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. 2024 Apr 17;14(4):801–812. doi: 10.1007/s13534-024-00376-1

Micro-current stimulation could inhibit IL-1β-induced inflammatory responses in chondrocytes and protect knee bone cartilage from osteoarthritis

Minjoo Lee 1,#, Hana Lee 1,#, Halim Chung 1, Jin-Ho Lee 2, Doyong Kim 1, Seungkwan Cho 3, Tack-Joong Kim 2, Han Sung Kim 1,✉
PMCID: PMC11208348  PMID: 38946809

Abstract

This study aimed to evaluate the inhibitory effects of micro-current stimulation (MCS) on inflammatory responses in chondrocytes and degradation of extracellular matrix (ECM) in osteoarthritis (OA). To determine the efficacy of MCS, IL-1β-treated chondrocytes and monosodium iodoacetate (MIA)-induced OA rat model were used. To evaluate the cytotoxicity and nitric oxide (NO) production in SW1353 cells, the presence or absence of IL-1β treatment or various levels of MCS were applied. Immunoblot analysis was conducted to evaluate whether MCS can modulate IL-1R1/MyD88/NF-κB signaling pathway and various indicators involved in ECM degradation. Additionally, to determine whether MCS alleviates subchondral bone structure destruction caused by OA, micro-CT analysis, immunoblot analysis, and ELISA were conducted using OA rat model. 25 and 50 µA levels of MCS showed effects in cell proliferation and NO production. The MCS group with IL-1β treatment lead to significant inhibition of protein expression levels regarding IL-1R1/MyD88/NF-κB signaling and reduction of the nucleus translocation of NF-κB. In addition, the protein expression levels of MMP-1, MMP-3, MMP-13, and IL-1β decreased, whereas collagen II and aggrecan increased. In animal results, morphological analysis of subchondral bone using micro-CT showed that MCS induced subchondral bone regeneration and improvement, as evidenced by increased thickness and bone mineral density of the subchondral bone. Furthermore, MCS-applied groups showed decreases in the protein expression of MMP-1 and MMP-3, while increases in collagen-II and aggrecan expressions. These findings suggest that MCS has the potential to be used as a non-pharmaceutical method to alleviate OA.

Keywords: Micro-current stimulation, Osteoarthritis, Chondrocytes, Articular cartilage, IL-1β/MyD88/NF-κB signaling

Introduction

The knee joint consists of the articular cartilage and subchondral bone. Firstly, the articular cartilage forms the joint surface in the knee and protects the joint [1]. The subchondral bone is the bony layer located just below the articular cartilage. It acts as a major factor in joint protection. Furthermore, articular cartilage is composed of chondrocytes and mostly extracellular matrix (ECM) [2]. Chondrocytes play an important role in synthesizing and maintaining the matrix of articular cartilage, while the ECM of cartilage regulates the metabolism and functions of chondrocytes [3].

The chondrocytes, the only cell type of articular cartilage, are involved in maintaining the shape of articular cartilage through the regulation of the synthesis pathway for producing ECM and the control of various matrix metalloproteinases (MMPs) involved in the degradation of articular cartilage [4]. The main degenerative factor of osteoarthritis (OA) is the increased activity of MMPs, which causes damage to articular cartilage. OA is known to aggravate due to increased pro-inflammatory cytokines such as interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) [5]. In particular, IL-1β has a crucial role in the pathogenesis of OA and triggers apoptosis of chondrocytes, and the activation of cells by IL-1β is mediated through interleukin-1 receptor I (IL-1RI) [6]. These pro-inflammatory cytokines stimulate chondrocytes, activating the cartilage degradation pathway which is a characteristic of OA is induced by the pro-inflammatory cytokine Interleukin-1 beta (IL-1β) that increases the expression of ECM-degrading enzymes called MMPs [7].

Damage to bone tissue is known to overexpress the protein IL-1β and inhibit bone regeneration by binding to receptors in the IL-1R1/MyD88 (Myeloid differentiation primary response 88) signaling [8]. The NF-κB signaling mediates inflammatory gene expression and contributes to the increased release of pro-inflammatory cytokines [9]. While numerous studies have indicated inhibition of the NF-κB signaling pathway involved in joint protection of rheumatoid arthritis animal models [10], research on its application in OA is limited. Not only in the progression of OA but also in the modulation of the signaling of inflammation could be considered valuable research.

Since there are no clinically approved disease-modifying osteoarthritis drugs (DMOADs), osteoarthritis is treated through pain management by non-steroidal anti-inflammatory drugs (NSAIDs), physical therapy, and surgical treatment [11]. However, long-term use and repeated drug treatment can lead to temporary relief of symptoms and damage to kidneys, and serious side effects such as joint stiffness after surgery [12]. Therefore, we would like to propose micro-current stimulation (MCS) as a way to protect and improve articular cartilage by inhibiting inflammatory cytokines such as IL-1β.

Micro-current stimulation (MCS), currents below 1 mA, utilized in recent clinical applications since it has shown significant effectiveness in pain relief and restoration of nerves and muscles [13]. In our previous study, it was studied that MCS suppresses inflammatory responses by TLR2/NF-κB signaling pathway [14]. Although there are differences depending on the type of cell and the intensity of stimulation, MCS has been reported to have an anti-inflammatory effect including TLR2, MyD88, TRAF6, TAK1, NF-κB, IκB, and pro-inflammatory cytokines. Furthermore, the TLR-2/NF-κB signaling pathway has been implicated in the progression of OA and mediates the expression of multiple inflammatory cytokines, resulting in cartilage protection and delaying cartilage degradation [15]. Several studies have shown that MCS has been reported to provide pain relief through physiological mechanisms, promote fracture healing, increase joint range of motion, inhibit chondrocyte destruction, promote wound and tissue regeneration, and inhibit bacterial growth, among other therapeutic effects [16]. Moreover, MCS has been shown to promote the synthesis of extracellular matrix glycoproteins and collagen and is one of the most favorable tools for cartilage healing and tissue repair [17]. Since MCS can reduce physical discomfort, recent studies suggest MCS as a new therapeutic method as a catalyst for chemical and electrical reactions that respond to the healing process [18]. However, research on whether MCS can inhibit the IL-1β-mediated inflammatory response, which is related to the main mechanisms of joint cartilage and subchondral bone regeneration, has not been conducted yet.

Therefore, in this study, changes in the factors of IL-1R1/MyD88/NF-κB signaling in IL-1β-treated SW1353 cells were observed. Additionally, the effects of MCS on the regulation of articular cartilage ECM components and subchondral bone improvement in monosodium iodoacetate (MIA)-induced OA rat model were evaluated.

Materials and methods

Materials

DMEM/Nutrient Mixture F-12 Ham (DMEM/F-12) and L-Glutamine solution, and interleukin-1β (IL-1β; SRP3083) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Penicillin-streptomycin (PS) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Invitrogen-Gibco-BRL, Grand Island, NY, USA). EZ-Cytox cell viability assay kit was purchased from the Daeil Labservice (Seoul, Korea). Lysis buffer was purchased from iNtRON Biotechnology Inc. (Seongnam, Korea). BCA assay kits were purchased from Thermo Scientific (Rockford, IL, USA). MMP13 ELISA Kit was purchased from USCN Life Science Inc. (Wuhan, China).

Micro-current stimulation (MCS)

In the in-vitro experiments, the parameters were set to a biphasic rectangular current pulse, a duty cycle of 50%, and a frequency of 10 Hz to compare the MCS effects of different intensities (Fig. 1A). To compare NO production according to the different intensities of 25, 50, 100, and 200 µA levels were applied to each well for 30 min. In further experiments, we only applied 25 and 50 µA of MCS, which showed relatively effective results in cell viability and NO production.

Fig. 1.

Fig. 1

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(A) Schematic diagram indicating experimental methods with the timeline for in-vitro experiment. (B) Schematic diagram indicating experimental methods with the timeline for in-vivo experiment

Subsequently, in the in-vivo experiment, we attached the custom-made electrodes made of conductive metal and hydrogels to the femur and tibia, using 25 and 50 µA of MCS (Fig. 1B). The pulse type and frequency were set to the same conditions, and MCS was applied for 30 min each day for 4 weeks.

SW1353 cell culture and induction of inflammation

SW1353 cells were incubated in DMEM/F-12 supplemented with 10% FBS, 1% PS, and 1% L-Glutamine at 37 °C in a humidified incubator under 5% CO2. SW1353 chondrocytes were cultured in a medium containing IL-1β (40 ng/mL) to induce inflammation. IL-1β was pre-treated for 30 min, followed by the application of MCS.

Cell viability and nitric oxide (NO) production

Cell viability of SW1353 cells was determined using the EZ-cytox cell viability assay kit. After pre-treating IL-1β (40 ng/mL) for 30 min in the 6-well plates containing serum-free medium, MCS at levels of 25, 50, 100, and 200 µA were applied. The EZ-cytox assay solution was then added to the medium after 24 h. After 30 min of incubation, the cell viability was measured at 450 nm using a microplate reader (Epoch, BioTek Instruments, Winooski, VT, USA).

The amount of NO accumulated in the culture medium was measured 24 h after the end of MCS. Griess reagent was mixed with the culture medium (1:1) in a 96-well plate and allowed to react for 10 min. The absorbance was measured at 540 nm using a microplate. The measured NO production was normalized for each cell viability, and it was expressed as % of the negative control.

Animals

All experimental protocols (YWCI-202102-002-01) were approved by the Institutional Animal Care and Use Committee, Yonsei University, and were conducted under the Guide for the National Institutes of Health guidelines.

A total of 24 male Sprague-Dawley rats at the age of 8 weeks were used for the experiment. The rats were randomly divided into the control group (CON), the osteoarthritis (OA) group, the OA group with 25 µA MCS (OA25), and the OA group with 50 µA MCS (OA50) with 6 rats for each group. The rats were provided with water and rodent-specific feed (Rodent NIH-41 Open Formula Auto, Zeigler Bros Inc., USA) ad libitum. After an adaptation period of 7 days, the MIA solution (3 mg/50 µL) was injected into the right knee joint to induce OA under respiratory anesthesia (Fig. 1B).

Immunoblot and immunofluorescence analysis

SW1353 cells and patellae were lysed using PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology Inc., Korea). Protein quantification was performed using the BCA assay kit (Thermo, Rock-ford, IL, USA). The protein was separated by electrophoresis on Sodium Dodecyl Sulfate (SDS) PAGE gel (Mini-PROTEAN® TGX Gels, Bio-Rad, Hercules, CA, USA) and then transferred to a PVDF membrane (Trans-Blot® Turbo™ Transfer pack, Bio-Rad) and blocked using Tris-Buffered Saline (TBS-t) containing blocking buffer (2% Skim milk and 0.1% Tween 20) for 1 h. Subsequently, primary antibodies IL-1R-1 (ab106278), MMP1 (ab134184), MMP3 (ab52915), MMP13 (ab39012), aggrecan (ab3773), IL-1β (ab9722), collagen II (ab34712), and TRAF6 (ab33915) purchased from abcam, MyD88 (#4283), p-TAK1 (#9339), TAK1 (#4505), p-NFκB (#3033), NF-κB (#8242), and IκB (#4812) purchased from Cell Signaling Technology, were applied at a ratio of 1:1,000, and β-actin (#4967, Cell Signaling Technology) at a ratio of 1:2,000, and incubated at 4 °C for over 12 h. After washing the membrane three times for 5 min with TBS-t, secondary antibody anti-Rabbit IgG (#7074, Cell Signaling Technology) and secondary antibody anti-Mouse IgG (#7076, Cell Signaling Technology) were applied at RT for 1 h. The membrane was washed with TBS-t and treated with Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA), followed by band visualization using ImageQuant LAS500 (GE healthcare, Buckinghamshire, UK). Protein expression levels were observed using Image J software (1.52a version, National Institutes of Health, Bethesda, MD, USA).

For immunofluorescence analysis, SW1353 cells were cultured on 6-well plates with coated cover slides. The cells were fixed with 3.7% paraformaldehyde/PBS at RT for 10 min. To permeabilize the cell membranes, 0.2% Triton X-100/PBS was applied and incubated for 25 min and then washed three times. After that, blocking was performed using 3% BSA/PBS at room temperature for 30 min. Then, the primary antibody anti-p65 (#8242, Cell Signaling Technology) was diluted 1:1000 and incubated at 4 °C for over 24 h. After two washes, Alexa Flour 488 (excitation/emission = 495/519 nm, green, Invitrogen, CA, USA) was diluted at 1:400 and incubated with the cells at RT for 1 h. After three additional washes, the cover slides were mounted onto slide glass using VECTASHIELD Antifade Mounting Medium with DAPI (LS-J1033, LSBio, Inc., Seattle, WA, USA). The immunofluorescence analysis was performed using an LSM700 confocal microscope (Carl-Zeiss, Oberkochen, Germany), and the images were merged using ZEN Lite software (Zeiss, Oberkochen, Germany).

Measurement of mechanical allodynia

The mechanical allodynia test for measuring pain avoidance threshold was performed by using von Frey filaments. Before starting the test, the experimental animals were acclimatized for more than 15 min in a transparent plastic box with a flat wire mesh. Then, von Frey filaments were applied to the plantar surface of the affected paw from low to high intensities vertically for 2–3 s. If the experimental animal exhibited a rapid avoidance response or licked the paw in more than 3 out of 5 trials, it was considered a positive response. If a positive response occurred, a lower filament was used, and if a negative response occurred, the next higher filament was applied for a total of 6 trials.

Micro-CT analysis

The subchondral bone was imaged using an in-vivo micro-CT (Skyscan1176, Bruker, Germany) (resolution: 18 μm, voltage: 65 kV, current: 278 µA, filter: 1.0 mm Al, exposure time: 520 ms, rotation step: 0.7 deg). The 0th-week imaging was performed one week after MIA injection, and the 4th week imaging was aimed to assess the effects of a total of 4 weeks of MCS. To minimize the movement of animals, respiratory anesthesia was conducted.

The raw images were converted into 2D gray-scale image slices using NRecon (Bruker micro CT, Kontich, Belgium). The bone mineral density (BMD), bone volume (BV), cross-sectional thickness (Cs.Th.), trabecular thickness (Tb.Th.), and trabecular separation (Tb.Sp.) were analyzed with the CT Analyzer (CT-AN, v1.10.9.0, Bruker microCT, Kontich, Belgium) to evaluate changes in the subchondral bone structure due to OA induction.

Enzyme-linked immunosorbent assay (ELISA)

The concentration of MMP13 in serum was measured with MMP13 ELISA kit (USCN Life Science Inc., Wuhan, China). ELISA was performed according to the manufacturer’s instructions.

Statistical analysis

All results were presented as mean ± standard deviation. For all statistical comparisons, the SPSS 25 program (IBM SPSS Statistics, SPSS Inc., Chicago, IL, USA) was used to evaluate the effect of MCS. The statistical analysis was determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. Differences were considered to be significant for values of p < 0.05.

Results

Micro-current stimulation could decrease the NO production and IL-1R1/MyD88-related protein expression

To determine cell cytotoxicity present in SW1353 cells in response to MCS, the WST-1 assay was performed. After 24 h of stimulation, MCS levels of 25, 50, 100, and 200 µA were applied and compared to the negative control. The results showed that at MCS levels of 25 and 50 µA, cell proliferation was similar to the control, while at MCS levels of 100 and 200 µA, cell proliferation was reduced (p < 0.0001). Furthermore, in the IL-1β-treated cells, the cell survival rate was reduced. However, when IL-1β-treated cells were applied to MCS levels of 25 and 50 µA, an increased cell survival rate was observed.

Subsequently, NO production was measured to see if MCS could inhibit the NO production in IL-1β-treated SW1353 cells. The results showed that NO production decreased at MCS levels of 25 and 50 µA. Therefore, MCS levels of 25 and 50 µA were selected for further experiments (Fig. 2A).

Fig. 2.

Fig. 2

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(A) Cell viability and NO production in IL-1β or MCS-treated SW1353 cells. **** p < 0.0001 vs. negative control. #### p < 0.0001 vs. positive control. The values shown represent the mean ± SD of triplicate measurements of separate experiments. Values are shown as % of the negative control. (B) Immunoblot analysis of IL-1R-1 related proteins. * p < 0.05 vs. CON. # p < 0.05 vs. IL-1β group

Immunoblot analysis results showed IL-1β treatment increases protein expression levels of IL-1R-1, MyD88, TRAF6, and TAK1 in SW1353 cells. On the other hand, the MCS group with IL-1β treatment lead to significant inhibition of the protein expression levels (p < 0.05) (Fig. 2B).

MCS could decrease the activity of NF-κB and related protein expression

Immunoblot analysis was performed to observe changes in protein expression levels of NF-κB and IκBα. In the case of NF-κB protein, it showed an increased expression level after IL-1β treatment, but in the MCS group, the expression level was significantly inhibited (p < 0.05). On the other hand, the expression level of IκB protein showed lower expression after IL-1β treatment, but it exhibited higher expression in the MCS group, particularly significant differences were observed at the intensity of 50 µA (p < 0.01) (Fig. 3B).

Fig. 3.

Fig. 3

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(A) The translocation of NF-κB (p65) to the nucleus was analyzed by confocal microscopy. Red arrows indicate the region with relatively high p65 intensity. (B) Immunoblot analysis of NF-κB-related proteins on SW1353 cells following the application of MCS. (C) Immunoblot analysis of MMP-1, MMP-3, MMP-13, IL-1β, Collagen II, and aggrecan. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON. # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. IL-1β

To investigate the reduction of NF-κB activity in the cytoplasm of SW1353 chondrocytes after IL-1β treatment followed by MCS, immunofluorescence staining of the p65 protein was conducted in the cytoplasm (Fig. 3A). In the IL-1β group, p65 expression in the nucleus showed high activity and was observed to surround the nuclear periphery (Fig. 3A, second panel). In contrast, the MCS group showed reduced p65 expression in the nucleus compared to the IL-1β group, and particularly at the intensity of 50 µA, it showed a similar level of expression as the CON group (Fig. 3A, third panel).

Cartilage ECM destruction and synthetic protein expression levels were additionally observed. The IL-1β-treated SW1353 cells resulted in increased protein expression levels of MMP-1, MMP-3, and MMP-13 compared to the CON group, but when treated with MCS, protein expression was significantly decreased (p < 0.05) (Fig. 3C). Moreover, IL-1β-treated SW1353 cells showed decreased protein expression levels of collagen II (p < 0.001) and aggrecan (p < 0.05) compared to the CON group, but when treated with MCS, protein expression was significantly increased (Fig. 3C).

Mechanical allodynia measurements with Von Frey filaments

Since active ‘foot lift’ occurs when the force exceeds 10% of body weight, in this study, the force required to bend a 26.0 g filament to its maximum was determined based on the weight after seven days of OA induction (at week 0). Rats with MIA-induction showed persistent pain and started exhibiting typical OA symptoms at week 0, while rats injected with PBS did not show any symptoms. Interestingly, the application of MCS at 50 µA significantly reduced pain (p < 0.05) compared to the OA group with increased pain at week 4 (Fig. 4A).

Fig. 4.

Fig. 4

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(A) Body weight was measured in each group. **** p < 0.0001 vs. 0th-week and 4th-week. Mechanical allodynia was conducted in 0th-week and 4th-week to measure hind paw mechanical thresholds. * p < 0.05 vs. 0th-and 4th-week. # p < 0.05, OA vs. OA25 or OA50. (B) Colorization images for representing BMD values of the subchondral bone in 2D image (C) 3D model images for subchondral bone. (D) Micro-CT analysis results indicated changes in BMD, BV, Cs.Th, Tb.Th, and Tb.Sp values. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CON. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. OA

Observation of subchondral bone using micro-CT analysis

At 7 days after OA induction and 4 weeks after the experiment, the subchondral bone was visualized and presented as 2D and 3D images for each group (Fig. 4B, C). In the OA, subchondral bone structure destruction was observed in the 4th week. In contrast, MCS groups showed a relatively restored subchondral bone shape compared to the OA.

Correspondingly, based on the Micro-CT analysis results comparing week 4 to week 0, the OA showed decrease in BMD, BV, Cs.Th, and Tb.Th values, along with increased Tb.Sp value, resulting in reduced thickness and weakening of the subchondral bone tissue and caused subchondral bone degeneration which is commonly seen in OA (Fig. 4D). On the other hand, MCS increased BMD, BV, Cs.Th, and Tb.Th values, along with decreased Tb.Sp values, indicate potential regenerative effects on subchondral bone.

Observation of changes in serum MMP-13 levels and ECM-related protein expressions

The concentrations of MMP-13 in serum, which is the primary MMP involved in cartilage degradation through its specific ability to cleave type II collagen [19], were compared. In the OA, an increase in MMP-13 levels induced cartilage degradation. In contrast, the OA25 and OA50 groups showed decreases in MMP-13, and especially, the OA50 significantly inhibited MMP-13 expression (p < 0.05) (Fig. 5A).

Fig. 5.

Fig. 5

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(A) ELISA result was conducted to measure serum MMP-13. (B) Immunoblot analysis of ECM anabolic and catabolic factors on the patella tissue. **** p < 0.0001 vs. CON. ### p < 0.001, #### p < 0.0001 vs. OA

Next, the protein expression levels of MMP-3, a cartilage ECM degradation enzyme, and the protein components of ECM synthesis, collagen II and aggrecan, were measured in MIA-induced rats. Consistent with the in-vivo results, the OA group exhibited increased expression of MMP-3 and decreased expression of collagen II and aggrecan, while MCS groups showed a decrease in MMP-3 expression and an increase in collagen II and aggrecan expression as the stimulation intensity increased (Fig. 5B).

Discussion

OA is caused by damage to the articular cartilage that makes up the knee bone cartilage, sclerosis of the subchondral bone, synovitis, and other factors. It also causes degeneration of articular cartilage and chronic pain which increases emphasis on the need for treatment of OA patients worldwide. However, pain management through drug therapy, physical therapy, and surgical treatment used in OA treatment can cause toxicity and side effects due to continuous medication and repeated drug therapy, posing risks of infection and joint destruction in the joint area [11]. Therefore, there is a need for the development of new treatment methods that are not solely reliant on drugs for OA.

According to recent studies, electrical stimulation is widely employed not only for pain management but also to prevent muscle atrophy and promote muscle function recovery. Duarte et al. studied a method of stimulating bone growth using 5 ~ 20 µA to promote cartilage tissue regeneration [20]. Another study evaluated the effects of 20 ~ 25 µA on the recovery of articular cartilage and the distribution of chondrocytes [21]. However, these studies only conducted morphological comparisons, and there are limitations in reaching conclusions regarding the synthesis of chondrocytes and ECM components in articular cartilage in OA.

Electrical stimulation has been reported as a useful method to change molecular mechanisms by inducing various cellular responses such as cell proliferation, apoptosis, and death. Since these changes can increase or decrease depending on the type of cell and the intensity of stimulation, pathological effects can be elicited by using electrical stimulation of appropriate intensity [22]. In particular, suppressing the apoptosis of chondrocytes is used as an important strategy to suppress OA [23]. In order to select an effective stimulation intensity, cell viability, and anti-inflammatory effects were evaluated based on the degree of cell proliferation and measurement of NO production according to the presence or absence of IL-1β treatment and MCS. According to our results, when only MCS was applied, MCS below 50 µA did not affect cell viability, but higher intensity of MCS decreased cell viability, suggesting that high intensity of MCS induced apoptosis. While cell viability decreased in IL-1β-treated chondrocytes, cell viability did not decrease when MCS of 50 µA or less was applied, suggesting the possibility that MCS may inhibit apoptosis. In particular, as cell viability decreased in IL-1β-treated chondrocytes applied with 200 µA MCS, NO production normalized to cell viability was increased compared to chondrocytes treated with IL-1β only. On the other hand, 25 and 50 µA MCS did not affect cell viability but showed inhibitory effects on NO production. Therefore, 25 and 50 µA MCS were further investigated.

When excessive expression of IL-1R-1 occurs, it leads to the inhibition of protein synthesis, resulting in cell death and functional impairment [24]. MyD88, an adaptor molecule in the intracellular signaling process of IL-1R, is known to facilitate cytokine activity by binding with TRAF6 and causing the degradation of IκB, allowing NF-κB to enter the nucleus [25]. In particular, when inflammation is triggered by IL-1β, chondrocytes activate the NF-κB signaling pathway, inducing the secretion of inflammatory factors associated with MMPs, leading to cartilage degradation [26]. Excessive expression of MMPs can directly degrade the ECM surrounding chondrocytes, damaging articular cartilage and progressively causing joint degeneration [27]. Based on the results of this study, it is predicted that 50 µA of MCS inhibits IL-1R1/MyD88/NF-κB signaling pathway activation, which can suppress the secretion of inflammatory factors. Additionally, it was confirmed that 50 µA of MCS suppresses the expression of MMPs and significantly increases the expression of collagen II and aggrecan, which are ECM components of cartilage. This study also confirmed the potential of regulating IL-1R1/MyD88/NF-κB signaling to promote articular cartilage regeneration. Furthermore, based on the results of NF-κB immunofluorescence staining, it is predicted that IL-1β promotes NF-κB activity in the IL-1β group, while in the 25 and 50 µA MCS groups, it can reduce IL-1β-induced NF-κB activity.

To evaluate the therapeutic effect of MCS in vivo, MCS with intensities of 25 and 50 µA was applied to an OA animal model. Body weight increased in all groups, but the increase in the OA25 and OA50 was smaller than that of CON and OA. It seems to be a result related to our previous studies representing that MCS can suppress obesity by modulating adipogenesis via Wnt/β-Catenin signaling and insulin signaling [28, 29]. The results of a mechanical allodynia test demonstrated that the pain caused by OA was improved with the application of 50 µA MCS. This aligns with previous research reporting the efficacy of low-intensity MCS in pain [16]. Subchondral bone remodeling also plays a key regulator in the development of OA [30]. It is mainly determined by the interaction of osteoclasts, osteoblasts, and osteocytes and includes the bone resorption by osteoclast and bone formation [31]. To investigate whether MCS application affects bone remodeling in subchondral bone, the microstructure of subchondral bone was analyzed with using micro-CT. Previous study has reported that osteoclasts cause abnormal bone remodeling in OA, resulting in a decrease in cartilage volume and plate thickness and an increase in trabecular separation in the microstructure of subchondral bone [31]. Consistent with these, our results showed that BV and Cs.Th decreased, while Tb.Sp increased in the OA-induced group. When compared to OA, both MCS groups appeared to improve in most structural parameters. In particular, 25µA MCS was effective in increasing bone density, contributing to improving bone quality. These findings are consistent with research suggesting that applying low-intensity current levels is necessary to suppress chondrocyte destruction and increase the range of joint motion [32].

OA is characterized by the imbalance between anabolic and catabolic processes within the articular cartilage. When an imbalance occurs, it leads to the deterioration of cartilage tissue due to its degradation. Anabolism involves molecules such as collagen II and aggrecan, while catabolism involves MMPs and cartilage-degrading enzymes [33]. The serum MMP-13 showed that MCS at the 50 µA level suppressed the expression of MMP-13, which is involved in the catabolic process within the cartilage tissue. This suggests that the degradation of articular cartilage is inhibited, leading to a protective effect on the articular cartilage. Furthermore, the increase in serum MMP-13 is associated with structural abnormalities in the knee of OA patients, indicating that blood MMP-13 can serve as a biomarker for OA [34].

We performed immunoblot analysis on the composition of the articular cartilage ECM components by extracting the patella from animals. MMP-3, which is involved in cartilage degradation, showed significantly higher expression levels in the OA compared to the CON, while in the MCS groups, it gradually decreased with increasing stimulation intensity. Furthermore, we also observed the expression levels of factors involved in articular cartilage anabolic processes, such as collagen II and aggrecan. The expression of collagen II decreased in the OA compared to the CON but showed an increasing trend in the MCS groups compared to the OA. Similarly, aggrecan showed a proportional increase in expression with stimulation intensity, following the same trend as collagen II expression. These results are consistent with the study by Hong et al. [35], suggesting that it is necessary to inhibit the degradation of collagen II and aggrecan by reducing MMPs to improve articular cartilage. Therefore, it can be expected that 25 µA can improve subchondral bone thickness as well as provide pain relief. Additionally, 50 µA demonstrates analgesic and cartilage ECM degradation enzyme inhibition effects, suggesting the potential for cartilage ECM regeneration based on the activation of chondrocytes.

Therefore, it is estimated that electrical stimulation at a range of 25 to 50 µA will show regenerative effects on OA articular cartilage and subchondral bone by modulating the IL-1R1/MyD88/NF-κB signaling (Fig. 6). It can also be proposed as a method for regulating ECM synthesis and degradation enzymes in chondrocytes in the OA.

Fig. 6.

Fig. 6

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Schematic diagram of regulating IL-1R1/MyD88/NF-κB signaling regarding osteoarthritis in chondrocytes

Author contributions

M. Lee, H. Lee, and H.S. Kim contributed to the study conception, data collection, and analysis. The first draft was written by M. Lee and H. Lee and all authors edited and supervised the revisions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A2C2093828) and "Regional Innovation Strategy (RIS)" through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2022RIS-005).

Declarations

Ethics approval

The protocols for all procedures were approved by the Yonsei University Animal Care Committee (YWCI-202102-002-01).

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Competing interests

The authors have no relevant financial or nonfinancial interests to disclose.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Minjoo Lee and Hana Lee contributed equally to this work.

References

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