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 (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.
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.
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
2. Micro-CT imaging16
3. Hematoxylin and eosin (H&E) staining
4. Immunohistochemistry
5. Enzyme-linked immunosorbent assay (ELISA) protocol
6. Statistical analysis
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&E staining
Histomorphological damage in ovariectomized rats was relieved after taking BZBS. As shown in Figure 2A,B, the H&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 17β-estradiol (Figure 4A) and Ca2+ (Figure 4B), and a higher level of phosphorus (Figure 4C), SOST (Figure 4D), TRACP-5b (Figure 4E), and β-CTX (Figure 4F). Nevertheless, BZBS treatment elevated serum 17β-estradiol and Ca2+ levels, but subdued the levels of phosphorus, SOST, β-CTX, and TRACP-5b.
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 < 0.05, compared with the SHAM group; *p < 0.05, compared with the OVX group. Please click here to view a larger version of this figure.
Figure 2: H&E staining images of the tibia (A) and L4 vertebra (B) sections. The number "1" represents bone marrow cells, the number "2" represents adipose cells, and the number "3" represents trabecular bone structure. n = 6. Please click here to view a larger version of this figure.
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 < 0.05, compared with the SHAM group; *p < 0.05, compared with the OVX group. Please click here to view a larger version of this figure.
Figure 4: Serum 17β-estradiol, Ca2+, phosphorus, SOST, TRACP-5b, and β-CTX expression in ovariectomized rats in different groups byELISA evaluation. Expression of 17β-estradiol (n = 5) (A), Ca2+ (n = 3) (B), phosphorus (n = 6) (C), SOST (n = 5) (D), TRACP-5b (n = 5) (E), and β-CTX (n = 3) (F). #p < 0.05, compared with the SHAM group; *p < 0.05, compared with the OVX group. Please click here to view a larger version of this figure.
Latin name | Pinyin | Amount in application (ratio) |
Cuscuta | Tusizi | 250 |
Fructus lycii | Gouqizi | 138 |
Fructus schisandrae | Wuweizi | 46 |
Fructus cnidii | Shechuangzi | 35 |
Fructus rosae laevigatae(Cherokee rose fruit) | Jinyingzi | 35 |
Raspberry | Fupenzi | 35 |
Semen Allii Tuberosi | Jiucaizi | 35 |
Toosendan fructus | Chuanlianzi | 23 |
Herba epimedii | Yinyanghuo | 70 |
Morinda officinalis | Bajitian | 35 |
Herba cistanches | Roucongrong | 35 |
Rehmannia glutinosa | Shengdihuang | 46 |
Medicinal cyathula root | Chuanniuxi | 35 |
Ginseng | Renshen | 25 |
Pilose antler | Lurong | 16 |
Hippocampus Kelloggi | Haima | 21 |
Table 1: Drug composition and proportion contained in prescription BZBS.
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&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&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.
The authors have nothing to disclose.
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).
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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