A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This study establishes a rat model of vascular calcification induced by a high-fat diet (HFD) combined with vitamin D3 (VD3). The model was used to evaluate the therapeutic efficacy of salidroside in preventing and treating vascular calcification, providing insights into its potential mechanisms of action through network pharmacology and in vivo experiments.

Abstract

Vascular calcification (VC) is a critical pathological condition associated with significant morbidity and mortality. This study employs a hybrid approach of network pharmacology and molecular biology to delineate the therapeutic mechanisms of salidroside (SAL), an active compound from Rhodiola crenulata, against VC. Through database mining and network analysis, 388 SAL targets intersecting with 2871 VC-associated targets were identified, resulting in 208 common targets. A protein-protein interaction (PPI) network constructed via the String database and topological analysis in Cytoscape 3.9.1 pinpointed 10 key targets, including IL6, TNF, TP53, IL1B, HIF1A, CASP3, and STAT3, among others. The identified genes were concentrated in the lipid and atherosclerosis pathways, indicating that the improvement of VC by SAL may occur through the regulation of abnormal expression of lipid and inflammatory factors. It was also found that SAL inhibits the abnormal expression of inflammatory factors, thereby activating the JAK2/STAT3 pathway to intervene in the progression of VC. The JAK2/STAT3 pathway is a key molecular mechanism by which SAL prevents further deterioration of VC. Functional enrichment analyses revealed the involvement of these targets in inflammatory responses and lipid metabolism, pivotal pathways in VC. In vivo studies in rats demonstrated SAL's efficacy in mitigating dyslipidemia and vascular inflammation, with improved serum lipid profiles and reduced vascular calcium deposition. The mechanistic exploration, grounded in Western blot analysis, demonstrated salidroside's ability to regulate the JAK2/STAT3 signaling pathway, highlighting its potential as a modulator in this critical molecular mechanism and offering a potential therapeutic target for VC. The strength of this research lies in its methodological rigor, integrating computational predictions with in vivo validations. This comprehensive approach establishes a robust framework for exploring the therapeutic mechanisms of natural compounds in combating VC.

Introduction

Vascular calcification (VC) refers to the abnormal deposition of calcium within the vessel walls, which leads to arterial stiffening and decreased elasticity, ultimately impairing vascular function. Traditionally, VC has been divided into two types: intimal calcification, linked to lipid buildup, and medial calcification. The former is closely associated with inflammatory infiltration, triggering an osteogenic transformation in the vascular wall, characterized by the migration, proliferation, and differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells1.

The ability of VSMCs to undergo osteogenic differentiation, influenced by factors such as aging, genetics, and environmental conditions like diabetes and chronic kidney disease, is a major contributor to age-related VC. This osteoblast-like transformation exacerbates arterial calcification and degeneration1.

VC is a multifaceted condition, driven by degenerative changes, metabolic imbalances, and various systemic conditions. Approximately 80% of vascular injuries and 90% of coronary artery disease cases exhibit VC, significantly increasing the risk of severe cardiovascular events1,2. Therefore, there is a pressing need to discover pharmacological treatments that effectively mitigate or reverse this condition.

Currently, treatment strategies for VC involve various pharmacological interventions, though no drugs are specifically designed for this purpose. For patients with mild calcification, statins are often prescribed to stabilize plaques. However, while they may reduce coronary artery stenosis by lowering lipid levels, their effect on calcification is limited2.

Given the complexities of atherosclerosis, many patients exhibit enhanced platelet activation, necessitating the use of antiplatelet drugs like aspirin or clopidogrel to inhibit platelet aggregation and reduce the risk of thrombosis. However, aspirin therapy is only beneficial for individuals with a high coronary artery calcium score and a low risk of bleeding3.

Additionally, research into supplements, such as vitamin K, suggests potential in preventing VC progression4. In severe cases, invasive interventions may be considered, although they are often unsuitable for widespread VC5. For individuals without existing VC, managing risk factors, such as blood pressure, lipid profiles, and lifestyle choices, remains critical6.

Rhodiola crenulata, a perennial herb of the Crassulaceae family, has been traditionally utilized in Chinese medicine. Its principal bioactive constituent, salidroside, commands significant attention due to its notable biological activities. Salidroside is renowned for its ability to inhibit apoptosis, exhibit robust antioxidant properties, and possess anti-inflammatory characteristics7,8. These attributes contribute to its potential to enhance vascular function, delay vascular aging, and safeguard the vascular endothelium. As a potential therapeutic agent for VC, salidroside holds substantial value for research. However, the precise mechanisms by which salidroside ameliorates VC remain to be fully elucidated and warrant further investigation to harness its therapeutic potential in the treatment of VC.

To explore these mechanisms, this study leverages network pharmacology, an innovative methodology that combines pharmacology, bioinformatics, and computer science to analyze biological systems and elucidate drug mechanisms. Compared to traditional single-target drug research, network pharmacology offers a more comprehensive approach by analyzing a drug's effects on multiple biological targets and signaling pathways. As a key tool in modern drug development, it constructs networks of drugs, targets, and pathways to reveal the underlying mechanisms of drug action9,10. Despite its extensive use in exploring therapeutic mechanisms, there has been limited research into the interactive mechanisms between salidroside and VC from the perspectives of bioinformatics and network pharmacology.

This research constructs a molecular network map of salidroside's potential impact on VC by identifying and analyzing key targets through extensive database mining. A protein-protein interaction (PPI) network is generated, and topological analysis is applied to highlight critical nodes in the calcification process.

To confirm the computational predictions, a rat model of VC is developed by administering a high-fat diet with vitamin D3 (VD3). This model replicates the pathological features of human VC. Vascular injury is assessed through histological techniques, serum lipid profiles and inflammation markers are evaluated to investigate the systemic effects of salidroside, and the expression of SAL anti-VC related proteins is measured using Western blotting to exploring the impact of salidroside on experimentally induced VC, this study aims to contribute valuable insights into the potential of this compound as a therapeutic strategy for combating VC.

Protocol

The protocol was approved by the Experimental Animals Committee of Changchun University of Chinese Medicine (Approval No. 2023091). This study adheres to international guidelines, including the European Community Guidelines and the EEC Directive of 1986, ensuring the ethical treatment of animals throughout the study. Male Wistar rats (8-10 weeks, weight 200-220 g) were used for the study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Network pharmacology prediction of potential salidroside-VC targets

NOTE: Network pharmacology utilizes computational methods and large-scale data analysis to investigate the complex interactions between drug molecules and biological targets such as pathways, genes, and proteins within an organism11,12. This approach helps to decipher the biological functions and relationships of the studied entities. The methodology encompasses database utilization, processing of chemical information, acquisition of bioactivity data, retrieval of protein data, analysis of gene expression profiles, construction of interaction networks, and enrichment analysis of pathways11. Figure 1 shows the interaction network of core targets between salidroside and vascular calcification.

  1. Construction of the "Ingredient" target database
    1. Use "Salidroside" as the keyword for ingredients to search databases13 (see Supplementary Table 1), such as HERB, TCMSP, PubChem, SwissTargetPrediction, CTD, PharmMapper, SEA, and STITCH.
    2. Review relevant literature to identify targets associated with salidroside, with the species set to Homo sapiens. After removing duplicates, standardize the target proteins using UniProt (see Supplementary Table 1) and establish a comprehensive Salidroside target database14.
  2. Construction of the "Disease" target database
    1. Use "Vascular calcification" as the keyword to search databases15 (See Supplementary Table 1), including GeneCards, OMIM, PharmGkb, and DrugBank, with the species set to Homo sapiens. After de-duplication, create a vascular calcification target database.
  3. Prediction of potential therapeutic targets
    1. Input the targets for salidroside and vascular calcification (see Supplementary Table 1) to identify common targets. Generate a Venn diagram to visualize the potential therapeutic targets for salidroside in treating vascular calcification.
  4. Construction of the "Salidroside-Vascular Calcification" Protein-Protein Interaction (PPI) network
    1. Compile the potential targets into a Multiple Proteins List and analyze them using STRING (see Supplementary Table 1), with the organism set to Homo sapiens and the interaction score set to medium confidence (>0.4)16. Extract the PPI data in TSV format for further analysis.
      NOTE: Given that 85% of rat genes are homologous to human genes, performing similar biological functions, rats were selected as experimental subjects for validating the effects of salidroside on vascular calcification17.
  5. Selection and network construction of key targets
    1. Import the PPI network data into Cytoscape 3.9.1 (see Supplementary Table 1) for analysis using the CytoNCA plugin to assess parameters such as Betweenness (BC), Closeness (CC), Degree (DC), Eigenvector (EC), Local Average Connectivity (LAC), and Network Centrality (NC). Select key targets with CC=1 to construct a core target action spectrum map for 'SAL-VC'.
    2. Use the CytoHubba plugin to identify the top 10 hub genes based on Maximal Clique Centrality (MCC), Maximum Neighborhood Component (MNC), and Degree calculations, generating a Hub-genes spectrum map.
  6. GO and KEGG pathway enrichment analysis
    1. Perform gene ID conversion using DAVID (see Supplementary Table 1), selecting ENSEMBL_GENE_ID for the gene type-9606 for species information. Analyze the converted gene list using Omicshare (see Supplementary Table 1) for GO functional and KEGG pathway enrichment, with significance set at P value < 0.0518.
      NOTE: GO functional analysis includes molecular function (MF), biological process (BP), and cellular component (CC). KEGG pathway analysis involves pathway enrichment and pathway classification enrichment19. The enrichment analysis results are depicted in Figure 2.

2. Animal experiment

  1. Acclimatization
    1. Acclimate Wistar rats under specific pathogen-free (SPF) conditions with a 12-h light/dark cycle. Ensure they have ad libitum access to food and water to maintain their health prior to the commencement of experiments. Perform adaptive feeding for 1 week.
  2. Model establishment
    1. Acclimate the rats to the environmental conditions and randomly assign them into five groups: Control group (Ctrl, ND + Vehicle), Model group (Model, HFD + Vehicle), SAL low-dose group (SAL-L, HFD + SAL 5mg/kg), SAL high-dose group (SAL-H, HFD + SAL 10mg/kg), and Simvastatin (SIM) group (HFD + SIM 5mg/kg).
    2. Administer a normal diet (ND) to the Ctrl group and a high-fat diet (HFD) to the remaining groups for the entire duration of the experiment. On the first day of HFD administration, inject a single subcutaneous dose of 600,000 IU/kg VD3 to all groups except the Ctrl group20,21. Follow with weekly subcutaneous injections of 100,000 IU/kg VD3 for the following 8 weeks (Figure 3).
    3. Monitor the health status and survival of the animals daily. Begin experimental interventions in the ninth week.
    4. Euthanize the animals at the conclusion of the study (following the institutionally approved protocols). Collect serum samples, allow them to stand for 30 min, and centrifuge to isolate the serum (following previously published reports20,21). Dissect vascular tissues (abdominal aorta), and rinse with phosphate-buffered saline (PBS) to remove blood.
    5. Fix one portion of the tissue in 4% paraformaldehyde for histological examination and store another portion in liquid nitrogen for molecular analysis.
      NOTE: Dilute salidroside with warm water before use.

3. Evaluation of vascular tissue injury using HE, VK, EVG staining

NOTE: Fix vascular tissue (abdominal aorta) in 4% paraformaldehyde, dehydrated in ethanol after 48 h, and embedded in paraffin. Cut the embedded paraffin blocks into 5 Β΅m slices for Hematoxylin-Eosin (HE), Elastica van Gieson (EVG), and Von Kossa (VK) staining, and observe the histological morphology under a light microscope. HE staining is used to assess changes in tissue morphology. In vascular tissue, it highlights structural alterations in the vessel wall, including smooth muscle cell proliferation, disorganized cell arrangement, and inflammation. EVG staining visualizes elastic and collagen fibers, which is essential for evaluating elastic fiber damage or remodeling in vascular tissue and helps in understanding the impact of calcification on vascular elasticity. VK staining detects calcium deposits, a key feature in VC, making it crucial for assessing the extent and distribution of calcification in vascular tissue22,23.

  1. HE staining for vascular tissue damage detection
    1. Deparaffinization and rehydration
      1. Deparaffinize sections in two changes of xylene for 8 min each and rehydrate through a graded ethanol series (100%, 95%, 85%, 75%) for 3 min per step. Follow with a 2-min rinse in running tap water.
    2. Hematoxylin staining
      1. Stain the sections with hematoxylin for 5-10 min and wash to remove excess stain, followed by a rinse with running tap water.
        ​NOTE: A light stain of 5 min is recommended to avoid overly dark staining, which can affect cytoplasmic color.
    3. Differentiation
      1. Differentiate the sections in a differentiation solution for 30 s, followed by two rinses in tap water for 3 min each.
    4. Eosin counterstaining
      1. Place the sections in eosin stain for 1 min. After removing the excess stain, perform rapid dehydration.
    5. Dehydration, clarification, and mounting
      1. For rapid dehydration, dip the sections in 75%, 85%, 95%, and 100% ethanol for 3 s each, followed by 100% ethanolΒ  for 1 min.
        NOTE: Rapid dehydration is recommended as eosin may lose color in water and ethanol gradients.
  2. VK staining for calcium detection
    1. Deparaffinization and rehydration
      1. Perform this step following step 3.1.1.
    2. Silver nitrate reaction
      1. Blot the sections dry, outline with a fine brush, and stain with Von Kossa (see Table of Materials). Expose them to ultraviolet light for 4 h, then thoroughly rinse with running tap water.
    3. Counterstaining with hematoxylin
      1. Stain the sections with hematoxylin for 5 min, rinse in running tap water, differentiate, and rinse again, followed by a running tap water rinse.
    4. Eosin counterstaining
      1. Dehydrate the sections through graded ethanol (85% and 95% for 5 min each), then stain with eosin for 5 min.
    5. Dehydration and mounting
      1. Dehydrate the sections in ethanol baths (100% Ethanol I, II, III for 5 min each) and clarify in xylene baths (Xylene I and II for 5 min each). Then, mount the sections with neutral balsam.
    6. Microscopic examination and image capture
      1. Examine the stained sections under a light microscope and capture images for analysis.
        NOTE: Ensure that calcium deposits appear brown-black to dark black, nuclei are blue, and the background is red. Prepare the silver nitrate solution for fresh VK staining immediately before use.
  3. EVG staining for elastic and collagen fibers
    1. Deparaffinization and rehydration
      1. Treat the paraffin sections with heat for 50 min, then immerse them in xylene for 20 min to remove paraffin. Proceed by subjecting the sections to a graded ethanol series (100%, 95%, 85%, 75%) for 5 min each, concluding with a rinse in running tap water for 5 min.
    2. EVG staining
      1. Apply the EVG staining solution (see Table of Materials) for 10 min, then rinse briefly in running tap water for 5 s to remove excess stain.
      2. Next, apply Verhoeff working solution (see Table of Materials) for 5 min, followed by another brief rinse in running tap water for 5 s. Apply Verhoeff's differentiating solution for 5 s until the elastin fibers are clearly visible, and then rinse with running tap water for 5 s.
        NOTE: Mix components A, B, and C (provided in the commercially available kit) in a 5:2:2 ratio to prepare the Verhoeff working solution before use. Use the solution within 2 h and replenish as needed during the staining process to prevent the sections from drying out.
    3. Dehydration and mounting
      1. Dehydrate the sections through a graded ethanol series (75%, 85%, 95%, and 100%) for 5 s each, followed by a clarification in two changes of xylene for 1 min each. After a brief air-drying period, mount the sections with neutral balsam.
    4. Microscopic examination and image analysis
      1. Examine the stained sections under a light microscope to visualize elastic and collagen fibers, and capture images for subsequent analysis.

4. Alkaline Phosphatase (ALP) assay

NOTE: Use ALP as a key indicator to evaluate the effectiveness of anti-calcification treatments.

  1. Reagent preparation
    1. Dissolve the color-developing substrate in ice-cold 0.05 M pH 9.6 carbonate buffer to a final volume of 2.5 mL.
      NOTE: Prepare the buffer by dissolving 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in 1,000 mL of double-distilled water.
  2. Substrate dilution
    1. Dilute 10 Β΅L of p-nitrophenol solution (10 mM) with 0.05 M pH 9.6 carbonate buffer to a final volume of 0.2 mL to achieve a final concentration of 0.5 mM.
  3. Sample preparation
    1. Homogenize the abdominal aorta in lysis buffer. Centrifuge the homogenate (~12,000 x g for 10 min at 4 Β°C) and collect the supernatant for ALP activity detection.
      NOTE: Ensure the lysis buffer is free of phosphatase inhibitors. Store pending test samples at -80 Β°C, avoiding repeated freeze-thaw cycles.
  4. Microplate preparation for assay
    1. Prepare a 96-well plate with blank, standard, and sample wells. Add 50 Β΅L of the standard solution to standard wells and 50 Β΅L of pending test samples to sample wells. Incubate the plate at 37 Β°C for 10 min.
  5. Reaction termination and absorbance measurement
    1. Add 100 Β΅L of stop solution to each well to terminate the reaction. Measure the absorbance at 405 nm and calculate ALP activity based on absorbance values (following the manufacturer's instructions, see Table of Materials).
      NOTE: Wells containing the standard or samples with ALP activity will show varying shades of yellow. The color is stable for up to 2 h.

5. Calcium content determination

NOTE: Calcium content determination is critical for assessing the extent of mineralization in biological tissues.

  1. Tissue preparation
    1. Mince the tissue into small pieces and homogenize in lysis buffer.
  2. Sample dilution and centrifugation
    1. Dilute the tissue in lysis buffer at a ratio of 1:10. Homogenize the mixture and centrifuge at 4 Β°C, 12,000 x g for 5 min. Collect the supernatant for analysis.
      NOTE: Prepare calcium standard dilutions (0-1.0 mM) using a 5 mM calcium standard solution (see Table of Materials).
  3. Plate setup and incubation
    1. Add 50 Β΅L of standard or test samples to each well of a 96-well plate. Add 150 Β΅L of assay working solution, mix thoroughly, and incubate the plate in the dark at room temperature for 10 min. Measure the absorbance at 575 nm and construct a standard curve.
  4. Calculation of calcium content
    1. Calculate the calcium content using the standard curve, incorporating the dilution factor, sample volume, and the atomic weight of calcium.

6. Enzyme-Linked Immunosorbent Assay (ELISA) for inflammatory cytokines (IL-6, TNF-Ξ±, IL-1Ξ²)

NOTE: IL-6, IL-1Ξ², and TNF-Ξ± are key pro-inflammatory cytokines that indicate the presence and severity of an inflammatory response. Measuring these cytokines is essential for understanding the inflammatory process and evaluating the effectiveness of anti-inflammatory treatments.

  1. Sample collection
    1. Collect serum from the rat's abdominal aorta. Allow it to coagulate at room temperature for 2 h. Centrifuge the sample at 3,000 x g for 10 min at 4 Β°C and collect the supernatant.
  2. Microplate preparation for the assay
    1. Prepare a 96-well plate with wells designated for standards and test samples. Add 50 Β΅L of standards (see Table of Materials) at varying concentrations to the appropriate wells.
  3. Sample preparation
    1. Add 40 Β΅L of sample diluent to each well designated for test samples. Add 10 Β΅L of the sample to the same wells, resulting in a 5-fold dilution.
  4. Incubation with enzyme-labeled reagents
    1. Add 100 Β΅L of enzyme-labeled reagents specific to IL-6, TNF-Ξ±, or IL-1Ξ² to all wells except the blank. Incubate the plate at 37 Β°C for 60 min.
  5. Washing
    1. Discard the liquid from all wells except the blank. Wash the wells with 1x wash solution (prepared as a 20-fold dilution in distilled water). Repeat this washing step five times and dry the wells.
  6. Color development and termination
    1. Add 50 Β΅L of substrate solutions A and B (from the commercially available kit, see Table of Materials) to each well. Mix gently and incubate the plate at 37 Β°C in the dark for 15 min. Add 50 Β΅L of stop solution to each well to terminate the reaction.
  7. Absorbance measurement and data analysis
    1. Set the blank well as zero. Measure the absorbance at 450 nm using a microplate reader. Generate a standard curve based on the OD values and standard concentrations. Calculate the sample concentrations using interpolation and adjust for the dilution factor.

7. Lipid profile assay

NOTE: The Lipid profile assay detects abnormal lipid levels, where elevated or imbalanced lipid levels can accelerate the risk of vascular calcification.

  1. Total cholesterol and triglycerides (TC and TG)
    1. Collect serum and prepare a 96-well plate with designated wells for blank, calibration, and pending test samples. Add 2.5 Β΅L of serum, calibration standard, or blank solution to each well.
    2. Add 250 Β΅L of the working solution to each well. Mix gently and incubate the plate at 37 Β°C for 10 min. Measure the absorbance at 500 nm using a microplate reader.
  2. Low-density lipoprotein and high-density lipoprotein (LDL-C and HDL-C)
    1. Collect serum and prepare a 96-well plate with wells designated for blank, calibration, and pending test samples. Add 2.5 Β΅L of serum, calibration standard, or blank solution to the respective wells.
    2. Add the appropriate reagent (Reagent One for 180 ΞΌL or Reagent Two for 60 ΞΌL, see Table of Materials) to each well as specified in the assay kit instructions. Incubate at the temperature (37 Β°C) and for the time (Reagent One for 5 min or Reagent Two for 10 min) recommended for each reagent.
    3. Measure the absorbance at the specific wavelength (600 nm) indicated for LDL-C or HDL-C using a microplate reader.

8. Western blotting

NOTE: Western blot (WB) is instrumental in assessing the expression levels of key proteins, allowing for the detection of both total and phosphorylated forms.

  1. Tissue preparation
    1. Weigh 0.05 g of tissue and rinse thoroughly with PBS to remove excess debris. Add 500 Β΅L of lysis buffer containing 1x phosphatase and protease inhibitors. Incubate the tissue at 4 Β°C for 10 min and homogenize it using a tissue grinder.
  2. Protein extraction
    1. Allow the homogenized sample to stand for 1 min, then centrifuge at 4 Β°C and 12,000 x g for 15 min. Collect the supernatant for protein analysis. Determine the protein concentration using the BCA method following the manufacturer's instructions (see Table of Materials). Use a standard curve to calculate the concentration.
      NOTE: Pre-cool the centrifuge to 4 Β°C before use. Measure protein concentration at 562 nm.
  3. Sample preparation
    1. Mix the protein sample with 5x loading buffer containing Ξ²-mercaptoethanol and SDS in a 4:1 ratio. Heat the mixture at 100 Β°C for 5 min to denature the proteins.
      ​NOTE: If 1x loading buffer is required, dilute the 5x buffer with lysis buffer.
  4. SDS-PAGE gel preparation
    1. Prepare a 12% SDS-PAGE gel and place it in the electrophoresis apparatus. Add 1x electrophoresis buffer up to the halfway mark as per the instructions (see Supplementary Table 2).
  5. Electrophoresis
    1. Retrieve the protein samples from -20 Β°C. Load 60 Β΅g of protein per well and run the gel.
    2. Set the current to 50 mA for 20 min for the stacking gel, then increase to 100 mA for 60 min for the separating gel until the dye front reaches the bottom.
  6. Membrane transfer
    1. Activate the PVDF membrane in methanol for 5 min.
    2. Assemble the transfer sandwich in the following order: filter paper β†’ SDS-PAGE gel β†’ PVDF membrane β†’ filter paper. Transfer proteins at 300 mA for 60 min.
      ​NOTE: Ensure no air bubbles are trapped between the SDS-PAGE gel and PVDF membrane.
  7. Blocking
    1. Incubate the PVDF membrane in 5% BSA (prepared in 1x TBST) at room temperature with gentle shaking for 60 min. Wash the membrane with 1x TBST three times for 5 min each.
  8. Primary antibody incubation
    1. Incubate the membrane in 5 mL of the appropriate primary antibody (e.g., p-JAK2, JAK2, p-STAT3, STAT3, p-NF-ΞΊB p65, NF-ΞΊB p65, IΞΊBΞ±) diluted in blocking buffer at room temperature for 2.5 h.
  9. Secondary antibody incubation
    1. Wash the membrane with 1x TBST three times for 5 min each after primary antibody incubation. Incubate with the appropriate secondary antibody at room temperature for 1 h. Wash the membrane again with 1x TBST three times for 5 min each.
  10. Detection
    1. Visualize protein bands using ECL chemiluminescence detection24 (see Table of Materials).
  11. Densitometry analysis
    1. Quantify protein bands using ImageJ software. Follow this workflow in ImageJ: Image β†’ 8-bit β†’ Process β†’ Subtract Background. Analyze β†’ Set Measurements β†’ Analyze β†’ Set Scale. Edit β†’ Invert β†’ Analyze β†’ Measure β†’ Results. File β†’ Save As β†’ IntDen.
    2. Generate statistical graphs using graphing and statistical analysis software.
      NOTE: Ensure results are consistent across replicates to validate findings.

9. Statistical analysis

  1. Collect and organize data using GraphPad Prism 9.0.
  2. Calculate and plot error bars using mean Β± SD from raw data.
  3. Perform statistical analysis using one-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons.
  4. Determine the statistical significance at P < 0.05, with smaller P-values indicating greater differences in the results.

Results

Network pharmacology analysis

Using databases such as HERB, TCMSP, Pubmed, SwissTargetPrediction, CTD, PharmMapper, SEA, and STITCH, 388 potential target genes for salidroside were identified. Additionally, 2871 potential target genes related to VC were retrieved from databases like GeneCards, OMIM, PharmGkb, and DrugBank. Intersection analysis via VENN diagrams revealed 208 overlapping targets, considered key targets for salidroside's intervention in VC (

Discussion

VC is characterized by degenerative changes in vascular cells and tissues, with pathological mineral deposits within blood vessels leading to stiffening of the vessel walls or the formation of atherosclerotic plaques, which can result in obstructive vascular diseases25. Studies show that about 85% of VC plaques may evolve into thrombosis, which can trigger acute cardiovascular episodes. Additionally, VC is a crucial indicator of potential acute cardiovascular events, strokes, and peripheral vascul...

Disclosures

Ensure that all authors have disclosed any and all conflicts of interest.

Acknowledgements

This work was financially supported by the Jilin Provincial Department of Science and Technology Project (YDZJ202301ZYTS460), and Jilin Provincial Department of Education Project (JJKH20230991KJ).

Materials

NameCompanyCatalog NumberComments
30% (29:1) Acrylamide/Bis SolutionBeijing Solarbio Science & Technology Co., Ltd ,ChinaA1010
4% Paraformaldehyde Fix SolutionBeyotime Biotech Inc (Beyotime) , ChinaP0099
5*loading bufferBeijing Solarbio Science & Technology Co., Ltd ,ChinaP1040
Alkaline Phosphatase Assay KitBeyotime Biotech Inc (Beyotime) , ChinaP0321S
AlphaView SoftwareProteinsimple Inc.USAAlphaView SA
BCA Protein Assay KitBeyotime Biotech Inc (Beyotime) , ChinaP0012
Bluing SolutionBeijing Solarbio Science & Technology Co., Ltd ,ChinaG1866
Calcium Colorimetric Assay KitBeyotime Biotech Inc (Beyotime) , ChinaS1063S
Collagen Fiber And Elastic Fiber Staining Kit(EVG-Verh eff Method)Beijing Solarbio Science & Technology Co., Ltd ,ChinaG1597
Dewatering machineDiapath BiosciencesΒ  Ltd, ItalyDonatello
Embedding machineWuhan Junjie Electronics Co., Ltd,ChinaJB-P5
Enzyme-labeled instrumentΒ Biotek Co., Ltd,USAEpoch
Ethanol absoluteGHTECHΒ  Co., Ltd, China64-17-5
Goat Anti-Mouse IgG (H+L) HRPBioworld technology, co, Ltd.,ChinaBS20242-Y
GraphPad Prism SoftwareGraphPad Software.,USAGraphPad Prism 9.0
Hematoxylin-Eosin Stain KitBeijing Solarbio Science & Technology Co., Ltd ,ChinaG1120
High-density lipoprotein cholesterol assay kitNanjing Jiancheng Bioengineering Research Institute Co., Ltd,ChinaA112
HRP-labeled Goat Anti-Rabbit IgG(H+L)Guangzhou saiguo biotech Co.,LTDA0208
Image J SoftwareNational Institutes of Health(NIH),USAImage JΒ 
IΞΊB Alpha Polyclonal antibodyProteintech Group, Inc.A,USA10268-1-AP
JAK2 AntibodyAffinity Biosciences Co., Ltd,ChinaAF6022
Low-density lipoprotein cholesterol assay kitNanjing Jiancheng Bioengineering Research Institute Co., Ltd,ChinaA113
NF-ΞΊB p65 AntibodyProteintech Group, Inc.A,USA10745-1-AP
Pathological microtomeLeica Biosystems,USARM2016
Phosphatase Inhibitor Cocktail TablesF. Hoffmann-La Roche, Ltd,Switzerland04906845001
Phospho-JAK2 (Tyr931) AntibodyAffinity Biosciences Co., Ltd,ChinaAF3024
Phospho-NF-ΞΊB p65(Ser276) AntibodyAffinity Biosciences Co., Ltd,ChinaAF2006
Phospho-STAT3(S727) AntibodyAbwaysΒ  Science & Technology Co., Ltd ,ChinaCY5291
Protease Inhibitor CocktailΒ F. Hoffmann-La Roche, Ltd,Switzerland11873580001
PVDF membraneF. Hoffmann-La Roche, Ltd,Switzerland3010040001
Rat IL-1Ξ² ELISA KitBeyotime Biotech Inc (Beyotime) , ChinaPI303
Rat IL-6 ELISA KitBeyotime Biotech Inc (Beyotime) , ChinaPI328
Rat TNF-Ξ± ELISA KitBeyotime Biotech Inc (Beyotime) , ChinaPT516
RIPA Lysis BufferBeyotime Biotech Inc (Beyotime) , ChinaP0013B
SalisorosideShanghai yuanye Bio-Technology Co., Ltd,ChinaS25475
SDSGuangzhou saiguo biotech Co.,LTD,China3250KG001
Sodium carbonateChina National Pharmaceutical Group Co., Ltd. , China1001921933
Sodium hydrogen carbonateChina National Pharmaceutical Group Co., Ltd. , China10018960
Sodium thiosulfateChina National Pharmaceutical Group Co., Ltd. , China20042518
STAT3 AntibodyProteintech Group, Inc.A,USA10253-2-AP
TBST (10Γ—)Beyotime Biotech Inc (Beyotime) , ChinaST673
Total cholesterol assay kitNanjing Jiancheng Bioengineering Research Institute Co., Ltd,ChinaA111
Triglyceride assay kitNanjing Jiancheng Bioengineering Research Institute Co., Ltd,ChinaA110
Tris BaseGuangzhou saiguo biotech Co.,LTD1115GR500
Upright optical microscopeNikon Corporation,JapanEclipse E100
Von Kossa SolutionΒ Wuhan servicebio technology CO.,LTD,ChinaG1043
Western Blotting Luminol ReagentSanta Cruz Biotechnology, Inc. ,USASC-2048
Ξ²-Actin antibodyΒ Cell Signaling Technology, Inc.,USAE4967

References

  1. Sutton, N. R., et al. Molecular mechanisms of vascular health: Insights from vascular aging and calcification. Arterioscler Thromb Vasc Biol. 43 (1), 15-29 (2023).
  2. Henein, M. Y., Owen, A. Statins moderate coronary stenoses but not coronary calcification: Results from meta-analyses. Int J Cardiol. 153 (1), 31-35 (2011).
  3. Ajufo, E., et al. Value of coronary artery calcium scanning in association with the net benefit of aspirin in primary prevention of atherosclerotic cardiovascular disease. JAMA Cardiol. 6 (2), 179-187 (2021).
  4. Vossen, L. M., Kroon, A. A., Schurgers, L. J., De Leeuw, P. W. Pharmacological and nutritional modulation of vascular calcification. Nutrients. 12 (1), 100 (2019).
  5. Kereiakes, D. J., et al. Principles of intravascular lithotripsy for calcific plaque modification. JACC Cardiovasc Interv. 14 (12), 1275-1292 (2021).
  6. Demer, L. L., Watson, K. E., BostrΓΆm, K. Mechanism of calcification in atherosclerosis. Trends Cardiovasc Med. 4 (1), 45-49 (1994).
  7. Chen, F., et al. Network pharmacology analysis combined with experimental validation to explore the therapeutic mechanism of salidroside on intestine ischemia-reperfusion. Biosci Rep. 43 (8), BSR20230539 (2023).
  8. Rong, L., et al. Salidroside induces apoptosis and protective autophagy in human gastric cancer ags cells through the pi3k/akt/MTOR pathway. Biomed Pharmacother. 122, 109726 (2020).
  9. Zhang, P., et al. Network pharmacology: Towards the artificial intelligence-based precision traditional Chinese medicine. Brief Bioinform. 25 (1), bbad518 (2023).
  10. Jiao, X., et al. A comprehensive application: Molecular docking and network pharmacology for the prediction of bioactive constituents and elucidation of mechanisms of action in component-based Chinese medicine. Comput Biol Chem. 90, 107402 (2021).
  11. Li, S., Zhang, B. Traditional Chinese medicine network pharmacology: Theory, methodology and application. Chin J Nat Med. 11 (2), 110-120 (2013).
  12. Wang, X., Hu, Y., Zhou, X., Li, S. Editorial: Network pharmacology and traditional medicine: Setting the new standards by combining in silico and experimental work. Front Pharmacol. 13, 1002537 (2022).
  13. Huang, Z., Yang, Y., Fan, X., Ma, W. Network pharmacology-based investigation and experimental validation of the mechanism of scutellarin in the treatment of acute myeloid leukemia. Front Pharmacol. 13, 952677 (2022).
  14. Zhang, R., Zhu, X., Bai, H., Ning, K. Network pharmacology databases for traditional Chinese medicine: Review and assessment. Front Pharmacol. 10, 123 (2019).
  15. Wang, C., Liu, X., Guo, S. Network pharmacology-based strategy to investigate the effect and mechanism of alpha-solanine against glioma. BMC Complement Med Ther. 23 (1), 371 (2023).
  16. Li, X., et al. Network pharmacology prediction and molecular docking-based strategy to explore the potential mechanism of Huang Lian jiedu decoction against sepsis. Comput Biol Med. 144, 105389 (2022).
  17. Holmes, R. S., et al. Recommended nomenclature for five mammalian carboxylesterase gene families: Human, mouse, and rat genes and proteins. Mamm Genome. 21 (9-10), 427-441 (2010).
  18. Geng, J., Zhou, G., Guo, S., Ma, C., Ma, J. Underlying mechanism of traditional herbal formula Chuang-ling-ye in the treatment of diabetic foot ulcer through network pharmacology and molecular docking. Curr Pharm Des. 30 (6), 448-467 (2024).
  19. Chen, X., et al. Puerarin inhibits emt induced by oxaliplatin via targeting carbonic anhydrase xii. Front Pharmacol. 13, 969422 (2022).
  20. Herrmann, J., Babic, M., Tolle, M., Van Der Giet, M., Schuchardt, M. Research models for studying vascular calcification. Int J Mol Sci. 21 (6), 2204 (2020).
  21. Zhou, H., X, W., Yuan, Y., Qi, X. Comparison of methods for establishing a rat model of atherosclerosis using three doses of vitamin D3 and atherogenic diet. Chin J Arterioscler. 20 (11), 995-998 (2012).
  22. Zhang, Y., et al. Il-18 mediates vascular calcification induced by high-fat diet in rats with chronic renal failure. Front Cardiovasc Med. 8, 724233 (2021).
  23. Kazlouskaya, V., et al. The utility of elastic verhoeff-van gieson staining in dermatopathology. J Cutan Pathol. 40 (2), 211-225 (2013).
  24. Tang, X., et al. Underlying mechanism and active ingredients of tianma gouteng acting on cerebral infarction as determined via network pharmacology analysis combined with experimental validation. Front Pharmacol. 12, 760503 (2021).
  25. Lee, S. J., Lee, I. K., Jeon, J. H. Vascular calcification-new insights into its mechanism. Int J Mol Sci. 21 (8), 2685 (2020).
  26. Magdic, J., et al. Intracranial vertebrobasilar calcification in patients with ischemic stroke is a predictor of recurrent stroke, vascular disease, and death: A case-control study. Int J Environ Res Public Health. 17 (6), 2013 (2013).
  27. Zhou, L., et al. Salidroside-pretreated mesenchymal stem cells contribute to neuroprotection in cerebral ischemic injury in vitro and in vivo. J Mol Histol. 52 (6), 1145-1154 (2021).
  28. Hutcheson, J. D., Goettsch, C. Cardiovascular calcification heterogeneity in chronic kidney disease. Circ Res. 132 (8), 993-1012 (2023).
  29. Zhang, X., et al. Salidroside: A review of its recent advances in synthetic pathways and pharmacological properties. Chem Biol Interact. 339, 109268 (2021).
  30. Zhang, P., Li, Y., Guo, R., Zang, W. Salidroside protects against advanced glycation end products-induced vascular endothelial dysfunction. Med Sci Monit. 24, 2420-2428 (2018).
  31. Li, Y., et al. Salidroside promotes angiogenesis after cerebral ischemia in mice through shh signaling pathway. Biomed Pharmacother. 174, 116625 (2024).
  32. Gao, X. F., Shi, H. M., Sun, T., Ao, H. Effects of radix et rhizoma Rhodiolae kirilowii on expressions of von Willebrand factor, hypoxia-inducible factor 1 and vascular endothelial growth factor in myocardium of rats with acute myocardial infarction. Zhong Xi Yi Jie He Xue Bao. 7 (5), 434-440 (2009).
  33. Li, X., Liu, C., Li, Y., Xiong, W., Zuo, D. Inflammation promotes erythropoietin induced vascular calcification by activating p38 pathway. Bioengineered. 13 (3), 5277-5291 (2022).
  34. Bessueille, L., Magne, D. Inflammation: A culprit for vascular calcification in atherosclerosis and diabetes. Cell Mol Life Sci. 72 (13), 2475-2489 (2015).
  35. Li, R., et al. Salidroside prevents tumor necrosis factor-alpha-induced vascular inflammation by blocking mitogen-activated protein kinase and nf-kappa B signaling activation. Exp Ther Med. 18 (5), 4137-4143 (2019).
  36. Xing, S. S., et al. Salidroside attenuates endothelial cellular senescence via decreasing the expression of inflammatory cytokines and increasing the expression of sirt3. Mech Ageing Dev. 175, 1-6 (2018).
  37. Hopkins, A. L. Network pharmacology. Nat Biotechnol. 25 (10), 1110-1111 (2007).
  38. Xin, P., et al. The role of jak/stat signaling pathway and its inhibitors in diseases. Int Immunopharmacol. 80, 106210 (2020).
  39. Fu, X., et al. Glycosides from buyang huanwu decoction inhibit atherosclerotic inflammation via jak/stat signaling pathway. Phytomedicine. 105, 154385 (2022).
  40. Macri, F., et al. High phosphate-induced jak-stat signalling sustains vascular smooth muscle cell inflammation and limits calcification. Biomolecules. 14 (1), 107328 (2023).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

SalidrosideVascular CalcificationNetwork PharmacologyTherapeutic AgentRhodiola CrenulataProtein protein InteractionInflammatory FactorsLipid MetabolismJAK2 STAT3 PathwayDyslipidemiaSerum Lipid ProfilesCalcium DepositionIn Vivo StudiesTherapeutic Mechanisms

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright Β© 2025 MyJoVE Corporation. All rights reserved