This work was supported by the Strategic Consulting Project of the Chinese Academy of Engineering: Strategic research on anti-aging effect of Traditional Chinese Medicine (Grant No.: 2022-XY-45), the Natural Science Foundation of Hebei Province in China (Grant No.:2022106065), S&#38;T Program of Hebei, China (Grant No.:22372502D) and High-level S&#38;T Innovation and Entrepreneurship Talent Project of Shijiazhuang, Hebei, China (Grant No.: 07202203).

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 &#176;C) and relative humidity (40%-70%).
1. Animal experiment
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Remove the fur of the rat&#39;s abdomen with a sterile razor and disinfect the skin of the rat&#39;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.</li>
	<li>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.</li>
	<li>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 &#176;C containers until the rats were awake and able to move freely.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;tweezers and ophthalmic scissors.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Store the tibia and L4 vertebra in 10% formalin tissue fluid (see Table of Materials) at room temperature for 3 days.</li>
	<li>Pack the carcass in a special cadaver bag, and place it in an animal carcass storage cabinet.</li>
</ol>
2. Micro-CT imaging16
<ol>
	<li>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.</li>
	<li>Create a database by clicking on a new one to build a file to save the data to obtain next.</li>
	<li>Set the data acquisition parameters in the software control window as follows: x-ray tube voltage (50 kV); CT x-ray tube current (100 &#181;A); live x-ray tube current (100 &#181;A); field of view (FOV) (18 mm); no gating technique; scanning technique (high resolution 4 min).</li>
	<li>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.</li>
	<li>Close the instrument door and turn on the live mode. Press the capture button to view the tibial tissue.</li>
	<li>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.</li>
	<li>Take the tibia out of the animal bed. Open the Analyze 12.0 software and import the objective CT image by clicking File &#62; Load &#62; Process. Click the Image Calculator... and Region Pad options to display the SubRegion dialog box.
	<ol>
		<li>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.</li>
		<li>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, &#181;m), and trabecular separation degree (Tb.Sp, &#181;m). 
		&#8203;NOTE: Micro-CT was not performed with L4 vertebrae.</li>
	</ol>
	</li>
</ol>
3. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C for 3 h and embed them in an automatic processor.</li>
	<li>Obtain the 4 &#181;m sections by using a rotary slicer. Place the sections in hematoxylin stain for 3-8 min and eosin stain for 1-3 min.</li>
	<li>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.</li>
	<li>Scan the slices with a slide scanner (see Table of Materials) at 40x magnification and use the viewing software to acquire the H&#38;E staining results of the entire tibia and L4 vertebra.</li>
</ol>
4. Immunohistochemistry
<ol>
	<li>Perform heat-induced epitope retrieval using the 4 &#181;m 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 &#176;C.</li>
	<li>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.</li>
	<li>Wash the sections in phosphate-buffered saline (PBS) (see Table of Materials) three times, 5 min each.</li>
	<li>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.</li>
	<li>Add 200 &#181;L 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 &#176;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.</li>
	<li>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.</li>
	<li>Dye the sections with hematoxylin for 1-2 min. After dehydrating and drying the sections, seal them with neutral gum.</li>
	<li>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).</li>
	<li>Open Imagepro Plus software (see Table of Materials).
	<ol>
		<li>Click File to import the analysis image. Click Measure &#62; Calibration &#62; Intensity Calibration for optical density correction. Click the Measure, IOD, Count/Size, and Select Colors options one by one to display the Segmentation interface.</li>
		<li>Click the Histogram Based option to select HIS and click Load File. Click Count to generate the IOD value and Area value.</li>
	</ol>
	</li>
</ol>
5. Enzyme-linked immunosorbent assay (ELISA) protocol
<ol>
	<li>Ensure that the ELISA kit for detecting 17&#946;-estradiol includes the anti-17&#946;-estradiol IgG-coated microplate (12 x 8 wells), 15 mL of stop solution, 22 mL of 17&#946;-estradiol-HRP conjugate, 15 mL of 3,3&#39;,5,5&#39; tetramethylbenzidine (TMB) substrate solution, 50 mL of 10x washing solution, cover foils, a strip holder, the 17&#946;-estradiol control, and a different concentration gradient of 17&#946;-estradiol standard (see Table of Materials).</li>
	<li>Remove the 17&#946;-estradiol kit from the refrigerator and place all the reagents at room temperature (18-25 &#176;C).</li>
	<li>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.</li>
	<li>Remove the serum sample from the refrigerator and thaw it at room temperature.</li>
	<li>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 &#176;C.</li>
	<li>Add 25 &#181;L of control, standard, or sample to each of their respective wells. Then, add 200 &#181;L of 17&#946;-estradiol-HRP conjugate to each well.</li>
	<li>Cover all the wells with foil and incubate the plate at 37 &#176;C for 2 h.</li>
	<li>Remove the foil and liquid from each well and wash the well three times with 300 &#181;L of 1x washing solution. 
	NOTE: Avoid overflowing of the reaction wells. Also, the soak time between each wash cycle should be &#62;5 s. At the end, carefully remove the remaining fluid by tapping tissue paper strips before the next step.</li>
	<li>Add 100 &#181;L of TMB substrate solution to each well and incubate in the dark for 30 min at room temperature.</li>
	<li>Add 100 &#181;L 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.</li>
	<li>Draw the average absorbance of the standards against concentration. Plot the best-fit curve by the plotted points.</li>
	<li>Obtain the corresponding concentration values expressed in pg/mL by interpolating the values of the samples on the standard curve. 
	&#8203;NOTE: The other protocols for detecting serum Ca2+, phosphorus, SOST, TRACP-5b, and &#946;-CTX are the same as the 17&#946;-estradiol measurement, according to the manufacturer&#39;s instructions.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Data were presented as mean &#177; standard deviation. Multiple comparisons were performed by one-way ANOVA followed by Tukey&#39;s test. p &#60; 0.05 was considered statistically significant.</li>
</ol>

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W., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sclerostin+on+the+functions+and+related+mechanisms+of+cementoblasts+under+mechanical+stress.">Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress.</a> West China Journal of Stomatology. 37 (2), 162-167 (2019).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+and+outlook+of+alternative+medicine+for+osteoporosis.">Therapeutic potential and outlook of alternative medicine for osteoporosis.</a> Current Drug Targets. 18 (9), 1051-1068 (2017).</li></ol>

The authors have nothing to disclose.

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 rats19,20. It has been reported that taking estrogen can help the metaphyseal fracture recovery of ovariectomized rats, and estrogen replacement therapy can also be used to maintain bone mass and prevent osteoporosis in postmenopausal women. Previous studies have indicated that using estradiol helps improve bone microarchitecture21. This experiment confirmed that BZBS could inhibit bone Ca2+ loss and improve the bone microstructure of ovariectomized rats, which is consistent with the previous study18. For the serum test, BZBS enhanced the decrease of estrogen in ovariectomized rats and regulated the levels of calcium and phosphate. Meanwhile, BZBS could lower the RANKL expression and increase the OPG expression in ovariectomized rats, which would effectively inhibit the rising of bone resorption markers. Furthermore, the micro-CT imaging and H&#38;E staining results showed that BZBS improved the bone microarchitecture of ovariectomized rats, which indicated that BZBS had the effect of preventing and treating osteoporosis, as well as lowering bone resorption markers and inhibiting bone loss. Compared with the model group, BZBS treatment increased serum estradiol levels and lowered bone loss, which suggested that BZBS might protect bone through an estrogen-like effect.
Osteoporosis induced by estrogen deficiency might be related to regulation of the RANKL/OPG signal. The ratio of RANKL to OPG could be a sign of osteoclast activation23, which is responsible for regulating the balance between bone formation and bone resorption. The binding of activated RANKL to RANK due to estrogen deficiency would promote osteoclast differentiation and improve bone resorption, which would lead to the temporary decrease of bone volume24. As a decoy receptor, OPG would competitively bind to RANK and directly block the activation of RANKL/RANK, which would lower osteoclast activity and inhibit bone resorption. In this experiment, increased OPG expression of ovariectomized rats in the BZBS group was found and RANKL was inhibited. It is reported that a raised SOST level could increase RANKL expression and lower OPG expression25. In addition, as the cause of bone loss in ovariectomized rats26, overexpression of sclerostin could be an indicator to accurately evaluate the bone microenvironment. Meanwhile, our results revealed that BZBS could prevent bone loss by inhibiting the overexpression of sclerostin and suppressing osteoclast activity.
In this study, a rat model of osteoporosis induced by ovarian removal was successfully constructed, and estrogen-like regulation of bone homeostasis by the Chinese herbal compound BZBS was further confirmed by integrating micro-CT, H&#38;E staining, and other various molecular biological techniques. This integrated methodological process provides a reference for the development of anti-osteoporosis drugs27. As the key imaging technique for evaluating bone health status, micro-CT can obtain nondestructive 3D imaging of bone trabecular microstructures to quantify micro-changes of bone structure and bone density. However, the following aspects should be paid attention to when performing micro-CT. First of all, in order to prevent the sample from moving, low-density media, such as plastic film, should be used to wrap the sample, and the sample should be placed parallel in the small animal bed. Secondly, it is worth noting that unstable voltage can easily cause malfunction during scanning. Finally, and more notably, the estrogen-deficient osteoporosis model established by ovarian removal cannot fully simulate clinical postmenopausal women with osteoporosis. More gene-edited animal models of hormone-deficient osteoporosis or actual clinical cases should be further tested.

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 prognosis9,10. 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 osteoporosis13,14.

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

estrogen-like effect of bazi bushen capsule in ovariectomized rats

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&amp;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 17&#946;-estradiol and serum calcium levels in the medication groups were elevated, but the levels of serum phosphorus, sclerostin, &#946;-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.

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 (Figure 1E), Tb.N (Figure 1F), and an increase in Tb.Sp (Figure 1G), compared with the SHAM group. Compared with the OVX group, BV/TV, Tb.N, and Tb.Th. of the right tibia in the BZBS treatment groups were significantly increased, and Tb.Sp. was markedly decreased.
Bone histology of the tibia and L4 vertebra was evaluated by H&#38;E staining 
Histomorphological damage in ovariectomized rats was relieved after taking BZBS. As shown in Figure 2A,B, the H&#38;E staining results indicated consistent changes between the tibia and L4 vertebra. Compared with the SHAM group, trabecular bone in the OVX group was sparser and thinner, characterized by a disordered, fractured, and irregular structure with more adipose cells, and a significantly decreased number of bone marrow cells. Compared with the OVX group, BZBS treatment showed an increased number and area of trabecular bone (yellow arrow), decreased number and area of adipose cells (blue arrow), and increased number of bone marrow cells (green arrow). All the above data indicated that BZBS could improve the bone histology of ovariectomized rats.
RANKL, OPG, and SOST protein expressions of the tibia were evaluated by immunohistochemistry 
The expression of osteogenic/osteoclast cytokines in ovariectomized rats was improved by BZBS treatment. Compared with the OVX group, the immunohistochemistry results showed a decreased expression of RANKL (Figure 3A), increased expression of OPG (Figure 3B), and reduced expression of SOST (Figure 3C) in the BZBS treatment groups.
Bone metabolic markers in serum were evaluated by ELISA 
Biochemical markers of bone metabolism in ovariectomized rats were also improved by BZBS. Compared with the SHAM group, the OVX group had a lower level of 17&#946;-estradiol (Figure 4A) and Ca2+ (Figure 4B), and a higher level of phosphorus (Figure 4C), SOST (Figure 4D), TRACP-5b (Figure 4E), and &#946;-CTX (Figure 4F). Nevertheless, BZBS treatment elevated serum 17&#946;-estradiol and Ca2+ levels, but subdued the levels of phosphorus, SOST, &#946;-CTX, and TRACP-5b.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64884/64884fig01.jpg" /> 
Figure 1: Micro-CT scanning resultson ovariectomized rats with osteoporosis after 4 months of treatment with BZBS. (A) 2D images in the vertical plane of the right tibia. (B) 3D images of the right tibia. The microstructure parameters include BMD (C), BV/TV (D), Tb.Th (E), Tb.N (F), and Tb.Sp (G). n = 3, #p &#60; 0.05, compared with the SHAM group; *p &#60; 0.05, compared with the OVX group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64884/64884fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64884/64884fig02.jpg" /> 
Figure 2: H&#38;E staining images of the tibia (A) and L4 vertebra (B) sections. The number &#34;1&#34; represents bone marrow cells, the number &#34;2&#34; represents adipose cells, and the number &#34;3&#34; represents trabecular bone structure. n = 6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64884/64884fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64884/64884fig03.jpg" /> 
Figure 3: Effect of BZBS on RANKL, OPG, and SOST expression in ovariectomized rats. The expression level of RANKL (A), OPG (B), and SOST (C) were evaluated with immunohistochemistry after 4 months of BZBS treatment. Quantitative analysis of RANKL (D), OPG (E), and SOST (F) expression. Red arrows represent positive expressions. n = 3, #p &#60; 0.05, compared with the SHAM group; *p &#60; 0.05, compared with the OVX group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64884/64884fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64884/64884fig04.jpg" /> 
Figure 4: Serum 17&#946;-estradiol, Ca2+, phosphorus, SOST, TRACP-5b, and &#946;-CTX expression in ovariectomized rats in different groups byELISA evaluation. Expression of 17&#946;-estradiol (n = 5) (A), Ca2+ (n = 3) (B), phosphorus (n = 6) (C), SOST (n = 5) (D), TRACP-5b (n = 5) (E), and &#946;-CTX (n = 3) (F). #p &#60; 0.05, compared with the SHAM group; *p &#60; 0.05, compared with the OVX group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64884/64884fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Latin name</td>
			<td>Pinyin</td>
			<td>Amount in application (ratio)</td>
		</tr>
		<tr>
			<td>Cuscuta</td>
			<td>Tusizi</td>
			<td>250</td>
		</tr>
		<tr>
			<td>Fructus lycii</td>
			<td>Gouqizi</td>
			<td>138</td>
		</tr>
		<tr>
			<td>Fructus schisandrae</td>
			<td>Wuweizi</td>
			<td>46</td>
		</tr>
		<tr>
			<td>Fructus cnidii</td>
			<td>Shechuangzi</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Fructus rosae laevigatae(Cherokee rose fruit)</td>
			<td>Jinyingzi</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Raspberry</td>
			<td>Fupenzi</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Semen Allii Tuberosi</td>
			<td>Jiucaizi</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Toosendan fructus</td>
			<td>Chuanlianzi</td>
			<td>23</td>
		</tr>
		<tr>
			<td>Herba epimedii</td>
			<td>Yinyanghuo</td>
			<td>70</td>
		</tr>
		<tr>
			<td>Morinda officinalis</td>
			<td>Bajitian</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Herba cistanches</td>
			<td>Roucongrong</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Rehmannia glutinosa</td>
			<td>Shengdihuang</td>
			<td>46</td>
		</tr>
		<tr>
			<td>Medicinal cyathula root</td>
			<td>Chuanniuxi</td>
			<td>35</td>
		</tr>
		<tr>
			<td>Ginseng</td>
			<td>Renshen</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Pilose antler</td>
			<td>Lurong</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Hippocampus Kelloggi</td>
			<td>Haima</td>
			<td>21</td>
		</tr>
	</tbody>
</table>
Table 1: Drug composition and proportion contained in prescription BZBS.

Watch this Scientific Journal Video about Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats at JoVE.com

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

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.

This study aims to show the estrogen-like effect of Bazi Bushen capsule (BZBS), a Chinese herbal compound, in ovariectomized mice. Female Sprague-Dawley ...

estrogen-like-effect-of-bazi-bushen-capsule-in-ovariectomized-rats

Nanyang Technological University

Research

JoVE Journal

Medicine

819 Views.  Hebei Medical University. Dit protocol kan intuïtief experimentele resultaten zoals botfenotype, metabole indicatoren, enzovoort. De belangrijkste toepassing van dit experiment is micro-CT, die een weefsel3D-reconstructie van botmorfologieanalyse kan uitvoeren, het groter op basis van huidmonsters. Om de procedure te starten, brengt u lokale analgesie aan met een plaatselijke injectie van bupivacaïne op een goed verdoofde rat.Gebruik vervolgens een steriel watje om het gescho...

Video: Oestrogeenachtig effect van Bazi Bushen-capsule bij ratten met ovariëctomie

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Behavior

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Biochemistry

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

Cancer Research

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Biology

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Neuroscience

Murine Orchiectomy and Ovariectomy to Reduce Sex Hormone Production

Sex hormone signaling plays a critical role in multiple organ systems as well as in the progression of various diseases, including neurodegenerative disease. The manipulation of sex hormone levels in the murine model system allows for the study of their impact on organs/tissues and within disease progression. Orchiectomy - the surgical removal of the testes - and ovariectomy - the surgical removal of the ovaries - provide a method to deplete the endogenous sex hormones so that the precise hormone levels can be provided through drug or other delivery methods. Here, we provide rapid and minimally invasive methods for both orchiectomy and ovariectomy in the murine model system for the reduction of sex hormones. This protocol details the surgical preparation and excision of the testes through the scrotal sac, and excision of the ovaries via two incisions in the right and left lateral dorsum.

This manuscript describes a consistent way to quickly perform survival rodent orchiectomies and ovariectomies.

Sex hormone signaling plays a critical role in multiple organ systems as well as in the progression of various diseases, including neurodegenerative ...

Polycystic Ovary Syndrome Induction in Mice Through Controlled Letrozole Release 

A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of PCOS and for related drug development. Letrozole-induced mouse models replicate the metabolic and reproductive phenotypes of patients with PCOS. The traditional method of letrozole treatment in PCOS mice requires daily dosing over a certain period, which can be labor-intensive and cause significant stress to the mice. This study describes a simple and effective method for inducing PCOS in mice by implanting a controlled letrozole-releasing mini-pump. A mini-pump capable of stable, continuous release of a quantitative amount of letrozole was fabricated and implanted subcutaneously in mice under anesthesia. This study demonstrated that the mouse model successfully mimicked PCOS features after letrozole mini-pump implantation. The materials and equipment used in this study are readily available to most laboratories, requiring no special customization. Collectively, this article provides a unique, easy-to-perform method for inducing PCOS in mice.

Author Spotlight: Investigating the Mechanisms and Inducing Models of Polycystic Ovary Syndrome

This protocol introduces a technique for inducing polycystic ovary syndrome (PCOS) models in mice through controlled letrozole release using mini-pumps. Under adequate anesthesia, the mini-pump was implanted subcutaneously, and PCOS was successfully induced in the mice after a certain period of the mini-pump release.

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of ...

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Therapeutic Effects of Xiaoyao Pills on Postmenopausal Osteoporosis in Mice

Studies on the Anti-Inflammatory Effect of Xiaoyao Pills in The Treatment of Postmenopausal Osteoporosis in Mice

Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of this condition, the patients are prone to fractures, and this can seriously affect their health and quality of life. The worldwide increase in life expectancy has made osteoporosis a global concern. The Xiaoyao pills were previously 
used in the treatment of depression. In addition, the drug appeared to have estrogen-like activity, which affected the expression of ALP, an early osteoblast-specific marker, and COL-1, a major component of bone extracellular matrix. Xiaoyao pills were assessed for their effects on postmenopausal osteoporosis (PMOM) in mice. The target information of each herbal component of Xiaoyao pills was accessed through the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Information from GeneCards, OMIM, PharmGkb, TTD, DrugBank, and other websites was used to construct the regulatory network of the herbal complex through Cytoscape and String network to assess the protein interactions. Mice were ovariectomized, and treated with high and low doses of Xiaoyao pills and these were compared to controls. Their symptoms were assessed by immunocytochemistry of bone tissues. The results suggested that Xiaoyao pills had the ability to alleviate the symptoms of PMOM in ovariectomized mice through the IL-17 signaling pathway. This drug has the potential to become a novel therapeutic agent for the treatment of osteoporosis.

Author Spotlight: Integrating Traditional Chinese Medicine with Modern Pharmacology and Genomics for Assessing Postmenopausal Osteoporosis in Mice 

Postmenopausal osteoporosis has become a global public health problem. The aim of this study is to explore the therapeutic effects and related mechanisms of the traditional Chinese medicine Xiaoyao pills on this condition.

Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of ...

Estrogen-Like Effect of Bazi Bushen Capsule in Ovariectomized Rats

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