Skip to main content
NCBI home page
Frontiers in Molecular Biosciences logoLink to Frontiers in Molecular Biosciences
. 2026 May 21;13:1764620. doi: 10.3389/fmolb.2026.1764620

Electroacupuncture alleviates osteoarthritis and is associated with activation of the Nrf2/HO-1 pathway and reduced ferroptosis-related changes

Mei Zhao 1,†, Lingxing Ouyang 2,†, Junjie Ma 1, Huimeng Zhu 1, Shengfang Zhu 1, Xuan Zhou 1, Bingyao Huang 1, Jun Yan 1,*
PMCID: PMC13233232  PMID: 42256315

Abstract

Background

Electroacupuncture (EA) has shown beneficial effects in osteoarthritis (OA), but the underlying mechanism remains unclear. This study examined whether the effects of EA on OA are associated with changes in ferroptosis-related markers and Nrf2/HO-1 signaling.

Methods

Rats were randomly assigned to the Control, OA, OA + ferrostatin-1, OA + EA, and OA + ferrostatin-1+EA groups. Gross cartilage appearance, paw withdrawal threshold, body weight, and knee joint width were evaluated. Cartilage injury was assessed by HE staining, Safranin O-fast green staining, and OARSI scoring. Inflammatory cytokines in serum and synovial fluid were measured by ELISA. TUNEL staining, immunofluorescence, biochemical assays, and western blot were used to assess apoptosis, ferroptosis-related markers, and Nrf2/HO-1 signaling in cartilage tissue.

Results

Compared with the Control group, OA rats showed obvious cartilage damage, lower paw withdrawal threshold, reduced body weight gain, and increased knee joint width. Histological injury and OARSI scores were markedly increased in the OA group. Serum and synovial fluid levels of IL-6, IL-1β, and TNF-α were also elevated. In cartilage tissue, OA was associated with increased apoptosis, decreased SLC7A11, GPX4, SOX9, and GSH levels, and increased Fe2+ and ACSL4 levels. Nrf2 and HO-1 expression was increased in OA cartilage. Treatment with ferrostatin-1, EA, or the combined treatment alleviated these changes to different degrees.

Conclusion

EA alleviated OA-related structural and inflammatory changes and was associated with reduced ferroptosis-related alterations and enhanced Nrf2/HO-1 signaling.

Keywords: electroacupuncture, ferroptosis, HO-1, Nrf2, osteoarthritis

Introduction

Osteoarthritis (OA) is the prevalent type of arthritis characterized by the degeneration of joint cartilage (Emami et al., 2023). It initiates with biochemical and cellular changes in the synovial joint tissues, leading to histological and structural alterations in the joints and eventually causing functional impairment (Yao et al., 2023). Recent perspectives on the pathogenesis of OA suggest that vascular dilation, bone remodeling, and disrupted bone turnover are influenced by various risk factors such as aging, obesity, and mechanical overload (Zhao et al., 2023). The goals of OA treatment include relieving joint pain and stiffness, preserving or increasing joint flexibility and stability, improving mobility and participation, and enhancing quality of life (Küçükdeveci, 2023). However, the current therapeutic prospects are insufficient in providing reliable pharmacological interventions and effective treatment approaches to address this increasingly prevalent issue (Sheng et al., 2023). Given the significant and expanding global burden, there is an urgent need for the development of novel treatment modalities.

In recent decades, acupuncture has gained increasing international attention (Ma et al., 2023). As a non-pharmacological treatment method, acupuncture has become increasingly important in pain management (Brinkhaus et al., 2020). By inserting fine metal needles into specific acupoints on the body and manipulating them, acupuncture has been proven to be an effective method for pain relief (Lin et al., 2022). Currently, electroacupuncture is extensively utilized in the clinical management of OA affecting the knee (Chen et al., 2021). In clinical settings, various electroacupuncture techniques have been extensively applied for the treatment of OA, not only alleviating pain and disability caused by OA but also reducing inflammation and the progression of OA pathogenesis, with equivalent or superior efficacy compared to conventional therapies. Furthermore, research on the mechanisms of electroacupuncture in relieving OA has mainly focused on improving pathological changes such as joint tissue histology, cartilage morphology, synovial inflammation, infiltration, and subchondral bone remodeling (Li et al., 2022). However, the exact mechanisms by which electroacupuncture improves cartilage damage remain unclear.

New research indicates that the onset of OA is controlled by various mechanisms of cell death and cytokine types (Yang et al., 2021). Ferroptosis, a newly discovered regulated cell death process, is distinguished by the iron-dependent buildup of lipid hydroperoxides that can reach lethal levels. However, the specific reversal of ferroptosis can be achieved through the use of ferroptosis inhibitors (Zhang S. et al., 2022). Recent studies have shown that iron-induced death of chondrocytes contributes to the progression of OA (Zhang X. et al., 2023). Previous research has demonstrated that chondrocytes undergo ferroptosis in inflammatory conditions, and inhibiting ferroptosis could alleviate chondrocyte damage (Guo et al., 2022). Therefore, targeting ferroptosis may be a promising approach for future OA treatment. However, the mechanisms of how electroacupuncture interacts with ferroptosis in OA are not yet clear.

Nrf2 is a transcription factor that is sensitive to changes in cellular redox status and has a vital role in controlling the expression of genes. It plays a critical role in regulating both basal and inducible gene expression. Specifically, Nrf2 can regulate the expression of antioxidant enzymes, thereby offering protection against oxidative damage (He F. et al., 2020). Studies have demonstrated that Nrf2 is involved in the regulation of the heme oxygenase-1 (HO-1) axis, which serves as an effective target for anti-inflammatory interventions (Saha et al., 2020). Wu J et al. reported that Stevioside, a compound found in sweeteners, alleviates OA by modulating Nrf2/HO-1/NF-κB pathway (Wu J. et al., 2023). Tan Z et al. reported that Echinacoside alleviates OA in rats by activating Nrf2/HO-1 pathway (Tan and Zhang, 2022). However, the mechanisms of electroacupuncture and Nrf2/HO-1 signaling pathway in OA are still unclear.

Based on the above background, it is plausible to hypothesize that the Nrf2/HO-1 pathway and ferroptosis may play a role in the therapeutic effects of electroacupuncture on the reduction of OA. To this end, we conducted in vivo experiments, established a rat model of OA, and treated it with electroacupuncture therapy and ferroptosis inhibitor ferrostatin-1 to explore the potential mechanism of electroacupuncture in OA treatment. Our study may provide new perspectives for further research on OA treatment.

Materials and methods

Animals

Thirty male Sprague-Dawley rats (200–250 g) were provided by Guangzhou Ruige Biotechnology Co., Ltd. and were randomly divided into 5 groups with 6 rats in each group. They were Control, OA, OA + ferrostatin-1, OA + EA, and OA + ferrostatin-1+EA groups. The OA model was established by intra-articular injection of 50 μL (equivalent to 3 mg) of 60 mg/mL sodium iodoacetate (Philpott and McDougall, 2020; Izumi et al., 2012). In the OA + ferrostatin-1 group, rats received intra-articular injections of 1 mg/kg of ferrostatin-1 on the second day after modeling (Wan et al., 2023), twice a week for 3 weeks. In the OA + EA group, rats underwent EA treatment on the second day after modeling, for 20 min each session, 5 days a week for 3 weeks. In the OA + ferrostatin-1+EA group, rats received intra-articular injections of 1 mg/kg of ferrostatin-1, followed by modeling, and then received EA treatment. The Control group received intra-articular injections of an equal amount of saline. The acupuncture points were ST36 (Zusanli), EX-LE5 (Neixiyan), and Heding. The rats were placed in a restrainer, and acupuncture needles (0.25 mm × 13 mm) were inserted at the designated points. Needle insertion depth was adjusted according to the anatomical location of each acupoint, with reference to previous studies (Tan et al., 2022; Huang et al., 2025). The needles were connected to an SDZ-II electronic acupuncture instrument. Sparse wave stimulation was administered at a frequency of 2 Hz and an intensity of 1 mA for a duration of 20 min (Li et al., 2020a; Zhang et al., 2019; Zhang W. et al., 2023). Behavioral changes and pain responses of the rats were observed once a week, and their body weight and knee joint width were measured. Anesthetized rats had their knee joint width measured using a caliper (N et al., 2016). After the rats were sacrificed, the changes in the color and size of the cartilage surface, as well as any ulceration, were observed and photographed. Blood and synovial fluid samples were collected.

Paw withdrawal threshold (PWT) detection

The pain behavior assessment was conducted using von Frey filaments. Rats were housed in modular cages equipped with a metal mesh floor that enabled direct contact with the plantar surface of each hind paw. Once the animals had sufficient time to acclimate to the environment and exhibited reduced levels of exploratory behavior (approximately 10 min), von Frey filaments were used for measurement. A single von Frey filament was applied perpendicularly to the plantar surface of the hind paw on the same side, taking care to avoid contact with the footpad. The force was applied gradually until the filament bent. After the filament was kept in place for 3 s, if a positive response such as withdrawal, shaking, or licking of the hind paw was observed, the next lower-intensity filament was applied for further testing. If no response was elicited, the next higher-intensity filament was applied until the filament with a bending force of 25 g was reached (Philpott and McDougall, 2020; Li et al., 2020a; Sun et al., 2022; He L. et al., 2020).

Hematoxylin eosin (HE) staining

The knee joint cartilage tissue was collected and fixed in a tissue fixative solution (G1101, Servicebio). The samples were then subjected to heat treatment at 60 °C for 1–2 h and subsequently paraffin-embedded and sectioned. The sections were dewaxed and rehydrated with distilled water. They were stained with Hematoxylin (G1003, Servicebio) for 5–10 min, followed by counterstaining with Eosin for 3–5 min. Sections were dehydrated using a graded series of ethanol (95%–100%) for 5 min at each concentration. Afterward, the sections were treated with xylene (10023418, Sinopharm Chemical Reagent Co., Ltd.) for 10 min, repeated twice, and finally mounted with neutral mounting medium (10004160, Sinopharm Chemical Reagent Co., Ltd.) for observation under a fluorescence microscope (BA210T, Motic).

Safranin O-fast green staining and OARSI scoring

For observation of cartilage degeneration, Safranin O-fast green staining was performed. Rat cartilage tissue was embedded in paraffin and cut into 2 μm sections. Sections were baked at 60 °C for 12 h. After dewaxing, the sections were stained with Safranin O and counterstained with Fast Green according to standard procedures. The sections were mounted with neutral mounting medium and observed and photographed under a microscope. Based on the Safranin O-fast green staining results, cartilage damage was further evaluated using the OARSI scoring system.

Enzyme linked immunosorbent assay (ELISA)

ELISA was utilized to evaluate inflammatory factors IL-6, TNF-α, and IL-1β levels in serum and synovial fluid. IL-6 (RX302856R, RUIXIN BIOTECH), TNF-α (RX302058R, RUIXIN BIOTECH), and IL-1β (RX302869R, RUIXIN BIOTECH) ELISA assay kits were applied to measure IL-6, TNF-α, and IL-1β according to the manufacturers’ instructions.

TdT-mediated dUTP nick-end labeling (TUNEL)

Knee cartilage tissue apoptosis was detected using a TUNEL kit (G1502, Servicebio). Cartilage tissues were sectioned and baked at 60 °C for 30–60 min. Sections were dewaxed to water. Subsequently, each sample was treated with 100 μL of Proteinase K (G1205, Servicebio), added dropwise, and incubated at 37 °C for 20 min. An appropriate amount of membrane-breaking solution (G1204, Servicebio) was added dropwise to the tissues, and the tissues were fully infiltrated and processed for 20 min, and samples were similarly washed with PBS after membrane-breaking treatment. The samples were washed with PBS. For each sample, dropwise addition of 100 μL of 1×Equilibration Buffer was performed to cover the region of the sample designated for examination, followed by incubation for 10–30 min. TdT enzyme incubation buffer was prepared. The excess 1×Equilibration Buffer was removed by gently blotting with absorbent paper around the equilibrated area, and then 50 μL of TdT enzyme incubation buffer was added to the targeted tissues for examination. The incubation process was carried out for 60 min at 37 °C, while being protected from light. Nuclei were subsequently stained with DAPI (G1012, Servicebio) for 10 min at 37 °C, followed by three rinses with PBS for 5 min each. Finally, sections were sealed with neutral gum and subjected to observation using a fluorescence microscope.

Immunofluorescence (IF)

IF was employed to assess co-localized expression of Nrf2, HO-1, SLC7A11, GPX4 and SOX9 in knee cartilage tissues. Sections were baked at 60 °C for 12 h and subsequently deparaffinized to water. Sections were then sequentially placed in 100%, 100%, 95%, 85% and 75% ethanol for 5 min at each concentration. To inactivate endogenous enzymes, 3% H2O2 was added for 10 min at room temperature. The sections were blocked with 5% BSA (BS114-100 g, Biosharp) for 60 min, followed by addition of Nrf2 (16396-1-AP, Proteintech), HO-1 (10701-1-AP, Proteintech), SLC7A11 (A13685, ABCLONAL), GPX4 (67763-1-Ig, Proteintech), and SOX9 (82630T, CST) and incubation at 4 °C overnight. Subsequently, secondary antibody (RC0086-23, Rutron) was incubated at 37 °C for 90 min, and the nuclei were stained with DAPI. Antifade mounting medium (G1401, Servicebio) was applied to seal sections. Images were captured by fluorescence microscopy.

Biochemical detection

According to the instructions, the GSH (BC1175, Solarbio) assay kit and the Fe2+ (BC5415, Solarbio) assay kit were employed to determine the levels of GSH and Fe2+ in rat cartilage tissue.

Western blot

Western blot was performed to determine ACSL4, Nrf2, and HO-1 levels in cartilage tissue. Total protein was extracted from cartilage tissue using RIPA (P0013B, Beyotime). After quantification with a BCA kit (BL521A, Biosharp), total proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with primary antibodies against ACSL4 (22401-1-AP, Proteintech), Nrf2 (16396-1-AP, Proteintech), HO-1 (10701-1-AP, Proteintech), and GAPDH (60004-1-Ig, Proteintech). The membranes were then incubated with Goat Anti-Rabbit IgG (111-035–003, JACKSON) or Goat Anti-Mouse IgG (115-035–003, JACKSON). Signals were visualized using an enhanced chemiluminescence substrate (K-12045-D50, Advansta) and analyzed with imaging software (Chemiscope 6100, CLINX). GAPDH was used as the internal control.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0. Data were presented as mean ± SD. Differences among groups were analyzed by one-way ANOVA followed by multiple comparisons. A value of P < 0.05 was considered statistically significant.

Results

Behavioral detection and characterization of rats

First, we established an OA model in rats and treated them with ferrostatin-1 and EA. Macroscopic examination of knee joint cartilage revealed cartilage damage and an irregular, rough surface in the OA group compared to the Control group. Treatment with ferrostatin-1 alone, EA alone, or the combination of ferrostatin-1 and EA improved the cartilage damage on the surface of the knee joint (Figure 1A). The PWT in the OA group significantly decreased compared to the Control group from day 7 to day 21. Treatment with ferrostatin-1 alone, EA alone, or the combination of ferrostatin-1 and EA increased the PWT in the rats (Figure 1C). Additionally, compared to the Control group, from day 7 to day 21, the body weight of the rats in the OA group gradually decreased, and the width of the knee joint increased. Treatment with ferrostatin-1 alone, EA alone, or the combination of ferrostatin-1 and EA partly restored body weight and decreased the width of the knee joint in rats (Figures 1B,D).

FIGURE 1.

Panel A shows photographs of dissected knee joints from five experimental groups: Control, OA, OA plus Fer-1, OA plus EA, and OA plus Fer-1 plus EA. Panel B presents a line chart comparing body weights over time for each group. Panel C displays a line chart of paw withdrawal thresholds across time points, with the control group maintaining higher thresholds. Panel D shows knee width measurements over time, with OA and treatment groups showing increased values compared to control.

Open in a new tab

Behavioral detection and characterization of rats. (A) Observation of knee joint cartilage. (B) Changes in body weight of rats. (C) PWT detection. (D) Knee joint width changes of rats.

Pathological analysis of cartilage tissue of knee joint in rats

Next, we analyzed the pathological conditions of knee cartilage tissue in rats. HE staining showed that the cartilage surface of the OA, OA + ferrostatin-1, OA + EA, and OA + ferrostatin-1+EA groups was rough, fibrotic, and erosive, with reduced numbers of chondrocytes, blurred tidemarks, and a more incomplete structure than in the Control group. These changes were most obvious in the OA group (Figure 2A). In comparison to the Control group, the OA, OA + ferrostatin-1, OA + EA, and OA + ferrostatin-1+EA groups showed cartilage erosion. This change was most obvious in the OA group (Figure 2B). OARSI scores were also significantly increased in the OA group compared with the Control group, and were reduced after treatment with ferrostatin-1, EA, or the combination of ferrostatin-1 and EA (Figure 2C). Based on the current data, no clear additional benefit of the combined treatment over the single-treatment groups was observed.

FIGURE 2.

Panel A shows representative histological images of cartilage stained with H&E for five groups: Control, OA, OA plus Fer-1, OA plus EA, and OA plus Fer-1 plus EA. Panel B presents corresponding Safranin O-stained cartilage images for the same groups, illustrating differences in cartilage integrity and proteoglycan content. Panel C is a bar graph comparing OARSI scores across groups, with statistical annotations indicating significant differences among treatments, showing that OA has the highest score while treatments reduce the score to varying degrees.

Open in a new tab

Pathological analysis of cartilage tissue of knee joint in rats. (A) HE staining of knee cartilage tissue. Magnification: ×100. (B) Safranin O-fast green staining of knee cartilage tissue. Magnification: ×100. (C) OARSI scores of cartilage lesions.

Levels of inflammatory cytokines IL-6, TNF-α, and IL-1β in serum and synovial fluid

In addition, inflammatory cytokine levels of IL-6, TNF-α, and IL-1β in the serum and synovial fluid of rats were significantly elevated in the OA group compared with the Control group. IL-6, TNF-α, and IL-1β levels were reduced with ferrostatin-1 and EA alone and in combination with ferrostatin-1 and EA (Figures 3A,B).

FIGURE 3.

Figure consists of two grouped bar chart panels labeled A and B, each displaying three bar plots for IL-6, IL-1β, and TNF-α concentrations across five experimental groups: Control, OA, OA+Fer-1, OA+EA, and OA+Fer-1+EA. Statistical comparisons are indicated with asterisks above bars, with OA groups showing elevated cytokine levels compared to Control, and intervention groups demonstrating reduced levels relative to OA. Error bars denote variability.

Open in a new tab

ELISA analysis of inflammatory cytokines in serum and synovial fluid. (A) IL-6, IL-1β, and TNF-α levels in serum. (B) IL-6, IL-1β, and TNF-α levels in synovial fluid. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Effects of acupuncture on apoptosis of knee cartilage and ferroptosis-related factors

Furthermore, TUNEL staining revealed a significant increase in apoptosis in the cartilage tissue and surrounding areas of the knee joints in the OA, OA + ferrostatin-1, OA + EA, and OA + ferrostatin-1+EA groups compared with the Control group. Notably, the OA group exhibited particularly pronounced apoptosis, as shown in Figure 4A. We further detected the co-localized expression of SLC7A11, GPX4, and SOX9 in knee cartilage tissues using IF staining. In contrast to the Control group, the expression of SLC7A11, GPX4, and SOX9 was found to be downregulated in the knee cartilage tissue of the OA group. However, the expression of SLC7A11, GPX4, and SOX9 in knee cartilage tissues increased after treatment with ferrostatin-1 and EA alone and the combined use of ferrostatin-1 and EA (Figures 4B,C). Detection of GSH and Fe2+ levels in rat cartilage tissues showed decreased GSH levels and increased Fe2+ levels in the OA group compared with the Control group. After the administration of ferrostatin-1 and EA alone, as well as the combination of ferrostatin-1 and EA, there was an increase in GSH levels and a decrease in Fe2+ levels in rat cartilage tissues (Figures 4D,E).

FIGURE 4.

Panel A shows immunofluorescent-stained tissue sections with blue nuclei across five treatment groups: Control, OA, OA plus Ferrostatin-1, OA plus EA, and OA plus both agents. Panel B shows staining for SLC7A11 (green) and SOX9 (orange) in the same groups, highlighting differences in expression. Panel C displays staining for GPX4 (green) and SOX9 (orange) across the groups, indicating changes in protein localization. Panel D is a bar graph measuring GSH levels in micrograms per gram for each group, with significant differences marked by asterisks. Panel E is a bar graph showing Fe²⁺ concentrations in micromoles per gram, with statistical significance indicated among groups.

Open in a new tab

Effects of acupuncture on apoptosis of knee cartilage and ferroptosis-related factors. (A) TUNEL staining was utilized to determine apoptosis in knee joint cartilage tissue. Magnification: ×200. (B) IF staining of SLC7A11 and SOX9 in knee joint cartilage tissue. Magnification: ×200. (C) IF staining of GPX4 and SOX9 in knee joint cartilage tissue. Magnification: ×200. (D) GSH levels in cartilage tissue. (E) Fe2+ levels in cartilage tissue. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Effect of acupuncture on nrf2/HO-1 pathway-related factors

Finally, we further evaluated Nrf2, HO-1, and SOX9 levels in knee cartilage tissues using IF staining. As depicted in Figures 5A,B, in comparison to the Control group, the expression of Nrf2 and HO-1 in knee cartilage tissue was found to be upregulated in the OA group, while the expression of SOX9 was downregulated. However, the expression of Nrf2, HO-1, and SOX9 in knee cartilage tissues increased after treatment with ferrostatin-1 and EA alone and the combined use of ferrostatin-1 and EA. Western blot analysis showed that ACSL4, Nrf2, and HO-1 levels in the cartilage tissues of rats in the OA group were significantly higher than those in the Control group. After treatment with ferrostatin-1 alone, EA alone, or the combination of ferrostatin-1 and EA, ACSL4 expression was reduced, whereas Nrf2 and HO-1 levels were further increased (Figure 5C).

FIGURE 5.

Panel A shows immunofluorescence staining of NRF2 (green) and SOX9 (red), while panel B shows HO-1 (green) and SOX9 (red) in tissue sections from Control, OA, OA+Fer-1, OA+EA, and OA+Fer-1+EA groups. Panel C presents Western blot results and quantitative bar graphs for ACSL4, NRF2, HO-1, and GAPDH protein levels, indicating statistical significance between groups.

Open in a new tab

Effect of acupuncture on Nrf2/HO-1 pathway-related factors. (A) IF staining of Nrf2 and SOX9 in knee joint cartilage tissue. Magnification: ×200. (B) IF staining of HO-1 and SOX9 in knee joint cartilage tissue. Magnification: ×200. (C) Western blot analysis of ACSL4, Nrf2, and HO-1 in cartilage tissue. **P < 0.01, ***P < 0.001.

Discussion

OA is a multifaceted process that involves various contributing factors such as mechanical load, inflammation, and metabolic factors (Yao et al., 2021). Current treatments, whether conservative or surgical, are designed to relieve symptoms and cannot delay or reverse the degenerative process of the joint (Wu C. et al., 2023). There is evidence that acupuncture could reduce pain, stiffness, and dysfunction in patients with OA, ultimately improving patient health (Luo et al., 2023). However, the mechanisms are unclear. In this study, we constructed a rat OA model and treated it with EA therapy and ferrostatin-1. We found that acupuncture was associated with activation of the Nrf2/HO-1 pathway, alleviation of OA, and reduced ferroptosis-related changes. To our knowledge, few studies have examined the relationship among acupuncture, ferroptosis, and the Nrf2/HO-1 pathway in OA, which highlights the novelty of the present study.

Acupuncture is a medical practice rooted in traditional Chinese medicine that involves the insertion of thin needles or the application of pressure to specific points on the body (Van Hal et al., 2023). There is evidence that acupuncture treatment combined with hyaluronic acid injection is more effective than hyaluronic acid injection alone in relieving pain and improving the function of the knee joint (Zheng et al., 2020). In addition, EA could significantly improve the clinical symptoms and quality of life of knee OA patients (Li et al., 2023). In this study, we first constructed a knee OA model by intra-articular injection of sodium iodoacetate, and then observed the effect of ferrostatin-1 and EA alone or combined with ferrostatin-1 and EA. We found that cartilage damage occurred on the knee surface of OA rats, the surface was rough and uneven, PWT decreased significantly, body weight decreased gradually, and knee width increased gradually. These conditions improved with ferrostatin-1 and EA alone and with ferrostatin-1 and EA together. In addition, our study showed that EA could reduce inflammatory factor levels in serum and synovial fluid of OA rats. Histological staining further showed that cartilage injury was accompanied by increased OARSI scores in the OA group, and these changes were attenuated after ferrostatin-1 or EA treatment. The combined treatment group also showed improvement, but no clear additional benefit over the single-treatment groups was observed in the present data. Taken together, our results suggest that EA exerted beneficial effects in OA, which may be related to the regulation of ferroptosis.

Iron is an essential element for many processes within cells. If cellular iron homeostasis is disrupted, iron will participate in the Fenton reaction (Fe2+ reduction of H2O2 to produce OH•), resulting in the formation of lipid peroxides and excessive accumulation of lethal ROS, thus leading to cell death (Yang and Stockwell, 2016; Dixon et al., 2012). Ferroptosis is a recently discovered form of cell death that is tightly regulated by iron metabolism, antioxidant processes, and lipid metabolism. It is known to play a crucial role in the development and progression of various diseases (Zhang Y. et al., 2022). Ferroptosis is manifested by the inactivation of GPX4, the regulatory core enzyme based on GSH depletion (Yang and Stockwell, 2016). GPX4 is responsible for transforming harmful lipid peroxides into harmless lipid alcohols (Ingold et al., 2018). Studies have shown that inhibiting ferroptosis could alleviate OA (Zhou et al., 2021). In this study, our findings suggested that EA may inhibit ferroptosis-related changes in OA. In addition to changes in GPX4, SLC7A11, GSH, and Fe2+, ACSL4 expression was increased in the OA group and decreased after ferrostatin-1 or EA treatment. This finding further supports the involvement of ferroptosis-related lipid metabolic changes in this process.

The Nrf2/HO-1 pathway serves as one of the vital endogenous defense systems in the body, and it represents the classical mechanism through which Nrf2 exerts its protective function (Li et al., 2020b). Nrf2 is a pivotal transcription factor that controls the expression of antioxidant enzymes. Its primary function is to safeguard against oxidative stress and protect tissues from damage (Chen et al., 2019). Indeed, the Nrf2/HO-1 signaling pathway is widely recognized as a protective molecular mechanism in situations of oxidative stress (Han et al., 2022). Guo Z et al. reported that deferoxamine alleviated OA by inhibiting ferroptosis and activating the Nrf2 pathway in chondrocytes (Guo et al., 2022). Wan Y et al. demonstrated that Baicalein inhibited chondrocyte ferroptosis by increasing the activity of AMPK/Nrf2/HO-1 signaling, thus alleviating the development of OA (Wan et al., 2023). In our study, Nrf2 and HO-1 expression was increased in the OA group and was further elevated after ferrostatin-1 or EA treatment. This pattern suggests that the upregulation of Nrf2/HO-1 in OA may reflect an endogenous compensatory response to oxidative stress, whereas its further enhancement after treatment may be associated with a protective effect. Taken together, these findings suggest that Nrf2/HO-1 activation in our model was mainly protective, while also showing compensatory features under OA conditions. This also suggests that the Nrf2/HO-1 pathway may be involved in the effects of EA. However, the present data do not establish a direct causal relationship between Nrf2/HO-1 activation and ferroptosis inhibition, which remains a limitation of this study.

Conclusion

We preliminarily assessed the effect of acupuncture on OA and explored whether it was related to ferroptosis and the Nrf2/HO-1 pathway. Our findings suggest that acupuncture alleviated OA and was associated with activation of the Nrf2/HO-1 pathway and reduced ferroptosis-related changes. Our findings may help to improve the understanding of the molecular changes associated with OA. They may also provide a basis for further studies on EA in OA.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the Research Project of Traditional Chinese Medicine Bureau of Guangdong Province, China (Grant number 20231282) received by Jun Yan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

Edited by: Palash Mandal, Charotar University of Science and Technology, India

Reviewed by: Gloria Riitano, Sapienza University of Rome, Italy

Jiangnan Ye, Chengdu University of Traditional Chinese Medicine, China

Data availability statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethics statement

The animal study was approved by the Guangzhou Forevergen Medical Laboratory Animal Center. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

MZ: Writing – original draft, Methodology, Writing – review and editing, Conceptualization. LO: Methodology, Writing – review and editing, Writing – original draft, Conceptualization. JM: Formal Analysis, Writing – original draft, Investigation, Writing – review and editing, Data curation, Methodology. HZ: Investigation, Writing – review and editing, Writing – original draft, Data curation, Methodology, Formal Analysis. SZ: Data curation, Formal Analysis, Writing – review and editing, Investigation, Writing – original draft, Methodology. XZ: Writing – original draft, Formal Analysis, Data curation, Writing – review and editing, Methodology, Investigation. BH: Methodology, Writing – review and editing, Formal Analysis, Data curation, Writing – original draft, Investigation. JY: Methodology, Writing – review and editing, Conceptualization, Writing – original draft.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

  1. Brinkhaus B., Ortiz M., Dietzel J., Willich S. (2020). Acupuncture for pain and allergic rhinitis-from clinical experience to evidence. Bundesgesundheitsblatt Gesundheitsforsch. Gesundheitsschutz 63 (5), 561–569. 10.1007/s00103-020-03127-6 [DOI] [PubMed] [Google Scholar]
  2. Chen Z., Zhong H., Wei J., Lin S., Zong Z., Gong F., et al. (2019). Inhibition of Nrf2/HO-1 signaling leads to increased activation of the NLRP3 inflammasome in osteoarthritis. Arthritis Res. Ther. 21 (1), 300. 10.1186/s13075-019-2085-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen J., Liu A., Zhou Q., Yu W., Guo T., Jia Y., et al. (2021). Acupuncture for the treatment of knee osteoarthritis: an overview of systematic reviews. Int. J. Gen. Med. 14, 8481–8494. 10.2147/IJGM.S342435 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dixon S. J., Lemberg K. M., Lamprecht M. R., Skouta R., Zaitsev E. M., Gleason C. E., et al. (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149 (5), 1060–1072. 10.1016/j.cell.2012.03.042 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Emami A., Namdari H., Parvizpour F., Arabpour Z. (2023). Challenges in osteoarthritis treatment. Tissue Cell 80, 101992. 10.1016/j.tice.2022.101992 [DOI] [PubMed] [Google Scholar]
  6. Guo Z., Lin J., Sun K., Guo J., Yao X., Wang G., et al. (2022). Deferoxamine alleviates osteoarthritis by inhibiting chondrocyte ferroptosis and activating the Nrf2 pathway. Front. Pharmacol. 13, 791376. 10.3389/fphar.2022.791376 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Han T., Zhang S., Wei R., Jia G., Wang B., Xu Q., et al. (2022). Synthesis and biological evaluation of scutellarein derivatives as neuroprotective agents via activating Nrf2/HO-1 pathway. Fitoterapia 160, 105207. 10.1016/j.fitote.2022.105207 [DOI] [PubMed] [Google Scholar]
  8. He F., Ru X., Wen T. (2020a). NRF2, a transcription factor for stress response and beyond. Int. J. Mol. Sci. 21 (13), 4777–4799. 10.3390/ijms21134777 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. He L., He T., Xing J., Zhou Q., Fan L., Liu C., et al. (2020b). Bone marrow mesenchymal stem cell-derived exosomes protect cartilage damage and relieve knee osteoarthritis pain in a rat model of osteoarthritis. Stem Cell Res. Ther. 11 (1), 276. 10.1186/s13287-020-01781-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huang L., Deng X., Jin Y., Li J., Wu X., Xiong Y., et al. (2025). Efficacy and potential mechanisms of Yi-Ping lin Circum-Knee acupuncture in the treatment of knee osteoarthritis. J. Pain Res. 18, 5891–5906. 10.2147/JPR.S547968 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ingold I., Berndt C., Schmitt S., Doll S., Poschmann G., Buday K., et al. (2018). Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis. Cell 172 (3), 409–422.e21. 10.1016/j.cell.2017.11.048 [DOI] [PubMed] [Google Scholar]
  12. Izumi M., Ikeuchi M., Ji Q., Tani T. (2012). Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis. J. Biomed. Sci. 19 (1), 77. 10.1186/1423-0127-19-77 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Küçükdeveci A. A. (2023). Rehabilitation interventions in osteoarthritis. Best. Pract. Res. Clin. Rheumatol. 37, 101846. 10.1016/j.berh.2023.101846 [DOI] [PubMed] [Google Scholar]
  14. Li B., Jing L., Jia L., Qian T., Jianyi C., Zhongsheng H., et al. (2020a). Acupuncture reduces pain in rats with osteoarthritis by inhibiting MCP2/CCR2 signaling pathway. Exp. Biol. Med. (Maywood) 245 (18), 1722–1731. 10.1177/1535370220952342 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Li B., Nasser M. I., Masood M., Adlat S., Huang Y., Yang B., et al. (2020b). Efficiency of traditional Chinese medicine targeting the Nrf2/HO-1 signaling pathway. Biomed. Pharmacother. 126, 110074. 10.1016/j.biopha.2020.110074 [DOI] [PubMed] [Google Scholar]
  16. Li Y., Xu Y., Chen B., Xu Z. F., Wang S. J., Fang Y. X., et al. (2022). Progress of researches on acupuncture-moxibustion treatment of cartilage damage of osteoarthritis. Zhen Ci Yan Jiu 47 (9), 837–842. 10.13702/j.1000-0607.20210750 [DOI] [PubMed] [Google Scholar]
  17. Li P., Zhang Y., Li F., Cai F., Xiao B., Yang H. (2023). The efficacy of electroacupuncture in the treatment of knee osteoarthritis: a systematic review and meta-analysis. Adv. Biol. (Weinh) 7 (10), e2200304. 10.1002/adbi.202200304 [DOI] [PubMed] [Google Scholar]
  18. Lin J. G., Kotha P., Chen Y. H. (2022). Understandings of acupuncture application and mechanisms. Am. J. Transl. Res. 14 (3), 1469–1481. [PMC free article] [PubMed] [Google Scholar]
  19. Luo X., Liu J., Li Q., Zhao J., Hao Q., Zhao L., et al. (2023). Acupuncture for treatment of knee osteoarthritis: a clinical practice guideline. J. Evid. Based Med. 16 (2), 237–245. 10.1111/jebm.12526 [DOI] [PubMed] [Google Scholar]
  20. Ma Y., Li B., Cui Y., Zhang Z., Jiang S., Yan X., et al. (2023). The top 100 cited articles on acupuncture in the last 20 years: a bibliometric analysis. Complement. Med. Res. 30 (5), 393–407. 10.1159/000530778 [DOI] [PubMed] [Google Scholar]
  21. Nwosu L. N., Mapp P. I., Chapman V., Walsh D. A. (2016). Relationship between structural pathology and pain behaviour in a model of osteoarthritis (OA). Osteoarthr. Cartil. 24 (11), 1910–1917. 10.1016/j.joca.2016.06.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Philpott H. T., McDougall J. J. (2020). Combatting joint pain and inflammation by dual inhibition of monoacylglycerol lipase and cyclooxygenase-2 in a rat model of osteoarthritis. Arthritis Res. Ther. 22 (1), 9. 10.1186/s13075-020-2096-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Saha S., Buttari B., Panieri E., Profumo E., Saso L. (2020). An overview of Nrf2 signaling pathway and its role in inflammation. Molecules 25 (22), 5474–5504. 10.3390/molecules25225474 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sheng W., Wang Q., Qin H., Cao S., Wei Y., Weng J., et al. (2023). Osteoarthritis: role of peroxisome proliferator-activated receptors. Int. J. Mol. Sci. 24 (17), 13137. 10.3390/ijms241713137 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sun J., Song F. H., Wu J. Y., Zhang L. Q., Li D. Y., Gao S. J., et al. (2022). Sestrin2 overexpression attenuates osteoarthritis pain via induction of AMPK/PGC-1α-mediated mitochondrial biogenesis and suppression of neuroinflammation. Brain Behav. Immun. 102, 53–70. 10.1016/j.bbi.2022.02.015 [DOI] [PubMed] [Google Scholar]
  26. Tan Z., Zhang B. (2022). Echinacoside alleviates osteoarthritis in rats by activating the Nrf2-HO-1 signaling pathway. Immunopharmacol. Immunotoxicol. 44 (6), 850–859. 10.1080/08923973.2022.2088384 [DOI] [PubMed] [Google Scholar]
  27. Tan Q., Li B., Li J., Li J., Xiang H., Cai G. (2022). Acupuncture combined with moxibustion regulates the expression of circadian clock protein in the synovium of rats with osteoarthritis. Chin. J. Tissue Eng. Res. 26 (11), 1714. 10.12307/2022.356 [DOI] [Google Scholar]
  28. Van Hal M., Dydyk A. M., Green M. S. (2023). StatPearls Publishing LLC: treasure Island (FL) ineligible companies. Disclosure: Alexander Dydyk declares no relevant financial relationships with ineligible companies. [Google Scholar]
  29. Wan Y., Shen K., Yu H., Fan W. (2023). Baicalein limits osteoarthritis development by inhibiting chondrocyte ferroptosis. Free Radic. Biol. Med. 196, 108–120. 10.1016/j.freeradbiomed.2023.01.006 [DOI] [PubMed] [Google Scholar]
  30. Wu J., Li H., Hu F., Luo P. (2023a). Stevioside attenuates osteoarthritis via regulating Nrf2/HO-1/NF-κB pathway. J. Orthop. Transl. 38, 190–202. 10.1016/j.jot.2022.05.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wu C., He Y., Yao Y., Yang H., Lu F. (2023b). Exosomes treating osteoarthritis: hope with challenge. Heliyon 9 (1), e13152. 10.1016/j.heliyon.2023.e13152 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang W. S., Stockwell B. R. (2016). Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 26 (3), 165–176. 10.1016/j.tcb.2015.10.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yang J., Hu S., Bian Y., Yao J., Wang D., Liu X., et al. (2021). Targeting cell death: pyroptosis, ferroptosis, apoptosis and necroptosis in osteoarthritis. Front. Cell Dev. Biol. 9, 789948. 10.3389/fcell.2021.789948 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yao X., Sun K., Yu S., Luo J., Guo J., Lin J., et al. (2021). Chondrocyte ferroptosis contribute to the progression of osteoarthritis. J. Orthop. Transl. 27, 33–43. 10.1016/j.jot.2020.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yao Q., Wu X., Tao C., Gong W., Chen M., Qu M., et al. (2023). Osteoarthritis: pathogenic signaling pathways and therapeutic targets. Signal Transduct. Target Ther. 8 (1), 56. 10.1038/s41392-023-01330-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang L., Yuan H., Zhang L., Li J., Li H. (2019). Effect of acupuncture therapies combined with usual medical care on knee osteoarthritis. J. Tradit. Chin. Med. 39 (1), 103–110. [PubMed] [Google Scholar]
  37. Zhang S., Xu J., Si H., Wu Y., Zhou S., Shen B. (2022a). The role played by ferroptosis in osteoarthritis: evidence based on iron dyshomeostasis and lipid peroxidation. Antioxidants (Basel) 11 (9), 1668. 10.3390/antiox11091668 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang Y., Huang X., Qi B., Sun C., Sun K., Liu N., et al. (2022b). Ferroptosis and musculoskeletal diseases: “Iron Maiden” cell death may be a promising therapeutic target. Front. Immunol. 13, 972753. 10.3389/fimmu.2022.972753 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang X., Hou L., Guo Z., Wang G., Xu J., Zheng Z., et al. (2023a). Lipid peroxidation in osteoarthritis: focusing on 4-hydroxynonenal, malondialdehyde, and ferroptosis. Cell Death Discov. 9 (1), 320. 10.1038/s41420-023-01613-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhang W., Zhang L., Yang S., Wen B., Chen J., Chang J. (2023b). Electroacupuncture ameliorates knee osteoarthritis in rats via inhibiting NLRP3 inflammasome and reducing pyroptosis. Mol. Pain 19, 17448069221147792. 10.1177/17448069221147792 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhao X. X., Xie W. Q., Xiao W. F., Li H. Z., Naranmandakh S., Bruyere O., et al. (2023). Perlecan: roles in osteoarthritis and potential treating target. Life Sci. 312, 121190. 10.1016/j.lfs.2022.121190 [DOI] [PubMed] [Google Scholar]
  42. Zheng Y., Duan X., Qi S., Hu H., Wang M., Ren C., et al. (2020). Acupuncture therapy plus hyaluronic acid injection for knee osteoarthritis: a meta-analysis of randomized controlled trials. Evid. Based Complement. Altern. Med. 2020, 4034105. 10.1155/2020/4034105 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhou X., Zheng Y., Sun W., Zhang Z., Liu J., Yang W., et al. (2021). D-mannose alleviates osteoarthritis progression by inhibiting chondrocyte ferroptosis in a HIF-2α-dependent manner. Cell Prolif. 54 (11), e13134. 10.1111/cpr.13134 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Articles from Frontiers in Molecular Biosciences are provided here courtesy of Frontiers Media SA

RESOURCES