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 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.
Protocol
Representative Results
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 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 …
Declarações
The authors have nothing to disclose.
Acknowledgements
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&T Program of Hebei, China (Grant No.:22372502D) and High-level S&T Innovation and Entrepreneurship Talent Project of Shijiazhuang, Hebei, China (Grant No.: 07202203).
Materials
| 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 |
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