** | 0.25 ± 0.01** | 0.26 ± 0.01** | 0.28 ± 0.01** |
BV/TV (%) | 58.71 ± 6.38 | 16.42 ± 3.44## | 34.52 ± 3.38** | 23.22 ± 4.07* | 26.12 ± 2.02** | 28.80 ± 2.53** |
Tb.N (1/mm) | 3.88 ± 0.25 | 2.18 ± 0.48## | 3.01 ± 0.10* | 2.76 ± 0.58 | 3.02 ± 0.38* | 3.12 ± 0.41* |
Tb.Sp ( |
106.52 ± 14.78 | 452.02 ± 65.48## | 219.83 ± 9.99** | 355.04 ± 41.53* | 321.98 ± 28.59* | 279.57 ± 23.15** |
Tb.Th ( |
152.28 ± 22.30 | 84.05 ± 7.19## | 114.24 ± 8.13** | 97.12 ± 7.47* | 112.06 ± 20.16* | 115.32 ± 10.79** |
SMI | 0.78 ± 0.12 | 2.77 ± 0.49## | 1.41 ± 0.25** | 2.11 ± 0.15* | 1.90 ± 0.43* | 1.86 ± 0.17* |
Maximum load (N) | 87.54 ± 5.86 | 60.00 ± 3.35## | 89.71 ± 6.45** | 83.05 ± 7.81** | 83.70 ± 6.49** | 93.31 ± 13.66** |
Stiffness (N/mm) | 190.98 ± 36.03 | 147.56 ± 12.54# | 189.81 ± 18.33** | 173.17 ± 24.52* | 178.67 ± 18,55** | 197.79 ± 32.05** |
Energy to ultimate load |
17.33 ± 2.58 | 12.00 ± 1.79## | 18.67 ± 3.01** | 25.67 ± 2.88** | 25.83 ± 3.13** | 34.00 ± 4.65** |
The data are expressed as mean ± SD,
Micro-CT scanning is a very accurate method of measuring the vertebral architecture because it quantifies the trabecular structure in three dimensions. The quantitative results of the metaphyseal region close to the growth plate of the distal femur from Micro-CT evaluation were expressed as BV/TV, Tb.N, Tb.Sp, Tb.Th, and SMI in Table
Representative Micro-CT images of trabecular bone microarchitecture in the distal femurs. (a) SHAM group, (b) OVX group, (c) E2 group, (d) ECH-L group, (e) ECH-M group, and (f) ECH-H group. The OVX rats presented notable reduction in the trabecular number, trabecular area compared with the SHAM rats. ECH and E2 partially prevented OVX-induced trabecular bone loss and significantly improved trabecular bone mass and microarchitecture.
The results of biomechanical three-point bending experiment are shown in Table
Effects on the lipid profile are summarized in Figure
Serum TC and TG levels were determined by assay kit after sacrifice at 12 weeks. Data were expressed as mean ± SD, error bars in the figure are presented as SD,
At the end of the protocol, measurements for serum CEA and CA-125 levels were collected as shown in Table
Serum CEA, CA-125 levels and incidence of principal microscopic pharmacologic effects of ECH and E2 on female rat’s uteri and mammary glands.
Group | SHAM | OVX | E2 | ECH-L | ECH-M | ECH-H |
---|---|---|---|---|---|---|
Serum parameters |
| |||||
Serum CEA (pg/mL) | 341.82 ± 32.94 | 331.08 ± 36.97 | 511.11 ± 62.39##** | 346.03 ± 28.62 | 342.22 ± 42.57 | 330.67 ± 60.93 |
Serum CA-125 (U/mL) | 1.89 ± 0.40 | 1.52 ± 0.39 | 2.43 ± 0.24##** | 1.37 ± 0.29* | 1.32 ± 0.41* | 1.08 ± 0.27#** |
Uteri |
| |||||
Endometrium thickness changes | ++ | + | +++ | + | + | + |
Immunohistochemical analysis of CEA | — | — | — | — | — | — |
Mammary glands |
| |||||
Proliferative changes | + | — | ++ | — | — | — |
Tubular changes | — | — | — | — | — | — |
Ductal intraepithelial neoplasia | — | — | — | — | — | — |
Carcinoma of breast | — | — | — | — | — | — |
Immunohistochemical analysis of CEA | — | — | + | — | — | — |
The data are expressed as mean ± SD,
+: minimal; ++: mild; +++: marked; —: not observed, the qualitative system according to [
The most common adverse events of uteri and mammary glands were not observed between all the groups but not the E2 group (Table
Osteoporosis is a disorder characterized by fragility fractures resulted from loss of bone mass and strength. The remodeling activity is essential to retain bone quality in healthy bone and to produce bones that can adapt appropriately to mechanical stimulus. Because the resorption phases of bone remodeling are short and the period required for osteoblastic replacement of the bone is long, any increase in the rate of bone remodeling will result in a loss of bone mass. Animals develop substantial osteoporosis after ovariectomy within several weeks [
Bone maintains its normal structural and functional integrity through continuous remodeling activity, characterized by the equilibrium between osteoblastic bone formation and osteoclastic bone resorption. However, the homeostasis could be disturbed by OVX, resulting in unbalance of bone formation and bone resorption. In a rat ovariectomy model of estrogen deficiency, OVX caused significant increase in bone remodeling after 12 weeks of treatment. This finding agrees with those of other investigators [
The trabecular bone microarchitecture is generally considered to be a good predictor of bone mass loss and bone structure deterioration [
BMD has been described as a surrogate measure of bone strength and the main contributor to bone quality [
Osteoporosis is mostly caused by increased bone resorption, resulting from increased recruitment, activation, and/or activity of osteoclasts driven by the RANKL signaling [
In this study, we observed lower OPG/RANKL ratio caused by increased RANKL levels and reduced plasma OPG in OVX rats. Ovariectomies have been shown to increase RANKL levels in various animal models which lead to the activation of osteoclasts [
Cancer Antigen 125 (CA-125) is a tumor marker or biomarker that may be elevated in the blood of some specific types of cancers, elevated marker not only for ovarian cancer in serum but also in other malignant cancers, including those originating in the endometrium, fallopian tubes, lungs, breast, and gastrointestinal tract; CEA, one of the first oncofetal antigens to be described and exploited clinically, is associated with the plasma membrane of tumor cells, from which it may be released into the blood, and elevated CEA levels were not only identified in colon cancer but also found in a variety of cancers including pancreatic, gastric, lung, and breast ones [
As implicated through our results, treatment of ECH can also significantly diminish the markers of cancer and cardiovascular disorders. These promising findings highlighted the great advantages of ECH in avoiding undesirable side effects in postmenopausal osteoporotic patients. Compared to the E2 group (50
Our study is the first to report that ECH administration could safely and effectively prevent OVX-induced bone loss through increasing OPG/RANKL ration, which was evidenced by the serum biochemical analysis, bone mineral density assay, Micro-CT analysis, biomechanical properties test, immunohistochemical evaluation, and histology assessment results. These findings shed some light on the potential of ECH, as a natural derived compound, to be developed into a safe and effective agent for prevention or treatment of osteoporosis in postmenopausal osteoporotic women.
Alkaline phosphatase
Bone mineral density
Bone volume fraction
Cancer antigen 125
Carcinoembryonic antigen
Carboxymethylcellulose sodium
17
Echinacoside
Enzyme-linked immunosorbent assay
Estrogen replacement therapy
Intragastric
Intraperitoneal
Microcomputed tomography
Osteoprotegerin
Ovariectomized
Phosphate buffered saline
Receptor activator of nuclear factor-
Receptor activator of nuclear factor-
Structure model index
Trabeculae number
Trabeculae separation
Trabeculae thickness
Total cholesterol
Triglycerides
Tartrate-resistant acid phosphatase 5b.
The authors listed above declare no conflict of interests.
X. Yang and F. Li contributed equally to this work and should be regarded as cofirst authors.
This research was supported by the Research and Innovation Project Foundation for College Graduates of Jiangsu Province (no. CXLX12-0329).