The combination of Herba Epimedii and Fructus Ligustri Lucidi has been used to treat osteoporosis for almost 50 years by Professor Shizeng Li, a famous doctor of traditional Chinese medicine (TCM). However, it is unclear whether the combination of the effective constituents of the two herbs may have a protective influence on the skeleton. In the present study, we investigated the effects of the combination extracts of Herba Epimedii and Fructus Ligustri Lucidi on rat model of osteoporosis induced by retinoic acid by gavage. With administrations of the combination extracts of the two herbs (50, 100, and 200 mg/kg/day) via oral gavage for 3 weeks, bone mineral density (BMD), femur histomorphometry, some sex hormones, and sex hormone receptors were measured. Results showed that the combined extracts could increase BMD, affect bone histomorphometry, coordinate the sex hormones at the level of hypothalamus-pituitary-gonad axis, and increase the protein and mRNA expressions of sex hormone receptors. The findings suggested that the combination extracts of Herba Epimedii and Fructus Ligustri Lucidi might be beneficial as an alternative medicine for the prevention and treatment of osteoporosis.
Osteoporosis is one kind of the most common bone remodeling diseases characterized by reduction of bone mass and microstructural deterioration of bone tissue. The essence of this disease is caused by an imbalance of bone remodeling, that is, less bone formation and more bone resorption which may ultimately result in skeletal fragility, an increased risk of hip and vertebral fracture [
According to the theories of TCM, bone health is considered to be closely related to kidney function, and “kidney deficiency” is thought to be the root of all pathologies related to bones and joints. Thus, the treatment of osteoporosis accordingly follows the principle of strengthening the kidney function. Herba Epimedii (
Researchers have found that hypervitaminosis A causes accelerated bone resorption, bone fragility, and spontaneous fractures [
Herba Epimedii, the dried leaf of
For the preparation of extracts, Herba Epimedii (1000 g) was extracted three times with 90% ethanol (10 000 mL) for 3 h, 2 h, and 2 h at 70°C in a reflux apparatus. The extracts were mixed, filtered, and concentrated under reduced pressure, until recovery to no alcohol precipitation. Then with petroleum ether extraction three times to remove chlorophyll, the extracts were washed with D-101 macroporous resin to colorless, eluted with 90% ethanol, and steamed to yield a dark yellow powder, namely, total flavonoids of Herba Epimedii (TFE). The yield of Herba Epimedii extract was 2.5%. Based on the phytochemical test (Pharmacopoeia of the People’s Republic of China, 2010 Edition), the content of TFE was 80% calculated by Icariin. Fructus Ligustri Lucidi (1000 g) was extracted three times with 75% ethanol (10 000 mL) for 3 h, 2 h, and 2 h at 70°C in a reflux apparatus. The extracts were mixed, filtered, and concentrated under reduced pressure, until recovery to no alcohol precipitation. Using AB-8 macroporous resin, the extracts were washed with distilled water and then with 75% ethanol after the water solution was discarded. The steamed extracts are total iridoid and flavonoids of Fructus Ligustri Lucidi (TIFL). The yield of Fructus Ligustri Lucidi extract was 5%. Based on the phytochemical test (Pharmacopoeia of the People’s Republic of China, 2010 Edition), the content of TIFL was more than 80% calculated by Oleanolic Acid and Rutin, respectively.
The extract combination of Herba Epimedii and Fructus Ligustri Lucidi was composed of TFE and TIFL with a ratio of 2 to 3 (equivalent to the raw herbs ratio of 4 to 3). Doses of Herba Epimedii and Fructus Ligustri Lucidi were chosen based on the clinical practice by Shizeng Li and our previous studies, which have demonstrated that combination of the two herbs has the effects on the prevention and treatment of osteoporosis [
Fifty male Wistar rats, weighing 230 to 270 g with the average age of three months, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The experiment complied with the Animal Management Rule of the Ministry of Public Health, China, and the experimental protocol was approved by the Animal Care Committee of Capital Medical University, Beijing, China. All the animals were cared for in the Experimental Animal Center of Capital Medical University. During the whole experiment, the animals were housed in stainless cages (three rats per cage) at conventional controlled conditions (temperature of
The rats were acclimated to conditions for one week before the experiment and randomly divided into 5 groups of 10 rats each, including normal control group, model control group, and three treatment groups [Extract-L (50 mg/kg), Extract-M (100 mg/kg), and Extract-H (200 mg/kg)]. All rats received intragastric administration of retinoic acid (70 mg/kg) daily for two weeks to establish osteoporosis model except those of normal control group. Rats in treatment groups were administered with the combined extracts via oral gavage at the dose of 50, 100, and 200 mg/kg body weight, respectively. At the same time, the rats in the normal control group and model control group were given the same volume of distilled water. Three weeks later, rats were anesthetized with ethyl carbamate (4 mL/kg, ip). Blood samples were collected and separated simultaneously using a centrifuge (Biofuge 15R, Heraeus Sepatech, USA), and serum was collected finally and stored at −80°C prior to assay. Each of the rats had the bilateral femurs and the left tibia dissected out and cleaned of any tissues excised. Each left femur was submerged in 4% neutral-buffered paraformaldehyde solution in PBS (pH 7.4) for 24 h and decalcified in a solution of 1% ethylene diamine tetraacetic acid solution (EDTA; PH 7.36) for 6 weeks. After that, each left femur sample was cut along the coronal plane, embedded in paraffin, and cut longitudinally into 5
The BMD of each right femur were measured
Staining with hematoxylin and eosin (HE), each left femur section was observed for microarchitectural changes under a microscope (ECLIPSE 80i, Nikon, Japan), especially the structure and morphology of trabecular bone. Histomorphometry variables were analyzed in trabecular bone using an image analyzing computer system (NIS-Elements BR 3.2, Nikon, Japan) linked to a microscope. The parameters measured included trabecular bone area (Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). All of the histomorphometric indices were reported according to the standardized nomenclature recommended by the American Society of Bone and Mineral Research Histomorphometry Nomenclature Committee [
The serum levels of estradiol (E2), testosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH) were determined. Serums E2, T, LH, and FSH were measured by rat E2, T, and LH radioimmunoassay kits (Sino-UK Institute of Biological Technology, Beijing, China) for control, standard, and duplicate tests. Serum GnRH was measured using a rat GnRH ELISA kit (BlueGene Biotech Co. Ltd., Shanghai, China) by enzyme-linked immunosorbent assay (Immunodiagnostic System Ltd., Boldon, UK), and absorbance was read using ELISA reader (Thermo, USA) at 450 nm.
Frozen sections were mounted on glass slides and used for immunohistochemical assessment. Primary antibodies [anti-rat androgen receptor (AR, 1 : 100) and anti-rat estrogen receptor (ER, 1 : 100)] were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The tissue slides were rinsed in PBS and incubated with the primary antibodies for 1 h at 37°C and then rinsed with PBS three times for 3 min. The slides were then incubated with the appropriated biotinylated antibody (EnVision, Dako, DEN) for 30 min at 37°C and then rinsed with PBS three times for 3 min, followed by incubation with a solution containing 3,3-diaminobenzidine and hydrogen peroxide at room temperature for 30 s, and then rinsed in running water. After that, the slides were counterstained with Harris hematoxylin and sealed for microscopic analysis. As negative control, nonimmune goat serum instead of the primary antibody was served as negative control. All measurements were performed with the Nikon ECLIPSE 80i biomicroscope and NIS-Elements BR 3.2 image analysis system (Nikon, Japanese). Five random images within tibiae from two sections were taken and further analyzed by using zoomed-in field at 400x magnification. We measured the integral optical density of AR or ER-positive cells under each examined field for each section and calculated the average number as the final result of this sample.
Total RNA was isolated from each left tibia using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. Total RNA (2
Primers used for qPCR analysis.
Primer | Forward primer | Reverse primer |
---|---|---|
rno-AR | 5′-ggcaaaggcactgaagagac-3′ | 3′-cccagagctacctgcttcac-5′ |
rno-ER | 5′-tccggcacatgagtaacaaa-3′ | 3′-tgaagacgatgagcatccag-5′ |
β-actin | 5′-agccatgtacgtagccatcc-3′ | 3′-accctcatagatgggcacag-5′ |
Results of all measurements were presented as means ± standard deviation (SD). The data analysis was performed using the SPSS 13.0 (SPSS Inc., Chicago, USA). All of the data were tested for normality, using the Kolmogorov-Smirnov test, and passed. A one-way analysis of variance (ANOVA) was performed to determine whether there were statistically significant differences (
To investigate whether the combination with TFE and TIFL has antiosteoporotic effects, the BMD of different parts of femur were measured by DXA. Figure
Effects of the combination with TFE and TIFL on BMD in whole of femur (a), femur head (b), and femur neck (c) in osteoporosis rats. BMD of the whole femur, femur head, and femur neck in osteoporosis rats was significantly lower than that in normal group. Mean ± SD,
HE staining showed that cancellous bone was formed of a dense network of trabecular bone in the normal group and osteocytes appeared in their lacunae. The endosteal surface of trabecular bone was lined by osteoprogenitor cells, osteoblasts, and osteoclasts in Howship’s lacunae. Bone marrow spaces were seen between the trabeculae. The results in model group revealed that the cancellous bone lost its normal architecture and trabecular bone became thinner and discontinuous, and osteoclasts apparently increased as compared with the normal group. After administration of the combination with TFE and TIFL for 3 weeks, pathological morphology of trabecular bone was improved significantly. The effects of medium dose and high dose group were more obvious. The cancellous bone partially regained near normal structure and the trabecular bone widened, its broken points lessened, and its Howship’s lacunae shallowed as compared to the normal group (Figure
Effects of the combination with TFE and TIFL on bone histomorphometry in osteoporosis rats. (a) Representative microscope images of trabecular bone microarchitecture in femurs (HE, ×100). Bone histomorphometry of the trabecular bone included Tb.Ar (b), Tb.Th (c), Tb.Sp (d), and Tb.N (e). Mean ± SD,
As shown in Figure
Effects of combination with TFE and TIFL on serum levels of gonadal hormones in osteoporosis rats. Gonadal hormones E2 (a), T (b), LH (c), FSH (d), and GnRH (e) were shown in (a), (b), (c), (d), and (e), respectively. Mean ± SD,
Immunohistochemical staining results showed that positive expressions of AR and ER protein in femur were located in cytoplasm and dyed pale brown. Positive areas are mainly located in bone marrow stroma. Proteins of AR and ER were strongly expressed in normal control group, while the two receptors were weakly expressed in osteoporosis rats. The level of AR or ER positive expression in each administration group was situated between the normal group and model group (Figures
Effects of the combination with TFE and TIFL on protein expressions of AR and ER in osteoporosis rats. Expression levels of AR and ER were estimated by immunohistochemical analysis. AR protein expression (a), ER protein expression (b), and quantitative analysis of protein expressions of AR and ER (c). Mean ± SD,
To further understand whether the combination with TFE and TIFL affect the mRNA levels of ER and AR, we analyzed mRNA expression levels of ER and AR in tibia of rats using qPCR. The mRNA expressions of AR and ER in the model group were significantly alleviated compared to the normal control group (
Effects of combination with TFE and TIFL on mRNA expressions of AR and ER in osteoporosis rats. (a) AR mRNA expression was determined by qPCR with β-actin as an internal control. (b) ER mRNA expression was determined by qPCR with β-actin as an internal control. (c) Amplification curves of the mRNA expressions of AR and ER. (d) Melt curves of the mRNA expressions of AR and ER. The mRNA expressions of AR and ER in osteoporosis rats were significantly lower than that in normal group. Mean ± SD,
To explore whether there were direct responses between E2 and T and between sex hormones and corresponding sex hormone receptors, we conducted a correlation analysis and the results were shown in Figure
Analysis of the correlation within sex hormones and sex hormone receptors. (a) The correlation between serum content of T and E2; (b) the correlation between serum T content and AR protein expression of femur; (c) the correlation between serum T content and AR mRNA expression of tibia; (d) the correlation between serum E2 content and ER protein expression of femur; (e) the correlation between serum E2 content and ER mRNA expression of tibia.
The kidney-tonifying Chinese herbal medicines, Herba Epimedii and Fructus Ligustri Lucidi, have been widely used to treat osteoarticular disease for thousands of years in China. Professor Shizeng Li, a famous doctor of TCM, has used the decoction compatibility of Herba Epimedii (as a kidney-
BMD and bone histomorphometry were used to evaluate the pharmacodynamics effects of the combination with TFE and TIFL on osteoporosis rats induced by retinoic acid. BMD has been known as the main contributor to bone quality and a surrogate measure of bone strength [
Sex hormones are an important regulator for the occurrence of osteoporosis. Androgen reduction was associated with bone loss not only in postmenopausal but also in male osteoporosis [
Osteoporosis was induced by not only a certain hormone change but also the entire hypothalamic-pituitary-adrenal axis at each level [
Sex hormone can start the response and regulate the bone remodeling only binding with the corresponding receptor. Studies suggest that both AR and ER are required for optimal cortical bone expansion via actions in immature osteoblasts and trabecular bone maintenance via actions in more differentiated osteoblasts and osteocytes [
This study demonstrated that the combination with TFE and TIFL had potential protective effects on osteoporosis rats induced by retinoic acid. The combination with TFE and TIFL is able to increase BMD, reverse pathological changes of bone tissue, coordinate hormones of the hypothalamic-pituitary-gonadal axis, and upregulate the expression of sex hormone receptors. The present data suggest that the combination with TFE and TIFL may be a reasonable natural alternative for the prevention and treatment of osteoporosis. However, further detailed mechanistic investigation of the antiosteoporotic effects of the combination with TFE and TIFL on bone metabolism is required.
Traditional Chinese medicine
Hormone replacement therapy
Bone mineral density
Total flavonoids of Herba Epimedii
Total iridoid and flavonoids of Fructus Ligustri Lucidi
Dual-energy X-ray absorptiometer
Trabecular bone area
Trabecular thickness
Trabecular number
Trabecular separation
Estradiol
Testosterone
Luteinizing hormone
Follicle stimulating hormone
Gonadotropin-releasing hormone
Androgen receptor
Estrogen receptor.
The authors declare that they have no competing interests.
R. Liu and X. Kang equally contributed to this work
This work was supported by the grants from the National Natural Science Foundation of China (nos. 30801494 and 81373814), Foundation of Beijing Educational Committee (no. KM201310025012), and Planned Project on Beijing Traditional Chinese Medicine “inheritance of 3 + 3 programme” of Beijing Chinese Medicine Administration Bureau (no. 2012-SZ-C-42).