Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

Summary

Presented here is a protocol to show that Bazi Bushen capsule (BZBS) can regulate the RANKL/OPG signaling pathway in the ovariectomized rodent model through its estrogen-like effect.

Abstract

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley (SD) rats were randomly divided into six groups: a sham-operated group, a model group (OVX), a progynova group, and BZBS groups (1, 2, and 4 d/kg/d). An ovariectomy was performed on all rats except those in the sham-operated group. Micro-computed tomography (micro-CT) scanning, hematoxylin and eosin (H&E) staining, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) detection were performed after 4 months of BZBS treatment. As a result, compared with the OVX group, rats treated with BZBS showed an increased number and area of trabecular bone and bone marrow cells, and a decreased number of adipose cells. The bone volume, trabecular number, and trabecular thickness of the right tibia in the medication groups increased and the trabecular space decreased. The 17β-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, β-CTX, and TRACP-5b were decreased. In the medication groups, the RANKL and sclerostin levels were decreased, while the osteoprotegerin (OPG) level was increased. In conclusion, this protocol systematically evaluated the therapeutic effects and potential molecular mechanisms of Chinese herbal compounds in ovariectomized rats with a variety of techniques.

Introduction

Postmenopausal osteoporosis (PMOP) is a skeletal system disease caused by inadequate ovarian function, decreased estrogen, and enhanced osteoclast activity¹, characterized by low bone mass, microarchitectural degeneration of the bone tissue, and damage of the bone trabecula. PMOP prone to cause fractures and can have serious effects on patients' quality of life. Osteoporosis affects about 200 million people worldwide²; about 40% of postmenopausal women suffer from osteoporosis³, and fractures occur in 33% of patients with PMOP⁴. The reduction of estrogen could lead to a relatively enhanced osteoclast activity⁵ and declined bone mass when bone resorption exceeds bone formation. Thus, osteoclast activation is usually considered a sign of bone loss⁶. Physiologically, RANKL/OPG serves as an important pathway involving bone remodeling by regulating osteoclast activation to promote bone resorption⁷. Meanwhile, estrogen can also mediate osteoclast formation and function by regulating RANKL/OPG signaling⁸.

Over recent years, traditional medicine has been increasingly used to treat different diseases with less adverse reactions and better prognosis^9,10. Bazi Bushen capsule (BZBS), a traditional Chinese medicine, could be an alternative approach to prevent and treat PMOP. It is composed of Cuscuta, Fructus lycii, Fructus schisandrae, Fructus cnidii, Fructus rosae laevigatae (Cherokee rose fruit), Raspberry, Semen Allii Tuberosi, Toosendan fructus, Herba epimedii, Morinda officinalis, Herba cistanches, Rehmannia glutinosa, Medicinal cyathula root, Ginseng, Pilose antler, and Hippocampus Kelloggi (Table 1), containing 11 kinds of phytoestrogen with hormone-like effects. A previous study has confirmed that BZBS can delay the formation of atheromatous plaque through an estrogen-like effect¹¹, which is similar to treating osteoporosis with estrogen¹². However, the underlying mechanisms of BZBS in the prevention and treatment of osteoporosis caused by the deficiency of estrogen are unclear. Therefore, the present study aimed to verify whether BZBS has a bone-protective effect on ovariectomy-induced rat osteoporosis^13,14.

Protocol

The animal experiments were approved by the Committee on Animal Research and Ethics of the Hebei Yiling Medical Research Institute (approval number: N2020150). The 36 Sprague-Dawley (SD) female rats (3 months old, weighing 180-200 g) (see Table of Materials) were fed with normal food and clean water in the new drug evaluation center of Hebei Yiling Medical Research Institute and were exposed to artificial light for 12 h per day in rooms with controlled temperatures (20-26 °C) and relative humidity (40%-70%).

1. Animal experiment

1. Divide the rats into six groups randomly, a sham-operated group (SHAM group, 10 mL/kg/d saline), model group (OVX group, 10 mL/kg/d saline), progynova¹⁵ group (0.2 mg/kg/d), low-dose BZBS group (BZBSL group, 1 g/kg/d), middle-dose BZBS group (BZBSM group, 2 g/kg/d), and high-dose BZBS group (BZBSH group, 4 g/kg/d).
   NOTE: The dose of the BZBSM group was determined from the recommended dosages for humans and calculated as follows: equivalent experimental dose for rats (mg/kg/d) = human dose (mg/kg)/body weight (60 kg) x 6.3.
2. Employ 3% and 1.5% isoflurane for the induction and maintenance of rat anesthesia throughout the operation, respectively.
   NOTE: If the rats do not blink and the pedal reflex is absent, the depth of anesthesia is confirmed and the maintenance of rat anesthesia is conducted, respectively.
3. Remove the fur of the rat's abdomen with a sterile razor and disinfect the skin of the rat's abdomen using alternative rounds of ethanol and povidone-iodine 3 times with a sterile cotton ball. Apply local analgesia with a topical injection of 1.5 mg/kg bupivacaine after anesthesia.
   NOTE: The disinfection method adopts a circular pattern centered around the incision and starting from the center outward.
4. Perform a 2 cm midline incision on the abdomen of the rat with a sterile scalpel in the SHAM group and suture the midline incision using a 4-0 surgical suture.
   NOTE: First, the internal muscle layer is sutured with absorbable suture, and the external/skin closure is sutured with monofilament. On the 10th day, use scissors to cut open the monofilament and use tweezers to extract the monofilament.
5. For the rats in the other groups, isolate and expose the intact ovary using sterile forceps and ligate at the root of the ovary using a 4-0 surgical suture. Remove the intact ovary using sterile surgical scissors. Suture the midline incision using a 4-0 surgical suture.
   NOTE: All postoperative rats were placed in isolation in 37 °C containers until the rats were awake and able to move freely.
6. After 4 months of intragastric administration of designated drugs (in step 1.1), first anesthetize the rat with 3% isoflurane, use hair removal cream to remove the fur from the back of the rat, and then disinfect the skin of the groin and entire lower limb using ethanol and povidone-iodine 3x with a sterile cotton ball.
   NOTE: The drug was administered once a day with a 10 cm stainless steel gavage needle. The gavage dose was adjusted weekly according to the weight of the rats.
7. Cut and peel the skin of the groin to expose the femoral artery using sterile surgical scissors and tweezers. Collect a blood sample from the femoral artery using a vacuum blood collection tube and then apply pressure to stop bleeding with a sterile cotton ball. Euthanize the rats with excessive inhalation of carbon dioxide.
8. Continue to cut and peel the skin of the groin and the lower limb to expose the intact tibia using sterile surgical scissors and tweezers. After removing the tibia, wash it three times with sterile saline and continue to remove excess muscle tissue in saline with tweezers and ophthalmic scissors.
9. Disinfect the back skin of the lumbar L4 using ethanol and povidone-iodine 3x with a sterile cotton ball. Use sterile surgical scissors and tweezers to cut the back skin and expose the lumbar L4.
10. Sever the upper and lower vertebrae of the lumbar L4 with a rongeur and cut off the sacrum around the lumbar L4 with sterile surgical scissors. Wash the isolated lumbar L4 with saline three times and continue to remove excess muscle tissue in saline with tweezers and micro scissors.
11. Store the tibia and L4 vertebra in 10% formalin tissue fluid (see Table of Materials) at room temperature for 3 days.
12. Pack the carcass in a special cadaver bag, and place it in an animal carcass storage cabinet.

2. Micro-CT imaging¹⁶

1. Press the green power button to turn on the micro-CT scanner (see Table of Materials) and start the software to heat the x-ray source.
   NOTE: To avoid scattering of the x-rays, move any metal objects near the imaging area before starting. Micro-CT examination of rat tibial tissue in step 1.11 with insufficient x-ray irradiation can affect the findings. The bone should be kept moist during the micro-CT scanning. The animal bed should be taken out while warming up. Make sure that the key is turned to the ON position on the SAFETY KEY in the lower left corner in front of the CT instrument.
2. Create a database by clicking on a new one to build a file to save the data to obtain next.
3. Set the data acquisition parameters in the software control window as follows: x-ray tube voltage (50 kV); CT x-ray tube current (100 µA); live x-ray tube current (100 µA); field of view (FOV) (18 mm); no gating technique; scanning technique (high resolution 4 min).
4. Wash the tibia with saline three times and blot out excess water with filter paper. Wrap the tibia tissue on the bed with plastic film to fix its position. Adjust the tibia tissue to the center of the imaging field by rotating the button in the instrument.
5. Close the instrument door and turn on the live mode. Press the capture button to view the tibial tissue.
6. Start the scan by clicking the CT scan button. Click Yes to produce the image.
   NOTE: Once the x-ray is turned on, the orange light at the top of the instrument will be illuminated and the sliding door will not open for the safety of the operator. When the scan is complete, a new window appears in the 2D Viewer software showing the reconstructed cross-axis, coronal plane, and sagittal plane. Click Paste to the drawing board to save.
7. Take the tibia out of the animal bed. Open the Analyze 12.0 software and import the objective CT image by clicking File > Load > Process. Click the Image Calculator... and Region Pad options to display the SubRegion dialog box.
   1. Click the Interactive option and select the bone in the 2 mm thickness area. Click Apply to select Change a Copy of the Loaded Volume. In the Analyze 12.0 screen, click the Apps option to select BMA.
   2. Click Segment Cortex and Segment Trabeculae, then click Save Final Object Map. Click Measure Bone to display the value of specific parameter indicators, including bone mineral density (BMD, g/cm³), bone volume fraction (bone tissue volume/tissue volume [BV/TV], %), number of bone trabeculae (Tb.N, 1/mm), bone trabecular thickness (TB.Th, µm), and trabecular separation degree (Tb.Sp, µm).
      ​NOTE: Micro-CT was not performed with L4 vertebrae.

3. Hematoxylin and eosin (H&E) staining

1. Take out the tibia and L4 vertebra samples of the rat in step 1.11 and store them in 20% formic acid solution for a 4 day decalcification.
   NOTE: The 20% formic acid solution is prepared by mixing 2,400 mL of formaldehyde solution and 600 mL of formic acid solution.
2. Put the processed tibia and L4 vertebra sample of the rat from step 3.1 in the embedding box and wash them with running water for more than 6 h.
3. Dehydrate the samples with gradient concentration alcohol solutions (60% ethanol for 1 h, 70% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 2 h, and 100% ethanol for 2 h) using an automated tissue processor.
4. Place the tissue specimens in xylene for 2 h to make them translucent. At the end of dehydration, place the permeabilized samples in paraffin wax at 60 °C for 3 h and embed them in an automatic processor.
5. Obtain the 4 µm sections by using a rotary slicer. Place the sections in hematoxylin stain for 3-8 min and eosin stain for 1-3 min.
6. Transfer the stained sections to pure alcohol and xylene, respectively. Seal and fix the stained sections with neutral gum for pathological examination under an optical microscope.
7. Scan the slices with a slide scanner (see Table of Materials) at 40x magnification and use the viewing software to acquire the H&E staining results of the entire tibia and L4 vertebra.

4. Immunohistochemistry

1. Perform heat-induced epitope retrieval using the 4 µm tibia sections obtained in step 3.5. Add the appropriate antigen repair buffer (citric acid buffer, pH = 6.0) to a microwaveable container. Place the carrier sheet into the microwaveable container and place the container in the microwave oven. Set the program to hold the antigen repair for 20 min at 98 °C.
2. Remove the container and rinse the interior with cold tap water for 10 min. Block endogenous peroxidase activity with hydrogen peroxide (3% H₂O₂) (see Table of Materials), then incubate the slice for 15 min at room temperature.
3. Wash the sections in phosphate-buffered saline (PBS) (see Table of Materials) three times, 5 min each.
4. Put the sections into normal goat serum blocking solution (see Table of Materials), then incubate the samples in a moist chamber for 15 min at room temperature.
5. Add 200 µL of RANKL antibody (1:200) (see Table of Materials), OPG antibody (1:300) (see Table of Materials), and sclerostin antibody¹⁷ (1:200) (see Table of Materials) into the slice samples and incubate for one night at 4 °C in the dark.
   NOTE: Sclerostin is a negative regulator of bone growth. It is a secreted glycoprotein with a C-terminal cysteine knot-like domain and has a sequence similarity to the DAN family of bone morphogenetic protein antagonists¹⁷. This protein is not a member of the RANKL pathway.
6. Add biotin-labeled goat anti-rabbit IgG (see Table of Materials) into the sections and incubate at room temperature for 30 min. Rinse the sections with PBS and use diaminobenzidine (DAB) (see Table of Materials) to stain the sections for 3-5 min at room temperature.
7. Dye the sections with hematoxylin for 1-2 min. After dehydrating and drying the sections, seal them with neutral gum.
8. Scan the sections at 40x magnification using a slide scanner (see Table of Materials) and capture digital images using an automatic microscope imaging system (see Table of Materials).
9. Open Imagepro Plus software (see Table of Materials).
   1. Click File to import the analysis image. Click Measure > Calibration > Intensity Calibration for optical density correction. Click the Measure, IOD, Count/Size, and Select Colors options one by one to display the Segmentation interface.
   2. Click the Histogram Based option to select HIS and click Load File. Click Count to generate the IOD value and Area value.

5. Enzyme-linked immunosorbent assay (ELISA) protocol

1. Ensure that the ELISA kit for detecting 17β-estradiol includes the anti-17β-estradiol IgG-coated microplate (12 x 8 wells), 15 mL of stop solution, 22 mL of 17β-estradiol-HRP conjugate, 15 mL of 3,3',5,5' tetramethylbenzidine (TMB) substrate solution, 50 mL of 10x washing solution, cover foils, a strip holder, the 17β-estradiol control, and a different concentration gradient of 17β-estradiol standard (see Table of Materials).
2. Remove the 17β-estradiol kit from the refrigerator and place all the reagents at room temperature (18-25 °C).
3. Reagent preparation: Dilute the 10x washing solution with deionized water to prepare a 1x washing solution. Mix 50 mL of 10x washing solution with 450 mL of deionized water to make 500 mL of 1x washing solution.
4. Remove the serum sample from the refrigerator and thaw it at room temperature.
5. Remove the excess microtiter strips from the plate frame, return them to the foil bag containing the desiccant pack, reseal them, and store at 4 °C.
6. Add 25 µL of control, standard, or sample to each of their respective wells. Then, add 200 µL of 17β-estradiol-HRP conjugate to each well.
7. Cover all the wells with foil and incubate the plate at 37 °C for 2 h.
8. Remove the foil and liquid from each well and wash the well three times with 300 µL of 1x washing solution.
   NOTE: Avoid overflowing of the reaction wells. Also, the soak time between each wash cycle should be >5 s. At the end, carefully remove the remaining fluid by tapping tissue paper strips before the next step.
9. Add 100 µL of TMB substrate solution to each well and incubate in the dark for 30 min at room temperature.
10. Add 100 µL of stop solution to each well and gently shake the microtiter plate for 30 s. Measure the absorbance of the sample at 450 nm within 30 min.
11. Draw the average absorbance of the standards against concentration. Plot the best-fit curve by the plotted points.
12. Obtain the corresponding concentration values expressed in pg/mL by interpolating the values of the samples on the standard curve.
    ​NOTE: The other protocols for detecting serum Ca²⁺, phosphorus, SOST, TRACP-5b, and β-CTX are the same as the 17β-estradiol measurement, according to the manufacturer's instructions.

6. Statistical analysis

1. Data were presented as mean ± standard deviation. Multiple comparisons were performed by one-way ANOVA followed by Tukey's test. p < 0.05 was considered statistically significant.

Results

The bone microstructure of the tibia was evaluated by micro-CT scanning
The treatment of ovariectomized rats with BZBS significantly reduced OVX-induced trabecular structural changes. As shown in the reconstructed micro-CT images of the right tibia (Figure 1A,B), trabecular bone in the OVX group showed a significant decrease in BMD (Figure 1C), BV/TV (Figure 1D), Tb.Th (Fi...

Discussion

After ovariectomy, estrogen levels decreased sharply due to the continuous reduction of cancellous bone and increased bone turnover. Essentially, decreased estrogen secretion after menopause could cause significant bone loss¹⁸, leading to Ca²⁺ loss in ovariectomized rats^19,20. It has been reported that taking estrogen can help the metaphyseal fracture recovery of ovariectomized rats, and estrogen replacement therapy can also be ...

Materials

Name	Company	Catalog Number	Comments
10% neutral buffered formalin	Proteintech Group, Inc.	23408-1AP-100
17-Beta-Estradiol ELISA kits	Proteintech Group, Inc.	21933-1-AP
3%H2O2	ShanDong LIRCON Medical Technology Incorporated Company	20221027
Anti-osteoprotegerin antibody	Abcam	ab203061
Bazibushen capsules	Shijiazhuang Yiling Pharmaceutical Co. Ltd.	XB2103001
Biotin goat anti-rabbit IgG	Abcam	ab207995
Calcium assay kits	Nanjing Jiancheng Bioengineering Institute	C004-2-1
Diaminobenzidine	ZSGB-BIO	ZLI-9018
Estradiol valerate tablets	Bayer AG	156A
Gene 1580R centrifuge	GENE Co. Ltd.	GZ422515090077
Image-Pro Plus	Media Cybernetics	IPP 6.0
Leica DM6000B Microscope (fully automated upright microscope system)	Leica	361715
Normal Goat Serum Blocking Solution	Vector Laboratories	S-1000-20
Phosphate assay kits	Nanjing Jiancheng Bioengineering Institute	C006-1-1
Phosphate buffered saline	Servicebio, Wuhan, China	CR10201M
Quantum GX2 microCT Imaging System	PerkinElmer	CLS149276
RANKL rabbit polyclonal antibody	Elabscience Biotechnology Co. Ltd.	E-EL-R3032-96T
Rat SOST (Sclerostin) ELISA kit	Elabscience Biotechnology Co. Ltd.	E-EL-R1405c-96T
Rat TRACP-5b (Tartrate Resistant Acid Phosphatase 5b) ELISA kit	Abcam	ab108667
Rat β-CTx (Beta Crosslaps) ELISA kit	Elabscience Biotechnology Co., Ltd.	E-EL-R0939c-96T
Sclerostin rabbit polyclonal antibody	Beijing Vital River Laboratory Animal Technology Co., Ltd.	SCXK(JING)2016-0006
Slide scanner	Hamamatsu Photonics K.K.	Nano Zoomer-SQ
Sprague Dawley female rats	Beijing Vital River Laboratory Animal Technology Co., Ltd.	ZLI-9381