Identification and preparation of herb decoctions
Radix Dipsaci (RD) is the dried roots of Dipsacus asperoides C. Y. Cheng et T. M. Ai produced in China, and was collected by the Zhonghong Herbal Drug Co. Ltd. (Sichuan, China) in March 2007, and identified and authenticated by an expert herbalist at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (CACMS).
Pyrola Herb (PH) is the dried aerial parts of Pyrola calliantha H. Andr produced in China, and was collected by the Huanghe Herbal Drug Co. Ltd. (Gansu, China) in March 2007, and identified and authenticated by an expert herbalist at the Institute of Chinese Materia Medica, CACMS.
Cynomorium songaricum (CS) is the dried fleshy stem of Cynomorium songaricum Rupr produced in China, and was collected by the Taizhou Herbal Drug Co. Ltd. (Zhejiang, China) in March 2007, and identified and authenticated by an expert herbalist at the Institute of Chinese Materia Medica, CACMS.
After collection, all the dried herbs were stored in a dry and sealed container at 4°C to protect them from moisture and moths.
Decoctions of RD, PH, and CS were extracted from the herbs by boiling 300 grams of the dried RD, PH, and CS in 6 liters of water at 100°C for 2 h. Each decoction was then concentrated to a final concentration of 1 crude drug gram per milliliter.
Animals and experimental procedures
Sixty-nine virgin Wistar rats (3 months old) in a body weight range of 250 ± 20.0 grams were obtained from The Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China) and housed in cages at the experimental animal room at the Institute of Basic Theory of Traditional Chinese Medicine in CACMS. These rats were maintained at 22°C with a 12 h light/dark cycle and fed with a standard rodent chow diet (produced by The Animal Center of The Fourth Military Medical University, Xi’an, China) containing 0.9% calcium and 0.7% phosphate, with distilled water available ad libitum.
The acclimatized rats either underwent sham surgery (a faked operative intervention that omits the step thought to be therapeutically necessary) (sham, n = 12) or bilaterally ovariectomized (OVX, n = 57) using the dorsal approach . Briefly, a surgical procedure on each of the anesthetized rats was performed with a single longitudinal skin incision on the dorsal midline at the level of the kidneys. Both ovaries were ligated and removed. Rats in the sham group underwent sham surgery, during which the ovaries were exposed and remained intact. The OVX rats were randomly divided into five groups: OVX control group (OVX, n = 12), positive control group treated with diethylstilbestrol (E2, n = 12), and three groups treated with RDD (n = 11), PHD (n = 11), and CSD (n = 11), respectively. Non-surgical rats (n = 12) were used as normal control (NC). The rats in the E2 group received diethylstilbestrol at a dose of 0.008 mg/ml (dissolved in distilled water) orally. The rats in the RDD, PHD, and CSD treatment groups were fed intragastrically with RDD at 5.6 ml per kg body weight a day, PHD at 5.6 ml per kg body weight a day, and CSD at 5.6 ml per kg body weight a day, respectively. This dose, about 40-fold lower than LD50 of these herbs (222.84 g/kg body weight), was optimized in our pre-experimental studies (data not shown). Rats in the NC, sham, and OVX control groups were administrated with an equal volume of distilled water to that in the decoction treatment groups. All the treatments were started four weeks after the surgery and continued for 12 weeks. The body weight of each rat was monitored weekly to assess the effect of the treatments.
On the fifteenth and third days before sacrifice, all the rats received tetracycline at 30 mg/kg by intraperitoneal injection. After sacrifice, the success of ovariectomy was confirmed at necropsy by failure to detect ovarian tissue and by observation of marked atrophy of the uterine horns.
All animals were maintained according to the Guide for Care and Use of Laboratory Animals, with the approval of the Institutional Ethics Committee of the CACMS.
After sacrifice, the tibiae were dissected for histomorphometrical analysis. Proximal right tibiae were fixed in 4% paraformaldehyde for 24 h, then dehydrated in an ethanol gradient of 80%, 90%, and 100% for two days at each step. Dehydrated samples were defatted in xylene for two days before embedding in plastic polymer solution I (100 ml Methyl Methacrylate Monomer, 35 ml Butyl Methacrylate, 5 ml Methyl Benzoate, and 1.2 ml Polyglycol 1.2 ml), solution II (solution I, 0.4 gram Drying Benzoyl Peroxide), and solution III (solution II, 0.8 gram Drying Benzoyl Peroxide) for three days at each step. Each undecalcified tissue was sliced into two longitudinal sections at 5 μm thickness using microtome. One section was stained with toluidine blue, and the other was used for fluorescence morphology observation. Proximal left tibiae were fixed in 4% paraformaldehyde for 24 h, then decalcified in a decalcifying solution of 10% EDTA (pH7.3) at 4°C for three weeks. After that, the decalcified samples were dehydrated in 15% sucrose solution for 10 h. Each sample was sliced into sections of 5 μm thickness each by freezing microtome. Frozen sections were fixed in acetone and ready for use for analysis of immunohistochemistry and in situ hybridization.
Undecalcified tibial slides were used for analysis of bone remodeling activity. All measurements were performed using the Qwin Image analysis system (Leica Corporation, Bensheim, Germany). Six bone histomorphometric parameters were analyzed, including percentage of trabecular bone volume (TBV%), percentage of trabecular resorption surface (TRS%), percentage of trabecular formation surface (TFS%), mineralization rate of trabecula (MAR), mineralization rate of bone cortex (mAR), and osteoid mean width (OSW)
Methylene blue staining
The undecalcified tibial sections were coated in Methyl Methacrylate Monomer solution for 2 h, then dehydrated in xylene solution for 1 h twice to remove resin. The samples were then rehydrated in an order of ethanol gradients, 100%, 90%, 80%, and 0% (in water). The rehydrated samples were stained with 1% toluidine blue, then rinsed with water. After that, the stained slides were dehydrated in an ethanol gradient of 80%, 90%, and 100%. Dehydrated slides were defatted in xylene, then sealed for microscopic analysis.
Frozen sections were mounted onto glass slides and used for immunohistochemical analysis. Protein expression level of RANKL and OPG in tissue sections was estimated using rat antibodies of either anti-RANKL (1:1000) or anti-OPG (1:1000) (Santa Cruz Biotechnology, CA, USA). The sections were rinsed with TBS, then immersed in 0.3% hydrogen peroxide for 5 min. The slides were then incubated with specific antibodies for 1 h at 37°C and rinsed with TBS three times for 3 min. Sections were then incubated with the appropriate unbiotinylated secondary antibody (Zhongshan Goldenbridge Biotechnology Co. Ltd, Guangzhou, China) for 30 min at 37°C. Slides were then incubated with a solution containing DAB (1, 4-dideoxy-1,4-imino-d-arabinitol-diaminobenzidine; Sigma, USA) for 3 min and rinsed with running water, then counterstained with Harris hematoxylin and sealed for microscopic examination. Slides incubated with nonimmune goat serum, instead of the primary antibody, served as negative control.
Image analysis was performed using the Qwin Image analysis system (Leica Corp, Bensheim, Germany). Five images from each tibial section of a rat were randomly captured with 400× magnification. The relative level of the osteoblasts (OB) and bone marrow stromal cells (bMSC) in trabecular area was calculated as a percentage based on the positive stained area of either OB or bMSC over the trabecular area in the selected images.
In situ hybridization
After a brief warm-up at room temperature, frozen sections were immersed in solution of 30% hydrogen dioxide and methanol for 30 min, then incubated with pepsin diluted by 3% citric acid at 37°C. After that, the sections were postfixed in 1% paraformaldehyde for 10 min. Sections were then incubated with the DIG-labeled antisense cRNA probes at 38°C-42°C overnight in a humidified chamber. After multiple washes in 4 × SSC at room temperature, slides were incubated in a blocking reagent for 30 min at 37°C, then with a biotinylated anti-digoxin antibody for 60 min, SABC for 20 min, and the biotinylated peroxydase for 20 min, at 37°C, followed by staining with DAB (Sigma-Aldrich Corp.). Finally, sections were covered with glycerol-gelatin and coverslips, and were ready for microscopic examination. The primer sequences used are listed below:
All values were expressed as mean ± standard deviations. All analyses were carried out using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The difference between the groups regarding the evaluated parameters was tested using the ANOVA test followed by the Tukey test. The data of all groups passed the Kolmogorov-Smirnov test of normality. All groups were carried out in triplicate and significance was accepted when P ≤ 0.05.