Erianin protects chondrocytes against IL-1β-induced oxidative stress and ferroptosis by activating GPX4/STING signaling in osteoarthritis

Abstract

Background

Erianin (Eri) has been known for its analgesic and antipyretic properties. This research focuses on impact of Eri on chondrocyte viability, inflammatory cytokine production, extracellular matrix (ECM) degradation, and ferroptosis, which are key factors in cartilage diseases.

Methods

The mouse model of osteoarthritis (OA) was induced by destabilization of medial meniscus (DMM). Chondrocytes were treated with different concentrations of Eri and exposed to IL-1β to simulate disease conditions. The chondrocytes were induced to undergo ferroptosis using erastin (Era), and ferroptosis was inhibited by Fer-1. This was done to form an intervention control group in combination with Era and to explore the synergistic effect. The effects of Eri on cell viability, proliferation, inflammatory responses, ECM degradation, and ferroptosis were assessed using CCK-8 analysis, EDU assay, Western blot, immunofluorescence, ROS staining, and flow cytometry. The Cellular Thermal Shift Assay (CETSA) was also employed to confirm the direct binding and thermal stability of GPX4 and STING in the presence of Eri.

Results

The findings indicate that Eri does not exhibit cytotoxic effects at certain concentrations and can actually enhance chondrocyte proliferation and viability. It also reduces the production of inflammatory cytokines and ECM degradation products, suggesting a protective role against cartilage damage. Furthermore, Eri was found to inhibit ferroptosis in chondrocytes, potentially through the activation of the GPX4/STING signaling pathway. Molecular docking combined with CETSA confirmed that Eri enhances the thermal stability of GPX4 and STING, indicating a stabilizing effect on this key enzyme. In the DMM mouse model, Eri significantly alleviated cartilage degeneration and improved chondrocyte function, as evidenced by reduced osteophyte formation and subchondral bone sclerosis. Eri can act independently or in combination with the ferroptosis inducer erastin (Era) and the ferroptosis inhibitor Ferrostatin-1 (Fer-1). By inhibiting lipid peroxidation, regulating cell proliferation and extracellular matrix degradation, it exerts an intervention effect on IL-1β-induced ferroptosis of chondrocytes. Moreover, when used in combination with Fer-1, it has a synergistic enhancing effect in reversing ferroptosis-related damage.

Conclusions

Eri demonstrates promising therapeutic potential in the treatment of OA by inhibiting chondrocyte ferroptosis and protecting against ECM degradation and inflammatory responses.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00011-025-02162-w.

Introduction

Osteoarthritis (OA) is currently the most common chronic joint disease, affecting millions worldwide and posing a significant medical and economic burden [1–3]. The pathogenesis of OA is mainly caused by the increase of inflammatory mediators, leading to the degradation of extracellular matrix (ECM) and subchondral bone remodeling [4–6]. Currently, conservative approaches for OA mainly focus on reducing inflammation and relieving pain, but they cannot effectively inhibit the progression of the disease, ultimately leading to surgical joint replacement [7, 8]. Therefore, it is valuable to further investigate new therapeutic targets and determine more effective treatment strategies for OA.

OA is characterized by the degeneration of articular cartilage, subchondral bone remodeling, and synovial inflammation. The pathogenesis of OA involves the upregulation of various inflammatory factors, including IL-6, TNF-α, and IL-1β. Among these, IL-1β is frequently used to simulate inflammation in OA models due to its potent pro-inflammatory effects and its ability to induce the expression of other inflammatory cytokines and matrix metalloproteinases (MMPs). This makes IL-1β a suitable candidate for creating an inflammatory environment similar to that observed in OA [9]. Chondrocyte death plays a pivotal role in OA progression, with various forms such as apoptosis [10, 11], necroptosis [12, 13], and autophagy [14, 15] contributing to cartilage degeneration. The disruption of cartilage matrix homeostasis by inflammatory mediators exacerbates these processes, highlighting the need for therapies targeting chondrocyte survival and matrix preservation [16]. Recent studies suggest that ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, may also be implicated in OA, offering new avenues for therapeutic intervention.

Ferroptosis, a distinct form of regulated cell death, has gained significant attention for its unique characteristics [17]. It is characterized by the disruption of redox balance due to the abnormal function of redox-active enzymes, leading to the production of free radicals and lipid peroxidation, which cause cell death independently of caspases and necroptosis [18–22]. A key feature is the accumulation of intracellular lipid hydroperoxides and iron, which trigger ferroptosis. Glutathione peroxidase 4 (GPX4) is pivotal in preventing ferroptosis by reducing harmful lipid hydroperoxides to alcohols using reduced glutathione (GSH) [23, 24].

Prior studies have unequivocally demonstrated that the progression of OA is often accompanied by DNA damage [25], which aggravates cartilage degeneration. The cGAS-STING pathway is integral to immune responses against pathogenic DNA and has roles in tumorigenesis and inflammation [26, 27]. Emerging evidence points to STING’s potential involvement in various diseases, hinting at a possible connection with OA cell death processes. Ferroptosis, a novel cell death mechanism, is increasingly associated with OA. While GPX4 is known to maintain redox balance and inhibit ferroptosis, the detailed mechanisms of GPX4’s regulation of STING activation and its impact on chondrocyte ferroptosis in OA are yet to be fully understood.

Therefore, this study aims to delve deeper into the mechanisms of how Erianin (Eri) inhibits ferroptosis in chondrocytes during OA by regulating the GPX4/STING pathway. By elucidating this mechanism, we hope to provide novel insights and approaches for the treatment of OA, paving new avenues for its prevention and management. Ultimately, our objective is to alleviate the suffering of OA patients and improve their quality of life.

Material and methods

Isolation and culture of primary chondrocytes

Chondrocytes were isolated from the cartilage tissue of C57BL/6 J mice. Under sterile conditions, articular cartilage tissue was collected and cut into small pieces. The tissue was then washed three times with PBS and incubated with 0.25% trypsin and 0.2% collagenase II (Sigma-Aldrich, USA) at 37 °C for 4 h as described [28]. The isolated cells were centrifuged at 1000 rpm for 3 min, transferred to a culture flask, and cultured in DMEM (Thermo, United States) containing 10% fetal bovine serum, penicillin, and streptomycin at 37 °C with 5% CO₂. Fresh complete medium was replaced 24 h later. Only cells from passages 0 to 5 were used in this study to ensure consistent cell phenotypes [29].

Cell viability assay and EDU assays (cell proliferation measurement)

The viability of chondrocytes following treatment was evaluated using the CCK-8 kit (Beyotime, Shanghai, China). Briefly, chondrocytes were treated by different concentrations of Eri (Cat. No. HY-N0517, MedChemExpress, USA) or 8 μM Ferrostatin-1 (Fer-1, S7243, Selleck. cn) or 10 μM erastin (Era, S7242, Selleck. cn) or 5 μM RSL3 (Cat. No.HY-100218A, MedChemExpress, USA) for 24 and 48 h. Briefly, chondrocytes were first quantified before culture to ensure consistent cell density across all experimental conditions. The cells were counted using a hemocytometer under a light microscope. For each experimental condition, a consistent number of 1 × 10^4 cells per well was seeded into a 96-well plate. Chondrocytes were treated with different concentrations of Eri for 24 and 48 h. Subsequently, the cells were gently washed three times with PBS solution maintained at a pH of 7.4. Following the washes, a combination of 90 μL of culture medium and 10 μL of CCK-8 reagent was dispensed into each well of a 96-well plate. The cells were then incubated at 37 °C for an additional 3.5 h to allow for colorimetric changes. The optical density of each well was measured at a wavelength of 450 nm using a spectrophotometer. Based on these measurements, the relative survival rate of the cells was calculated as a percentage.

Chondrocytes were seeded into a 96-well plate, and EdU (Cat. No.HY-118411, MedChemExpress, USA) was added to the culture medium followed by incubation for 2 h. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min. After washing the cells with PBS three times, 100 μL Apollo staining solution was added to each well and incubated for 30 min. Finally, the cells were stained with DAPI (Invitrogen, USA) for 60 s in the dark. Quantitative analysis of the positive cell rate was performed using the ImageJ software (Bethesda, MD, USA).

Western blot analysis

The proteins of mouse chondrocytes were extracted using RIPA lysis buffer containing 1% PMSF. The protein concentration in each sample was analyzed using a BCA protein assay kit (Beyotime, China). 30 mg of cellular proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, United States) via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membrane was blocked with 5% non-fat milk powder at room temperature for 1 h, followed by incubation with the corresponding primary antibody overnight at 4 °C. After washing with TBST (Cat. No.: HY-K1027, MedChemExpress, USA) three times, the membrane was incubated with the secondary antibody for 1 h at room temperature. The protein blots were then detected using an ultra-sensitive ECL chemiluminescence kit (Beyotime, China). The source of the antibodies used in the experiment see the Table 1.

Table 1.

The source of the antibodies used in the Western Blotting experiment

Name	Item number	Manufacturer
Anti-iNOS	HY-P80725	MedChemExpress
Anti-COX2	HY-P86578	MedChemExpress
Anti-IL-6	HY-P80189	MedChemExpress
Anti-TNF-α	HY-P80914	MedChemExpress
Anti-Collagen II	HY-P81047	MedChemExpress
Anti-Aggrecan	HY-P81088	MedChemExpress
Anti-MMP13	HY-P83398	MedChemExpress
Anti-ADAMTS-5	HY-P81193	MedChemExpress
Goat Anti-Mouse IgG H&L	HY-P87169	MedChemExpress

Immunofluorescence

Chondrocytes were seeded onto glass coverslips and cultured for 24 h. After being rinsed three times with PBS, the cells were fixed with 4% paraformaldehyde (Cat. No. HY-Y0333, MedChemExpress, USA) for 15 min at room temperature, followed by another rinse with PBS. Subsequently, the cells were treated with 0.1% Triton X-100 solution for 5 min at room temperature to facilitate the permeation of cell and nuclear membranes. Cells were then blocked with 5% protease-free bovine serum albumin (BSA, Product No. A3059, Sigma Aldrich, USA) for 1 h at room temperature. After further washed with PBS, the cells were incubated overnight with the corresponding primary antibodies at 4 °C. The cells were washed with PBS and incubated with fluorescein-conjugated goat anti-rabbit IgG antibody (Product No. SAB370023, Sigma Aldrich, USA) for 1 h at room temperature. Subsequently, the nuclei were labeled with DAPI (Invitrogen, USA) for 60 s in the dark. The slides were observed under a confocal laser scanning microscope, and the fluorescence intensity was analyzed using ImageJ software (Bethesda, MD, USA).

Malonaldehyde (MDA), SOD and iron level

The relative MDA of cell supernatant was assessed by Lipid Peroxidation (MDA) assay kit (Product No. MAK568, Sigma Aldrich, USA) with according to the manufacturer’s protocol as previously reported. Briefly, the reaction of the MDA in the sample with thiobarbituric acid (TBA) generated an MDA-TBA adduct. The absorbance of MDA-TBA adduct was measured at 532 nm with a microplate reader (Thermo Fisher Scientific, Vantaa, Finland). Total SOD activities were measured using a Total Superoxide Dismutase Assay Kit with NBT (Beyotime Biotechnology). The assays were performed according to the instructions provided by the manufacturer. We determined intracellular ferrous iron content by Cell Ferrous Iron Colorimetric Assay Kit (Elabscience, E-BC-K881-M) according to manufacturer’s instructions. The assays were performed according to the instructions provided by the manufacturer.

Assessment of intracellular ROS and lipid-ROS

1 × 10⁵ cells were planted in 12-well plates and intervened as described above after the cells were adhered to the plates. The cells were washed three times with serum-free culture medium and then the medium was replaced with one containing either 10 μM dichloro-fluorescein diacetate DCFH-DA (Cat. No.HY-118411, MedChemExpress, USA) or 5 μM C11 BODIPY (Cat. No.HY-D1301, MedChemExpress, USA), and incubated at 37 °C for 20 min. After washing the cells with serum-free medium, ROS and lipid-ROS levels were observed using a fluorescence Leica microscope (Leica DMI 3000 M, German), or assessed using a FACS LSRFortessa™ flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Measurement of intracellular Fe²⁺ levels

1 × 10⁵ cells were seeded in a 12-well plate and treated as previously described. The cells were washed three times with serum-free medium and then the medium was replaced with one containing 1 μM FerroOrange (F374, Dojindo, Japan). The cells were stained for exactly 30 min at 37 °C with 5% CO2. Fluorescence microscopy (Leica DMI 3000 M, German) imaging was performed immediately thereafter.

Transmission electron microscopy (TEM)

In brief, cells were fixed with a 2.5% glutaraldehyde solution at 4 °C, followed by fixation with a 1% osmium acid solution after washed with PBS. The samples were then dehydrated using a gradient ethanol solution, and embedded at 37 °C for overnight polymerization by Epoxy embedding medium (Product No. 45345, Sigma Aldrich, USA). Ultrathin sections of the samples were prepared and stained consecutively with uranyl acetate and lead citrate. The stained sections were placed in a transmission electron microscope for observation and imaging.

Animal model and in vivo experiments

Eight-week-old male C57BL/6 J (wild-type, WT) mice were obtained from Cyagen Biosciences (Suzhou) Inc.. To establish an OA model, destabilization of the medial meniscus surgery (DMM) was performed on the mice [28, 30], with six animals per group. Briefly, under anesthesia, a medial articular incision was made to expose the left joint cavity. Subsequently, the tibial collateral ligament was transected, and the articular incision was closed. In the control group, only the joint cavity was accessed without further manipulation. One week postoperatively, the mice received weekly intra-articular injections of 1 mg/kg Eri, 1 mg/kg RSL3 (Cat. No.HY-100218A, MedChemExpress, USA), or a combination of both, into the knee joint. The control group received saline injections. Ten weeks after the operation, all the mice were sacrificed and their knee joints were exposed and dissected. Then, the surrounding muscles and connective tissues were carefully removed. The femoral condyles, tibia, joint cartilage and the bone beneath the joint were retained for subsequent tissue staining and molecular detection. The animal protocol adhered to the ethical guidelines of Bioethics Committee of The First Affiliated Hospital of the University of Science and Technology of China.

Evaluation Criteria for the Success of OA Animal Model:

The success of the OA animal model was evaluated based on several criteria: Histological Analysis: The extent of cartilage degradation was assessed using the OARSI OA cartilage histopathology assessment method (Supplementary Data). Tissue sections were stained with Hematoxylin and Eosin (H&E) kit (C0105S, Beyotime, China) and Safranin O-fast green kit (C0621S, Beyotime, China) to evaluate cartilage structure and matrix integrity. Micro-CT Analysis: The degree of osteophyte formation and subchondral bone sclerosis was measured using micro-CT scans. Parameters such as bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were quantified. Immunohistochemical Analysis: The expression levels of key proteins related to chondrocyte inflammation and ferroptosis, such as Collagen II, Aggrecan, MMP-13, and ADAMTS-5, were assessed using immunohistochemical staining.

Immunohistochemistry analysis

The immunostained sections were first dewaxed and then treated with 3% H₂O₂ solution for 10 min to effectively inhibit endogenous peroxidase activity. After thorough washing with PBS, the samples were incubated with the primary antibody at 4 °C overnight to ensure sufficient binding of the antigen–antibody complex. Subsequently, the samples were treated with HRP-labeled goat anti-rabbit secondary antibody (Product No.A9044, Sigma Aldrich, USA) at 37 °C for 1 h for signal amplification. Finally, the positive cell rates of the various slice observation groups were statistically counted through a blinded counting method, and images were captured and saved using a microscope.

Hematoxylin and eosin (H&E) and Safranin O-fast green staining

Knee joints were preserved in formalin and decalcified for 4 weeks using 0.5 M EDTA at pH 8.0. Tissue sections were cut sagittally at a thickness of 4–6 µm for H&E and Safranin O-Fast Green staining. The OARSI OA cartilage histopathology assessment method was employed to determine the extent of cartilage degradation.

Micro-CT analysis

The scanning range was determined to be from distal femur to middle femur, and the number of scanning layer was set to 400, with each layer being 9 μm thick. Scanning parameters included a voltage of 46 kV, a current of 75 uA, and a scanning time of 10 min. Three-dimensional (3D) visualization images of the distal metaphysis were constructed by Nrecon software.

Cellular thermal shift assay (CETSA)

Briefly, after the cells were treated with Eri or DMSO for 4 h in 37 °C, cell lysates were collected. The lysate was distributed and each sample was heated for 3 min at the indicated temperature in the range of 43 to 64 °C, followed by 3 min of cooling at room temperature. The soluble fraction from precipitate was separated by centrifugation at 12,000 rpm for 20 min, followed by subsequent examination using western blot analysis.

Statistical analysis

GraphPad Prism 9.0 software was used for all statistical analyses. Data are presented as the mean ± standard error of the mean (n ≥ 3). Specific statistical tests were chosen based on the nature of the data and the experimental design. For comparisons between two groups, a nonparametric t-test (Mann–Whitney U test) was conducted due to the small sample size and potential non-normal distribution of the data.To compare multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was employed. This method was chosen because it allows for the identification of specific group differences while controlling for type I error.

All statistical tests were performed at a significance level of 0.05. The detailed justification for each statistical test was based on the experimental conditions and the distribution characteristics of the datasets, ensuring clarity and reproducibility of the analyses.

Results

Eri promotes chondrocyte viability and proliferation

Eri, a natural low-molecular-weight compound derived from the traditional Chinese medicine Dendrobium chrysotoxum Lindl, has attracted widespread scientific attention due to its unique biphenyl ring structure and multiple methoxy substituents [31] (Supplementary Fig. 1A). In ancient medical literature, Eri has been recorded for its analgesic and antipyretic effects. In recent years, the potential of Eri in disease treatment has gradually emerged, and its role in controlling diseases such as tumor angiogenesis, diabetic retinopathy, and Staphylococcus aureus infection has been extensively studied [32–34].

To investigate the impact of Eri on chondrocyte viability, cells were incubated with various concentrations of Eri (0, 10, 25, 50, 100, 200 and 400 nM) for 24 and 48 h. Subsequently, CCK-8 analysis was employed to assess the effects on cell viability. As depicted in Supplementary Fig. 1B, no significant cytotoxic effects of Eri on chondrocytes were observed at concentrations of 10, 25, 50 and100 nmol/L, either at 24 or 48 h. Hence, the concentrations of 10, 50, and 100 nM were chosen for subsequent in vitro experiments.

Eri reduced the production of inflammatory cytokines and ECM degradation in chondrocytes treated with IL-1β

In cartilage diseases such as arthritis, the degradation of ECM and inflammatory response are the key factors leading to damage and dysfunction. IL-1β, as an inflammatory mediator, can trigger a series of pathological reactions in chondrocytes. Eri, a natural component with therapeutic potential, remains to be studied for its impact on ECM degradation and inflammatory response. Therefore, this experiment utilizes an IL-1β-induced chondrocyte injury model to explore the protective effects and mechanisms of Eri.

The EDU assay demonstrated that following IL-1β induction, the proliferative activity of chondrocytes was significantly suppressed. However, upon treatment with Eri, a notable increase in the fluorescence intensity of chondrocytes was observed. As the concentration of Eri increased, a gradual enhancement in cell proliferative activity was evident, indicating that Eri can attenuate the reduction in proliferative activity caused by IL-1β in chondrocytes (Fig. 1A). Similar experimental results were observed in the toluidine blue experiment (Fig. 1B).

Fig. 1.

Eri Reduces the Production of Inflammatory Cytokines and ECM Degradation in Chondrocytes Treated with IL-1β (#401-ML, R&D systems). A The chondrocytes were preconditioned with either IL-1β (10 ng/mL) alone or in combination with varying concentrations of Eri for a duration of 1 h. Subsequently, the cell viability and, B toluidine blue staining were evaluated. C The protein expression levels of Collagen II, Aggrecan, MMP13, and ADAMTS5 were quantitatively determined through Western blot analysis. D An immunofluorescence analysis of the expression of collagen II and MMP-13 in chondrocytes. E iNOS, COX2, IL-6, and TNF-α proteins were quantitatively assessed using Western blot analysis. *p < 0.05, **p < 0.01, ***p < 0.001

Subsequently, in order to further investigate the role of Eri in IL-1β-induced ECM degradation, we conducted Western blot and immunofluorescence analysis. We specifically focused on the effect of Eri on the expression levels of collagen II and Aggrecan (ECM key components) as well as MMP13 and ADAMTS5 (ECM degradation enzymes) in chondrocytes. The results showed that IL-1β induction significantly inhibited the expression of collagen II and Aggrecan proteins, while promoting the expression of MMP13 and ADAMTS5 proteins. However, it is exciting to note that Eri can effectively attenuate this induction effect of IL-1β, both alleviating the inhibition of collagen II and Aggrecan proteins and inhibiting the promotion of MMP13 and ADAMTS5 proteins (Fig. 1C). These findings suggest that Eri has the potential to inhibit ECM degradation processes. Consistent with this, immunofluorescence analysis further showed that compared with the IL-1β treatment group, Eri treatment significantly enhanced the expression of collagen II protein and inhibited the expression of MMP13 (Fig. 1D). These results collectively reveal the positive role of Eri in protecting chondrocytes from IL-1β-induced ECM degradation. To investigate the impact of Eri on inflammatory responses, cells were pretreated with IL-1β and Eri, followed by Western blot analysis to assess the protein expression of iNOS, COX2, and inflammatory cytokines IL-6 and TNF-α. These factors play pivotal roles in inflammatory reactions, and their expression levels serve as indicators of the intensity of the inflammatory response. IL-1β significantly stimulates the expression of these inflammatory factors. Conversely, Eri exhibits protective activity by mitigating the effects of IL-1β (Fig. 1E), thereby reducing the production of these inflammatory cytokines in chondrocytes.

Eri inhibits IL-1β-induced ferroptosis in chondrocytes

To elucidate the role of erianin (Eri) in IL-1β-induced ferroptosis in chondrocytes, in Fig. 2, we designed experimental groups to assess Eri’s mechanism. The EDU assay showed that IL-1β or IL-1β + erastin (Era) drastically reduced cell proliferation, while Eri notably restored it, especially in the IL-1β + Era + Eri group, leading to a substantial proliferation increase (Fig. 2A). For ROS, IL-1β significantly elevated ROS levels in chondrocytes, and Eri effectively reversed this trend (Fig. 2B, C). Using the C11-BODIPY probe to detect lipid peroxidation, we found that IL-1β + Era groups showed significantly augmented lipid peroxidation, but Eri intervention gradually reduced it (Fig. 2D). Ferroptosis-specific staining showed that IL-1β + Era groups exhibited distinct ferroptosis features, which were significantly alleviated by Eri (Fig. 2E, F). Western blot analysis revealed that IL-1β + Era increased ECM degradation markers and decreased structural protein expression (Fig. 2G), indicating ECM degradation and cellular damage, while Eri reversed these changes. Compared to the IL-1β group, co-treatment with IL-1β and Fer-1 partially restored cell proliferation, and combining Eri with Fer-1 significantly enhanced this restoration (Fig. 3A), reflecting their synergistic effect in inhibiting ferroptosis. In ROS staining, when Fer-1 was combined with Eri, the IL-1β-induced increase in ROS levels was reduced to a greater extent (Fig. 3B). For lipid peroxidation detected by the C11-BODIPY probe, combining Fer-1 with Eri more significantly reduced IL-1β-induced lipid peroxidation (Fig. 3C), and ferroptosis characteristics were also significantly improved (Fig. 3D). Western blot analysis showed that when Eri was combined with Fer-1, the reversal of ECM degradation and cellular injury caused by IL-1β was more pronounced (Fig. 3E), validating their combined effectiveness in inhibiting chondrocyte injury.

Fig. 2.

Eri inhibits ferroptosis of chondrocytes induced by IL-1β. A EDU assay showed that IL-1β and Era promoted chondrocytes death, while Eri inhibited chondrocytes death caused by IL-1β or Era. B Eri effectively reversed the elevation of ROS levels caused by IL-1β and Era by Immunofluorescence or, C flow cytometer. D After 24-h treatment with IL-1β (10 ng/ml) in combination with or without Era (5 μM) or Eri (100 nM), representative images captured under a C11 BODIPY-labeled fluorescence microscope are presented. E Detecting intracellular active iron ions using FerroOrange fluorescent probe. F After 24 h of treatment as described in method (A), the level of MDA, SOD and Fe²⁺ were measured in Chondrocytes. G The expression levels of collagen II, Aggrecan, MMP13, and ADAMTS5 proteins were quantitatively assessed via Western blot analysis. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3.

Eri and Fer-1 synergistically inhibit ferroptosis of chondrocytes induced by IL-1β. A The EDU assay revealed that IL-1βaccelerated chondrocyte death, whereas Eri and Fer-1 effectively suppressed chondrocyte death induced by IL-1β. B Eri and Fer-1 synergistically reversed the elevation of ROS levels caused by IL-1β. C After 24 h of treatment with IL-1β (10 ng/mL) in combination with or without Eri (100 nM) or Fer-1 (1 μM), C11 BODIPY-labeled representative images were captured to assess lipid peroxidation. D Utilizing FerroOrange fluorescent probe to detect intracellular active iron ions. E Western blot analysis was used to quantitatively assess the protein expression levels of collagen II, Aggrecan, MMP13, and ADAMTS5. *p < 0.05, **p < 0.01, ***p < 0.001

Eri mediates the activation of the GPX4/STING signaling pathway to inhibit ferroptosis

We further investigated the mechanism of Eri in inhibiting ferroptosis. In the control group, the mitochondrial structure remained intact, and the expression of signaling pathway proteins did not change abnormally. However, under IL-1β induction, through TEM observation, we found that the mitochondria in chondrocytes cells significantly shrank and the membrane density increased (Fig. 4A), which is the unique morphological characteristic of ferroptosis, indicating that the mitochondrial structure was damaged. When cells were treated with both IL-1β and Eri, the mitochondrial structure was significantly improved. GPX4, as a key antioxidant enzyme against ferroptosis, consumes lipid hydroperoxides by reducing GSH. The downregulation of GPX4 usually indicates the induction of ferroptosis. Meanwhile, P-TBK1 and P-IRF3, as key components of the STING signaling pathway, are mainly involved in regulating immune response, while GPX4 is closely linked to the process of ferroptosis.

Fig. 4.

Eri mediates the activation of the GPX4/STING signaling pathway to inhibit ferroptosis. A Transmission electron microscopy (TEM) images show the morphology of mitochondria in proximal tubular cells. Scale bar, 1 μm. B Molecular docking analysis of GPX4 with its potential target. C Molecular docking analysis of GPX4 with its potential target. D The CETSA was performed on both intact cells and cell lysates in the presence of Eri. GPX4 protein stability across the temperature range of 43–64 °C was assessed by Western Blot and quantified using Image J software. (E) The expression levels of STING, GPX4, P-TBK1 and P-IRF3 proteins were evaluated following a 24-h treatment with IL-1β (10 ng/mL), both alone and in combination with Eri (100 nM). F Immunofluorescence images of GPX4 and STRING. G Western blotting was used to detect the expression of GPX4 and STING. H Use EdU staining to detect cell proliferation. I Fluorescence staining used to detect the level of ROS. J Fluorescence staining was used to detect the redox state. K Use fluorescence staining to detect the level of Fe²⁺. L Western blotting was used to detect the expression of Collagen Ⅱ, Aggrecan, ADAMTS5, and MMP13. *p < 0.05, **p < 0.01, ***p < 0.001

The binding relationships of Eri with GPX4 and STING were analyzed using the molecular docking software Autodock vina 1.5.6, we identified potential direct molecular targets of GPX4 and STING (Fig. 4B, C). CETSA was used to demonstrate the binding stability of Eri with the GPX4 and STING proteins. As the temperature rises, the bands of the GPX4 and STING protein in the control group gradually become lighter, indicating that the protein is gradually undergoing thermal denaturation and precipitation. Eri can combine with GPX4 and STING, significantly enhancing its thermal stability, enabling GPX4 and STING to maintain its protein structural integrity at higher temperatures. Further quantitative analysis shows that Eri can enhance the thermal stability of the GPX4 and STING protein, allowing the GPX4 and STING protein to retain a relatively higher degree of solubility at higher temperatures (Fig. 4D). The protein expression levels of STING, P-TBK1, and P-IRF3 were significantly increased in both the IL-1β treatment group and the group treated with a combination of IL-1β and Eri. However, the GPX4 protein level in IL-1β treatment group was decreased compared to the control group, and yet this decrement was mitigated following treatment with Eri (Fig. 4E). These results clearly indicate that Eri can mediate the GPX4/STING signaling pathway, thereby effectively inhibiting ferroptosis. In addition, the results of cell immunofluorescence staining were consistent with the Western blot results (Fig. 4F), further supporting our findings. To better understand the mechanism of action of Eri, we also introduced the GPX4 inhibitor RSL3.

The results of Western Blotting showed that Sh-GPX4 could effectively knock down the expression of GPX4 protein, while Oe-STING could significantly upregulate the expression of STING protein. The transfection efficiency met the requirements for subsequent experiments (Fig. 4G). EdU staining and statistics indicated that IL-1β inhibited chondrocyte proliferation, and knockdown of GPX4 or overexpression of STING would exacerbate this inhibition. Eri could improve the cell proliferation inhibition caused by knockdown of GPX4 or overexpression of STING, and promote chondrocyte proliferation (Fig. 4H). ROS fluorescence staining and statistics showed that IL-1β induced the production of a large amount of ROS in chondrocytes, and knockdown of GPX4 or overexpression of STING would increase ROS production. Eri could reduce the excessive production of ROS caused by knockdown of GPX4 or overexpression of STING, and alleviate oxidative stress (Fig. 4I). Redox staining and statistics indicated that IL-1β disrupted the redox balance of chondrocytes, making oxidation dominant. Knockdown of GPX4 or overexpression of STING would aggravate this imbalance. Eri could regulate the redox imbalance caused by knockdown of GPX4 or overexpression of STING, and restore the reduced state of cells (Fig. 4J). Fe²⁺ fluorescence staining and statistics showed that IL-1β promoted the accumulation of Fe²⁺ in chondrocytes, and knockdown of GPX4 or overexpression of STING would exacerbate the Fe²⁺ accumulation. Eri could reduce the excessive accumulation of Fe²⁺ caused by knockdown of GPX4 or overexpression of STING (Fig. 4K). Western Blotting and statistics showed that IL-1β inhibited chondrocyte synthesis (Collagen II, Aggrecan expression decreased) and promoted chondrocyte degradation (ADAMTS5, MMP13 expression increased), and knockdown of GPX4 or overexpression of STING would exacerbate the inhibition of chondrocyte synthesis and the promotion of degradation. Eri could promote the inhibited chondrocyte synthesis caused by knockdown of GPX4 or overexpression of STING, and inhibit the corresponding chondrocyte degradation (Fig. 4L).

Eri alleviates cartilage degeneration and ferroptosis in OA mouse models

When chondrocytes come from C57BL/6 J mice were treated with IL-1β and RSL3 together, the expression of GPX4 was significantly inhibited, while STING, P-TBK1, and P-IRF3 were activated. However, when Eri was added on top of this, the effect was alleviated to some extent (Fig. 5A). This suggests that Eri can counteract the inhibitory effect of RSL3 on GPX4 by activating the GPX4/STING signaling pathway. In addition, we also detected indicators closely related to the function of chondrocytes, such as CollangeII, Aggrecan, MMP-13, and ADAMTS-5. The results showed that Eri could significantly improve the functional damage of chondrocytes caused by IL-1β and RSL3 (Fig. 5B), further confirming the protective effect of Eri on chondrocytes.

Fig. 5.

Eri alleviates the development of osteoarthritis by inhibiting ferroptosis in a mouse model of destabilization of the medial meniscus (DMM). A The expression levels of STING, GPX4, P-TBK1, and P-IRF3 proteins were assayed after a 24-h treatment with IL-1β (10 ng/mL), administered either alone or in conjunction with RSL3 (5 μM) and Eri (100 nM). B The expression levels of collagen II, Aggrecan, MMP13, and ADAMTS5 proteins were quantitatively assessed via Western blot analysis. C Flowchart of the mouse model of osteoarthritis, which was created by surgical instability of the medial meniscus (DMM). One week after surgery, Eri, RSL3, and a combination of both were injected into the joint cavity of the mice. n = 6. D The micro-CT scans were performed on sham mice that were treated with Eri, RSL3, or a combination of both drugs. E The OARSI score, along with measurements of bone mineral density (BMD), bone volume fraction (BV/IV), trabecular thickness (Tb.Th), trabecular number (Tb.n), and trabecular separation (Tb.SP), were all carefully assessed. F Cartilage degradation was evaluated through the utilization of Safranin O/fast green staining and HE staining. Sham: Healthy control group without any surgical intervention. This serves as the baseline for comparison. DMM: Mice that underwent destabilization of the medial meniscus (DMM) surgery to induce osteoarthritis (OA), showing significant cartilage degradation. DMM + Eri: Mice treated with Eri following DMM surgery, demonstrating the protective effects of Eri against cartilage degeneration. DMM + RSL3: Mice treated with RSL3, a GPX4 inhibitor, following DMM surgery, which intensifies cartilage damage through enhanced ferroptosis. DMM + Eri + RSL3: Mice treated with a combination of Eri and RSL3 following DMM surgery, showing the combined effects on cartilage structure and integrity. G IHC was performed to detect key proteins related to chondrocyte inflammation (Col-II, Aggrecan, MMP13 and ADAMTS-5) and ferroptosis (STING and GPX4) in Sham, DMM, DMM + Eri, DMM + RSL3 and DMM + Eri + RSL3 group respectively. *p < 0.05, **p < 0.01, ***p < 0.001

Given the ability of Eri to alleviate cartilage degeneration and iron death in vitro experiments, we further explored its effect on the progression of OA in vivo. We used a surgical-induced DMM mouse model to simulate the in vivo environment of OA, and ensured the standardization and reproducibility of experimental procedures through detailed flow charts (Fig. 5C). With the help of micro-CT technology, we measured the degree of osteophyte formation and bone sclerosis in subchondral bone (Fig. 5D, E). The experimental results showed that the joint cartilage injury in DMM group mice was significant, manifested as the reduction of osteophyte size, osteophyte maturity, bone density BMD, and trabecular thickness Tb.Th. The intervention of Eri significantly alleviated cartilage degeneration, and the OARSI score also decreased accordingly (Fig. 5D, E, Supplementary Data). It is particularly noteworthy that the injection of Eri in RSL3 group mice significantly alleviated cartilage degeneration, osteophyte formation, and subchondral bone sclerosis (Fig. 5D, E). To further verify this finding, we conducted Safranin O/fast green and HE staining analysis. These analyses visually demonstrated the inhibitory effect of Eri on cartilage degeneration. Compared with the control group, the joint cartilage structure treated with Eri was more complete, and the number of cells was relatively higher (Fig. 5F).

In addition, we also used immunohistochemical staining to assess the levels of inflammation and ferroptosis in chondrocytes. The experimental results showed that the expression of CollangeII, Aggrecan, and GPX4 in the joint cartilage of mice in the DMM group and the RSL3 injection group decreased, while the expression of MMP-13, ADAMTS-5, and STING increased, further confirming the impairment of chondrocyte function. However, it is gratifying that the expression of these indicators was significantly improved after injection of Eri (Fig. 5G), which is consistent with our previous in vitro experimental results. This finding not only strengthens the therapeutic effect of Eri on OA, but also further reveals its mechanism of inhibiting ferroptosis by activating the GPX4/STING signaling pathway. In summary, our study fully demonstrates that Eri can alleviate the development of OA by inhibiting ferroptosis in vivo, providing a new potential strategy for the treatment of OA (Fig. 6).

Fig. 6.

A schematic diagram illustrates the mechanisms underlying the action of Erianin in inhibiting the IL-1β-induced ECM degradation and ferroptosis of chondrocytes. This figure summarizes the proposed pathways and effects of Erianin based on the experimental findings

Discussion

OA, a degenerative joint disease, is experiencing a continuously rising incidence among the middle-aged and elderly population, significantly affecting the quality of life of patients [35]. The pathological process of OA is intricate, with chondrocyte death closely associated with the loss of articular cartilage structure and function [36], serving as a critical factor in OA progression. In recent years, ferroptosis, a novel form of cell death, has gradually garnered attention for its role in various diseases. However, the specific mechanisms underlying ferroptosis in OA remain elusive, and effective therapeutic strategies are lacking. Therefore, exploring drugs capable of inhibiting chondrocyte ferroptosis holds significant importance for the treatment of OA.

Eri, a natural compound with extensive biological activities, has preliminarily demonstrated its role in anti-inflammatory, antioxidant, and cytoprotective functions [37–40]. Although previous studies have not directly addressed the relationship between Eri and OA, given its positive effects in other cellular death and inflammatory processes, we believe that Eri may has potential therapeutic value in OA. The results of this research clearly indicate that Eri does not adversely affect chondrocyte viability at certain concentrations, which is a critical first step in considering its use as a therapeutic agent. Moreover, Eri’s ability to enhance chondrocyte proliferation is particularly noteworthy, as it suggests a potential for promoting cartilage repair and regeneration, which are essential for combating the degenerative processes associated with OA.

This study not only demonstrates the ameliorative effects of Eri in on ECM degradation in chondrocytes but also uncovers its unique capacity in suppressing inflammatory responses. As a fundamental component maintaining the structural integrity and functional proficiency of cartilage, ECM degradation plays a crucial role in the pathogenesis of OA [41]. By modulating the expression of proteins involved in ECM degradation, Eri effectively shields chondrocytes from degradation, offering a novel perspective for cartilage cell protection and presenting fresh opportunities for the development of therapeutic agents targeting ECM degradation in OA. Furthermore, inflammatory responses play a pivotal role in the progression of OA [42]. Eri’s ability to modulate these factors could represent a novel approach to managing inflammation and preserving cartilage integrity. This is particularly important given the limited options currently available for these aspects of OA management.

Given Eri’s multifaceted effects, considering potential synergies with current OA treatments could be an exciting area for further investigation. Existing treatments for OA often include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and hyaluronic acid injections, which primarily address pain and inflammation but do not significantly alter the disease course. Eri, with its anti-inflammatory and chondroprotective properties, could potentially complement these treatments by providing additional protective effects against cartilage degradation and chondrocyte death. The combination of Eri with current therapies might enhance overall therapeutic outcomes, reduce the dosage requirements of conventional drugs, and minimize their associated side effects.

For instance, the combination of Eri with NSAIDs could offer a dual approach by not only managing inflammation more effectively but also by protecting chondrocytes from oxidative stress and ferroptosis. Similarly, combining Eri with corticosteroids could mitigate the long-term detrimental effects of steroids on cartilage while enhancing the anti-inflammatory response [43, 44]. The synergistic effects of Eri with hyaluronic acid injections might improve joint lubrication and further protect the cartilage matrix from degradation. Future studies should focus on evaluating these combination therapies in clinical settings to fully understand their potential benefits and mechanisms of action. By exploring the synergistic effects of Eri with existing OA treatments, we can broaden the scope and implications of our findings, paving the way for more comprehensive and effective treatment strategies for OA.

Ferroptosis, a novel type of cell death, is closely associated with the pathogenesis of various diseases. In OA, ferroptosis of chondrocytes may exacerbate disease progression. The GPX4/STING signaling pathway plays a pivotal role in regulating ferroptosis [45]. STING, as a pivotal signaling molecule, activates the downstream antioxidant enzyme GPX4, thereby inhibiting the onset of ferroptosis. In the context of OA pathology, the accumulation of iron in chondrocytes may initiate ferroptosis, and the disruption of the GPX4/STING signaling pathway could potentially intensify this process. Our study has identified that Eri can suppress ferroptosis by activating the GPX4/STING signaling pathway, highlighting its therapeutic potential for maintaining cartilage integrity.The STING pathway, recognized for its role in innate immunity and inflammatory responses, intersects with GPX4 in the regulation of redox balance and ferroptosis. This interaction extends to other pathways implicated in OA, such as NF-κB, Wnt/β-catenin, and MAPK. By modulating both GPX4 and STING, Eri may mitigate inflammation and oxidative stress, presenting a comprehensive strategy for OA treatment. While beneficial, STING activation could provoke inflammatory responses, and its prolonged activation might lead to risks of chronic inflammation. Additionally, an overabundance of GPX4 upregulation could perturb cellular redox states, potentially affecting processes like autophagy and apoptosis. Therefore, it is crucial to optimize the dosing of Eri to strike a balance between therapeutic efficacy and possible adverse effects. Results from in vivo experiments further validated the protective effect of Eri on OA. In the DMM mouse model, Eri significantly reduced the degree of articular cartilage damage and improved the function of chondrocytes. This discovery not only provides strong support for the clinical application of Eri in the treatment of cartilage-related diseases such as OA, but also offers new insights into the role of the GPX4/STING signaling pathway in the pathogenesis and treatment of OA.

Conclusion

This study reveals the potential value of Eri in the treatment of OA. We found that Eri can slow down OA progression by inhibiting chondrocyte ferroptosis. In vitro experiments further demonstrated its ability to reduce inflammatory responses and ECM degradation induced by IL-1β. Additionally, Eri exhibits protective effects on chondrocytes by regulating the expression of relevant proteins, thereby maintaining the structure and function of cartilage. Therefore, Eri holds promise as a novel and promising drug candidate for the treatment of OA, offering a new therapeutic option for patients suffering from this condition.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Eri

Erianin

Era

Erastin

ADAMTS5

A disintegrin and metalloproteinase with thrombospondin motifs 5

ANOVA

Analysis of variance

ATG7

Autophagy related 7

CCK-8

Cell counting Kit-8

cGAS

Cyclic GMP-AMP synthase

COX2

Cyclooxygenase-2

DCFH-DA

Dichloro-fluorescein diacetate

DMM

Destabilization of the medial meniscus

ECM

Extracellular matrix

EDTA

Ethylenediaminetetraacetic acid

EDU

5-Ethynyl-2′-deoxyuridine

GPX4

Glutathione peroxidase 4

HIFs

Hypoxia-inducible factors

HRP

Horseradish peroxidase

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

iNOS

Inducible nitric oxide synthase

JNK

C-Jun N-terminal kinase

MDA

Malondialdehyde

MMP

Matrix metalloproteinase

MMP13

Matrix metalloproteinase-13

MTOR

Mechanistic target of rapamycin

NF-kappaB

Nuclear factor kappa-light-chain-enhancer of activated B cells

OA

Osteoarthritis

OARSI

Osteoarthritis Research Society International

ROS

Reactive oxygen species

RSL3

RSL3 (RAS-selective lethal small molecule 3)

SOD

Superoxide dismutase

STING

Stimulator of interferon genes

TEM

Transmission electron microscopy

TLR4

Toll-like receptor 4

TNF-α

Tumor necrosis factor alpha

Authors’ contributions

F.C., L.C.Y. and J.W. was the overall principal investigator who conceived the study, revised the manuscript and obtained financial support. L.C.Y. and J.W. performed the experiments and wrote the manuscript. L.S. and W.Y., collected the data and drew the figures and tables. All authors critically reviewed the article and approved the final manuscript.

Funding

This study was supported by Postdoctoral Research Foundation of China (2023M733424), and Doctoral Research Initiation Foundation of the First Affiliated Hospital of the USTC (RC2021133).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethical approval

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the Bioethics Committee of The First Affiliated Hospital of the University of Science and Technology of China No:(2024-N(A)-127).

Consent for publication

Not applicable.