Baicalein inhibits IL-1β-induced extracellular matrix degradation with decreased MCP-1 expression in primary rat chondrocytes

Abstract

Baicalein is a flavonoid extracted from the roots of Scutellaria baicalensis and Scutellaria lateriflora. This compound exerts various biochemical activities, including antioxidant and anti-inflammatory effects. The study aimed to investigate the effect of baicalein on articular cartilage cells and elucidate its underlying mechanism. In primary rat chondrocyte cultures, treatment with baicalein demonstrated a reduction in the loss of proteoglycan and extracellular matrix degradation induced by interleukin (IL)-1β. Baicalein suppressed IL-1β-induced catabolic responses, including the expression and activation of matrix metalloproteinase (MMP)-13, MMP-3, and MMP-1. In addition, baicalein effectively reduced nitric oxide and prostaglandin E2 production, and it downregulated the expression of inducible nitric oxide synthase and cyclooxygenase-2 in primary rat chondrocytes. Furthermore, baicalein downregulated IL-1β-induced inflammatory chemokines and cytokines, such as GM-CSF and MCP-1. These findings suggest that baicalein could potentially mitigate the catabolic responses of IL-1β in chondrocytes, making it a promising candidate for both the prevention and treatment of osteoarthritis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43188-024-00225-4.

Introduction

Osteoarthritis (OA) is one of the most widespread age-associated conditions, characterized by persistent joint discomfort, progressive deterioration of the articular cartilage, emergence of osteophytes, sclerosis of the subchondral bone, and impaired mechanical functioning of the impacted joints [1, 2]. The metabolism of the articular cartilage is intricately regulated by maintaining a delicate balance between extracellular matrix synthesis and degradation. The disturbance of this equilibrium, resulting in an imbalance between anabolism and catabolism of the cartilage tissue, is considered as a contributing factor in the development of OA [3]. Inflammation in the joints stimulates the catabolic degradation of the extracellular matrix with an increase in expression and activation of matrix-degrading enzymes [4]. Therefore, the inhibition of cartilage-degrading factors has been considered as a strategy to alleviate OA symptoms.

The proinflammatory cytokine interleukin (IL)-1β is a major contributing factor in OA or articular cartilage degeneration [3]. Under inflammatory conditions, synoviocytes or articular chondrocytes produce IL-1β, upregulating matrix-degrading enzymes such as metalloproteinases (MMPs) and aggrecanase and increasing the production of nitric oxide (NO) and prostaglandin E (PGE) [5]. These changes contribute to catabolic responses in chondrocytes and joints.

Recently, compounds derived from herbal plants have been found to exhibit strong anti-inflammatory, anticatabolic, and anabolic effects on chondrocytes, implying their potential for alleviating OA symptoms [4, 6]. For example, treatment with curcumin has been found to inhibit the production of IL-1β, TNF-α, IL-8, NO, and MMPs by reducing the activation of NF-κB, Akt, and MAPK signaling pathways[7, 8].

Baicalein (5,6,7-trihydroxy-2-phenyl-chromen-4-one) is a major flavonoid derived from the roots of Scutellaria baicalensis and Scutellaria lateriflora and frequently utilized in traditional Chinese herbal medicine. Baicalein has been showed to have diverse pharmacological effects, including anti-inflammatory, anti-tumor, and anti-oxidant properties [9]. Additionally, baicalein exhibits anti-apoptotic properties against IL-1β-mediated cell death in chondrocytes [10]. In this study, we showed that baicalein could effectively inhibit IL-1β-induced catabolic inflammation in chondrocytes by regulating MMPs and other inflammatory cytokines. Our findings have led to the hypothesis that baicalein could have preventive or therapeutic potential in mitigating articular cartilage damage caused by IL-1β-induced catabolic inflammation.

Materials and methods

Isolation and cultivation of primary chondrocytes from rats

Primary chondrocytes were isolated from the articular cartilage of 4-day-old Sprague Dawley rats using an enzymatic digestion technique. Specifically, the articular cartilage was digested using 0.025% collagenase P and 0.2% pronase in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Paisley, Scotland, UK) following a previously established protocol [11, 12]. For long-term culture in alginate beads, the isolated chondrocytes were suspended in alginate solution (1.2%) and added dropwise to CaCl₂ solution (105 mM) for bead formation. Subsequently, the chondrocytes were cultured in DMEM/F-12 supplemented with penicillin (50 U/mL) and ascorbic acid (50 μg/mL) for 21 days. For long-term culture, the chondrocytes were treated with lower concentrations of IL-1β (1 ng/mL; ProSpec Tany TechnoGene, Rehovot, Israel) and baicalein (5 μM, Sigma-Aldrich, St. Louis, MO, USA) to assess their physiological responses, which could minimize potential toxic effects due to extended exposure. For short-term monolayer culture, the chondrocytes were seeded at a density of 1 × 10⁶ cells/mL. In these experiments, higher concentrations of IL-1β (10 ng/mL) and baicalein (10/20 μM) were used for shorter treatment durations to observe the cellular responses to more robust stimuli, which could also reduce the risk of toxicity associated with longer exposure times. For siRNA transfection, the cells were cultured in serum-free Opti-MEM medium along with a mixture of 100 nmol/L monocyte chemoattractant protein-1 (MCP-1) siRNA and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Cytotoxicity assay

To assess the potential cytotoxic effects of baicalein on primary rat chondrocytes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted. The chondrocytes were seeded at a density of 1 × 10⁴ cells/well in a 96-well culture plate and treated with baicalein at various concentrations (5, 10, 20, and 50 μM) in triplicate. After incubation at 37 °C with 5% CO₂ for 24 h, the cells were reacted with MTT solution for 4 h following the manufacturer’s instructions. The absorbance of the resulting reaction products was measured at an incident wavelength of 570 nm using a spectrophotometer (Epoch Spectrophotometer; BioTek, Winooski, VT, USA).

Live/dead cell imaging assay

To validate the effects of baicalein on cell viability, chondrocytes were exposed to two different concentrations of baicalein (10 μM and 20 μM). Following exposure, the cells were stained with green-fluorescent calcein AM to identify live cells and red-fluorescent ethidium homodimer-1 to identify dead cells according to the manufacturer’s instructions (Molecular Probes, Leiden, Netherlands). Fluorescent images were taken using a fluorescence microscope (Eclipse TE200; Nikon Instruments, Melville, NY, USA), and the number of cells stained with green or red fluorescence was counted. The viability of the chondrocytes was then determined by calculating the ratio of live cells to dead cells.

Measurement of proteoglycan content

During the experimental period of 21 days, cells were cultured in alginate beads containing a combination of 5 μM baicalein and/or 1 ng/mL IL-1β. The proteoglycan content in the extracellular matrix was assessed by dimethylmethylene blue (DMMB) assay and normalized to the number of cells present in the alginate beads. To determine the relative number of cells, the total DNA content was measured by PicoGreen assay (Molecular Probes, Carlsbad, CA, USA) as reported in previous studies [11, 12].

Particle exclusion assay for matrix assessment

To visualize cultured cells along with their surrounding matrix, particle exclusion assay was carried out, according to a previously described method [13]. In brief, alginate beads containing the cells were exposed to sodium citrate (55 mM, pH 6.8) to dissolve the beads. The cells were then harvested by centrifugation and plated onto a chamber slide using DMEM/F-12. After incubating the cells for 16 h, cold ethanol-fixed erythrocytes (red blood cells) were introduced and allowed to settle for 30 min. The matrix surrounding the cells was examined using an inverted phase-contrast microscope (Eclipse 2000; Nikon).

Polymerase chain reaction (PCR) analysis

Total RNA was extracted using the Cell&Tissue RNA Extraction Kit (Infusion Tech, Gyeonggi-do, Korea) and reverse-transcribed into cDNA using the SuperScript II Reverse Transcriptase PCR kit (Invitrogen, Madison, WI, USA). For quantitative PCR (qPCR), cDNA was amplified using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, CA, USA) and Power SYBR Green PCR Master Mix (Life Technologies Ltd., Warrington, UK). Relative gene expression was determined using the ^ΔΔCT method following the manufacturer’s instructions (StepOne Software v2.1; Applied Biosystems). The primer sequences used are listed in Table 1. 18S rRNA was used as the internal control for qPCR, and GAPDH served as the internal control for conventional reverse transcription PCR (RT-PCR).

Table 1.

RT-PCR primer sequences used in this study

Gene	Primer sequences	NCBI gene No
MMP-13	Forward: 5′-GGCAAAAGCCATTTCATGCTCCCA-3′ Reverse: 5′-AGACAGCATCTACTTTGTCGCCA-3′	NM_133530.1
MMP-1	Forward: 5′-AAACGTGGATGCCAAGGAGG-3′ Reverse: 5′-GCTCTCTCGATGGCGTTTTC-3′	NM_001134530.1
MMP-3	Forward: 5′-ATGGGCCTGGAATGGTCTTG-3′ Reverse: 5′-TGTGGGAGGTCCATAGAGGG-3′	NM_133523.3
iNOS	Forward: 5′-GCATCGGCAGGATTCAGTGG-3′ Reverse: 5′-TAGCCAGCGTACCGGATGAG-3′	NM_012611.3
COX-2	Forward: 5′-AGGAGCATCCTGAGTGGGAT-3′ Reverse: 5′-AGAAGCGTTTGCGGTACTCA-3′	NM_017232.3
GAPDH	Forward: 5′-TGATGCTGGTGCTGAGTATG-3′ Reverse: 5′-GGATGCAGGGATGATGTTCT-3′	XM_214281.5

Western blotting

Total protein samples were extracted with lysis buffer (Cell Signaling Technology, Danvers, MA, USA). The protein concentration was determined using the BCA Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The extracted proteins were loaded onto 10–14% SDS-PAGE gels and separated, after which they were transferred to polyvinylidene difluoride (PVDF) membranes with a pore size of 0.2 μm. The membrane was left to incubate overnight at a temperature of 4 °C with primary antibodies against matrix MMPs from Santa Cruz Biotechnology (Dallas, TX, USA), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) from Cell Signaling Technology. Subsequently, immunoreactive bands were visualized using a chemiluminescent HRP substrate (Millipore, Darmstadt, Germany).

Measurement of total NO production and PGE2 concentration

To measure NO production and PGE2 concentration, a spectrophotometric method was employed. Primary chondrocytes were cultured on a 12-well plate and treated with baicalein for 24 h in the presence or absence of 10 ng/mL IL-1β. After treatment, 50 μL of the conditioned medium was mixed with 50 μL of sulfanilamide and N-1-naphthylethylenediamine dihydrochloride (NED) to measure NO production. Then, the absorbance was measured at 540 nm using a spectrophotometer (BioTek). The PGE2 concentration in the conditioned medium was measured using the PGE2 Parameter Assay Kit from R&D Systems (Minneapolis, MN, USA) according to the manufacturer’s instructions.

Cytokine array

To evaluate changes in inflammatory cytokines, the Rat Cytokine Antibody Array C1 Kit (Cat#AAR-CYT-1-4; RayBiotech, Norcross, GA, USA) was used following the manufacturer’s instructions. The isolated primary rat chondrocytes were seeded at a density of 1 × 10⁶ cells/mL in a 6-well culture plate. Subsequently, they were treated with 20 μM baicalein for 24 h in the presence or absence of 10 ng/mL IL-1β.

Histology and immunohistochemical analyses

In this study, knee joints were obtained from 5-day-old rat pups and decalcified using ethylenediaminetetraacetic acid (EDTA) before embedding in paraffin. Then, the paraffin-embedded tissue blocks were sectioned into 5-μm-thick slices and placed on microscope slides. Standard hematoxylin and eosin staining was performed for general examination, and safranin-O staining and fast-green staining were conducted to assess the proteoglycan content in the articular cartilage matrix. Changes in proteoglycan content were evaluated based on the stained color density.

Furthermore, immunohistochemical analysis was performed to evaluate MCP-1 protein expression. MCP-1 antibodies were incubated with the tissue sections overnight at 4 °C, and protein expression was visualized using the VECTASTAIN^® Universal Quick Kit (PK-7800; Burlingame, CA, USA).

Data analysis

Statistical analysis was conducted using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple comparison test. Statistical significance was defined as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. The results are presented as the mean ± standard deviation.

Results

Effect of baicalein on the viability of primary rat chondrocytes

To investigate the effects of baicalein on cell viability, we performed MTT and live/dead cell imaging assays. In MTT assay, compared with the control group, treatment with baicalein at concentrations of 5, 10, 20, and 50 μM resulted in cell viability rates of 101 ± 3%, 108 ± 3%, 104 ± 3%, and 110 ± 3%, respectively (Fig. 1a). Live/dead cell imaging assay showed relative cell survival rates of 103 ± 2% and 102 ± 4% following treatment with baicalein at concentrations of 10 and 20 μM, respectively (Fig. 1b). These results suggest that baicalein concentrations below 50 μM may not affect the viability of rat primary chondrocytes.

Fig. 1.

Suppression of the IL-1β-dependent loss of proteoglycan and matrix degradation in primary rat chondrocytes by baicalein. a, b Cells were cultured with 5–50 μM baicalein for 24 h, and MTT assay and microscopic analysis were performed to determine cell viability based on the live-to-dead-cell ratio. c Proteoglycan content was determined by DMMB assay. d Particle exclusion assay was performed to visualize the extracellular matrix surrounding the chondrocytes. e Changes in proteoglycan content in the rat articular cartilage ex vivo were evaluated by safranin-O and fast-green staining. Each value represents the mean ± standard error (n = 3). **p < 0.01 indicates a significant difference relative to the IL-1β control

Inhibitory effect of baicalein on the IL-1β-dependent loss of proteoglycan and matrix degradation

To investigate the inhibitory effects of baicalein, chondrocytes were embedded in 1.5% alginate and exposed to 5 μM baicalein for 21 days with or without 1 ng/mL IL-1β. Treatment with 1 ng/mL IL-1β resulted in a significant reduction in proteoglycan content to 77.8 ± 0.6% compared with that of the control group. However, treatment with 5 μM baicalein abrogated the IL-1β-induced decrease in proteoglycan content (Fig. 1c). Furthermore, particle exclusion assay revealed that treatment with 1 ng/mL IL-1β substantially reduced the pericellular matrix of primary rat chondrocytes. However, treatment with 5 μM baicalein alleviated the reduction of the pericellular matrix caused by IL-1β (Fig. 1d). Moreover, ex vivo safranin-O and fast-green staining of the isolated knee joints revealed that treatment with IL-1β resulted in extracellular matrix degradation in the articular cartilage of the knee joints. However, treatment with baicalein mitigated the IL-1β-induced loss of the extracellular matrix (Fig. 1e). These findings suggest that baicalein may have inhibitory effects on the IL-1β-induced catabolism of the extracellular matrix in primary rat chondrocytes.

Inhibitory effect of baicalein on the expression of cartilage-degrading enzymes induced by IL-1β

IL-1β plays a crucial role in promoting the degradation of the articular cartilage by regulating cartilage-degrading enzymes such as MMPs [14, 15]. In this study, we also examined the effects of baicalein on the expression of MMP-13, MMP-1, and MMP-3 in primary rat chondrocytes through RT-PCR and western blot analyses. Treatment with 10 ng/mL IL-1β significantly increased the mRNA and protein expression levels of these cartilage-degrading enzymes. However, treatment with baicalein at concentrations of 10 and 20 μM dose-dependently suppressed the mRNA and protein expression levels of MMP-13, MMP-1, and MMP-3 (Fig. 2a–c). These findings suggest that baicalein may exert an inhibitory effect on IL-1β-induced matrix degradation by suppressing the expression of matrix-degrading enzymes.

Fig. 2.

Downregulation of the expression of matrix MMPs in the presence of IL-1β by baicalein. Cells were treated with 10 μM (denoted as +) and 20 μM (denoted as ++) baicalein in the presence or absence of IL-1β (10 ng/mL, indicated as +) for 24 h. The expression levels of MMP-1, MMP-3, and MMP-13 were quantified by qPCR (a), RT-PCR (b), and western blotting (c). Each value represents the mean ± standard error (n = 3). Western blotting was repeated three times independently. Statistical significance was determined by t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Baicalein-mediated suppression of the production of the catabolic mediators NO and PGE2 induced by IL-1β

NO and PGE2 are known to mediate the catabolic responses induced by IL-1β in chondrocytes [16]. Treatment with 10 ng/mL IL-1β significantly increased NO production by approximately 8.6 times in primary rat chondrocytes, and the addition of 10 or 20 μM baicalein effectively inhibited IL-1β-induced NO production (Fig. 3a). Similarly, IL-1β increased PGE2 production by approximately 2.2 times, and the addition of 10 or 20 μM baicalein dose-dependently suppressed IL-1β-induced PGE2 production (Fig. 3b).

Fig. 3.

Suppression of the IL-1β-induced production of NO and PGE2 by baicalein. a, b Cells were treated with baicalein at concentrations of 10 μM (indicated as +) and 20 μM (indicated as ++) in the presence or absence of IL-1β (10 ng/mL, indicated as +) for 24 h. The production of NO and PGE2 was assessed following the protocol outlined in the materials and methods section. c, d The expression levels of iNOS and COX-2 were evaluated by RT-PCR (c) and western blotting (d). All experiments were performed in triplicate, and each value represents the mean ± standard error (**p < 0.01; ***p < 0.001)

Furthermore, treatment with 10 ng/mL IL-1β upregulated the expression of the iNOS and COX-2 enzymes, which are involved in the production of NO and PGE2, respectively. However, treatment with 10 and 20 μM baicalein inhibited the mRNA and protein expression levels of these enzymes in a dose-dependent manner (Fig. 3c, d). These results suggest that baicalein may protect chondrocytes from IL-1β-induced catabolic responses by suppressing the production of NO and PGE2 through the regulation of iNOS and COX-2 expression.

Baicalein-mediated suppression of IL-1β-induced proinflammatory cytokines and chemokines

In chondrocytes, IL-1β can induce catabolic inflammatory responses by increasing the production of various cytokines and chemokines [12]. In this study, the effects of baicalein on the expression of proinflammatory cytokines and chemokines in the presence of 10 ng/mL IL-1β were examined using the Rat Cytokine Antibody Array C1 Kit.

As shown in Fig. 4, compared with treatment with 10 ng/mL IL-1β alone, treatment with baicalein significantly decreased the protein levels of fractalkine, granulocyte–macrophage colony-stimulating factor (GM-CSF), LIX, and MCP-1. These results indicate that the anticatabolic effects of baicalein may also be attributed to the inhibition of the IL-1β-induced expression of inflammatory cytokines and chemokines.

Fig. 4.

Suppression of the IL-1β-induced production of catabolic proinflammatory cytokines in primary rat chondrocytes by baicalein. Cells were treated with 20 μM baicalein in the presence of 10 ng/mL IL-1β for 24 h. The relative levels of cytokines were assessed using the Rat Cytokine Antibody Array C1 Kit. Each value represents the mean ± standard error (n = 3). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01

Attenuation of proteoglycan loss by MCP-1 inhibition in the articular cartilage

We further examined whether inhibiting MCP-1 expression could prevent proteoglycan loss in joint cartilage. Primary chondrocytes were treated with IL-1β and/or MCP-1 siRNA for 14 days. Particle exclusion assay confirmed that MCP-1 siRNA treatment effectively suppressed the reduction of cell peripheral substrates induced by IL-1β. Notably, as shown in Fig. 5a, quantitative analysis demonstrated that treatment with baicalein significantly mitigated the loss of the pericellular matrix area in primary rat chondrocytes, indicating its potential in protecting against IL-1β-induced matrix degradation. Immunohistochemistry was also performed to examine the upregulation of MCP-1 expression in rat knee joints induced by IL-1β, and the results showed that treatment with IL-1β increased MCP-1 expression in the articular cartilage. However, treatment with baicalein attenuated IL-1β-induced MCP-1 expression, as shown in Fig. 5b. Furthermore, baicalein decreased the expression levels of iNOS and COX-2, which were induced by MCP-1 in chondrocytes (Fig. 5c). These results suggest that baicalein may inhibit extracellular matrix degradation and the expression of iNOS and COX-2 by regulating MCP-1 expression.

Fig. 5.

Effects of MCP-1 siRNA on IL-1β-induced catabolic responses in primary rat chondrocytes. a Particle exclusion assay was conducted to visualize the extracellular matrix of cultured chondrocytes. MCP-1 siRNA treatment suppressed the reduction of cell peripheral substrates induced by IL-1β. b Immunohistochemistry was performed to evaluate MCP-1 protein expression in rat knee joints. c Western blotting was performed with specific antibodies against MCP-1, iNOS, and COX-2. All experiments, including western blotting, were repeated three times, and the results are expressed as the mean ± standard error. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3)

Discussion

In this study, the effects of baicalein on IL-1β-induced catabolic responses in articular chondrocytes were investigated. The results demonstrated that treatment with baicalein effectively prevented the IL-1β-induced degradation of the extracellular matrix in primary rat chondrocytes. Additionally, it inhibited the production of catabolic mediators, including MMPs, NO, and PGE2, which play critical roles in cartilage degradation and inflammation. Treatment with baicalein was also found to suppress the expression of other inflammatory cytokines, such as MCP-1 and LIX, which are associated with inflammatory responses in chondrocytes.

IL-1β is vital in inducing inflammatory responses and connective tissue destruction in joint diseases such as OA [17]. In both OA patients and animal models, the concentrations of IL-1β in the blood and intra-articular fluid have been found to be higher than those of control groups. IL-1β administration causes chondrocyte death and cartilage matrix destruction, along with the upregulation of inflammatory mediators. The catabolic effects of IL-1β on chondrocytes are closely associated with the increased production of MMPs, NO, and PGE2 [16]. Notably, in the present study, we observed that treatment with 1 ng/mL IL-1β in rat primary chondrocytes resulted in the loss of the proteoglycan and extracellular matrix degradation. Furthermore, the production of matrix MMPs, NO, and PGE2 was upregulated. Based on the results, 1 ng/mL IL-1β may be considered to experimentally provide the catabolic conditions of OA.

Baicalein has a wide range of pharmacological properties, such as anti-inflammatory, antineoplastic, and antioxidant effects; thus, it is being studied as a potential therapeutic agent for various diseases [18]. To examine the therapeutic applications of baicalein, we first determined a nontoxic concentration in primary rat chondrocytes (Fig. 1). Baicalein at a concentration of less than 50 μM did not affect the viability of chondrocytes, and baicalein showed an inhibitory effect on catabolic responses in chondrocytes induced by IL-1β. OA is partly characterized by the progressive destruction of the extracellular matrix in the articular cartilage, which is driven by the activity of matrix-degrading enzymes and inflammatory mediators. In chronic arthritis, the degradation of the extracellular matrix is mediated by a variety of cartilage-degrading enzymes, including MMPs and aggrecanases [19]. Specifically, the roles of MMP-1, MMP-3, and MMP-13 have been identified [15]. MMP-13 is regarded as a potent enzyme in cartilage damage because of its capacity to degrade collagen type II, a crucial component of the extracellular matrix in the articular cartilage [20]. In the present study, we observed that IL-1β treatment resulted in the upregulation of cartilage-degrading enzymes in primary rat chondrocytes. However, when treated with baicalein, IL-1β-induced catabolic responses were inhibited. In addition, treatment with baicalein consistently prevented the IL-1β-induced decrease in proteoglycan content and extracellular matrix degradation at the level of both isolated cells and tissues. The results strongly suggest that the flavonoid baicalein may function as an effective agent for preventing cartilage damage in OA.

IL-1β is known to induce the excessive expression of iNOS and COX-2 in chondrocytes. Consequently, this can lead to an increase in the production of NO and PGE2. Both NO and PGE2 play significant roles in promoting cartilage degradation by inhibiting proteoglycan biosynthesis and promoting catabolic responses in articular cartilage cells [21]. Therefore, targeting NO and PGE2, which are present at elevated levels in the synovial fluid of OA patients, could be a promising approach for managing OA [22]. Our study demonstrated that baicalein effectively suppressed the expression of inflammatory mediators (iNOS, COX-2, NO, and PGE2) induced by IL-1β in primary rat chondrocytes. These findings support the hypothesis that baicalein may be a useful therapeutic agent for mitigating IL-1β-induced cartilage destruction and managing conditions such as OA.

IL-1β can stimulate the expression of various proinflammatory cytokines that play crucial roles in cartilage destruction and joint inflammation. For example, IL-1β stimulation has been found to increase the expression levels of various proinflammatory cytokines, including IL-1β itself, TNF-α, IL-6, leukemia inhibitory factor (LIF), and aggrecanases (MMP-13 and ADAMTS-4), in OA chondrocytes [23]. In this study, we conducted cytokine array experiments to further examine whether baicalein effectively inhibits the IL-1β-induced expression of inflammatory cytokines and chemokines (Fig. 4). Baicalein significantly inhibited the IL-1β-induced expression of proinflammatory fractalkine (CX3CL1), LIX (CXCL5), and MCP-1 (CCL2). MCP-1, a member of the C-C class of the beta-chemokine family, is important for inflammation initiation. It facilitates chemotaxis and the migration of monocytes to inflammatory sites by binding to CCR2 receptors on the cell membrane [24]. MCP-1 expression can be observed in chondrocytes, and it plays a stimulatory role in cartilage degradation and OA progression [25]. In our experiments, treatment with MCP-1 siRNA inhibited the IL-1β-induced loss of the primary rat chondrocyte pericellular matrix (Fig. 5). These findings suggest that MCP-1 may play a specific role in OA pathogenesis, and they support the hypothesis that baicalein may prevent OA progression by inhibiting the IL-1β-mediated induction of inflammatory cytokines and chemokines, such as MCP-1.

Recent studies have uncovered the molecular mechanism underlying the therapeutic efficacy of baicalein in treating inflammatory diseases and cancers [26]. In human mast cell cultures, baicalein was found to suppress the expression of IL-8, IL-6, and MCP-1 via NF-κB pathway regulation [27]. In HeLa cell cultures, baicalein was shown to inhibit the expression of NF-κB target genes induced by TNF-α, which are implicated in anti-apoptosis, proliferation, invasion, and major inflammatory cytokines. Additionally, baicalein was observed to block the activation of the p38 and ERK1/2 pathways [28]. In our primary chondrocyte cultures, treatment with baicalein inhibited IL-1β-induced p65 phosphorylation (Supplementary Fig. 1). Based on the findings, we hypothesize that baicalein may exert anticatabolic effects in chondrocytes against IL-1β stimulation via the inhibition of NF-κB pathways.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was supported by National Research Foundation of Korea (NRF) grants (NRF-2019R1A6A3A01090239, NRF-2019R1A5A2027521) and a Korean Fund for Regenerative Medicine (KFRM) grant (Ministry of Science and ICT, Ministry of Health and Welfare, 22A0104L1).

Data availability

All data generated or analyzed during this study, including supplementary material files, are included in this article. For further inquiries, please contact the corresponding author.

Declarations

Conflict of interest

The authors declare no conflict of interest.