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Method Article

Integrating Network Pharmacology to Investigate Cinnamaldehyde's Protective Effect Against Oxaliplatin-induced Neurotoxicity

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DOI:

10.3791/69013

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November 28th, 2025

 ,  ,  , 
1Department of Integrated Traditional Chinese and Western Medicine Medical Oncology, Cancer Hospital of China Medical University, Cancer Hospital of Dalian University of Technology, Liaoning Cancer Hospital & Institute, 2China Medical University, 3Liaoning University of Traditional Chinese Medicine

In This Article

Summary

This study investigates the protective effects of cinnamaldehyde (CA) against oxaliplatin (OXA)-induced damage in rat dorsal root ganglion (DRG) cells. The underlying mechanism involves suppression of the JAK2/STAT3 signaling pathway and activation of the SLC7A11-GSH-GPX4 pathway, as revealed through network pharmacology and experimental validation.

Abstract

This study investigates the protective effects of cinnamaldehyde (CA) against oxaliplatin (OXA)-induced damage in rat dorsal root ganglion (DRG) cells. The pharmacological activities of CA were analyzed, and its potential targets, along with those of its metabolites, were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP), and PharmMapper platforms. The CCK-8 assay is used to determine the optimal concentrations of drugs, while malondialdehyde (MDA) assays are applied to assess the level of lipid peroxidation. Intracellular levels of Fe2+ and reactive oxygen species (ROS) were quantified using fluorescent probes. The impact of CA on protein expression levels, including p-STAT3, XCT, and GPX4, in OXA-treated DRG cells was examined via immunoblotting. Network pharmacology analysis identified 14 overlapping targets among "CA-neuropathic pain (NP)-ferroptosis". Experimental results demonstrated that a 24 h treatment with 4.0 µmol/L CA yielded optimal effects, significantly reducing MDA, Fe2+, and ROS levels in DRG cells (P < 0.05). Furthermore, CA treatment downregulated the expression of JAK2, STAT3, p-STAT3, TFRC, ErbB-1, and nuclear factor-kappa B (NF-κB) (P < 0.05) while upregulating XCT, GPX4, and FTH1 expression (P < 0.05). These findings suggest that CA mitigates OXA-induced damage in DRG cells by inhibiting the JAK2/STAT3 signaling pathway and activating the SLC7A11-GSH-GPX4 axis.

Introduction

In recent years, ferroptosis, as a non-apoptotic cell death mode, has been proven to be involved in the tissue damage induced by various chemotherapeutic drugs. The core mechanism of ferroptosis is the accumulation of iron-dependent lipid peroxidation products1. Research on the mechanisms of cisplatin neurotoxicity reveals that this drug induces neuronal ferroptosis through multiple pathways, with this process being directly linked to the development of neurotoxicity. Specifically, cisplatin inhibits glutathione peroxidase 4 (GPX4) activity. As a critical "brake molecule" in ferroptosis, the inactivation of GPX4 prevents the clearance of lipid hydroperoxides (L-OOH), thereby causing oxidative damage to neuronal cell membranes2. Meanwhile, oxaliplatin can promote the release of ferroptosis in nerve cells and exacerbate the generation of reactive oxygen species (ROS) through the Fenton reaction, further amplifying the ferroptosis cascade3. This evidence suggests that targeted regulation of neurodegeneration may be a potential strategy to alleviate the neurotoxicity of oxaliplatin.

Oxaliplatin (OXA), a widely used third-generation platinum-based chemotherapeutic agent for colorectal cancer, offers robust antitumor efficacy but is frequently associated with significant neurotoxicity. Chronic administration often leads to OXA-induced neuropathic pain (OINP), with an incidence rate reaching 60%4, characterized by mechanical allodynia, thermal hyperalgesia, and cold hypersensitivity5. Despite its prevalence, current treatment strategies for OINP remain inadequate, substantially diminishing patient quality of life. Consequently, elucidating the molecular mechanisms underlying OINP and identifying novel therapeutic targets remain critical areas of ongoing research6.

Cinnamaldehyde (CA), the primary active constituent of cinnamon essential oil, is a yellow, viscous liquid7,8 with diverse biological activities, including insecticidal9, antimicrobial10, antifungal11, antioxidant12, antidiabetic13, anticancer14, anti-inflammatory, and neurovascular protective effects15,16. Emerging evidence suggests that CA may inhibit the activation of the JAK/STAT signaling pathway17. The chemical formula of CA is C9H8O, and its chemical structure is shown in Figure 1. As a natural monomer of traditional Chinese medicine, CA exhibits a wide range of pharmacological activities. In terms of neuroprotection, a number of studies have confirmed that CA can enter the central nervous system through the blood-brain barrier, promote the expression of antioxidant enzymes (such as HO-1 and NQO1) by activating the Nrf2/ARE signaling pathway, reduce the accumulation of ROS in nerve cells, and thus alleviate the neuro-oxidative damage in ischemic stroke, Alzheimer's disease, and other diseases18,19. At the same time, CA can inhibit microglia hyperactivation, reduce inflammatory factor (such as TNF-α, IL-1β) release, and reduce neuroinflammatory response20. However, it has not been reported whether CA can alleviate the neurotoxicity induced by oxaliplatin by regulating the ferroptosis pathway.

The JAK2/STAT3 signaling pathway is an important intracellular signal transduction mechanism that transmits signals received at the cell membrane to the cell nucleus, and is closely related to key biological processes such as cell proliferation, differentiation, apoptosis, oxidative stress, and inflammatory responses21. As a pathway closely related to inflammatory responses, the JAK3/STAT3 signaling pathway can trigger inflammatory responses in an activated state, leading to tissue inflammatory damage. Studies indicate that inhibiting the JAK3/STAT3 signaling pathway can reduce the inflammatory response and oxidative damage in rats with vascular dementia, thereby improving the cognitive function of the rats22. In addition, inhibiting the JAK3/STAT3 signaling pathway can also alleviate the inflammatory response in epileptic rats, reduce the rate of neuronal apoptosis, and mitigate brain injury23. Although caffeic acid (CA) has a certain protective effect on the nervous system, whether its improvement effect is related to the regulation of the JAK3/STAT3 signaling pathway is currently not clearly concluded.

This study aims to validate whether the in vitro inhibition of JAK2/STAT3 signaling and the activation of the LC7A11-GSH-GPX4 axis can mitigate OXA-induced damage in rat DRG cells. Additionally, it explores the potential anti-ferroptotic effects of CA and examines the role of key targets identified through network pharmacology in modulating inflammation in DRG cells.

Current therapeutic approaches for oxaliplatin-induced neurotoxicity primarily focus on chelating free platinum ions to mitigate nerve damage, such as using calcium magnesium complexes, with the main goal of alleviating acute peripheral neuropathies (e.g., numbness and tingling in hands and feet)24. However, the exact mechanisms remain unclear. These treatments lack capacity to reverse chronic conditions like axonal degeneration and nerve fiber deterioration caused by oxaliplatin, and demonstrate limited efficacy in preventing cumulative neurotoxicity. In this experiment, the neuroprotective effect of 4 µM CA under 5 µM OXA treatment was explored by combining anti-inflammatory and anti-ferroptosis mechanisms. The findings confirm that activation of this mechanism serves as a critical initiating step in OINP. The discovery of specific inhibitors could replace the existing nonspecific approaches, thereby enhancing both precision and effectiveness of therapeutic interventions.

The in vitro model used in this experiment involves isolated rat dorsal root ganglion (DRG) neurons for studying OINP. However, this model differs significantly from human primary cells in genetic background, metabolic characteristics, and toxicity sensitivity, which may render research conclusions difficult to directly apply clinically. Moreover, the functional maintenance of in vivo neural tissues relies on precise regulation of multicellular coordination and dynamic microenvironments - complex factors that are challenging to fully replicate in vitro models.

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Protocol

NOTE: The cell line was derived from rat cell lines.

1. Network pharmacology analysis

NOTE: The links can be found in the Table of Materials.

  1. Finding possible targets of CA
    1. Retrieve structural information from the PubChem database and explore the potential biological mechanisms of CA. Subsequently, conduct the target prediction analysis using SwissTargetPrediction, leveraging its advanced computational capabilities.
    2. To enhance the accuracy and comprehensiveness of the analysis, cross-reference the predicted targets with Traditional Chinese Medicine-specific databases, including TCMSP and TCMIP.
    3. Review the relevant literature to identify additional potential targets. Duplicate the consolidated target list to create a comprehensive dataset, serving as a critical reference for further biological activity studies and drug development involving CA.
  2. Identification of potential targets of neuropathic pain (NP)
    1. By utilizing the GeneCards database and the TTD database, conduct an extensive search using the keywords "neuropathic pain" and "ferroptosis." Prioritize the gene targets from the GeneCards database with a "Relevance score" exceeding the median, as these genes demonstrate higher pertinence to the research focus.
    2. Integrate the previously selected gene targets with data retrieved from the TTD (Therapeutic Target Database). Ensure comprehensive identification of potential targets during the integration process by cross-referencing all selected gene targets with TTD database entries. Systematically remove duplicate entries from the integrated dataset of gene targets. Verify that the resulting dataset contains a unique list of potential targets.
  3. Identification of the intersection targets of "CA-NP- Ferroptosis"
    1. Determine the intersection of targets for CA, NP, and ferroptosis using a Venn diagram generated online.
    2. These intersection targets were designated as potential candidates for the "CA-regulated NP-ferroptosis pathway."
  4. Building the Protein-Protein Interaction (PPI) network
    1. To further investigate, submit the common targets for "CA-NP-ferroptosis," "CA-NP," and "CA-ferroptosis" to the STRING database.
    2. Set the interaction score threshold at "highest confidence > 0.4" and restrict the maximum number of interactors to 10 for both the first and second interaction layers. Apply default settings for all other parameters, and construct a 4.5 PPI network for "CA-NP-ferroptosis".
  5. GO and KEGG enrichment analyses
    1. Upload the intersection targets for "CA-NP-ferroptosis" to the DAVID platform with the following parameters: "select identifier: official gene symbol," "list type: genelist," and "species: Homo sapiens." Gene Ontology (GO) enrichment analysis was conducted across three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF).
    2. Perform the KEGG pathway enrichment analysis, and visualize the resultant data using bar plots created on the Hiplot platform.

2. Molecular docking

  1. To elucidate the interaction between active compounds and their disease-associated targets, obtain structural information for active substances from the PubChem database as small molecules, while retrieve the 3D structures of target proteins from the Protein Data Bank (PDB) as protein models.
  2. Utilize PyMOL to preprocess the protein receptor structures by removing water molecules and non-essential ligands. Subsequently, prepare the target proteins and active substances for docking using AutoDock Tools 1.5.7, and execute semi-flexible molecular docking using AutoDock Vina.
  3. Analyze the binding energies and active substance binding sites on the receptor proteins. For the JAK2 protein, its structure (PDB ID: 3e64) was selected from the PDB database based on X-ray crystallography data and optimal resolution.
  4. Use the PyMOL software to prepare the JAK2 structure by eliminating ligands, removing water molecules, and adding hydrogens. The small-molecule structures of active compounds were sourced from PubChem.
  5. Load both protein and compound structures into AutoDock Tools 1.5.7, define the docking boxes, and perform molecular docking with AutoDock Vina.

3. Cell culture

NOTE: The following steps will be repeatedly used in the validation experiment. Adhere to strict aseptic techniques. This part of the experiment used a low-speed horizontal rotor centrifuge.

  1. Disinfect the outer packaging and culture bottles of sterile cells purchased with 75% alcohol, and then transfer to culture dishes on a super-clean workbench for subculture until the cells are spread over five 10 cm diameter petri dishes to ensure sufficient cell stock.
  2. Cryopreserve the cells at -80 °C for subsequent experiments.
    1. Prepare DMEM, cell culture-grade DMSO, fetal bovine serum (FBS), 15 mL centrifuge tubes, pipettes, cryovials, and an ice bath. Mix 1 mL of DMSO, 1 mL of FBS, and 8 mL of DMEM to achieve a total volume of 10 mL. Cool the DMSO on ice for 15 min. First, transfer the base medium to the centrifuge tube, then slowly add the pre-cooled DMSO while rotating. Mix thoroughly with a pipette, then gently add FBS and mix again. Store the mixture in the ice bath for later use.
      NOTE: A concentration of 20%-50% DMSO resulted in a significant increase in LDH release, indicating that high concentrations of DMSO are cytotoxic, while 10% DMSO is relatively safe. This did not affect our experiment25.
    2. Take logarithmically growing cells, wash them 3 times with PBS, then add 0.25% trypsin at 400 µL/10 cm dish.
      NOTE: Once cytoplasmic retraction occurs, connections become loose, or if there are signs of large-scale floating, the digestion process is immediately stopped.
    3. Pre-digestion preparation: Preheat key reagents (0.25% trypsin-EDTA solution and DRG complete medium) to 37 °C. Remove the cell culture flask from the incubator and discard the old medium using a sterile pipette to prevent residual medium from affecting digestion efficiency. Gently rinse the single-layer cultured cells twice with 5 mL sterile PBS to remove residual serum while avoiding cell elution.
    4. Add 2 mL of preheated 0.25% trypsin-EDTA solution to the culture vessel, ensuring it completely covers the single cell layer. Incubate at room temperature for 1-3 min. During this period, observe the cells under an inverted microscope every 30 s. Immediately stop digestion when most cells begin to contract, become round, and show distinct intercellular spaces (to prevent over-digestion and cell damage).
    5. Quickly add 4 mL of fully preheated medium to the culture vessel. Gently pipette the mixture 5 times with a sterile pipette to disperse the cell cluster into a single-cell suspension. Transfer the entire single-cell suspension to sterile 15 mL centrifuge tubes and collect the cell pellet by centrifugation at 300 x g 5 min using a low-speed horizontal rotor centrifuge at 25 °C.
    6. Discard the supernatant and add 5 mL of the prepared cryopreservation medium. Gently blow with a pipette to mix the cells evenly. Distribute them evenly into 5 vials.
    7. Label the cryovial with the cell name, freezing date, and operator; place it in a freezing container and store it in a -80 °C freezer, then transfer to liquid nitrogen after overnight storage.
  3. Cell resuscitation
    1. Wear gloves, remove the vial from the liquid nitrogen container, and immediately immerse it in 37 °C warm water while shaking it occasionally to ensure rapid thawing (within 1 min).
    2. Remove the vial from the 37 °C water bath, open the cap, aspirate the cell suspension with a pipette, transfer it to a centrifuge tube, add 10 times the volume of culture medium, mix well, and centrifuge at 300 x g for 5 min (25 °C).
    3. Discard the supernatant, add 5 mL of culture medium supplemented with 10% fetal bovine serum (the complete medium for this cell culture condition), inoculate the culture dish, and incubate statically in a 37 °C, 95% humidity, 5% CO2 incubator.
    4. Replace the culture medium the next day (to remove residual DMSO and dead cells), and continue observing the culture for growth conditions. If the cell density is high, perform subculture promptly for subsequent experiments.
  4. Cell counting
    NOTE: This part of the experiment used a low-speed horizontal rotor centrifuge.
    1. Prepare 75% alcohol in advance to clean the counting chamber and cover slip, then dry them for later use.
    2. Resuspend the digested and centrifuged cells in 1 mL of medium, and dilute 50 µL of the cell suspension with 150 µL of medium in a 1.5 mL EP tube to make a 4-fold dilution.
    3. Oscillate evenly, take 10 µL, and inject between the coverslip and counting chamber to fully fill the chamber while avoiding bubbles. Place under the microscope for observation and counting. Counting principles: Do not omit or recount; count cells touching the top and left borders, but exclude those touching the bottom and right borders.
    4. According to the experimental requirements, calculate the cell volume based on the desired cell count for plating or dish seeding and the counting results, then supplement the total medium volume with complete medium.

4. Cell viability assay protocol

NOTE: Duloxetine was selected as the western medicine control agent because it is a widely used and clinically validated drug for the treatment of peripheral neuropathic pain. Its established efficacy in this indication (supported by clinical practice guidelines and randomized controlled trials) ensures that the control group can provide a reliable reference for evaluating the relative effectiveness of the test intervention.

  1. First, discard the old culture medium from the culture vessel and rinse the cell surface with 5 mL PBS to remove residual serum. Then, add 2 mL of 0.25% trypsin-EDTA solution and incubate at room temperature for 1-3 min. When microscopic observation reveals cell shrinkage, rounding, and detachment, immediately add 4 mL DRG Complete Medium to terminate digestion and resuspend the cells by pipetting.
  2. Transfer the cell suspension to a centrifuge tube and collect the cell pellet by centrifuging at 300 x g for 5 min using a low-speed horizontal rotor centrifuge at 25 °C. Discard the supernatant and resuspend the cell pellet in a 15 mL centrifuge tube, then thoroughly resuspend and mix the suspension.
  3. Take a 1.5 mL EP tube, and first add 150 µL of culture medium, followed by 50 µL of cell suspension. Mix well to achieve a 4-fold dilution.
  4. Then use 75% alcohol to clean the counting chamber and cover glass, dry them, and shake the EP tube evenly again.
  5. Take out 10 µL of cell suspension and inject it between the cover glass and counting chamber, ensuring the liquid fills the counting chamber without generating bubbles.
  6. Observe under the microscope and count the number of cells according to the counting principle, avoiding omissions or duplicates.
    NOTE: If the counted number of cells is n, then the cell concentration in the centrifuge tube is n/mL.
  7. According to the experimental requirements, seed the cells in a 96-well plate at a density of 2,000 cells per well as needed.
  8. First, inject 200 µL of PBS into each well of the peripheral circle of a 96-well plate, then add cell-containing culture medium according to the grouping.
  9. After incubating in the 37 °C, 95% humidity, 5% CO2 incubator for 24 h, aspirate the old medium and wash twice with 200 µL of PBS. According to the experimental setup, dilute the CA stepwise (0 µM, 2 µM, 4 µM, 8 µM, 16 µM, 32 µM) and add 200 µL to a 96-well plate.
  10. Pretreat the cells with OXA (5 µM) for 24 h, then treat DRG cells with varying concentrations of CA (0 µM, 2 µM, 4 µM, 8 µM, 16 µM, 32 µM) for 24 h, respectively.
    NOTE: This section of the experiment aims to determine the optimal drug concentration for CA.
  11. Wash the 96-well plate three times with PBS buffer, then add 200 µL of freshly prepared CCK-8 working solution to each well. This step is carried out in the dark, and the experiment time is controlled to be completed within 10 min. Incubate the solution for 4 h in a 37 °C, 95% humidity, 5% CO2 incubator. Finally, measure the absorbance values using a microplate reader. Repeat all experiments at least three times under identical conditions.

5. MDA

  1. Seed 10 × 106 DRG cells in a 15 cm dish and culture overnight in a 37 °C, 95% humidity, 5% CO2 incubator. Remove the culture medium, add drugs, and incubate for 24 h in a 37 °C, 95% humidity, 5% CO2 incubator.
  2. Remove the culture medium and wash once with 5 mL of PBS. After digestion, collect the cells into a conical tube using DMEM medium.
  3. Add the treated DRG cells to 5 mL microtubes, respectively. Centrifuge at 300 x g for 5 min with a low-speed horizontal rotor centrifuge, and remove the supernatant. Then, add 1 mL of PBS, mix by pipetting, and transfer to a new 1.5 mL microtube. Centrifuge at 300 x g for 5 min with a low-speed horizontal rotor centrifuge, and remove the supernatant.
  4. Add 100 µL of Antioxidant PBS solution, pipette to mix, and prepare the cell suspension. Prepare the MDA standard solution.
    NOTE: See the preparation of the standard MDA solution using the fluorescence method; refer to the kit's "Kit Components" section for detailed procedures.
  5. Add 100 µL of Lysis Buffer to the sample tube and standard tube, respectively, and mix thoroughly using a vortex oscillator. Let it stand at room temperature for 5 min.
  6. Add 250 µL of the prepared working solution to each tube, and mix thoroughly using a vortex mixer. Heat the water bath to 95 °C in a natural light environment for 15 min, and then put it into the ice bath for 5 min to cool. High-speed angular rotor centrifuge at 10,000 x g for 10 min (4 °C).
  7. Take 100 µL of supernatant and add it to a black 96-well plate. Measure the fluorescence intensity using a fluorescence microplate reader (Ex: 540 nm, Em: 590 nm). Calculate the MDA concentration in the sample based on the standard curve.

6. Fe2+ content in cells measured using a Fe2+ detection kit

  1. Preheat the water bath in advance and raise the temperature to 37 °C. Wear gloves, retrieve the DRG cells from liquid nitrogen, wrap them in a layer of plastic bag, then submerge them in 37 °C warm water while gently shaking intermittently to ensure complete thawing within 1 min.
  2. After thawing, remove the vial from the 37 °C water bath, open the cap, aspirate the cell suspension, transfer it to a 15 mL centrifuge tube, and add 10 mL of culture medium dropwise, mixing thoroughly. Low speed horizontal rotor centrifuge at 300 x g for 5 min (25 °C).
  3. After centrifugation, discard the supernatant, add 10 mL of fresh culture medium, pipette to mix thoroughly, and centrifuge once more. Repeat this procedure 3 times before inoculating onto new culture dishes.
  4. Culture the cells statically in a 37 °C, 95% humidity, 5% CO2 incubator. Replace the medium the next day to remove residual DMSO and dead cells, then continue cultivation. If the cell density is high, subculture timely. Subsequently, collect cells in the logarithmic growth phase, digest them, centrifuge, and resuspend. Seed the resuspended cells into 12-well plates at a density of 70%-80%, then place the plates back into the 37 °C, 95% humidity, 5% CO2 incubator for further cultivation.
  5. Observe the cell state the next day, provide corresponding treatments according to the experimental setup, and then place them back into the 37 °C, 95% humidity, 5% CO2 incubator for continued cultivation.
  6. After 24 h of drug administration, discard the original culture medium and wash the cells once with PBS. Add 300 µL/well of FerroOrange probe (1 µM) diluted in HBSS, and incubate at 37 °C, 95% humidity, 5% carbon dioxide for 30 min.
  7. Then add Hoechst 33342 live cell staining solution (100x) to the cell plate at a ratio of 1:100, incubate at room temperature in the dark for 5 min to stain the cell nuclei.
  8. Observe the staining effect under a fluorescence microscope and take photographs for documentation. Subsequently, use ImageJ to calculate the average fluorescence intensity, with the experiment repeated three times for statistical analysis.

7. ROS detection kit

  1. Take cells in the logarithmic growth phase, digest, centrifuge, and resuspend them, then seed the cells in a 12-well plate at a density (70%-80%). After thoroughly mixing the cells, place them in the 37 °C, 95% humidity, 5% CO2 incubator for continued cultivation.
    NOTE: For the digestion, centrifugation, and resuspension steps, refer to step 4.1.
  2. Observe the cell status the next day, administer corresponding treatments according to the experimental setup, and then place the cells back into the 37 °C, 95% humidity, 5% CO2 incubator for continued culture. After 24 h of drug administration, discard the original culture medium and wash the cells twice with HBSS.
  3. Add 200 µL of Highly Sensitive DCFH-DA Dye working solution, then incubate at 37 °C, 95% humidity, 5% CO2 incubator for 30 min.
  4. Discard the supernatant, wash twice with HBSS, and stain with 2 µL of Hoechst 33342 live cell staining solution (100x) for 5 min, then examine under a fluorescence microscope. Repeat the experiment three times with statistical analysis.

8. Mitochondrial morphology of the cells observed using TEM

  1. Discard the culture medium from the well-cultured cells, add 1 mL of 2.5% room-temperature glutaraldehyde fixative solution, and fix the cells in the dark at room temperature for approximately 5 min. Gently scrape the cells off in one direction using a cell scraper.
  2. Transfer the cell suspension to a centrifuge tube and centrifuge at 25 °C, 600 x g for 3 min using a low-speed horizontal rotor centrifuge.
  3. After discarding the supernatant of the fixative solution, add fresh electron microscopy fixative (use 1 mL per well for a 12-well plate), and fix at room temperature away from light for 30 min, then transfer to 4 °C for storage.
  4. Dehydration infiltration: Insert the sample into the embedding plate and place it in a 37 °C oven overnight.
  5. Polymerize the embedding medium in a 60 °C oven for 48 s. Cut ultrathin sections of 60-80 nm using an ultramicrotome.
  6. Stain the sections with 2% uranyl acetate saturated alcohol solution for 15 min, then with lead citrate for another 15 min. Dry the stained sections overnight at room temperature. Observe and photograph the sections under a transmission electron microscope.

9. Western blotting

  1. Extract the total protein of the cells
    1. Treat and inoculate the cells into a 6 cm petri dish until they reach 70% density.
      NOTE: Refer to step 3.3 for instructions on cell revival and inoculation.
    2. In the culture dish, add 3 mL of OA (5 µM) and after 4 h, add CA (4 µM).
    3. Prepare a box of ice, pre-cool PBS, cell scrapers, prepared lysis buffer, and several labeled EP tubes in advance.
    4. After drug treatment, remove the culture medium from the cells and wash them twice with pre-chilled PBS. Perform this process on ice, and aspirate any residual PBS using a vacuum centrifuge.
    5. Label each petri dish, then add 300 µL of lysis buffer and let it stand on ice for 5 min with constant agitation to prevent incomplete lysis and freezing.
    6. Then use a pre-cooled cell scraper to detach the adherent cells, collecting 300 µL into an EP tube after each scraping. Use a new cell scraper for each scraping and do not reuse it.
    7. Hold the top of the EP tube and mix thoroughly on a vortex mixer, then continue to place it on ice for 25 min. At this time, turn on the 4 °C high-speed angular rotor centrifuge in advance, as pre-cooling the centrifuge requires a certain amount of time.
    8. After the timer ends, vortex once more, then high-speed angular rotor centrifuge at 4500 x g for 15 min in a 4 °C centrifuge.
    9. Now turn on the dry bath heater and set the temperature to 100 °C.
    10. Take out the BCA protein quantification kit, add samples and reagents to the 96-well plate, mix well, and react in a 37 °C, 95% humidity, 5% CO2 incubator for 30 min. Measure the extracted protein content using a multifunctional microplate reader, then adjust with the remaining lysis buffer based on the results to ensure the concentration of each sample is consistent.
    11. Finally, based on the content of each supernatant, add 1/5 volume of 6× Loading Buffer to prepare the protein loading system. Carefully aspirate 150 µL of supernatant into a new EP tube. Add 30 µL of 6× loading buffer to the supernatant and mix thoroughly (typically at a 1:5 ratio). Store the mixture on ice.
    12. Then heat at 100 °C on a heater for 6 min, making sure to hold down the cap of the EP tube to prevent it from popping open. After heating, cool it down in an ice bath.
  2. Prepare a 10% concentration protein gel.
    1. Rinse the gel plate with pure water, air-dry it, wipe off excess moisture, and avoid water stains and dust as much as possible.
    2. Take out the gel casting stand, with the short plates of the two glass plates in front and the long plates at the back, aligning the bottoms to ensure the gap between the glass plates tightly fits with the sealing strip at the bottom of the stand to prevent leakage, then clamp the plates securely to prepare for gel casting.
    3. Prepare the lower gel with 6 mL of pure water, 3.75 mL of 1.5 M Tris-HCl (pH 8.8), 0.15 mL of 10% SDS, 5 mL of 30% Acry/bis, 75 µL of 10% APS, and 7.5 µL of TEMED.
    4. Gently swirl to avoid foaming, then quickly and evenly add between the two glass plates, leaving space for the upper gel. Subsequently, flatten the liquid surface with 1 mL of isopropanol and let it stand for approximately 30 min.
    5. Rinse the comb for gel preparation with pure water, air-dry, and set aside. After the lower gel has completely solidified, pour out the isopropanol, carefully rinse 2-3 times with pure water, absorb the liquid, and invert the gel plate to drain any remaining liquid.
    6. Prepare the upper stacking gel with 6 mL of pure water, 2.5 mL of 0.5 M Tris-HCl (pH 6.8), 0.1 mL of 10% SDS, 1.34 mL of 30% Acry/bis, 50 µL of 10% APS, and 10 µL of TEMED.
    7. Fill the upper layer and carefully insert a 15-well, 1.5 mm thick comb. After 1-2 min, replenish the gaps, taking care to minimize bubble formation during this process. Use a fully polymerized gel.
  3. Protein gel electrophoresis
    1. Pre-configure the Running Buffer and Transfer Buffer, with the Transfer Buffer pre-chilled in a 4 °C refrigerator.
    2. Rinse the protein gel with ultrapure water, take out a clean electrophoresis tank, clamp the two short plates of the protein gel inward and opposite to each other, pour in the Running Buffer, and check for any leakage.
    3. Carefully remove the comb, load 10 µL per well with a total of 4 sample groups. Load 3 sample groups per gel, separated by 4 µL protein marker between each group, totaling 15 wells.
    4. After loading the samples, pour all the remaining Running Buffer into the electrophoresis tank, cover it with the lid, set the voltage to 140 V, and perform constant voltage electrophoresis for 20 min.
    5. When the protein bands enter the separation gel after concentration, adjust the voltage to 120 V for constant voltage electrophoresis. During this process, soak the NC membrane in Transfer Buffer. Stop electrophoresis when the bromophenol blue indicates that the sample has reached the bottom edge of the gel, and prepare for membrane transfer.
    6. Fill a foam box halfway with ice, take out the transfer clamp, pre-chilled Transfer Buffer, and soaked NC membrane. Pour half of the Transfer Buffer into a flat square container.
    7. Take out the protein gel, rinse it thoroughly with ultrapure water, place the black side facing down, and the transparent side facing up. In the middle, arrange one layer of sponge, one layer of filter paper, the protein gel, NC membrane, then place another layer of filter paper and sponge.
    8. After pressing to remove air bubbles, clamp the transfer cassette and place it into the transfer tank. Once both protein gels are fully inserted, pour all the Transfer Buffer into the transfer tank, then add two ice packs for cooling and cover with the lid.
    9. Surround the transfer cassette with ice and transfer at a constant voltage of 120 V for 90 min. The transfer process generates significant heat, so an ice bath is required for cooling.
    10. Take out the NC membrane, immerse and stain it in 20 mL of Ponceau S staining solution, mark the marker size with a pencil, and cut the membrane with a pencil according to the size of the target protein to be detected.
    11. Recover the Ponceau S staining solution. Wash away the staining solution with PBS and incubate the NC membrane with 20 mL of blocking buffer, then block at room temperature on a shaker at low speed for 1-2 h.
    12. Discard the blocking solution, quickly rinse the membrane twice with PBS on a decolorizing shaker to thoroughly remove the blocking solution, then add the corresponding antibodies according to the Western Blot usage ratios of each antibody in the antibody incubation box, and incubate overnight at 4 °C.
    13. Separately recover the primary antibodies and store at -20 °C. The membranes were quickly washed 4 times with TBST on a decolorizing shaker, 5 min each time, to thoroughly remove the primary antibodies. Then, add 4 mL of the blocking buffer with secondary antibodies, and incubate the membranes at room temperature on a slow shaker for 1 h.
    14. Wash the secondary antibody four times with TBST buffer for 5 min each, then replace with PBS buffer to fully moisten the NC membrane.
    15. According to the ECL chemiluminescence reagent instruction manual, prepare the luminescent solution and drop it onto the membrane surface, then incubate for 1-2 min before developing with a gel imaging system.
    16. Process the raw data in the Image J imaging system, then transfer it to the Photoshop software for figure creation.

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Results

Network Pharmacology and Molecular Docking
Identification of potential targets of CA: In the comprehensive investigation of CA's pharmacological activity, 60 potential targets were meticulously curated from an extensive review of relevant literature. To further expand the scope of the analysis, an additional 12 potential targets associated with CA were identified through the TCMSP database. For enhanced accuracy and thoroughness, the advanced predictive capabilities of the SwissTargetPrediction platf...

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Discussion

Ferroptosis, a recently identified mode of cell death, is characterized by excessive intracellular iron accumulation and lipid peroxidation. Morphologically, ferroptosis manifests as mitochondrial shrinkage, loss or reduction of cristae structures, and increased membrane density, while the cell membrane and nuclear morphology remain unaffected26. Mechanistically, ferroptosis is driven by glutathione (GSH) depletion and impaired GPX4 activity, preventing the metabolism of lipid peroxides via

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Disclosures

There are no financial conflicts of interest to disclose

Acknowledgements

We express our heartfelt gratitude for the superior conditions provided by the hospital and its departments, which have enabled us to achieve these results. At the same time, we also extend our sincere thanks to the many members of the laboratory for their tireless efforts and valuable contributions over the years. The hard work of every participant is an indispensable part of our success, and in the future, we will continue to work together to advance progress in the field of medicine. China National Natural Science Foundation (82104838), China Promotion Foundation Spark Program (XH-D001), Liaoning Provincial Key Research and Development Programme (2024JH2/102500062), and Liaoning Provincial Natural Science Foundation (2025-MSLH-490).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
 GlycineBiofroxx1275KG001
 Tris-baseBiofrooxx1115KG001
0.5  M  Tris-HCl buffer (pH  6.8)Biorad1610799
1 M Tris-Hcl (PH 7.5)BeyotimeST775
1.5  M  Tris-HCl buffer (pH  8.8)Biorad1610798
APSBeyotimeST005
Basic electrophoresis power supplyBIO-RAD PowerPac Basic
BCA Protein Quantification KitBeyotimeP0012
Beta Actin Recombinant antibodyHuabio81115-1-RR
Carbon dioxide incubatorThermo150L
CCK-8 assay kitApexbioK1018
Cell Activity and Cytotoxicity Assay KitBeyotimeC2015M
Cinnamaldehyde(CA)MacklinC805134purity 98%
Decolorizing shakerKylin-BellTS-100
DMEM (Dulbecco's Modified Eagle Medium)BasalMediaL110KJ
DMSOSigmaD2650
dorsal root ganglion (DRG)Fuheng BioFH165Serial number: 20211227-1
DRG complete mediumFuheng BioFH-DRG
ECL luminescent solutionThermo34580
ErbB-1Proteintech06-8471:2000
Fetal  Bovine  SerumMacklinFO193
Ferrous ion detection probe - FerroOrangeDojindoF374
FTH1Cell Signaling Technology4393S1:1000
Gel imaging systemBIO-RAD ChemiDoc Touch
Hanks' Balanced Salt Solution(HBSS)SolarbioH1025
High-speed refrigerated centrifugeEppendorf5804R
Hoechst 33342 Live Cell Staining SolutionBeyotimeC1028
Immunohistochemistry kitBiosharpBS-24-PB
Inverted fluorescence microscope systemLeicaLeica Dmi 8
JAK2 Recombinant Rabbit Monoclonal Antibody [SY0245]HuabioET1607-351:2000
Laboratory inverted phase contrast microscopeLeicaLeica Dmi 1
Low-speed centrifugeEppendorf5804
MDA Content Detection KitSangonD799762
Microplate readerSUNRISE TECAN
Multifunctional microplate readerBMGLABTECH PHERAstar FS
NF-κB p65 Polyclonal antibodyProteintech10745-1-AP1:1000
ParaformaldehydeMacklinP804536
Ponceau staining solutionBeyotimeP0022
Protein MarkerThermo26617
P-STAT3 (Ser727)Cell Signaling Technology9134S1:1000
P-STAT3 (Tyr705)Cell Signaling Technology9145S1:2000
Recombinant Anti-Glutathione Peroxidase 4 Antibody[EPNCIR144]abcamab1250661:1000
Recombinant Anti-XCT antibody[EPR27115-64]abcamab3076011:1000
ROS Assay KitDojindoR252
SDS-PAGE Protein Loading Buffer (6X)BeyotimeP0289
Skimmed milk powderBiosharpBS102-500g
Sodium dodecyl sulfate SDSBiofrooxx3250GR500
STAT3 (F-2)Santa CruzSc-80191:200
TEMEDSolarbio   T8090  
TFRCInvitrogen13-68000.5 µg/mL
Tween80SelleckS6702
Ultra-clean workbenchHDLDL-CJ-2NDI
Upright microscopeOlympusCX 31
Vertical electrophoresisBIO-RAD Mini Protean Tetra
Wet transfer tankBIO-RAD Mini Trans Blot
α-Tubulinα-TubulinER130905
GraphPad PrismStatistical analysis
ImageJ softwareObtain the gray value of the protein blot

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Cinnamaldehyde NeuroprotectionOxaliplatin NeurotoxicityDorsal Root GanglionFerroptosis PathwayJAK2 STAT3 SignalingGPX4 ExpressionLipid Peroxidation AssayReactive Oxygen SpeciesProtein Expression Analysis
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