Abstract Background Macrophage immunomodulation has emerged as a novel intervention and therapeutic strategy for temporomandibular joint osteoarthritis (TMJOA), potentially serving as a key approach for reducing synovial inflammation and promoting cartilage repair. The soluble epoxide hydrolase inhibitor (sEHi), TPPU, has shown potential therapeutic effects against inflammatory diseases and osteogenesis by elevating endogenous Epoxyeicosatrienoic acids (EETs). However, it remains largely unknown whether TPPU can reduce inflammation and cartilage degradation in the TMJOA. Methods In vivo, the effects of TPPU on articular cartilage and synovial tissue pathology were assessed using H&E, Masson, Safranin-O/Fast Green staining and immunohistochemistry in a mouse model of TMJOA induced by unilateral anterior crossbite (UAC). RNA-seq and Western Blot was employed to investigate the key signal pathway of TPPU on M1 macrophage polarization. Subsequently, a co-culture system of macrophages and ATDC5 chondrocytes was established, and the influence of TPPU-treated macrophages on chondrogenesis was evaluated through Alcian Blue staining and RT-qPCR. Results In vivo, we observed that in UAC-induced TMJOA mice, TPPU significantly reduced the infiltration of inflammatory cells in the synovium and the positive expression of inflammatory factors TNF-α and IL-1β. It also mitigated the degradation of cartilage matrix and increased the positive expression of chondrogenic markers SOX9 and COL II. In vitro experiments revealed that TPPU inhibited the polarization of M1 macrophages, reduced inflammatory responses, and subsequently increased the expression of chondrogenic markers (SOX9 and COLII) in chondrocytes. RNA-seq data indicated that the NF-κB/IL-17 pathway as a putative target following TPPU treatment in macrophages. Further experiments confirmed that the addition of TPPU to macrophages inhibited the reduction in chondrogenesis induced by IL-17 and NF-κB agonists in the co-cultured cells. Conclusions Our study elucidates a novel role of TPPU in inhibiting M1 macrophage polarization and modulating inflammatory immune responses via the EETs/NF-κB/IL-17 axis, thereby inhibiting cartilage damage in TMJOA. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-025-07003-2. Keywords: TMJOA, EETs, TPPU, Macrophage, IL-17, NF-κB Introduction Temporomandibular joint osteoarthritis (TMJOA) is a progressive degenerative disease marked by pain, joint crepitus or noise, tenderness of the facial and neck muscles, and reduced mandibular function [[36]1]. Previous studies have suggested that among individuals with temporomandibular disorders (TMD), the incidence of TMJOA ranges from 18.01 to 84.47%, affecting 8–16% of the global population [[37]2, [38]3]. Progressive synovitis, condylar cartilage degradation, and bone destruction may play crucial roles in the pathological progression of the TMJOA [[39]4, [40]5]. Articular cartilage can effectively distribute and cushion the impact forces applied to the joint surface. However, it lacks intrinsic self-repair capabilities, making it difficult to heal after injury, which ultimately results in irreversible damage to the joint [[41]6]. Therefore, investigating the molecular mechanisms underlying articular cartilage repair in patients with TMJOA is essential. There is an increasing amount of evidence showing that macrophages, which are main constituent cells of synovial tissue, exert a significant influence on modulating synovial inflammation and the severity of OA by secreting a variety of pro-inflammatory cytokines [[42]7–[43]9]. This process releases high levels of inducible nitric oxide synthase (INOS), interleukin (IL-1β), and tumor necrosis factor-alpha (TNF-α), all of which contribute to inflammation and initiate a cascade of pathological changes, including extracellular matrix (ECM) degradation and a reduction in chondrocytes [[44]10, [45]11]. Therefore, controlling the polarization state of proinflammatory macrophages in TMJOA is particularly important and may become a key method for hindering the cycle of latent inflammation and promoting cartilage repair [[46]12, [47]13]. At present, the clinical treatment options for patients with TMJOA primarily encompass occlusal splints, non-steroidal anti-inflammatory drugs (NSAIDs) and medication joint injections. However, the effectiveness of these non-surgical approaches, particularly oral medications, is often suboptimal. While they may alleviate symptoms in most cases, their long-term therapeutic effects are generally unsatisfactory, and they fail to fully reverse existing degenerative changes [[48]14, [49]15]. Consequently, the development of endogenous, safe, and effective drugs aimed at modulating macrophage polarization to prevent cartilage degeneration in TMJOA is critically important. Such advancements could pave the way for new treatment strategies that promote TMJ regeneration. Epoxyeicosatrienoic acids (EETs), synthesized by cytochrome P450 enzymes during arachidonic acid metabolism, are endogenous bioactive molecules that exert a significant impact on alleviating inflammatory immune responses under pathological conditions such as diabetes, sepsis, lung injury, and arthritis [[50]16, [51]17]. Additionally, research has indicated that EETs can promote growth and regeneration in a variety of tissues and organs, including bone regeneration in cranial defect areas, neuronal axon growth, wound healing, liver regeneration, and compensatory lung growth [[52]18–[53]21]. In the cardiovascular system, EETs can modulate macrophage polarization by activating the PPARα/γ and HO-1 pathways, thereby reducing the activity of the NF-κB signaling pathway and decreasing the release of inflammatory factors [[54]22, [55]23]. IL-17, predominantly secreted by activated T cells, prompts immunocytes to generate an increased number of inflammatory mediators, thereby creating a feedback loop that amplifies inflammatory responses [[56]24]. The IL-17 signaling pathway involves the intricate modulation of various signaling pathways, including the roles of molecules such as A20, Act1, AP-1, and TRAF6. This pathway primarily induces the expression of downstream inflammatory genes by the activation of pathways like NF-κB and MAPKs [[57]25, [58]26]. In osteoarthritis, IL-17 induces the polarization of M0 macrophages towards the M1 phenotype [[59]13]. Under certain circumstances, this feedback loop may lead to the persistence and exacerbation of inflammatory responses, playing a significant role in the onset and progression of the disease, making the IL-17 axis as a new potential target for OA treatment. EETs possess significant anti-inflammatory and tissue-protective effects. However, their short half-life and susceptibility to degradation by soluble epoxide hydrolase (sEH) into biologically inactive dihydroxyeicosatrienoic acids (DHET) limit the efficacy of direct EET administration. Therefore, stabilizing endogenous EETs via sEH inhibitors (sEHi) emerges as an excellent candidate strategy. 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), an effective inhibitor of sEH, reduces EET hydrolysis by inhibiting sEH, thereby increasing endogenous EETs [[60]19]. In the treatment of inflammatory diseases, TPPU has demonstrated potential therapeutic effects across various inflammatory conditions by reshaping the body’s pro-inflammatory and anti-inflammatory balance through the stabilization of endogenous EETs [[61]20, [62]21]. However, it remains uncertain whether TPPU influences macrophage polarization in TMJOA cells and whether it can effectively halt OA progression. In this study, we analyzed the effects of TPPU on cartilage degeneration in a model of UAC-induced TMJOA. We subsequently co-cultured RAW264.7 macrophages with ATDC5 chondrogenic cells to investigate the role of TPPU in M1 macrophage polarization and the associated signaling pathways, as well as to evaluate the impact of TPPU-treated macrophages on chondrogenic differentiation. Our findings suggest that TPPU may mediate the M1 macrophages via the NF-κB/IL-17 signaling pathway, thereby inhibiting bone and cartilage damage in TMJOA. Methods Animals and UAC treatment Forty-five female C57BL/6J mice (6 weeks old, 18–20 g) were purchased from the SPF Animal Experiment Center of Dalian Medical University. The animal research protocol was approved by the Ethics Committee of Dalian Medical University (AEE.22004). The mice were randomly assigned to three distinct experimental groups: Control, UAC and UAC + TPPU. One week after modeling, the TPPU treatment group was administered TPPU (3 mg/kg) via oral gavage every other day. The control and UAC groups received an equivalent volume of saline. The UAC model was constructed using metal tubes according to the previously study [[63]27, [64]28]. And the metal tubes were prepared by a syringe needle (1.6 mm, Shuguang, Henan, China). The maxillary metal tube was 0.3 mm in length, and the mandibular metal tube was bent to form a 135° labially inclined guide. The metal tubes were then bonded with glass ions to the right maxillary and mandibular mesial incisors of mice under anesthesia to form a functional stimulus for a unilateral anterior crossbite. They were observed every alternate day to ensure that they were not dislodged. Mice were euthanized at 3, 7, and 11 weeks post-modeling, with fifteen mice per time point. Bilateral temporomandibular joint tissues were harvested for histological staining and RT-qPCR experiments. Additionally, fresh blood samples were collected at the terminal time point and analyzed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS). Histological detection and analysis TMJ samples from all of the mice were immersed in 4% paraformaldehyde solution for a duration of 24 h, followed by a rinse under running water for 2 h. After 4 weeks of decalcification with 10% EDTA, all tissues were embedded in paraffin for 6 μm serial sagittal sections. According to the operative experimental protocols, H&E (G1120, Solarbio, Beijing, China), Safranin-O/Fast Green (G1371, Solarbio) and Masson staining (G1150, Solarbio) were used to assess synovial inflammation and the changes in bone and cartilage tissue. Cartilage degradation was assessed using the modified Mankin OA score [[65]29] with Safranin-O/Fast Green staining (Table S1). Immunohistochemistry & immunofluorescence TMJ tissue sections were subjected to IHC staining after xylene deparaffinization, gradient hydration, and antigen repair. First, the endogenous peroxidase activity blocker was inactivated (PV9000, ZSGB-BIO, Beijing, China). Then the membrane was broken by PBST for 10 min, and the primary antibody was used at 4° overnight. The primary antibodies are summarized in Table S2. Subsequently, the reaction enhancement solution and enzyme-labelled sheep anti-mouse/rabbit IgG polymer were applied for incubation and hematoxylin staining of the nuclei was performed for 2 min after staining with DAB (ZLI-9018, ZSGB-BIO). Immunofluorescence staining antibodies are listed in Table S2. Followed by secondary antibody for 1.5 h at 37 °C. Nuclei were stained with an anti-quenching blocker containing DAPI (82100, COOLABER, Beijing, China). Positive expression in the condylar and synovial tissues was quantified using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). LC-MS analysis Fresh blood samples were centrifuged at 4 °C to obtain the supernatant (1510×g, 15 min). Subsequently, the serum samples were each added to centrifuge tubes containing 1.3 mL of methyl tert-butyl ether, which were vortexed for 1 min and then centrifuged at 4 °C (1510×g, 10 min). The organic phase (1 ml) was transferred to a new centrifuge tube, followed by the addition of 200 µL of acetonitrile solution. The mixture was then centrifuged at 4 °C at 3778×g for 10 min. Subsequently, the supernatant was then transferred to an autosampler vial for analysis by liquid chromatography-tandem mass spectrometry (API3000, AB Sciex, USA). Data analysis was performed using Analyst v1.6.2 software. Cell culture and chondrogenic differentiation The RAW264.7 and ATDC5 cell line were purchased from Cellverse (Shanghai, China), cultured in DMEM supplemented with 10% FBS at 37 ℃. The RAW264.7 were treated with lipopolysaccharide (LPS) at a concentration of 100 ng/ml (L4391, Sigma, San Luis, MO, USA) for 24 h to elicit M1 polarization. And chondrogenic differentiation was induced using induction medium containing 1% Insulin, Transferrin, Selenium (ITS-G, 41400045, Gibco) for 7–14 d. The effect of TPPU on M1 macrophage inflammation was analyzed by pretreatment with TPPU (10µM, HY-101294, MCE, Shanghai, China) for 1 h. After chondrogenic induction and differentiation, the chondrocytes were co-cultured with M0 and M1 macrophages for 7–14 d (with or without TPPU). The induction medium was changed every three days. CCK-8 assay The proliferative effect of TPPU on ATDC5 chondrogenic cells was assessed using the CCK-8 assay (KTA1020, Abbkine). ATDC5 cells were seeded in 96-well plates with a density of 2 × 10^3 cells per well and subsequently exposed to TPPU over a period of 12–72 h. Subsequently, 10ul CCK-8 reagent and 90ul normal medium were added dropwise to each well, and the OD value at 450 nm was detected by using an enzyme labelling equipment (Flash Spectrum Biotechnology, Shanghai, China) after incubation for 2 h. Alcian blue stain ATDC5 cells, after a 7 d or 14 d induction period, were fixed with a 4% paraformaldehyde solution for 20 min, stained with Alcian Blue solution (ALCB-10001, Oricell, Guangzhou, China) for 1 h, and then rinsed twice. Images of the sections were captured using a stereo microscope (Olympus Corp, Tokyo, Japan), and the percentage of Alcian Blue positivity was assessed using Image-Pro Plus (Media Cybernetics, Rockville, MD, USA). RT-qPCR Total RNA was extracted from RAW264.7 and ATDC5 using an RNA extraction reagent (SM139-02, Seven, Beijing, China). Reverse transcription was performed utilizing the SevenFast Two-Step RT-qPCR Kit (SM143-01, Seven), which was succeeded by quantitative real-time PCR employing a SYBR PCR kit for real-time fluorescence quantification (SM143-01, Seven). And mRNA expression levels were standardized against GAPDH expression, and RT-qPCR results were calculated using the 2^−ΔΔCq method. The sequences of the primers are detailed in Table S3. Western blotting Total cellular proteins of RAW264.7 and ATDC5 cells were extracted using RIPA lysis buffer (RIPAR0010, Solarbio) containing 0.1% PMSF (G2008-1ML, Servicebio, Wuhan, Beijing) enhancement, respectively. After the separation and concentration gels were configured, electrophoresis was started using a rapid electrophoresis solution (G2081-1 L, Servicebio) after consecutive samples were taken. At the end of the electrophoresis, the gel was blotted onto a PVDF membrane (Millipore, Merck KGaA, Darmstadt, Germany), followed by a 15 min incubation in a blocking solution, and then mixed with a primary antibody at 4°overnight (Table S1). On the subsequent day, the membrane underwent three washes with TBST buffer and incubated with the secondary antibody for a period of 1 h at room temperature. Finally, gray values were obtained using Enhanced Chemiluminescence (ECL, PE0010, Solarbio) and detected using the ProteinSimple FluorChem system (Bio-Rad Laboratories, Hercules, CA, USA). RNA sequencing and transcriptome analysis RNA sequencing was performed by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). Total RNA was extracted, and sequencing libraries were constructed using the NEBNext Ultra™ RNA Library Prep Kit (NEB, MA, USA) following the manufacturer’s protocol. Quality-checked libraries were pooled based on desired sequencing depth and subjected to paired-end sequencing on an Illumina platform (Illumina NovaSeq X plus, Illumina, CA, USA). Raw reads were quality-filtered using fastp (v 0.23.2) [[66]30]. Clean reads were aligned to the GRCm39 mouse reference genome (Ensembl release 109: Mus_musculus. GRCm39.109) using HISAT2 (v 2.2.1) [[67]31]. Gene expression quantification was performed with Feature Counts (v 2.0.3) [[68]32], counting reads mapped to exonic regions. Enrichment analysis Both Overrepresentation Analysis (ORA) and Gene Set Enrichment Analysis (GSEA) were conducted employing the R package “clusterprofile” (v 4.12.0) in R (v 4.4.0). In this context, a threshold of P < 0.05 was utilized to denote statistical significance for an enriched pathway. Differential gene analysis The “limma”(3.60.2) package was used for differential analysis of gene expression in R (v 4.4.0). And a P-value below 0.05, which enabled the identification of differentially expressed genes, was set as the criterion for statistical significance. Flow cytometry Digest and centrifuge 1.5 × 10^6 cells were digested and centrifuged, followed by washing with PBS twice at 350–500 ×g for 5 min each. One hundred microliters of PBS was used as the staining and washing buffer and incubated with the antibody at 4 °C in the dark for 30 min to facilitate optimal antibody-antigen interactions (Table [69]S1). Data were collected using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and subsequently analyzed using FlowJo X10 software for comprehensive data interpretation. IL-17 assay The ELISA kit (ml037866, mlbio, Shanghai, China) was used for the detection of IL-17. Briefly, 50 µl of the cell culture supernatant and different concentrations of standards were added to the corresponding wells, respectively. The experimental procedures were carried out according to the manufacturer’s instructions. The OD value at 450 nm was detected by using an enzyme labelling equipment (Flash Spectrum Biotechnology). A standard curve was plotted with the standard concentration on the y-axis and the OD value on the x-axis, and the IL-17 concentration was calculated based on this standard curve. Statistical analysis Each experimental set was conducted a minimum of three times, with results presented as the mean ± SD. Statistical analyses were performed using GraphPad Prism version 8.0. The two-tailed t-test was employed for pairwise comparisons, and a p-value of less than 0.05 was deemed to indicate statistical significance. One-way ANOVA followed by Tukey’s post-hoc test was used to assess differences among groups, and Pearson’s correlation coefficient was used for correlation analysis, with a significance level set at p < 0.05. Results In vivo experiments demonstrate TPPU reduces cartilage degeneration and bone destruction in TMJOA A mouse model of UAC-induced TMJOA was constructed. Then TPPU was administered orally every other day, and samples were collected at 3, 7, and 11 weeks after modeling (Fig. [70]1A and B). HE staining revealed the temporal progression of chondrodysplasia and bone destruction in the condyles of TMJOA mice induced by UAC, characterized by a reduction in cartilage thickness, atrophy, and a decreased number of chondrocytes, especially at 7 and 11 weeks. Masson staining indicated a significant enlargement of the subchondral bone marrow cavity and bone collagen fiber damage in the UAC group. Both the HE and Masson staining results collectively demonstrated that TPPU treatment prevented the destruction of cartilage and subchondral bone induced by UAC. Likewise, Safranin-O/Fast Green staining revealed that, compared to the control group, the expression of aggrecan, a key proteoglycan, was decreased in the articular cartilage of the UAC group, but was increased in groups treated with TPPU. Furthermore, IHC indicated that the expression of SOX9 and COLII, which are anabolic markers, was significantly increased following TPPU treatment, compared to the UAC group (Fig. [71]1C). Collectively, these results suggested that TPPU exerts a protective influence against bone and cartilage damage in TMJOA. Fig. 1. [72]Fig. 1 [73]Open in a new tab In vivo experiments demonstrate that TPPU reduces cartilage degeneration and bone destruction in TMJOA. (A) 7-week-old C57 mice were selected to establish a TMJOA model induced by UAC. (B) Schematic of the treatment protocol for TMJOA mice with TPPU. Mice were randomly assigned into three groups: Control, UAC, and UAC + TPPU. According to the timeline, the TPPU group was administered TPPU via gavage every other day (3 mg/kg) starting 1 week post-modeling, while the other two groups received an equivalent volume of normal saline. Subsequently, mice were euthanized at 3, 7, and 11 weeks post-modeling. (C) Representative images of H&E and Masson staining of TMJ tissue sections, as well as IHC staining for SOX9 and COL II. The black line segments represent the maximum thickness of cartilage. The red arrows indicate the collagen fibers of cartilage and subchondral bone. The orange arrows denote safranin staining of cartilage, and the yellow arrows indicate fast green staining of subchondral bone. (D) HE staining analysis of joint cartilage thickness changes and quantitative analysis (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, *p < 0.05, **P < 0.01, ***P < 0.001; # vs. UAC, # P < 0.05, ## P < 0.01). (E) Quantitative analysis of collagen volume fraction (CVF) in Masson staining (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, *p < 0.05, **P < 0.01, ****P < 0.0001; # vs. UAC, ## P < 0.01, ### P < 0.001). (F) Modified Mankin OA scoring to verify the inhibiting effect of TPPU treatment on condylar cartilage degeneration in TMJOA mice (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, **P < 0.01, ***P < 0.001; # vs. UAC, ## P < 0.01, ### P < 0.001). (G) Quantitative analysis of SOX9 and COL II expression by IHC (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, **P < 0.01; # vs. UAC, ## P < 0.01, ### P < 0.001; ns indicates no significant). Data are presented as mean ± SD TPPU inhibits macrophage polarization towards the M1 phenotype and reduces TMJOA Inflammation Studies have shown that the infiltration of macrophages into the synovium is a key step in the inflammatory response induced by osteoarthritis [[74]33]. Activated M1 macrophages can lead to synovitis or directly participate in the degradation and destruction of the articular cartilage [[75]34]. Records from the GEO database indicate that the expression of M1 phenotype markers, including INOS and TNF-α, within the synovial tissues of patients with TMJOA are higher than those in normal individuals (Fig. [76]2A and B). However, there was no significant variation in the expression of CD163 and CD206 (M2 phenotype markers) between the healthy and OA samples (Fig. [77]2A). To further investigate whether TPPU can inhibit the M1 phenotype macrophages, we conducted RT-qPCR analysis and found that the expression of CD86 and INOS increased after 24 h of LPS stimulationin in the M0 group, while pretreatment with TPPU significantly inhibited their expression. In the M0 group, the expression levels of CD86 and iNOS did not show any significant differences between the conditions with and without TPPU treatment (Fig. [78]2C). Flow cytometry revealed a similar phenomenon, and the results indicated that the number of CD86/INOS double-positive cells in the M1 group decreased after TPPU treatment (Fig. [79]2D). Accordingly, we conducted validation in TMJOA tissue sections. IF showed increased expression of INOS/CD68 in the synovial tissue and subchondral bone of the UAC group compared to the control group, along with increased infiltration of M1 macrophages, which was reduced following TPPU treatment (Fig. [80]2E and F; Fig. [81]S1 and S2). Fig. 2. [82]Fig. 2 [83]Open in a new tab TPPU inhibits macrophage polarization towards the M1 phenotype. (A) The [84]GSE205389 dataset displays a heatmap of the differential macrophage gene expression in the synovial tissue of TMJOA patients when compared to the normal (log[10] of normalized counts). (B) Gene levels retrieved from the GEO database in normal and OA synovial tissue samples. (C) RT-qPCR detection of CD86 and INOS mRNA expression in M0 and M1 macrophages following TPPU treatment for 24 h (n = 3, unpaired two-tailed t-test; ***P < 0.001; ns indicates no significant). (D) Representative flow cytometry analysis images in M0 and M1 macrophages following TPPU treatment for 24 h. (E and F) Identification of M1 macrophages (CD68/INOS) in the synovial and subchondral bone using IF in 7 weeks TMJOA mice tissue section (n = 5, one-way ANOVA followed by Tukey’s multiple comparisons; * vs. Control, *p < 0.05, **P < 0.01, ****P < 0.0001; # vs. UAC, ### P < 0.001; ns indicates no significant). Data are presented as mean ± SD Based on these results, we hypothesized that TPPU may initially mitigate OA progression through its anti-inflammatory effects. H&E-stained tissue sections at 7 weeks clearly displayed an inflamed synovium in the UAC group, accompanied by infiltrating inflammatory cells and lymphoid follicle formation, whereas TPPU treatment resulted in less synovial inflammation (Fig. [85]3A). RT-qPCR analysis showed that TNF-α and IL-1β were upregulated in the UAC models when compared to the control group. However, treatment with TPPU significantly reduced the expression levels of these cytokines in the UAC group (Fig. [86]3B). The IHC results were consistent with these findings (Fig. [87]3C and D). To assess the ability of TPPU to inhibit sEH in vivo, the concentration of the EETs’ metabolite DHET in mouse serum was measured using LC-MS. The results showed that the levels of DHET in the serum of TPPU-treated mice were significantly lower than those in the control and UAC groups (Fig. S3), indicating that TPPU can inhibit the metabolism of EETs to DHET in mice, thereby increasing the levels of endogenous EETs. To further confirm whether the effect of TPPU is associated with the polarization of M1 macrophages, we selected CD86 as a marker for M1 macrophages and IF showed an increase in co-staining for CD86 and IL-1β in the UAC group, which was also decreased by TPPU treatment (Fig. [88]3E). Collectively, these results revealed that TPPU could inhibit the M1 macrophage phenotype and reduce synovial and joint inflammation induced by UAC in TMJOA mice, exerting an anti-inflammatory effect. Fig. 3. [89]Fig. 3 [90]Open in a new tab TPPU reduces joint inflammation and exerts anti-inflammatory effects. (A) Representative images of the H&E staining of inflammatory synovial tissue from TMJOA mice induced by UAC at 3 weeks, 7 weeks, and 11 weeks, along with quantitative analysis of synovial inflammation scores (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, **P < 0.01, ***P < 0.001; # vs. UAC, # P < 0.05, ## P < 0.01; ns indicates no significant). (B) RT-qPCR detection of TNF-α and IL-1β mRNA expression in joint tissue of mice at the 7-week time point (n = 3, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, ***P < 0.001, ****P < 0.0001; # vs. UAC, ## P < 0.01). (C and D) IHC and quantitative analysis of TNF-α and IL-1β in joint tissue sections (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; # vs. UAC, ## P < 0.01, ### P < 0.001; ns indicates no significant). (E) IF staining and correlation analysis of CD86 and IL-1β in joint tissue sections (n = 15, two‑tailed Pearson’s correlation). Data are presented as mean ± SD TPPU reduces chondrocyte degradation and exerts chondroprotective effects by targeting M1 macrophage polarization in vitro To evaluate whether TPPU suppresses M1 polarization and further regulates cartilage remodeling, we established a co-culture model to study the effects of M0, M1, and TPPU-treated M1 macrophages on ATDC5 cells (Fig. [91]4A). Our previous studies have shown that TPPU exhibits osteogenic effects at a concentration of 10 µM [[92]35]. Initially, we assessed the toxicity of various conditioned media (CM) on RAW264.7 macrophages using the CCK-8. We found that treatment with TPPU (10 µM) did not significantly affect RAW264.7 proliferation after 24 h in terms of the various CM (Fig. [93]4B). Alcian Blue staining revealed that intervention with TPPU did not exert a statistically significant effect on the chondrogenic differentiation process of chondroprogenitor cells. Similar phenomena were observed in the RT-qPCR results, implying that the TPPU’s protective effects were not achieved by directly affecting chondrogenic cell function. These findings led us to speculate that TPPU can mediate the immune regulation of macrophages, thereby reducing OA cartilage destruction. In contrast to the M0 macrophage group, the synthetic metabolism of ATDC5 cells was inhibited when they were cultured with inflammation-activated M1 macrophages. However, when M1 macrophages were treated with TPPU, this effect was partially reversed, and TPPU exhibited a tendency to promote and enhance chondrogenesis (Fig. [94]4C). We subsequently examined the transcriptional levels of the anabolic indicators Acan, SOX9, and COL II, as well as the catabolic markers MMP-13 and Adamts5, in chondrogenic cells. RT-qPCR results indicated that the M1 macrophage group suppressed the mRNA expression of chondrocyte anabolic markers on days 7 and 14, whereas this trend was inhibited in the TPPU-treated M1 macrophage group (Fig. [95]4D). The IF results were consistent with these findings (Fig. [96]4E). These results suggest that M1 macrophages can suppress chondrocyte function and that TPPU possess the ability to reduce the suppressive influence of M1 macrophages on chondrocyte function. Fig. 4. [97]Fig. 4 [98]Open in a new tab TPPU reduces chondrocyte degradation and exerts chondroprotective effects by targeting M1 macrophage polarization in vitro. (A) A co-culture system of Raw264.7 macrophages with ATDC5 cells was used to investigate the effect of TPPU on chondrogenesis. (B) The impact of M0/M1 macrophage-conditioned medium with or without TPPU treatment on the proliferation of ATDC5 cell proliferation was assessed using the CCK8 assay. (C) After inducing chondrocytes with M0/M1 macrophage-conditioned medium treated with or without TPPU for 7 and 14 days, Alcian Blue staining and quantitative analysis were utilized to assess the accumulation of proteoglycans in the cartilage formation matrix (n = 5, unpaired two-tailed t-test; **P < 0.01, ***P < 0.001; ns indicates no significant). (D) RT-qPCR was used to assess the expression of the anabolic markers (COLⅡ, Acan, SOX9) and catabolic markers (Adamts, MMP-13) in ATDC5 cells after 7 and 14 days of induction (n = 3, unpaired two-tailed t-test; *p < 0.05, **P < 0.01, ***P < 0.001; ns indicates no significant). (E) IF was employed to examine the effect of TPPU-treated macrophages on the expression of the anabolic COL II in chondrocytes. Data are presented as mean ± SD The transcriptome reveals that TPPU inhibits the IL-17 signaling pathway in M1 phenotype macrophages We conducted transcriptome sequencing of M1 macrophages in the TPPU (-) and TPPU (+) treatment groups to elucidate the molecular mechanisms underlying their polarized states in immune and inflammatory responses ([99]GSE285011). After differential analysis, we identified 626 upregulated and 553 downregulated genes relative to the expression levels observed in the control group. (Fig. [100]5A). Subsequently, we conducted GO Enrichment Analysis on the genes exhibiting differential expression, and the findings suggested that TPPU may mediate biological processes, including the regulation of response to stimuli, intracellular signal transduction, and regulation of apoptotic processes (Fig. [101]5B). Next, we used the KEGG dataset to perform pathway enrichment analysis and found that the genes were primarily enriched in the IL-17, HIF-1α, and TNF-α signaling pathways (Fig. [102]5C). Elevated IL-17 have been detected in patients with osteoarthritis (OA) and are implicated in cartilage matrix synthesis and degradation [[103]36]. Additionally, we performed GSEA to validate the enrichment of the IL-17 pathway. This indicated that IL-17-related biological processes were significantly active among our differentially expressed genes, potentially linking them to immune responses and inflammatory processes (Fig. [104]5D). Finally, we utilized a heatmap to illustrate the differentially expressed genes enriched in the IL-17 pathway (Fig. [105]5E). Our results suggested that TPPU inhibited M1 macrophage polarization by downregulating the IL-17 signaling pathway. Fig. 5. [106]Fig. 5 [107]Open in a new tab The transcriptome reveals that TPPU inhibited the IL-17 signaling pathway in M1 phenotype macrophages. (A) Heatmap showing the top 10 differentially expressed genes. (B) Bubble chart displaying 5 GO enrichment terms for Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). (C) A bar chart displaying 10 KEGG pathways obtained from pathway enrichment analysis. (D) The GSEA enrichment for IL-17 signaling pathway (NES = 1.708293, p value = 3.427046^e−02). (E) The heatmap displays the genes that show changes in the IL-17 signaling pathway TPPU inhibits M1 macrophage polarization through the lL-17 signaling pathway Next, we detected the expression levels of pivotal factors in the IL-17 signaling pathway both in vivo and in vitro. Initially, the IHC results revealed that IL-17 levels in the subchondral bone and synovial tissues were significantly elevated in the UAC group compared to the control group. In contrast, treatment with TPPU effectively reduced its expression (Fig. [108]6A). ELlSA was employed to assess IL-17 expression across different experimental groups, including M0 macrophages, M1 macrophages, and TPPU-treated M0 and M1 macrophages. No notable differences were observed among the M0 groups, whereas IL-17 expression was markedly decreased in the TPPU-treated M1 group in comparison to the untreated M1 group (Fig. [109]6B). Fig. 6. [110]Fig. 6 [111]Open in a new tab TPPU inhibits macrophage polarization to the M1 phenotype through the lL-17 signaling pathway. (A) IHC detection of lL-17 expression among the three groups in TMJOA mice tissue sections (n = 5, one-way ANOVA followed by Tukey’s post-hoc test; * vs. Control, ***P < 0.001; # vs. UAC, ## P < 0.01, ### P < 0.001; ns indicates no significant). (B) ELISA for lL-17 expression in M0 and M1 macrophages with and without TPPU treatment (n = 3, unpaired two-tailed t-test; ***P < 0.001; ns indicates no significant). (C) RT-qPCR detection of mRNA expression of key factors of the lL-17 signaling pathway in M0 and M1 macrophages with and without TPPU treatment (lL-17 A, p-NF-κB, FOS, A20, Act1) (n = 3, unpaired two-tailed t-test; *p < 0.05, **P < 0.01; ns indicates no significant). (D) IF detection of the expression of the M1 macrophage marker CD86/INOS in M0 macrophages stimulated by lL-17 agonist (n = 5, unpaired two-tailed t-test; **P < 0.01; ns indicates no significant). (E) RT-qPCR detection of mRNA expression of the M1 macrophage marker CD86/INOS in M0 macrophages stimulated by lL-17 agonist (n = 3, unpaired two-tailed t-test; **P < 0.01, ***P < 0.001; ns indicates no significant). (F) Western blot detection of NF-κB pathway associated protein p-p65 expression and quantitative analysis (n = 3, unpaired two-tailed t-test; ***P < 0.001; ns indicates no significant). Data are expressed as mean ± SD Subsequently, we used RT-qPCR to detect the key factors in the lL-17 signaling pathway, incorporating IL-17 A, p-NF-κB, FOS and A20, which also showed that the expression of the above factors in the M1 macrophages treated with TPPU were lower than those in the untreated group (Fig. [112]6C). This indicates that TPPU can negatively regulate the lL-17 signaling pathway in M1 macrophages, further confirming the transcriptomic sequencing results. We used lL-17 agonists acting on M0 cells. RT-qPCR and IF showed that the lL-17 agonist increased the expression of INOS and CD86, indicating that lL-17 agonist treatment promotes the polarization of M0 macrophages towards M1 macrophages. However, the expression of these markers was reduced by TPPU treatment, suggesting that the polarization of M1 macrophages is inhibited to some extent (Fig. [113]6D and E). EETs can inhibit the NF-κB signaling pathway and reduce tissue inflammation [[114]22]. Our RT-qPCR results indicated that TPPU significantly reduced the level of p-NF-kB. The application of the lL-17 agonist enhanced the expression of p-p65 in M0 macrophages, whereas its expression was diminished following TPPU treatment (Fig. [115]6F). These findings suggest that TPPU inhibits IL-17 signaling by targeting the NF-kB pathway. TPPU targets the NF-κB/IL-17 pathway in M1 macrophages to promote chondrogenic differentiation To explore the inhibitory effect of TPPU on the IL-17 signaling pathway via NF-κB, M0 macrophages were initially treated with IL-17 and NF-κB agonists. The RT-qPCR results demonstrated a markedly elevated expression of inflammatory mediators in M0 macrophages treated with both agonists. Conversely, treatment with TPPU resulted in the downregulation of these inflammatory factors (Fig. [116]7A). Next, we co-cultured TPPU-treated M0 macrophages and ATDC5 cells for chondrocyte induction for 7 and 14 days, utilizing either the IL-17 agonist or the NF-κB agonist. Alcian Blue staining revealed that the synthesis of chondrocytes in ATDC5 cells was inhibited following treatment with the IL-17 agonist and the NF-κB agonist. However, this inhibition was alleviated by TPPU treatment, with the TPPU-treated group exhibiting a trend towards increased chondrogenic synthesis (Fig. [117]7B). The mRNA expression of Acan, SOX9, COL II, Adamts5, and MMP-13 was further examined by RT-qPCR after 7 and 14 days of induction. We observed that TPPU treatment reduced the agonist-induced reduction in COL II, Acan, and SOX9 mRNA expression following agonist treatment. Conversely, TPPU mitigated the agonist-induced increase in Adamts5 and MMP-13 mRNA levels (Fig. [118]7C), (Fig. [119]8) COLⅡ is the main collagen in the cartilage tissue, and its synthesis changes when the cartilage is defective and repaired [[120]37]. We conducted IF analysis to assess cartilage anabolism following induction and observed effects similar to those described earlier. Specifically, the use of lL-17 and NF-κB agonists inhibited the expression of COLⅡ on days 7 and 14, and the TPPU-treated group could effectively reduce this inhibition (Fig. [121]7D and Fig. S4). The results suggest that TPPU exerts a potent anabolic effect on chondrogenic cell function by targeting M1 macrophage via the NF-κB/lL-17 pathway (Fig. [122]8). Fig. 7. [123]Fig. 7 [124]Open in a new tab TPPU targets the NF-κB/IL-17 pathway in macrophages to promote chondrogenic differentiation and exert chondroprotective effects. (A) RT-qPCR for mRNA expression of inflammatory factors lL-1β, TNF-α, and lL-6 in macrophages treated with lL-17 agonists, NF-κB agonists, and TPPU (n = 3, unpaired two-tailed t-test; *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns indicates no significant). (B) Chondrocyte induction after 7 and 14 days of co-culture of M0 macrophages and ATDC5 cells treated with lL-17 agonists, NF-κB agonists, and TPPU, and observation of cartilage anabolism by Alcian blue staining and quantitative analysis (n = 5, unpaired two-tailed t-test; *p < 0.05, **P < 0.01; ns indicates no significant). (C) RT-qPCR detection for mRNA expression of anabolic markers (COLⅡ, Acan, SOX9) and catabolic markers (Adamts, MMP-13) in ATDC5 after 7 and 14 days of induction (n = 3, unpaired two-tailed t-test; *p < 0.05, **P < 0.01, ***P < 0.001; ns indicates no significant). (D) IF was used detected the expression of anabolic marker COLⅡ in ATDC5 after 7 days of induction (n = 5, unpaired two-tailed t-test; *p < 0.05, **P < 0.01; ns indicates no significant). Data are presented as mean ± SD Fig. 8. [125]Fig. 8 [126]Open in a new tab Graphic abstract. TPPU inhibiting M1 macrophage polarization and reducing cartilage degradation through the EETs/NF-KB/IL-17 signaling pathway Discussion The potent anti-inflammatory effects of EETs and their ability to promote the growth and regeneration of various tissues and organs have garnered significant attention [[127]38–[128]41]. In this study, we uncovered several pivotal findings that not only revealed the anti-inflammatory effects of TPPU but also elucidated its potential mechanisms in reducing cartilage damage. Our study indicates, for the first time, that oral administration of TPPU can effectively inhibit TMJOA progression in regard to cartilage degeneration and bone destruction induced by UAC, mitigating the inflammatory response associated with osteoarthritis. M1 macrophages exert a significant influence in the inflammatory process of OA. Upon activation by local signaling molecules and cytokines, these macrophages polarize toward the M1 phenotype, establishing a pro-inflammatory environment that secretes a variety of inflammatory factors, which can lead to symptoms of synovitis [[129]42]. In addition, activated M1 macrophages produce proteases and ROS, which directly contribute to degradation and destruction of articular cartilage [[130]43, [131]44]. Previous studies have reported that EETs can inhibit macrophage polarization to the M1 phenotype and alleviate inflammation [[132]45]. Our results indicated that the number of M1 macrophages in the synovium and condylar cartilage was elevated in the UAC group compared to the control group, which is closely associated with the exacerbation of inflammatory responses. TPPU not only suppresses the polarization of M1 macrophages but also mitigates the production of TNF-α and IL-1β. Synovitis is an early symptom of osteoarthritis and is ubiquitous throughout the entire disease trajectory. Changes in the levels of inflammatory factors caused by synovial inflammation are highly correlated with cartilage degeneration [[133]7, [134]46]. Therefore, this study offers compelling evidence supporting the potential therapeutic benefits of TPPU in controlling macrophage activity, which may reduce joint inflammation and cartilage damage by inhibiting M1 macrophages. Additionally, our in vitro experimental data indicated that TPPU does not significantly affect chondrogenic differentiation. The protective effects of TPPU in OA are not achieved by directly affecting the function of chondrogenic cells. These results led us to hypothesize that TPPU mediates the immune modulation of macrophages, thereby reducing OA cartilage destruction. We subsequently investigated the effects of TPPU-mediated M1 macrophage polarization on chondrogenic cell functionality and discovered that, compared to the M0 macrophage group, M1 macrophages could inhibit anabolic mediators and induce the production of catabolic mediators MMP13 and Adamts5. TPPU treatment partially restrained this regulatory effect. These results indicate that TPPU enhances the functionality of ATDC5 cells through modulating macrophage polarization. Several studies have shown that the IL-17 pathway is pivotal in immune regulation in autoimmune diseases, including rheumatoid arthritis (RA), ankylosing spondylitis, and psoriatic arthritis [[135]26, [136]47]. IL-17 has been shown to promote the macrophage polarization from M0 to M1 phenotypes, and targeted IL-17 therapy can inhibit PINK1/Parkin autophagy and M1 macrophage polarization in rheumatic heart disease [[137]48]. In this study, the RNA-seq results indicated that TPPU effectively suppressed the activity of the IL-17 signaling pathway in M1 macrophages. IHC results from tissue sections further revealed significantly elevated expression of IL-17 in synovial tissue and subchondral bone of OA samples, which was markedly reduced following TPPU intervention. Concurrently, we observed that the application of IL-17 agonists led to an upregulation of INOS and CD86 expression in M0 macrophages, facilitating their transition from the M0 to the M1 phenotype. However, TPPU was able to inhibit the regulatory effects of IL-17, indicating that TPPU can modulate M1 polarization by inhibiting the activation of the IL-17 pathway. Studies have shown that IL-17 can modulate the expression of inflammatory genes such as IL-6, IL-1β and TNF-α, through activating the NF-κB pathway [[138]49–[139]51]. It was demonstrated that EETs could suppress the phosphorylation levels of NF-κB by upregulating its receptor levels of PPAR-γ [[140]52, [141]53]. In models of myocarditis and rheumatoid arthritis, EETs reduced the activation of M1 macrophages and inhibited the activation of the NF-κB pathway, thereby playing an anti-inflammatory role [[142]54, [143]55]. In our study, IL-17 stimulation enhanced the phosphorylation level of p65 in M0 macrophages and this effect was attenuated by TPPU treatment. Furthermore, TPPU also decreased the pro-inflammatory cytokines that were elevated by the addition of an NF-κB agonist in M0 macrophages, indicating that TPPU can help alleviate inflammatory responses by inhibiting p-NF-κB. Co-culture experiments involving chondrogenic cells and macrophages demonstrated that TPPU mitigated the inhibitory effects of an NF-κB agonist on chondrogenesis and promoted the synthesis of cartilage matrix. These findings suggest that TPPU may provide protective effects on articular cartilage in TMJOA by modulating the NF-κB/IL-17 pathway in macrophages. Although this study provides valuable insights into the promotion of cartilage repair in TMJOA, it also has certain limitations. Firstly, the ATDC5 cell line and animal models used in this study are of murine origin, which may not be entirely applicable to human clinical scenarios. Therefore, it is necessary to conduct further research using human temporomandibular joint cells in the future. Additionally, in vivo studies should delve deeper into the potential side effects of TPPU treatment to ensure its biosafety and to determine the efficacy of local TPPU application. Moreover, EETs are susceptible to degradation by sEH into DHET, which results in a loss of their anti-inflammatory properties. The exogenous administration of EETs has a short duration of action in vivo, making it less than ideal for therapeutic applications. Consequently, most current studies on EETs utilize sEHi to enhance their endogenous levels [[144]19–[145]21]. TPPU has shown significant effects in various models, including those related to pain, inflammation, and autoimmune responses, and is recognized as a standard sEHi in the field [[146]32–[147]35]. In our study, we employed LC-MS analysis to demonstrate that TPPU treatment in UAC mice significantly reduced the levels of EET degradation products, DHET, which indirectly suggests that TPPU effectively increases EET levels. However, to directly elucidate the mechanism of EETs, further validation is necessary to assess whether EET alone possesses significant anti-inflammatory and cartilage-protective effects on TMJOA, as well as to explore the specific mechanisms by which EETs regulate the NF-κB/IL-17 pathway in macrophages. In summary, this study marks a significant step in our understanding of the potential of TPPU to promote cartilage repair in TMJOA. However, before its translation into clinical therapeutic outcomes, more in-depth research is necessary. Conclusions To summarize, this study indicated that TPPU administration reduced bone and cartilage damage in the TMJOA by inhibiting sEH and stabilizing endogenous EET levels. Mechanistically, EETs inhibit M1 phenotype macrophages polarization by the modulation of the NF-κB/IL-17 signaling pathway, alleviating macrophage inflammatory responses and subsequently regulating the anabolic and catabolic processes of chondrocytes. This study offers new insights into the therapeutic analysis of patients with TMJOA and presents novel strategies for developing treatments that target the regulation of inflammatory immune responses in TMJOA. Supplementary Information [148]Supplementary Material 1^ (569.2KB, docx) [149]Supplementary Material 2^ (19.1KB, docx) Acknowledgements