Name: Studies on the Anti-Inflammatory Effect of Xiaoyao Pills in The Treatment of Postmenopausal Osteoporosis in Mice (Video) | JoVE Journal
Uploaded: 2024-08-23T14:00:00.000+00:00
Duration: 7 min 20 s
Description: Explore the therapeutic effects of Xiaoyao pills on postmenopausal osteoporosis in mice using histological analysis and immunohistochemistry. This study demonstrates improved bone density and trabecular structure.

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The Baise City Scientific Research and Technology Development Program (20224128) supported this work. The authors thank Dr. Dev Sooranna of Imperial College London and YMUN for editing the manuscript. YYX and ZYW contributed equally to this study.

Therapeutic Effects of Xiaoyao Pills on Postmenopausal Osteoporosis in Mice

The authors declare there is no conflict of interest.

According to statistics, osteoporosis causes 1.5 million fractures each year in the United States, and the vast majority of these occur in postmenopausal women27. With an increase in the aging population, it is predicted that the majority of the world&#39;s future hip fractures will occur in Asia and that by 2050, the total number of these worldwide will reach 8.2 million28. The cost of preventing fractures is almost equal to that of treating them, and the use of medications will inevitably have some side effects29. Therefore, there is an urgent need for a more cost-effective way to address this problem in a responsible society.
Xiaoyao pills consist of eight ingredients. Studies on the effects of its ingredients on osteoporosis are ongoing. Poria cocos inhibits osteoclastogenesis and osteolytic activity in an ovariectomized osteoporosis-induced mouse model30. The combination of two components, licorice, and Shu-di-huang, may exert an antioxidant effect by inhibiting the NF-&#954;B signaling pathway31. The traditional Chinese herbal tonic, Danggui buxue tang, may be useful in the treatment of osteoporosis by its ability to inhibit bone turnover and modulate the regulation of the hypothalamic-pituitary-gonadal (HPG) axis and thereby prevent bone loss in ovariectomized rats32. All of these results suggest that Xiaoyao pills may have a therapeutic effect on osteoporosis. The results from this study also suggest that Xiaoyao pills may exert an anti-osteoporosis effect by modulating the activity of the IL-17 signaling pathway in an anti-inflammatory manner.
A total of 168 relevant signaling pathways were obtained from the regulatory network of drug targets and osteoporosis-related genes constructed in this study, and this study explored the IL-17 signaling pathway further. The IL-17 family consists of six members, including IL-17A-F, of which IL-17A was the first and most important to be discovered33. In early studies, IL-17 was thought to be associated with inflammatory and autoimmune diseases in humans. Later, studies suggested it had a role in maintaining barrier integrity, and it was also found to be associated with cancer15. However, although it does not play a physiological role in bone remodeling, it behaves differently during abnormal bone conditions. In addition, IL-17 can act as a weak activator, and it synergizes with other cytokines to elevate energy output. This is demonstrated by its co-activation with TNF&#945;15,34.
In vitro experiments have demonstrated that IL-17 can induce proliferation and differentiation of osteoblastic MSCs35, and it can also inhibit BMP-2-induced differentiation of primary rat osteoblasts36. IL-17 can stimulate osteoblasts to express RANKL, which, in turn, promotes osteoclastogenesis, but this effect seems to be dependent on the presence of osteoblasts37,38. In this study, the different concentrations of the Xiaoyao pills produced therapeutic effects on PMOM model mice, especially when used at higher concentrations. IL-17, Act1, and IL-6, which are correlated with the IL-17 signaling pathway, were significantly decreased in these mice when compared to controls. When the results of the enrichment analysis were assessed, these suggested the involvement of the IL-17 pathway when the animals were treated with Xiaoyao pills.
This study also tested some of the bone conversion markers. ALP is a tetrameric membrane-bound enzyme that can be secreted by osteoblasts and plays an important role in bone-like formation and mineralization by degrading the mineralization inhibitor, pyrophosphate39. About 90% of the organic fraction in the extracellular matrix of bone tissues is composed of COL-1, and during bone mineralization, collagen plays a key role in this process40. In the result of this study, the levels of ALP and COL-1 protein expression, as observed by immunohistochemistry of mice in the drug group, were higher than those in the model group, suggesting that the effect of Xiaoyao pills in ameliorating osteoporosis could be achieved by increasing the indicators of the osteogenic genes. Some scholars have demonstrated that the expression and transcriptional activity of the hepatocyte estrogen receptor (ER&#945;) can be upregulated by Xiaoyao san41. The extracts of Angelica sinensis, White Paeonia lactiflora, and Licorice in the Xiaoyao san have been shown to have estrogen-like activity. The same ingredients are also present in the Xiaoyao pills used in this study, which are made from the prescription of Xiaoyao san by modern methods of preparation. Therefore, it can be hypothesized that Xiaoyao pills may also increase the expression of osteogenic genes such as ALP and COL-1 by exerting estrogen-like effects on osteoblasts to increase their activity and improve bone quality.
This study successfully established a model of PMOM in mice by removing both ovaries by dorsal ovariectomy. The animal model is the basis for all subsequent experiments in this study. When surgically removing the ovaries of the mice, it was important to ensure that the ovaries of the mice in the OVX and drug groups were completely removed, whereas in the sham group, only the fat near the ovaries was removed, and also it was important&#160;to avoid&#160;wound infections.
The number of bone trabeculae and connection density of mice was improved after treatment of ovariectomized with Xiaoyao pills, which suggested that this drug had a therapeutic effect on osteoporosis. Immunohistochemistry and immunocytochemistry were used to evaluate the efficacy of the treatment in this study; the action time of various reagents is not absolute, especially in the process of color development, which requires real-time observation and timely reaction to ensure the best results. During antibody incubation, the antibody concentration gradient is first set, and after observation and comparison, choose the most suitable concentration for the subsequent experiments.
However, there were some limitations in this study. The web pharmacology data only had a single source of data, and the analysis with respect to the drug composition was only from one database. In addition, the efficacy of Xiaoyao pills was not compared with positive drugs in this study, and it was not possible to assess the specific differences between Xiaoyao pills and current treatment modalities.
For PMOM, the available first-line treatment option is bisphosphonates. However, the use of these drugs may lead to gastrointestinal contraindications and even more serious atypical femur fractures and osteonecrosis of the jaws42,43. The second-line treatment options include the use of denosumab and hormone therapy. Denosumab has been shown to reduce hip, vertebral, and non-vertebral fractures. However, rebound can occur once the drug is discontinued, and the longer the duration of discontinuation, the greater the loss of bone density42,44,45. Menopausal hormone therapy reduces the risk of osteoporotic fractures of the spine, but its effects are age-limited, and its long-term risks appear to outweigh the benefits46. Sequential therapy is a long-term treatment option for PMOM, in which the use of bone-forming medications followed by anti-resorptive medications can achieve the greatest increase in bone mass. Specific medications are usually determined on a patient-by-patient basis42.
In this respect, it can be assumed that traditional Chinese herbs can have an advantageous potential in the treatment of osteoporosis. The mode of action of herbal medicine is multi-pathway and multi-target rather than acting via a single mechanism47. Some scholars have evaluated the efficacy and safety of some Chinese herbal medicines, and they have shown that the number of fractures when using a combination of Chinese and Western medicines as compared to only administering Chinese medicine was lower than that in the Western medicine alone group48. Compared with chemical drugs, Chinese herbal remedies have fewer side effects, are relatively inexpensive, and are suitable for long-term use. Chinese herbal remedies cannot only repair the bone microstructure and increase bone volume, but they can also reduce or eliminate the symptoms of lumbar spine weakness and back pain49. The current study may help provide a possible approach to uncovering novel drugs and treatments.
The application of Xiaoyao pills to treat PMOM appears to be promising. In the present study, the pills can improve bone mass and bone quality in a mouse model of PMOM by suppressing inflammation. Some scholars have even suggested that a combination of Chinese medicine and acupuncture can be effective in the treatment of PMOM50. At the same time, a combination of Chinese and Western medicine has been proven to be effective51. Both these possible remedies are valid, although any recommended treatment using the Xiaoyao pills in conjunction with acupuncture and Western medicine should be validated by clinical trials. The clinical significance of osteoporosis lies in bone fractures, and future research should also focus on preventing these.
In summary, this study demonstrated through network pharmacology and animal experiments that PMOM mice showed significant improvement in bone metabolism and increased bone mass after treatment with Xiaoyao pills. The mechanism of action for this phenomenon may be related to the inhibition of the IL-17 signaling pathway and the enhancement of osteogenic factors, ALP, and COL-1 activity. This study lays a theoretical foundation and experimental basis for the clinical application of Xiaoyao pills in the treatment of osteoporosis-associated diseases.

The World Health Organization (WHO) defines osteoporosis (OP) as a disease characterized by a decrease in bone mass and deterioration of the microarchitecture of bone tissue, leading to an increase in bone brittleness and, thus an increased risk of fracture1. The clinical significance of osteoporosis is that it can lead to fractures, which are associated with high mortality, morbidity, and economic costs2. Postmenopausal osteoporosis (PMOP) is caused by a decrease in estrogen levels in women after menopause, which leads to an increase in osteoclast activity, resulting in bone loss and destruction of bone microstructure. This often causes osteoporosis with a serious impact on health3. Current therapies for PMOP include estrogen replacement therapy, bisphosphonates, and parathyroid hormone, but they can have varying degrees of adverse effects, insufficient long-term compliance, and high costs4. Therefore, affordable herbal medicine is a viable alternative for a large proportion of the population.
Xiaoyao pills are included in the Chinese Pharmacopoeia5, and these contain eight herbal components, including Chai Hu, Angelica sinensis, White paeonia lactiflora, Atractylodes macrocephala, Poria cocos, Menthae Herba, licorice, and fresh ginger. All these herbs are known to be effective in detoxifying the liver and strengthening the spleen, nourishing the blood, and regulating the menstrual cycle, and the mixture has also been used for treating depression6. However, the role of Xiaoyao pills in osteoporosis is unclear.
Early studies have suggested that inflammation can lead to bone loss7, and that the decline in bone density associated with this process may be accelerated by menopause. In addition, there is a strong relationship between the development of osteoporosis and inflammation. The inflammatory factor, interleukin-17 (IL-17), is a pro-inflammatory factor secreted by Th17 cells, a subset of CD4+ T lymphocytes. These cells are associated with several chronic inflammatory conditions, and they play an important role in the development of bone destruction in rheumatoid arthritis8. Additionally, IL-17 stimulates nuclear factor-&#954; B ligand receptor activator (RANKL), which regulates osteoclastogenesis, leading to greater bone resorption than bone formation9. IL-17 stimulates the expression of other osteoclastogenic cytokines such as TNF&#945;, IL-1, IL-6, and IL-8. It has the ability to synergize with other inflammatory factors, making it an important inflammatory effector10.
Studies have also shown a&#160;link between Xiaoyao pills and inflammation. Shi et al.11 and Fang et al.12 have recently confirmed that Xiaoyao pills can reduce the levels of IL-6 and TNF-&#945;, respectively. In another study of metabolism-associated steatohepatitis, it was reported that Xiaoyao pills could upregulate the expression of propionic acid, which in turn inhibits the expression of TNF-&#945; and exerts an anti-inflammatory effect13. However, at present, it is not known whether Xiaoyao pills can regulate the development of PMOM by mediating an inflammatory response through IL-17, which was the aim of this study.
This study predicted the intersection of the targets of Xiaoyao pills and osteoporosis-related genes through network pharmacology and bioinformatics analysis and analyzed the intersecting genes for protein interactions, GO, and KEGG. Based on the predicted results, the expression of Act1 and IL-6, which are key proteins in the IL-17 signaling pathway, can be observed14,15, as well as the bone turnover markers alkaline phosphatase (ALP) and collagen type I (COL-1), to observe the therapeutic efficacy of the Xiaoyao pills in the PMOM mice model.

<table><tbody><tr><td>1ml Sampler</td><td>Guangxi Beilunhe Medical Industrial Group Co.</td><td>JYQ001</td><td>For anesthesia in mice</td></tr><tr><td>4%polyformaldehyde</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>P1110</td><td>For tissue fixation</td></tr><tr><td>6-0 absorbable suture (angled needle)</td><td>Shanghai Pudong Jinhuan Medical Supplies Co.</td><td>HZX-06</td><td>For postoperative suturing</td></tr><tr><td>Absorbent cotton ball</td><td>Winner</td><td>MIANQIU-500g</td><td>For sterilization and hemostasis</td></tr><tr><td>Adhesive slides</td><td>Jiangsu Shitai Experimental Equipment Co.</td><td>188105</td><td>For tissue sectioning</td></tr><tr><td>Amobarbital</td><td>Sigma Aldrich (Shanghai) Trading Co.</td><td>A-020-1ML</td><td>For anesthesia in mice</td></tr><tr><td>Bluing Solution</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G1866</td><td>Blue coloration of the nuclei of cells after the action of hematoxylin differentiation solution</td></tr><tr><td>C57BL/6 mice</td><td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td><td>(SCXK 2033-0063)</td><td>For use in animal experiments</td></tr><tr><td>Carbon steel surgical blades</td><td>Premier Medical Equipment Co.</td><td>SP239</td><td>For mouse surgery</td></tr><tr><td>CIKS/TRAF3IP2 Rabbit pAb</td><td>BIOSS ANTIBODIES</td><td>bs-6202R</td><td>Binds to Act1 in tissues</td></tr><tr><td>Cole&#039;s Hematoxylin Solution (For Conventional Stain)</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G1140</td><td>For staining paraffin sections</td></tr><tr><td>Collagen Type I Polyclonal antibody</td><td>proteintech</td><td>14695-1-AP</td><td>Binds to COL-1 in tissues</td></tr><tr><td>DAB Substrate kit,20x</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>DA1010</td><td>For tissue color development</td></tr><tr><td>Disposable surgical sheet 50*60CM</td><td>Nanchang Xuhui Medical Equipment Co.</td><td>SP4529777</td><td>For mouse surgery</td></tr><tr><td>EDTA decalcification solution (pH 7.2)</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>E1171-500ml</td><td>For tissue decalcification</td></tr><tr><td>Enhanced Endogenous Peroxidase Bloching Buffer</td><td>Beyotime Biotechnology</td><td>P0100B</td><td>Sequestration of tissue or cellular endogenous peroxidases</td></tr><tr><td>Environmentally friendly dewaxing clear liquid</td><td>Servicebio</td><td>G1128-1L</td><td>For dewaxing paraffin sections</td></tr><tr><td>Ethyl Alcohol</td><td>CHRON CHEMICALS</td><td>64-17-5 (CAS)</td><td>For dehydration of paraffin sections</td></tr><tr><td>General Purpose Antibody Diluent</td><td>Epizyme Biotech</td><td>PS119L</td><td>For antibody dilution</td></tr><tr><td>Hematoxylin Differentiation Solution</td><td>Servicebio</td><td>G1039-500ML</td><td>For differentiation after hematoxylin staining and removal of excessively bound and non-specifically adsorbed dye from tissues</td></tr><tr><td>Hematoxylin Eosin (HE) Staining Kit</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G1120-3*100ml</td><td>For tissue staining</td></tr><tr><td>High quality stainless steel surgical knife handle</td><td>Premier Medical Equipment Co.</td><td>SP0088</td><td>For mouse surgery</td></tr><tr><td>HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)</td><td>proteintech</td><td>SA00001-2</td><td>Binds to primary antibody and amplifies signal</td></tr><tr><td>IL-17A Polyclonal antibody</td><td>proteintech</td><td>13082-1-AP</td><td>Binds to IL-17 in tissues</td></tr><tr><td>IL-6 Polyclonal antibody</td><td>proteintech</td><td>21865-1-AP</td><td>Binds to IL-6 in tissues</td></tr><tr><td>Immunohistochemistry pen</td><td>Beijing Zhongshan Jinqiao Biotechnology Co.</td><td>ZLI-9305 (YA0310)</td><td>For drawing circles in immunohistochemistry</td></tr><tr><td>Medical surgical suture Non-absorbent (ball) 5-0 3.5m</td><td>Yangzhou Yuanlikang Medical Equipment Co.</td><td>FHX-5-2</td><td>For postoperative suturing</td></tr><tr><td>Medical Suture Needles Angle Needles 4*10 3/8</td><td>Chaohu Binxiong Medical Equipment Co.</td><td>FHZ612-4</td><td>For postoperative suturing</td></tr><tr><td>Neutral Balsam</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G8590</td><td>As a slice sealer</td></tr><tr><td>PBS(1X)</td><td>Shenzhen Mohong Technology Co.,Ltd</td><td>B0015</td><td>Buffer for slide washing and partial solution dilution</td></tr><tr><td>Protein Free Rapid Blocking Buffer (1X)</td><td>Epizyme Biotech</td><td>PS108P</td><td>Avoiding non-specific binding of proteins</td></tr><tr><td>Rabbit Anti-Bone Alkaline Phosphatase antibody</td><td>BIOSS ANTIBODIES</td><td>bs-6292R</td><td>Binds to alkaline phosphatase in tissues</td></tr><tr><td>Saline</td><td>Affiliated Hospital Youjiang Medical University For Nationalities</td><td>LHN500</td><td>For animals by gavage</td></tr><tr><td>Shaving/Electric clippers</td><td>HANGZHOU HUAYUAN PET PRODUCTS CO., LTD.</td><td>DTJ-002</td><td>For shaving mice</td></tr><tr><td>Stainless Steel Medical Needle Holder 14cm Coarse Needle</td><td>Premier Medical Equipment Co.</td><td>SP784</td><td>For mouse surgery</td></tr><tr><td>Stainless Steel Ophthalmic Forceps 10.5cm Curved (No Hook)</td><td>Zhuoyouyue</td><td>YKNWW-10.5</td><td>For mouse surgery</td></tr><tr><td>Stainless Steel Ophthalmic Scissors/Surgical Scissors 10CM Straight Tip</td><td>Premier Medical Equipment Co.</td><td>ZYJD-10-ZJ</td><td>For mouse surgery</td></tr><tr><td>Stainless Steel Tip Gastric Needle 12 Gauge 55mm Elbow</td><td></td><td>GWJ-12-55W</td><td>For use in mice by gavage</td></tr><tr><td>Tris-EDTA Antigen Repair Fluid (50x)</td><td>proteintech</td><td>PR30002</td><td>For antigen repair of paraffin sections</td></tr><tr><td>Wooden dissecting board 25*16cm</td><td></td><td>JP16*24</td><td>For mouse surgery</td></tr><tr><td>Xiaoyao pills</td><td>Jiuzhitang Co.,Ltd.</td><td>YPG-041</td><td>For animal drug delivery</td></tr><tr><td>&lt;strong&gt;Others&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>R 4.3.1</td><td></td><td></td><td>Data processing</td></tr><tr><td>Cytoscape3.9.1</td><td>National Resource for Network Biology</td><td></td><td>Building a regulatory network for traditional Chinese medicine</td></tr><tr><td>ImageJ 1.54f</td><td>National Institutes of Health</td><td></td><td>Image processing for immunohistochemistry results</td></tr><tr><td>Adobe Photoshop 24.0.0</td><td>Adobe</td><td></td><td>For image combination</td></tr><tr><td>GraphpadPrism 9.5</td><td>GraphPad Software</td><td></td><td>Statistical analysis of data</td></tr><tr><td>cellsens Dimension</td><td>OLYMPUS</td><td></td><td>For slicing and photographing</td></tr><tr><td>OLYMPUS BX53</td><td>OLYMPUS</td><td></td><td>For HE staining and immunohistochemical section photography</td></tr></tbody></table>

studies on the anti-inflammatory effect of xiaoyao pills in the treatment of 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.

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 ...

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Explore the therapeutic effects of Xiaoyao pills on postmenopausal osteoporosis in mice using histological analysis and immunohistochemistry. This study demonstrates improved bone density and trabecular structure.

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

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