Bone-Protective Role of Bupleurum in a Rat Model of Postmenopausal Osteoporosis

Summary

In this study, we used an ovariectomized rat model of postmenopausal osteoporosis to demonstrate that Bupleurum (Chai hu) oral liquid improves bone microstructure by suppressing pro-inflammatory factors and enhancing anti-inflammatory ones. This suggests that its anti-inflammatory mechanism partially mitigates estrogen deficiency-induced osteoporosis.

Abstract

The protocol aims to investigate the effects of Chai hu administration on postmenopausal osteoporosis in ovariectomized (OVX) rats. Eight-week-old female Sprague-Dawley rats underwent bilateral ovariectomy using the dorsal approach to establish a postmenopausal osteoporosis model. Different doses of Chai hu oral liquid were administered by oral gavage for 12 consecutive weeks, followed by evaluations of bone microstructure and measurements of inflammatory cytokine levels. Medium- and high-dose Chai hu treatment partially inhibited bone loss, reduced pro-inflammatory cytokine expression, and upregulated anti-inflammatory cytokine levels in OVX rats. There was a significant reduction in the severity of bone destruction from low to high doses, and dose-related improvements in bone microstructural reconstruction were observed. Chai hu mitigated estrogen deficiency-induced bone loss and microstructural deterioration, thereby suppressing OP progression in this OVX model. This was likely to have resulted from the anti-inflammatory effects of the pathways involved.

Introduction

Osteoporosis (OP) is a common metabolic bone disease characterized by decreased bone mass, reduced bone strength, and destruction of bone microarchitecture¹. OP can manifest as either primary or secondary, and primary OP is classified as postmenopausal OP (PMOP), senile OP, and idiopathic OP (which often occurs in adolescents). Among them, type I PMOP occurs primarily in PM women, where there is a marked decrease in bone density after menopause due to reduced circulating levels of estrogen². It is a systemic metabolic bone disease, which results in increased osteoclast activity and intense differentiation³. In addition, the intensity of bone resorption is much greater than its formation, resulting in decreased bone mass as well as increased bone fragility and risk of fracture⁴.

Current research has found that many chronic inflammatory diseases, such as osteoarthritis and periodontitis, are associated with significant bone loss, suggesting that inflammation may be closely related to OP⁵. As PM women age, in addition to endocrine changes, physiological and immune functions decline, and the body is left in a state of chronic inflammation for long periods of time. Many of these inflammation-promoting factors and T-cell-derived cytokines play important roles in bone loss. Therefore, PMOP is also recognized as a chronic inflammatory disease^6,7.

Traditional Chinese medicine (TCM) has a long history and rich clinical experience in the prevention and treatment of PMOP, focusing on holistic management and individualized diagnosis and treatment, and can regulate the endocrine and immune systems at the same time, which is a blessing for postmenopausal women⁸. It allows a large proportion of women to have access to medical treatment. Modern pharmacological studies and clinical trials have shown that TCM has the advantages of multi-targeting, low toxicity and side effects, and is cheap. The risk of complications can be greatly reduced in the TCM treatment of PMOP⁹.

Chai hu itself has anti-inflammatory¹⁰ and antidepressant¹¹properties, and it can regulate hormone secretion in the body¹² as well as aiding good health¹³. Chai hu is the main ingredient that plays a major role in the TCM compound formula, Xiaoyao Pills, which has the effect of soothing emotions, promoting digestion, and absorption in the stomach and intestines. It is also known for replenishing qi and blood energy as well as regulating the symptoms of the menstrual cycle and its associated discomfort¹⁴. The main constituent of Chai hu is Chai hu saponin, which has strong anti-inflammatory activity, mainly manifested in inhibiting the expression of pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β, and IL-6¹⁵. In addition, it enhances the expression of anti-inflammatory cytokine, IL-10, thereby alleviating inflammation, such as colitis, endometritis, and hepatitis¹⁶.

Compared with standard OP treatments (including the use of bisphosphonates¹⁷, selective estrogen receptor modulators^18, and denosumab¹⁹), Chai hu, as a commonly used TCM, has the potential advantage of a multi-component, multi-target mechanism of action. Standard therapies usually target specific bone metabolic pathways²⁰ (inhibition of osteoclast activity), which may be associated with certain side effects such as gastrointestinal discomfort, increased risk of osteonecrosis of the jaws, and flu-like symptoms, as well as high long-term drug costs²¹.

A review of the relevant literature found that the use of Chai hu to intervene in PM rats with OP could have therapeutic alleviating effects²². Thus, based on bioinformatics and pharmacological analysis as well as molecular docking simulation to predict docking sites²³, we hypothesized that Chai hu could intervene in the inflammatory process, modulate the immune status of the body, and restore bone immune homeostasis. These will favor the stability of the bone reconstruction state and achieve the desired therapeutic effect of alleviating OP.

This study employed Chai hu oral liquid (concentration: 1 g/mL) administration over 12 consecutive weeks. The dosing regimen was designed based on clinically common doses in TCM and pharmacokinetic studies²⁴.

The overall goal of this administration method is to establish a stable and clinically relevant dosing protocol, thereby systematically evaluating the sustained intervention effect of Chai hu in the target model (a rat model of postmenopausal osteoporosis). This administration approach ensures that the drug maintains effective plasma concentration throughout the experimental period, providing a reliable intervention basis for subsequent assessments (such as bone metabolism indicators and inflammatory factor levels). Furthermore, it helps clarify the role of Chai hu in related diseases as well as its dose-effect relationship, while offering a reference paradigm for the standardized administration of traditional Chinese medicines in experimental research.

Protocol

All experimental procedures involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (8th edition) and were specifically approved by the Animal Ethics Committee of Youjiang Medical University for Nationalities (Approval No. 2024071001).

1. Analyzing network pharmacology

NOTE: Cyberpharmacology is an approach that combines systems biology, computational biology, and pharmacology and aims to understand the mechanism of action of drugs and drug-drug interactions by analyzing biomolecular networks²⁵.

1. Screen active compounds with good oral bioavailability (OB) and drug-likeness (DL) using tools such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP: http://ibts.hkbu.edu.hk/LSP/tcmsp.php). Set the OB at ≥30%, and the DL at ≥0.18²⁶. Search Chai hu and click Herb Details and download all compound data. Name the downloaded file Chaihu.
2. Obtain the target name of the core ingredient by pasting the MOL ID of the core ingredient into the TCMSP database. Paste into TCMSP batch search and select Target Prediction, then click Submit.
3. Open the UniProtsh database, copy the Target Name column into the search, and find the bolded protein name, which is exactly the same as the pasted target protein name. After finding the corresponding protein name, collect the corresponding gene names in the column after it.
4. Filter the osteoporosis-related genes in the GeneCards (https://www.genecards.org) database according to the Relevance score ≥ 1 and obtain all the (5741) genes²⁷.
   NOTE: The reasons for selection of genes with a Relevance score ≥ 1 from the GeneCards database are as follows: A score ≥ 1 indicates that the gene has documented associations with the target disease, clearly distinguishing it from genes with no reported associations (score = 0). This threshold ensures the retention of genes with credible disease relevance while avoiding excessive interference from unrelated genes. A lower threshold (<1) might include genes without validated associations, thereby increasing false positives.
5. Obtain the intersecting genes after combining disease-related gene sets with drug targets for intersection. Based on the interactions between disease targets and drug active ingredients, construct a traditional Chinese medicine-active ingredient-target-disease intersection network using Cytoscape software.
6. Obtain protein-protein interaction (PPI) information through the String database (https://cn.string-db.org/) and construct a PPI network. Open Cytoscape (https://www.cytoscape.org), load the downloaded TSV file at the upload network, then select Calculate > TOP10 or TOP20, and click Submit.
7. Create two tables for importing into Cytoscape. Create an electronic spreadsheet file named Network. In Column A, input the same MOL IDs (CH1, CH2...) that have been replaced with the abbreviation of the traditional Chinese medicine. Label Column B as MOL ID and sort it in ascending order by MOL IDs. Label Column C as Symbol. Copy component IDs (MOL IDs) and gene names from Chaihu-related files into Columns B and C, respectively. Then delete Column B and rename Column A as MOL ID.
8. Extend the table to the end of the text. In the Symbol column, input the MOL IDs (CH1, CH2...), replaced with the abbreviation of the traditional Chinese medicine. In the MOL ID column, input the abbreviation of the traditional Chinese medicine: CH. With the above steps, the information supplementation and the construction of the Traditional Chinese Medicine - Component - Target network is completed.
9. Next, do the second form, the TYPE file. Create a new spreadsheet and name it TYPE. Label column A as Term and column B as TYPE. Copy the gene name to the term column and give it the gene attribute in the type column (the purpose is to search for the gene; all the gene names will be selected, so that it is easy to operate). Input the ingredients into the Term column and give the ingredient the attribute drug1.
10. Import into the Cytoscape software. Import Network to upload the network file and import table to upload the type file. In the search box, type gene, press Enter, so that all the genes are selected, pull the selected genes to the left, and then click Layout > Attribute() > Selected (choose round or square in the parentheses).
11. Combine the results of network pharmacology analysis with experimental data to identify the key compounds and molecular mechanisms of drug action.

2. Molecular docking

NOTE: Molecular docking is a computational technique for predicting the optimal binding mode and affinity between a small molecule (ligand) and a target protein or biomolecule²⁸.

1. Receptor and ligand preparation
   1. Receptor (Protein) source: Obtain the target proteins' PDB file from the RCSB Protein Data Bank (https://www.rcsb.org). Search 3Q4B (IL-6 receptor example), click Download files > PDB format.
   2. Remove crystallographic water molecules by clicking Edit > Delete > Water, extraneous ions, and non-essential ligands (retain critical cofactors like metal ions) by clicking Edit > Delete > Heteroatoms. Add missing hydrogen atoms by clicking Edit > Hydrogens > Add. Save as protein.pdbqt. by clicking Edit > Charges > Compute Gasteiger then Grid > Macromolecule > Choose > Save as PDBQT.
   3. Ligand (Small Molecule) source: Download ligand structure from PubChem (https://pubchem.ncbi.nlm.nih.gov). Search Saikosaponin A (CID:6443568), click 3D Conformer > Download SDF.
   4. Use Open Babel software to convert the Saikosaponin A file in SDF format into a PDB format file with low-energy three-dimensional conformations, and name it saikosaponinA.
2. Docking parameter setup (AutoDock tools)
   1. Load receptor and ligand files. Import the small molecule file saikosaponin A and save it as a PDBQT file. Subsequently, load the receptor and ligand: click Docking > Macromolecule > Open, select IL6_receptor.pdbqt; then click Docking > Ligand > Open, select saikosaponinA.pdbqt.
   2. Configure the grid box: Click Grid > Grid Box, adjust the box size to fully encompass the active site (use the control dial on the left panel to fine-tune), then save the current settings vby clicking File > Close Saving Current and name the file grid.gpf.
   3. Set up the AutoGrid running program: Click Run AutoGrid, select the grid.gpf file, and then click Launch. Next, configure the docking parameters: In sequence, under the Docking menu, click Macromolecule and select the protein file IL6_receptor.pdbqt; click Ligand and select the small molecule file saikosaponin A.pdbqt; set Genetic Algorithm and confirm it; adjust Docking Parameters and confirm them. Finally, in Output, select the Lamarckian (4,2) algorithm, name the file dock.dpf, and save it.
   4. Execute AutoGrid and save the results: Click Run AutoDock, select the dock.dpf file, and launch the simulation. Upon completion, click Write Complex, name the complex structure, and save it in PDBQT format.
3. Result Analysis using PyMOL
   1. Convert the PDBQT format file obtained from the previous molecular docking step to PDB format using the Open Babel software. Then import the PDB format file into the PyMOL software for subsequent visualization operations.
   2. Locate the ligand and separate it individually: After selecting the ligand, change its color by clicking Sele and then choosing a Color from Color. In the Sele column, click Extract Object under Action to extract the ligand as a new independent object.
   3. Hydrogen bond distance measurement: In the sele column, click Action, find Polar Contacts, and click To Other Atoms in Object; hydrogen bonds will be displayed as yellow lines. Click the Measurement option in the Wizard column, click the Atoms at Both Ends of the Hydrogen Bond in sequence, and then click Done to complete the measurement.
4. Export the image: Click Export Image as PNG in File. If high-definition rendering is needed, enter ray 300 (pixels adjustable) and press Enter.

3. Micro-CT analysis

1. Inject female SD rats (32 weeks old, 320-350 g) intraperitoneally with pentobarbital sodium (40 mg/kg) for deep anesthesia, confirmed by absence of corneal reflex (no blinking upon cotton swab touch) and pain reflex (no hindlimb contraction to forceps pinch).
   1. Perform hindlimb isolation surgery. Euthanize the rats by intraperitoneal injection of pentobarbital sodium at a dose 3-5 times the surgical anesthetic dose (40 mg/kg), place them supine, and make a 3 cm transverse incision, 1 cm above the pubic symphysis along the inguinal ligament. Bluntly dissect the gap between the gracilis and sartorius muscles to expose the femoral artery (pulsatile tubular structure). Ligate both ends of the femoral artery using 4-0 sutures, leaving an unligated section in between, before truncating the vessel with ophthalmic scissors and transect it.
   2. Rotate the lower limb internally to dislocate the femoral head from the acetabulum and cut the joint capsule and round ligament, preserving femoral head integrity. Immerse the intact hindlimb in pre-cooled saline, then fix in 4% formaldehyde solution for 24-48 h.
2. Imaging setup: Deliver the fixed femur to the facility for micro-CT scanning. Turn on the Micro-CT device and select the Small Animal Bone Scanning preset program. Use the following scan parameters: X-ray voltage = 90 kV, X-ray current = 80 µA, Field of view (FOV) = 36 mm, Pixel size = 72.0 µm, Rotation = 360°, Scan duration = 14 min.
3. Place the fixed rat's left leg femur in the sample holder, fix the femoral epiphysis to be photographed, start the device, and wait for the machine self-test to complete. Initiate a preview scan (low-resolution fast scan) to confirm the sample is centered, then adjust the region of interest to cover the entire femur.
4. When the scanning and photographing is completed, the X-ray light source will be turned off automatically. At this time, the radiation value inside the machine exceeds the standard, and it is absolutely forbidden to take out the sample without protective equipment.
5. Image analysis: Analyze bone parameters (bone mineral density [BMD], trabecular number) using the scanner's built-in software. Save and export reconstructed images for further evaluation.

4. Quantitative real-time PCR for RNA extraction from rat bone tissue

1. Remove adherent muscles and fascia from the right femur. Cut 100-200 mg of bone tissue into small pieces. Add 2-3 grinding beads and 500 µL of RNA extraction buffer. Homogenize using a tissue grinder at 70 Hz for 5 min at 4 °C.
2. Phase separation: After centrifugation, the solution separates into an upper aqueous phase (contains RNA) and a lower dark precipitate (contains proteins, DNA, and polysaccharides). Carefully transfer 500 µL of the supernatant to a new RNase-free 1.5 mL tube.
3. Process the upper solution obtained from the isolation for total RNA extraction according to the manufacturer's instructions.
4. After adding isopropanol to the lysis solution, gently invert the tube to mix the solution and let it stand at room temperature for 10 min. Perform a visual check at this point; if white flocculent RNA precipitates are observed (adhering to the tube wall), it indicates that RNA has been effectively precipitated.
5. Use a 200 µL pipette tip to aspirate residual liquid. Retain the white RNA pellet at the tube bottom/wall. Resuspend RNA in 50-100 µL of RNase-free water and store at -80 °C.
6. Mix 2 µL of RNA template, 10 µL of master mix, 2 µL of DNase, and 5 µL of Nuclease-Free Water in a nuclease-free tube. Mix gently by pipetting.
7. Incubate at 37 °C for 2 min to remove genomic DNA contamination. Incubate at 55 °C for 15 min. At the end of the reaction, incubate at 85 °C for 5 min to abort the reaction.
8. Configure the real-time fluorescence quantitative PCR system by placing the reverse-transcribed DNA on ice. Pre-mix all components: 10 µL of qPCR SYBR Green Master Mix (No Rox), 0.4 µL each of Forward Primer and Reverse Primer, and 8.2 µL of RNase-free water, divide and add 1µL of DNA, manipulate on ice, and protect from light.
9. Run the qPCR program as follows: Pre-denaturation at 95 °C for 5 min, 1 cycle followed by denaturation at 95 °C for 10 s for 40 cycles. Perform annealing, extension, fluorescence acquisition at 60 °C for 30 s for 40 cycles in the SYBR Green channel. Obtain a melting curve for temperature from 65-95 °C with 0.5 °C increment/step and 5 s/step. The primers used are provided in Table 1.

5. Animal preparation

1. Obtain 48 female nulliparous SD rats (SPF-grade, aged 8 weeks, body weight 200-220 g).
2. Prior to experimental procedures, acclimatize all rats with a 7-day quarantine period in an SPF-grade isolation facility and house in an SPF-controlled laboratory environment (temperature: 22-24 °C, humidity: 45%-55%).

6. Preparation of test drug

1. Obtain Chai hu oral liquid (China National Medical Product Approval No. Z41021539). The formulation contains Chai hu (Northern source) as the active ingredient, with excipients including flavoring agent, sucrose, and propylene glycol. Each 10 mL vial contains an extract equivalent to 10 g crude herb.

7. Preparation of ovariectomized rat model

1. Preoperative preparation: Fast rats for 12 h with ad libitum access to water. Perform instrument sterilization by autoclaving all surgical tools.
2. Anesthesia and preparation: Administer pentobarbital sodium by intraperitoneal injection (40 mg/kg). Check the depth of anesthesia by assessing corneal reflex absence (no blink on corneal touch with cotton swab) and toe-pinch reflex absence (no hindlimb retraction on clamping). After 15-20 min, shave the surgical site, disinfect with iodine, and cover with a fenestrated drape. Apply vitamin A eye ointment (containing 15,000 IU/g) during the operation to prevent the animal's eyes from drying out (Table 2).
3. Ovariectomized rat model: Perform the surgery in a dedicated sterile operating room. All personnel wore full isolation gowns. Wipe the countertop and surrounding equipment with 75% ethanol (Table 2) before surgery, and turn on the ultraviolet lamps for at least 30 min to eliminate microbes in the air and on surfaces.
   1. Make a 1 cm incision on the dorsal skin, bluntly dissect the subcutaneous tissue, pinch up the muscle layer, and make a 1 cm incision. Locate the fat pad and exteriorize it to identify the ovary (bright red in color). Ligate the fallopian tube with 4-0 suture, excise the ovarian tissue, and place it into a 50 mL centrifuge tube containing 4% paraformaldehyde (Table 2).
4. Wound closure and disinfection: Sprinkle penicillin powder into the wound prior to suturing. Suture the muscle layer with No. 6 non-absorbable suture and close the skin with No. 2 suture. Disinfect the closed incision with iodine and apply topical penicillin powder.
5. Postoperative care: Place rats in fresh cages with warm water bottles and towels to maintain body temperature. Fast (water allowed) animals for 24 h post-surgery to prevent intestinal obstruction. Apply penicillin powder to the incision site daily for 3 consecutive days.
   1. Inject lidocaine (1%-2% concentration, maximum 5 mg/kg) around the surgical incision to reduce immediate postoperative pain. Provide continuous analgesia for 3-5 days, tapering the dose gradually based on pain severity to avoid abrupt discontinuation.
   2. Keep the animal in lateral position or head-down position before waking to prevent tongue fall and airway obstruction; allow autonomous comfortable positioning after waking. Isolate postoperative animals from others until full recovery to avoid fighting or cross-infection (Figure 1).

8. Experimental grouping and drug administration

1. Group allocation: Divide a total of 40 SD rats into five groups (n = 8 per group), namely, normal control, model control, low concentration Chai hu, medium concentration Chai hu, and high concentration Chai hu.
2. For the medium dose (5 mL/kg) group, select dosage based on guidelines found in^29,30. Follow a concentration gradient ration of 1:2:4 for low concentration group: 2.5 mL/kg, medium concentration group: 5 mL/kg and high concentration group: 10 mL/kg.
3. Administration of drug: Use oral gavage and administer the drug once daily. For the normal control group and model control group (animals subjected to bilateral ovariectomy), perform oral gavage with an equal amount of saline. Commence treatment at week 1 after surgery and continue for 12 weeks.

9. Post-experiment analysis and data collection

1. Perform the following analyses on five animal groups: micro-CT of bone structure and determination of inflammatory cytokine mRNA expression in rat femoral tissues.
2. Collect data which include visualized images of femoral trabecular microstructure; quantitative bone parameters (including bone mineral density, bone volume fraction, trabecular thickness standard deviation, trabecular spacing, trabecular number, and connectivity density); and relative mRNA expression levels of pro-inflammatory cytokines (IL-6, IL-17) and anti-inflammatory cytokines (IL-4, IL-10) in femoral tissues.
3. Perform data analyses using GraphPad Prism 10.1.2 software by one-way analysis of variance (ANOVA). Statistical significance for inter-group differences in means was defined as p < 0.05. Post-hoc multiple comparisons were executed using Tukey's test.

Results

GO function and KEGG pathway enrichment analysis

We relied on GO and KEGG enrichment analysis in bioinformatics to predict the potential mechanism of action of Chai Hu on PMOP. The GO enrichment analysis of target genes intersecting with the active ingredients of Chai hu for diseases yielded 2138 significant entries, and the top ten significant results are listed for each group (Figure 2D). With respect to biologi...

Discussion

PMOP is a systemic disorder of bone metabolism that often occurs in PM women, and it is caused by a lack of estrogen that results in a disruption of the osteoblast-osteoclast balance, leading to enhanced osteoclast bone resorption and diminished osteoblast bone production³¹. A growing number of studies have shown that the levels of inflammatory factors in the body also affect the differentiation between osteoclasts and osteoblasts, and that overexpression of pro-inflammatory factors contributes to...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% sodium chloride injection	Huarenpharmaceutical	h1712059
1×PBS	Thermofisher	10010023
75% alcohol	Nanjingreagent	C069156931
95% ethanol	Nanjingreagent	C0691520075
Anhydrous ethanol	Macklin	E809056
Bupleurum chinense	Henan Baiquan Pharmacy Co.	Z41021539
decalcification solution	solarbio	E1171
erythromycin ointment	Guangzhou Baiyunshan Pharmaceutical Group Company Limited Baiyunshan Hejigong Pharmaceutical Factory	H44023088
Isopropyl alcohol	Jinan Jinrihe Chemical Co.	23727-29-9
penicillin V potassium capsules	Sichuan Pharmaceutical	H20103156
polyformaldehyde (CH2O)n	Beyotime	P0099
povidone-iodine disinfectant	Guangdong Hengjian Pharmaceutical Co.	H44023924
Real-Time Fluorescence PCR Kit	Yeasen Biotechnology	11201ES03
RNA Extraction Kit	Monadbiotech	MI20201S
RNA Reverse Transcription Kit	YuGongBiotech	EG15123S
Sodium pentobarbital	Shanghai Jixiang Biotechnology Co.	JX082085
Trizol	Thermofisher	15596026CN