Studies on the Anti-Inflammatory Effect of Xiaoyao Pills in The Treatment of Postmenopausal Osteoporosis in Mice
Summary
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.
Abstract
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.
Introduction
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-κ 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 TNFα, 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 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-α, 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-α 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.
Protocol
Representative Results
Discussion
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'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 medication…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 1ml Sampler | Guangxi Beilunhe Medical Industrial Group Co. | JYQ001 | For anesthesia in mice |
| 4%polyformaldehyde | Beijing Solarbio Science & Technology Co.,Ltd. | P1110 | For tissue fixation |
| 6-0 absorbable suture (angled needle) | Shanghai Pudong Jinhuan Medical Supplies Co. | HZX-06 | For postoperative suturing |
| Absorbent cotton ball | Winner | MIANQIU-500g | For sterilization and hemostasis |
| Adhesive slides | Jiangsu Shitai Experimental Equipment Co. | 188105 | For tissue sectioning |
| Amobarbital | Sigma Aldrich (Shanghai) Trading Co. | A-020-1ML | For anesthesia in mice |
| Bluing Solution | Beijing Solarbio Science & Technology Co.,Ltd. | G1866 | Blue coloration of the nuclei of cells after the action of hematoxylin differentiation solution |
| C57BL/6 mice | Beijing Vital River Laboratory Animal Technology Co., Ltd. | (SCXK 2033-0063) | For use in animal experiments |
| Carbon steel surgical blades | Premier Medical Equipment Co. | SP239 | For mouse surgery |
| CIKS/TRAF3IP2 Rabbit pAb | BIOSS ANTIBODIES | bs-6202R | Binds to Act1 in tissues |
| Cole's Hematoxylin Solution (For Conventional Stain) | Beijing Solarbio Science & Technology Co.,Ltd. | G1140 | For staining paraffin sections |
| Collagen Type I Polyclonal antibody | proteintech | 14695-1-AP | Binds to COL-1 in tissues |
| DAB Substrate kit,20x | Beijing Solarbio Science & Technology Co.,Ltd. | DA1010 | For tissue color development |
| Disposable surgical sheet 50*60CM | Nanchang Xuhui Medical Equipment Co. | SP4529777 | For mouse surgery |
| EDTA decalcification solution (pH 7.2) | Beijing Solarbio Science & Technology Co.,Ltd. | E1171-500ml | For tissue decalcification |
| Enhanced Endogenous Peroxidase Bloching Buffer | Beyotime Biotechnology | P0100B | Sequestration of tissue or cellular endogenous peroxidases |
| Environmentally friendly dewaxing clear liquid | Servicebio | G1128-1L | For dewaxing paraffin sections |
| Ethyl Alcohol | CHRON CHEMICALS | 64-17-5 (CAS) | For dehydration of paraffin sections |
| General Purpose Antibody Diluent | Epizyme Biotech | PS119L | For antibody dilution |
| Hematoxylin Differentiation Solution | Servicebio | G1039-500ML | For differentiation after hematoxylin staining and removal of excessively bound and non-specifically adsorbed dye from tissues |
| Hematoxylin Eosin (HE) Staining Kit | Beijing Solarbio Science & Technology Co.,Ltd. | G1120-3*100ml | For tissue staining |
| High quality stainless steel surgical knife handle | Premier Medical Equipment Co. | SP0088 | For mouse surgery |
| HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) | proteintech | SA00001-2 | Binds to primary antibody and amplifies signal |
| IL-17A Polyclonal antibody | proteintech | 13082-1-AP | Binds to IL-17 in tissues |
| IL-6 Polyclonal antibody | proteintech | 21865-1-AP | Binds to IL-6 in tissues |
| Immunohistochemistry pen | Beijing Zhongshan Jinqiao Biotechnology Co. | ZLI-9305 (YA0310) | For drawing circles in immunohistochemistry |
| Medical surgical suture Non-absorbent (ball) 5-0 3.5m | Yangzhou Yuanlikang Medical Equipment Co. | FHX-5-2 | For postoperative suturing |
| Medical Suture Needles Angle Needles 4*10 3/8 | Chaohu Binxiong Medical Equipment Co. | FHZ612-4 | For postoperative suturing |
| Neutral Balsam | Beijing Solarbio Science & Technology Co.,Ltd. | G8590 | As a slice sealer |
| PBS(1X) | Shenzhen Mohong Technology Co.,Ltd | B0015 | Buffer for slide washing and partial solution dilution |
| Protein Free Rapid Blocking Buffer (1X) | Epizyme Biotech | PS108P | Avoiding non-specific binding of proteins |
| Rabbit Anti-Bone Alkaline Phosphatase antibody | BIOSS ANTIBODIES | bs-6292R | Binds to alkaline phosphatase in tissues |
| Saline | Affiliated Hospital Youjiang Medical University For Nationalities | LHN500 | For animals by gavage |
| Shaving/Electric clippers | HANGZHOU HUAYUAN PET PRODUCTS CO., LTD. | DTJ-002 | For shaving mice |
| Stainless Steel Medical Needle Holder 14cm Coarse Needle | Premier Medical Equipment Co. | SP784 | For mouse surgery |
| Stainless Steel Ophthalmic Forceps 10.5cm Curved (No Hook) | Zhuoyouyue | YKNWW-10.5 | For mouse surgery |
| Stainless Steel Ophthalmic Scissors/Surgical Scissors 10CM Straight Tip | Premier Medical Equipment Co. | ZYJD-10-ZJ | For mouse surgery |
| Stainless Steel Tip Gastric Needle 12 Gauge 55mm Elbow | GWJ-12-55W | For use in mice by gavage | |
| Tris-EDTA Antigen Repair Fluid (50x) | proteintech | PR30002 | For antigen repair of paraffin sections |
| Wooden dissecting board 25*16cm | JP16*24 | For mouse surgery | |
| Xiaoyao pills | Jiuzhitang Co.,Ltd. | YPG-041 | For animal drug delivery |
| Others | |||
| R 4.3.1 | Data processing | ||
| Cytoscape3.9.1 | National Resource for Network Biology | Building a regulatory network for traditional Chinese medicine | |
| ImageJ 1.54f | National Institutes of Health | Image processing for immunohistochemistry results | |
| Adobe Photoshop 24.0.0 | Adobe | For image combination | |
| GraphpadPrism 9.5 | GraphPad Software | Statistical analysis of data | |
| cellsens Dimension | OLYMPUS | For slicing and photographing | |
| OLYMPUS BX53 | OLYMPUS | For HE staining and immunohistochemical section photography |
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