Abstract Purpose To investigate the pathological changes of the synovium in mice with post-traumatic osteoarthritis (PTOA) treated with 4-octyl itaconate (4-OI) and evaluate the therapeutic effects of 4-OI. Methods In the phenotypic validation experiment, the mice were randomly divided into 3 groups: wild-type (WT) group, sham group, and destabilization of the medial meniscus (DMM) group. Through MRI, micro-CT, and histological analysis, it was determined that the DMM surgery induced a mouse PTOA model with significant signs of synovitis. At 12 weeks post-DMM surgery, synovial tissues from the DMM group and WT group mice were collected for ribonucleic acid sequencing analysis. In the 4-OI treatment experiment, mice were randomly divided into the sham group, DMM group, DMM + 4-OI (50 mg/kg) group, and DMM + 4-OI (100 mg/kg) group. Von Frey tests and open field tests were conducted at intervals during the 12 weeks following the DMM surgery. After 12 weeks of surgery, the efficacy of 4-OI treatment on PTOA in mice was evaluated using MRI, micro-CT, histological analysis, and quantitative real-time polymerase chain reaction. Finally, we utilized network pharmacology analysis to predict the mechanism of 4-OI in treating PTOA synovitis and conducted preliminary validation. Statistical analysis was performed using one-way ANOVA and the Kruskal-Wallis test. Difference was considered statistically significant at p < 0.05. Results The DMM surgery effectively induced a PTOA mouse model, which displayed significant symptoms of synovitis. These symptoms included a notable increase in both the number of calcified tissues and osteophytes (p < 0.001), an enlargement of the calcified meniscus and synovial tissue volume (p < 0.001), and thickening of the synovial lining layer attributable to M1 macrophage accumulation (p = 0.035). Additionally, we observed elevated histological scores for synovitis (p < 0.001). Treatment with 4-OI inhibited the thickening of M1 macrophages in the synovial lining layer of PTOA mice (p < 0.001) and reduced fibrosis in the synovial stroma (p = 0.004). Furthermore, it reduced the histological scores of knee synovitis in PTOA mice (p = 0.006) and improved the inflammatory microenvironment associated with synovitis. Consequently, this treatment alleviated pain in PTOA mice (p < 0.001) and reduced spontaneous activity (p = 0.003). Bioinformatics and network pharmacology analyses indicated that 4-OI may exert its therapeutic effects by inhibiting the differentiation of synovial Th17 cells. Specifically, compared to the lipopolysaccharide stimulation group, 4-OI reduced the levels of positive regulatory factors of Th17 cell differentiation (IL-1: p < 0.001, IL-6: p < 0.001), key effector molecules (IL-17A: p < 0.001, IL-17F: p = 0.004), and downstream effector molecules in the IL-17 signaling pathway (CCL2: p < 0.001, MMP13: p < 0.001). Conclusion 4-OI is effective in inhibiting synovitis in PTOA, thereby alleviating the associated painful symptoms. Keywords: 4-Octyl itaconate, Synovitis, Post-traumatic osteoarthritis, Pain 1. Introduction Post-traumatic osteoarthritis (PTOA) is a subtype of osteoarthritis (OA) induced by mechanical joint injuries. Common etiological factors include ligament and meniscus tears, cartilage degeneration, bone fractures resulting from high-impact landings, and joint dislocations.[33]^1^,[34]^2 The pathological changes of PTOA are consistent with those of OA, including the degeneration of articular cartilage, synovitis, subchondral bone injury, osteophyte formation, and production of pro-inflammatory mediators, but the progress of PTOA is more rapid.[35]^3^,[36]^4 Due to the early onset and rapid progression of PTOA, it can severely impair joint function and reduce quality of life, leading to substantial socioeconomic burdens.[37]^5 Because patients with joint injuries are highly predisposed to developing PTOA, early therapeutic interventions may have the potential to prevent the onset or progression of the disease. Synovium acts as a semipermeable membrane, controlling the flow of molecules into and out of the joint space and maintaining the composition of synovial fluid, which is essential for preserving the normal physiologic state of articular cartilage.[38]^6 Studies demonstrated the presence of synovitis in PTOA by ultrasound and MRI.[39]^7 A growing body of scientific evidence demonstrates that synovitis is not a bystander in the development of OA but rather a participant in joint structural damage.[40]^8 Inflammatory synovium can produce inflammatory factors that directly damage and degrade the cartilage matrix. Additionally, it can generate cytokines and metalloproteinases that promote the catabolism of chondrocytes, accelerating the degeneration of articular cartilage and facilitating the progression of OA.[41]^9^,[42]^10 Besides, synovitis is the main source of PTOA pain.[43]^11^,[44]^12 Most of available studies demonstrate that synovitis is a rationale target for early therapeutic intervention to control joint symptoms and progression of PTOA.[45]^9^,[46]^13 Pain is a predominant symptom in individuals with PTOA and requires potent analgesic interventions, with the current use of nonsteroidal anti-inflammatory drugs (NSAIDs) and weak opioids.[47]^14 Therefore, the long-term use of NSAIDs is associated with significant adverse effects, particularly gastrointestinal and cardiovascular complications, and the risk increases with age and the presence of comorbidities.[48]^15 Long-term use of weak opioids such as tramadol is also fraught with safety concerns, and reliable clinical trials have shown that long-term use of this drug leads to an elevated risk of all-cause mortality.[49]^16^,[50]^17 Consequently, therefore, there is an urgent need to develop novel, effective, and safe drugs for more effective PTOA treatment strategies. Itaconate is an endogenous metabolite produced primarily by pro-inflammatory macrophages with well-recognized anti-inflammatory and antioxidant properties for the treatment of PTOA synovitis and improvement of the inflammatory milieu.[51]^18 As an intrinsic cellular metabolite, itaconate can reduce the risk of therapeutic side effects compared to NSAIDs and opioids. Moreover, itaconate modulates the pro-inflammatory differentiation of immune cells, encompassing macrophages and T cells, and inhibits the expression of a spectrum of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, thus enhancing the condition of synovitis and the inflammatory environment. Itaconate may exert a greater impact on reducing systemic inflammation and inflammatory microenvironment than biological agents targeting individual inflammatory cytokines, such as diacerein for IL1-β and etanercept for TNF-α.[52]^19^,[53]^20 Current research suggests that itaconate confers singular therapeutic benefits in the treatment of conditions like renal fibrosis, neuroinflammation, acute lung injury, and hepatic damage.[54]21, [55]22, [56]23, [57]24 Given its hydrophilic nature, itaconate's absorption by cell membranes is impeded. As a result, the permeable analog of 4-octyl itaconate (4-OI) has been selected for investigation. 4-OI can traverse cell membranes and is intracellularly converted into itaconate, eliciting effects analogous to those of endogenous itaconate.[58]^18^,[59]^21 Previous studies have described the therapeutic effects of 4-OI in animal models of PTOA, focusing on its ability to reduce cartilage damage and maintain the integrity of the cartilage matrix, but there is limited research on the effects of 4-OI on the pathologic changes of synovitis and pain relief in PTOA.[60]^25^,[61]^26 In this study, we hypothesized that 4-OI may reduce synovitis and alleviate pain in the mouse PTOA model. MRI, micro-CT, histological analysis, quantitative real-time polymerase chain reaction (qRT-PCR), and behavioral assessments were used to evaluate the effect of 4-OI on synovitis in PTOA. The mechanism of 4-OI in treating synovitis in PTOA was studied by ribonucleic acid sequencing (RNA-Seq) and network pharmacology analysis. 2. Methods 2.1. Source of animals All experiments conducted in this study were approved by the Animal Research Ethics Committee of our university (ethic cord: AMUWEC20232275). Eight-week-old male C57BL/6 mice were purchased from the Animal Centre of the Army Medical University and housed 6 mice per cage, provided with unlimited cage activities, and maintained on a 12 h-light/dark cycle. Every effort was made to minimize animal suffering and reduce the total number of animals used per the guidelines of the Animal Research Ethics Committee. 2.2. Animal model The mouse PTOA model was established through surgical destabilization of the medial meniscus (DMM). The mice were anesthetized with isoflurane and positioned in a prone position on a foam operation plate, and then the mouse model in the DMM group was induced by the DMM surgery of the right knee. The main steps of the procedure were as follows: firstly, the medial side of the right knee joint skin and capsule was incised. Next, the medial tibiofibular ligament was cut from the patellar side along the medial meniscus using a sharp blade. Finally, the joint capsule and skin were sutured.[62]^27^,[63]^28 Conversely, a sham operation, consisting of a skin incision and medial capsulotomy followed by capsule and skin closure as described above, was performed on the right knee of a separate group of mice. 2.3. Study design To verify whether the DMM surgery can effectively model the phenotype of PTOA in mice, we randomly divided 18 mice into 3 groups: wild-type (WT) group, sham group, and DMM group. Twelve weeks post-DMM surgery, MRI, micro-CT, and histological section analysis were performed on the right knee joints of the mice. After confirming that the DMM surgery successfully models the pathological features of PTOA in mouse knee joints, we collected the synovial tissue from the right knee joints of the WT group and DMM group mice for RNA-seq. To evaluate the therapeutic effects of 4-OI, we randomly divided 64 mice into 4 groups: sham surgery group, DMM group, DMM + 4-OI (50 mg/kg) group, and DMM + 4-OI (100 mg/kg) group, with 16 mice in each group. Mice in the DMM group, DMM + 4-OI (50 mg/kg) group, and DMM + 4-OI (100 mg/kg) group underwent DMM surgery on their right knee joints. Mice in the DMM + 4-OI (50 mg/kg) group and DMM + 4-OI (100 mg/kg) group were injected intraperitoneally with the appropriate dose of 4-OI 3 times per week at least 24 h apart starting on the first day after surgery. A total of 12 weeks of treatment was administered. During the 12 weeks following the DMM surgery, von Frey tests were conducted every 3–5 days, and open field tests were performed every 4 weeks. After 12 weeks, MRI, histological analysis, and qRT-PCR detection of tissues were conducted on the right knee joints of all groups. The above experimental process can be illustrated by a flowchart ([64]Fig. 1). Finally, we conducted a preliminary exploration and validation of the mechanisms of 4-OI treatment for PTOA synovitis using bioinformatics and network pharmacology analyses. Fig. 1. [65]Fig. 1 [66]Open in a new tab Experimental flow chart. DMM: destabilization of the medial meniscus; MRI: magnetic resonance imaging; RNA: ribonucleic acid; PCR: polymerase chain reaction. 2.4. Histological staining and analysis Total right knee joints were fixed in 4% paraformaldehyde for 24 h, decalcified in 0.5 M EDTA at a pH of 7.4 for 14 days, and subsequently embedded in paraffin. Sections with a thickness of 7 μm were prepared for staining with hematoxylin and eosin (H&E) and picrosirius red and safranin o/fast green (Saf-O), respectively. H&E staining facilitated the evaluation of synovial lining layer cellularity and the extent of synovitis. Picrosirius red staining was employed to assess the types and abundance of collagen fibers in the synovium. Saf-O staining was used to assess the degree of cartilage degeneration in PTOA. 2.5. Immunohistochemistry and immunofluorescence For immunohistochemical staining, the specimens were processed and sectioned as previously described. The paraffin sections were dewaxed and rehydrated, followed by immersion in 20 × Tris-EDTA antigen retrieval solution (pH 9.0) for antigen masking. The sections were then immersed in 3% hydrogen peroxide and incubated for 25 min to quench endogenous peroxidase activity. They were blocked with 1% goat serum at 37 °C for 1 h. Subsequently, the primary antibody (1:100, Servicebio, INOS, GB11119, China) was added and incubated overnight at 4 °C. After that, a horseradish peroxidase-conjugated secondary antibody (Servicebio, hrp-conjugated goat anti-rabbit IgG (H + L), GB23303, China) was added and incubated at room temperature for 50 min. The sections were visualized using 3,3′-diaminobenzidine staining and counterstained with hematoxylin. Finally, the sections were dehydrated and mounted for microscopic observation. For immunofluorescence staining, after dewaxing and rehydrating the paraffin sections, the slides were immersed in 20 × Tris-EDTA antigen retrieval solution (pH 9.0) for antigen masking. The sections were blocked with 1% goat serum at 37 °C for 1 h, followed by overnight incubation at 4 °C with the primary antibody (1:100, Affinity, F4/80, DF2789, USA). The sections were then incubated in the dark for 50 min with a dye-conjugated secondary antibody (Servicebio, CY3 Goat Anti-Rabbit, GB21303, China). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Servicebio, G1012, China) for 10 min. After treating the sections with a fluorescence quenching agent (Servicebio, G1221, China) for 5 min, they were mounted. Imaging was performed using a confocal laser scanning microscope (Zeiss, Germany). 2.6. Microscopic analysis A vertical fluorescence microscope (BX53 LED, Olympus, Japan) was used to observe and analyze the mouse right knee joint sections stained with chemical staining and immunohistochemistry at different magnifications of 10×, 20×, and 40×. Detailed imaging and analysis of the immunofluorescence-stained mouse right knee joint sections were performed using a confocal laser scanning microscope (LSM900, Zeiss, Germany). To evaluate the presence of macrophages and quantify the average thickness of macrophages in the synovial lining layer, we examined the anterior, anteromedial, and posterolateral regions of the synovium. For analysis, random section coding and blinded scoring were conducted by 2 observers on 3 sections from each joint. 2.7. Micro-CT and MRI analysis For micro-CT, imaging analysis of the fixed knee joint specimens was performed (SKYSCAN1276, Bruker, Belgium). The scanning parameters were as follows: tube current of 200 μA, voltage of 85 kV, scanning resolution of 10.141270 μm, exposure time of 384 ms, and a scanning angle of 180°. Three-dimensional reconstruction and image acquisition were conducted using Mimics-Medical-21.0 software, and the regions of interest surrounding the knee joint were delineated to isolate and count osteophytes and calcified tissues. The volume of osteophytes and calcified tissues around the knee joint was assessed by blinded quantification and the mean values were calculated for subsequent statistical analysis. The right knee joints of mice were imaged by MRI - a 7.0 T vertical bore Bruker Biospec 70/30 scanner (Bruker BioSpin MRI GmbH, Rheinstetten, Germany). Scan parameters were optimized for gray-white matter contrast. Analysis of the fixed knee joints was conducted using T2-weighted images in the sagittal plane to assess effusion and synovial edema. 2.8. RNA-seq analysis After the phenotypic verification experiments, the synovial tissue from the right knee joints of WT and DMM groups of mice was collected. RNA was extracted and purified from the collected samples employing TRIzol reagent (Thermo Fisher, 15596018). Then, the quantity and purity of total RNA were quality controlled, and the integrity of the RNA was assessed. A concentration > 50 ng/μL, a RIN value > 7.0, and total RNA >1 μg were deemed acceptable for downstream experiments. Messenger RNA (mRNA) was specifically isolated using Dynabeads Oligo (dT) magnetic beads (Thermo Fisher, USA, cat. 25–61005), followed by complementary DNA (cDNA) synthesis from the mRNA template using the SuperScript™ II Reverse Transcriptase (Invitrogen, CA, USA, cat. 1896649). Subsequently, E. coli DNA polymerase I (NEB, USA, cat. m0209) and RNase H (NEB, USA, cat. m0297) converted the RNA-DNA hybridization into double-stranded DNA with the addition of dUTP Solution (Thermo Fisher, CA, USA, cat. R0133) at the blunt ends. At both ends of the double-stranded DNA, an adenine (A) base was added, followed by selection and purification of the DNA fragments using magnetic beads. Subsequently, the double-stranded DNA was digested with UDG enzyme (NEB, MA, USA, cat. m0280), and the fragments were amplified via PCR to create a library with a size of (300 ± 50) bp. Library sequencing was conducted on the Illumina NovaSeq™ 6000 system (LC Bio Technology CO, Ltd., Hangzhou, China) in PE150 mode. Sequencing data underwent quality filtering to yield high-quality reads, which were then mapped to the reference genome for gene expression quantification, differential gene expression analysis, and enrichment analysis. 2.9. Behavioral assessment The mechanical allodynia test was conducted utilizing a calibrated set of von Frey filaments (North Coast Medical Inc., CA, USA). Mice were first acclimated for 15 min on a wire mesh grid and then underwent the von Frey hind paw test. The filaments (exerting forces ranged from 0.04 to 2.0 g and initial force was 0.4 g) were applied perpendicularly to the plantar surface of the hind paw to determine the 50% force withdrawal threshold using the classical up-down iterative method. The 50% force withdrawal threshold was determined using the classical up-down iterative method. A nociceptive response was noted if the mouse displayed brisk paw withdrawal, licking, or shaking of the paw during or immediately following the stimulus. This procedure was carried out by an investigator who was blinded to the animal study groups to ensure objectivity.[67]^29 The evaluation of spontaneous behavior was conducted via the open field test. A 40 × 40 × 40 cm^3 box made of polyvinyl chloride was sanitized with 70% ethanol and a paper towel before each test. Mice were placed at the center of the box to explore freely for 30 min, while their movements were recorded by an overhead camera. The analysis of behavioral parameters, including travel distance, average speed, active time, and motion trail, was performed using EthoVision XT 11 behavioral tracking software.[68]^30 2.10. qRT-PCR From each group, the right knee synovial tissue was collected and cut into small pieces, and the synovial cells were gathered. Then, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA). The extracted RNA was then reverse-transcribed into cDNA utilizing PrimeScript RT Master Mix (Takara, Japan). qRT-PCR was performed on the cDNA samples using mouse-specific primers and the thermal cycler dice real-time system (Takara) was used under the following conditions: an initial denaturation at 95 °C for 3 min, followed by 40 amplification cycles at 95 °C for 5 s and 60 °C for 30 s. Gene expression levels were quantified by densitometry and normalized against the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. Relative expression changes were calculated using the 2–ΔΔCt method, with the analysis replicated independently 3 times. 2.11. Network pharmacology and bioinformatics analysis The chemical structure of 4-OI was obtained from the PubChem database, along with the corresponding SMILES notation. Using the SMILES notation, a query was conducted on the Swiss target prediction database to identify potential therapeutic targets of 4-OI in both mice and humans, revealing 100 and 107 targets, respectively. Subsequently, RNA-Seq data from synovial membranes of DMM and WT mice were analyzed for differential expression, with thresholds set at log2 (fold change) > 1 or < −1 and p < 0.05. This analysis identified a total of 2099 targets associated with synovitis in DMM mice. Additionally, targets related to human synovitis were compiled from the DisGeNET and GeneCards databases, yielding 197 and 973 targets, respectively. After removing duplicate data, 928 unique targets related to human synovitis were retained. The drug targets of 4-OI were then crossed with the identified targets for DMM mice and human synovitis to predict relevant therapeutic targets for 4-OI treatment in both mice and human synovitis. Further, gene ontology (GO) functional enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were conducted using the database for annotation, visualization, and integrated discovery (DAVID). The results were visualized using Cytoscape software, providing insights into the potential mechanisms and pathways through which 4-OI may exert its effects in the treatment of synovitis. 2.12. Statistical analysis All quantitative data in our study are expressed as mean ± standard deviation (SD). For normally distributed data, we employed a one-way analysis of variance with a test for homogeneity of variance, followed by Tukey's HSD post hoc test for intergroup comparisons. A p < 0.05 was considered to indicate statistical significance. For data not conforming to a normal distribution, non-parametric tests were utilized for statistical analysis. SPSS 25.0 (SPSS, Inc., IL, USA) was used for statistical analysis, and GraphPad Prism 9.0 (GraphPad Software Inc., CA, USA) was employed for generating graphs and charts. 3. Results 3.1. Synovitis manifestation obviously in mouse PTOA model In the phenotypic validation experiment, 12 weeks after the DMM surgery, micro-CT, MRI examinations, and histological section analyses were performed on the right knee joints in WT, sham, and DMM groups ([69]Fig. 2). Micro-CT analysis revealed a significant increase in both the number of calcified tissue and osteophytes and the volume of calcified meniscus and synovial tissue (CAL Tis.V) in the right knee joints in the DMM group compared to the WT and sham groups ([70]Fig. 2A). In addition, Saf-O staining confirmed the severe degeneration of knee cartilage in the DMM group ([71]Fig. 2B), indicating that the DMM procedure successfully induced PTOA in the mouse knee joints. Next, we analyzed synovitis in the knee joints of the mouse PTOA model. MRI assessments revealed that the knee joint synovium in the DMM group exhibited elevated signal intensity on T2 fat-suppressed images compared with the WT and sham groups, indicating the increased joint cavity fluid and the presence of synovitis ([72]Fig. 2C). Histological assessments revealed a significant increase in synovial lining layer cellularity in the DMM group compared to the WT and sham groups. Additionally, a decrease in sub-lining layer cell density and an increase in angiogenesis were observed ([73]Fig. 2D). Synovitis severity was semi-quantitatively assessed using a chronic synovitis grading system, revealing significant synovitis in the DMM group compared to the WT and sham groups ([74]Fig. 2F and D).[75]^31 Immunohistochemical staining revealed a significant increase in M1 macrophages in the synovial lining layer of the DMM group ([76]Fig. 2E). Fig. 2. [77]Fig. 2 [78]Open in a new tab Synovitis manifestation in the mouse PTOA model induced by DMM. (A) Three-dimensional micro-CT images illustrating pathological structural changes in mouse knees and the corresponding quantification of CT&OP.N and CAL Tis.V in the right knee joints 12 weeks post-surgery; (B) Safranin O/Fast Green staining of knee joint 12 weeks post-surgery, with a scale bar of 500 μm (top panel) and 100 μm (bottom panel) to demonstrate cartilage degeneration; (C) MRI analysis of knee joints revealing sagittal T2-weighted images 12 weeks post-surgery; (D) Representative images of sagittal sections of synovial membrane stained with H&E and the corresponding quantitative evaluation of the synovial lining cell layer, and the semi-quantitative evaluation of synovitis according to the grading system, were obtained 12 weeks post-surgery; (E) Representative immunohistochemical images of sagittal sections of the synovial membrane stained with INOS 12 weeks post-surgery. n = 6 for all groups, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. WT: wild-type; DMM: destabilization of the medial meniscus; CT&OP.N: the number of calcified tissue and osteophytes; CAL Tis.V: the volume of calcified meniscus and synovial tissue.; H&E: hematoxylin and eosin; INOS: inducible nitric oxide synthase; PTOA: post-traumatic osteoarthritis. 3.2. Differential gene expression analysis of synovium between PTOA and WT mice Following RNA-Seq of the synovium from PTOA and WT mice, GO enrichment analysis of differential gene expression revealed significant alterations in genes related to biological processes, such as the inflammatory response, behavioral response to pain, sensory perception of pain, collagen fibril organization, positive regulation of fibroblast proliferation, and collagen catabolic process ([79]Fig. 3A and B). Subsequently, genes exhibiting significant differential expression in these categories were selected for further analysis. Gene expression heatmap was generated, demonstrating significant upregulation of genes associated with the mentioned processes in PTOA mice compared to WT mice ([80]Fig. 3C). Fig. 3. [81]Fig. 3 [82]Open in a new tab Clustering, GO, and KEGG analysis of differential genes with synovial tissue between PTOA and WT mice. (A) Differential genes volcano map of synovial cells in DMM group and WT group; (B) GO enrichment analysis of differential genes function in synovial cells of DMM group and WT group; (C) Differential genes cluster analysis heatmap of synovial cells in DMM group and WT group; (D) KEGG pathways enrichment analysis of differential genes in synovial cells of DMM group and WT group; (E) FC Barplot of some special differential genes in synovial cells of DMM group and WT group. WT: wild-type; DMM: destabilization of the medial meniscus; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes; PTOA: post-traumatic osteoarthritis; FC: fold change. Moreover, KEGG pathway enrichment analysis of significantly differentially expressed genes revealed notable enrichment in the Th17 cell differentiation signaling pathway, T cell receptor signaling pathway, cell adhesion molecule signaling pathway, and calcium ion signaling pathway ([83]Fig. 3D). Interestingly, the latter 3 pathways were closely related to the Th17 cell differentiation signaling pathway. Therefore, we hypothesized that positive regulatory factors produced by the synovium in PTOA mice induce an increase in Th17 cell differentiation, and the primary effector molecule IL-17 further activates downstream signaling pathways, leading to increased expression of synovial chemokines (CCL2, CCL3, CCL7, CXCL2, etc.), inflammatory factors (IL-1β, IL-6, TNFα, etc.), fibrosis-associated (COL1A1, CCN2, TGFβ, etc.), and catabolic metabolism-related genes ([84]Fig. 3E). 3.3. 4-OI can effectively inhibit synovitis in the mouse PTOA model and slow down cartilage degeneration In the 4-OI treatment experiment, 12 weeks after the DMM surgery, micro-CT, MRI examinations, and histological section analyses were conducted on the right knee joints of the sham, DMM, and DMM + 4-OI groups to evaluate the efficacy of 4-OI ([85]Fig. 4). In the DMM group, MRI T2-weighted/fat-suppressed imaging showed high signal intensity of synovitis-effusion, which was significantly lower in the DMM + 4-OI group ([86]Fig. 4A), suggesting that 4-OI inhibits synovitis in the mouse PTOA model. The administration of 4-OI caused a partial but significant reduction in cartilage degradation ([87]Fig. 4B). H&E staining further indicated the reduction of inflammatory cell infiltration, fibrosis, and angiogenesis in the DMM + 4-OI group compared to the DMM group. Additionally, H&E staining showed that 4-OI resulted in a significant decrease in the aggregation of synovial lining cells ([88]Fig. 4C and D). Immunofluorescence staining revealed a significant decrease of F4/80+ macrophages in the synovial lining layer of the DMM + 4-OI group compared with the DMM group ([89]Fig. 4D). Similarly, immunohistochemical staining revealed a significant decrease in iNOS-positive cells (M1 macrophages markers) in the DMM + 4-OI group compared to that in the DMM group ([90]Fig. 4E), indicating the role of 4-OI in suppressing the M1 macrophages. Based on a chronic synovitis grading system for semi-quantitative assessment,[91]^31 4-OI was found to effectively inhibit the infiltration of M1 macrophages compared to the DMM group, leading to a reduction in synovitis scores ([92]Fig. 4F). Picrosirius red staining revealed a predominance of type I collagen fibers in the DMM group, indicating alterations in extracellular matrix fibrosis in the synovium ([93]Fig. 4G). Nevertheless, as assessed by the fibrosis grading system,[94]^32^,[95]^33 4-OI treatment significantly reduced fibrosis compared with the DMM group ([96]Fig. 4G). Fig. 4. [97]Fig. 4 [98]Open in a new tab The effect of 4-OI on the pathological changes of mouse PTOA model. (A) Representative MRI analysis of knee joints 12 weeks after surgery showing sagittal T2-weighted images; (B) Representative images of sagittal sections of cartilage stained with safranin O/Fast green 12 weeks after surgery; (C) Representative images of sagittal sections of synovial membrane stained with H&E 12 weeks after surgery; (D) Quantitative evaluation of the synovial lining cell layer and Semi-quantitative evaluation of synovitis according to the grading system 12 weeks after surgery; (E) Immunofluorescence staining assay of F4/80 in synovium 12 weeks after surgery; (F) Immunohistochemical staining assay of INOS in synovium 12 weeks after surgery; (G) Sections were stained with Picrosirius Red to show fibrotic changes in synovium 12 weeks after surgery, and a semi-quantitative evaluation of the distribution range of synovial fibrosis was performed. n = 6 for all groups, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. DMM: destabilization of the medial meniscus; 4-OI: 4-octyl itaconate; PTOA: post-traumatic osteoarthritis; DAPI: 40,6-diamidino-2-phenylindole; INOS: inducible nitric oxide synthase; AC: articular cavity; L: synovial lining; SL: synovial sublining; F: femur; M: meniscus. 3.4. 4-OI suppresses mRNA expression of synovitis-related genes in mouse PTOA model and alleviates pain During the 4-OI treatment experiment, the Von Frey test indicated that the withdrawal threshold of the right hind paw in mice significantly decreased after the DMM surgery. The 4-OI treatment increased the withdrawal threshold of the right hind paw, suggesting a reduction in pain sensitivity in the mice. Interestingly, in the 4-OI treatment group, there was no significant difference in pain relief between the 50 mg/kg and 100 mg/kg doses of 4-OI ([99]Fig. 5A). Additionally, an assessment of spontaneous activity in the sham, DMM, and 4-OI treatment groups of mice in the open-field experiment showed that mice in the DMM group walked significantly less distance, speed, and duration of locomotion due to pain after surgery. The 4-OI treatment effectively alleviated the decrease in spontaneous activity caused by postoperative pain in the DMM group. Similarly, at both 50 mg/kg and 100 mg/kg doses, there was no significant difference in the effects of 4-OI on spontaneous activity ([100]Fig. 5B–D). Furthermore, we verified through qRT-PCR that 4-OI significantly reduced the mRNA transcription levels of monocyte/macrophage chemotactic-related genes (CCL2, CCL7, CXCL2) ([101]Fig. 5E), inflammation and pain-related genes (IL-1, IL-6, TNF-α) ([102]Fig. 5F), and fibrosis-related genes (COL1A1, CCN2, TGFβ) ([103]Fig. 5G) in the synovium of PTOA mice. Fig. 5. [104]Fig. 5 [105]Open in a new tab 4-OI reduces mRNA Levels of synovitis-related genes and alleviates pain. (A) Mechanical sensitivity was measured using von Frey filaments two times a week after DMM surgery. Statistical analysis was conducted using a two-way analysis of variance, p values were compared between the DMM or DMM + 4-OI group and sham group. Changes in spontaneous activity, including (B) travel distance, (C) average speed were evaluated 0, 4, 8, and 12 weeks after DMM surgery; (D) Representative track plots show decreased spontaneous activity of different groups' mice in open field tests at 12 weeks after DMM surgery; (E) Expression levels of certain monocyte chemoattractant factor mRNAs in mouse synovial tissue; (F) Expression levels of certain inflammation and pain-related mRNAs in mouse synovial tissue; (G) Expression levels of certain fibrosis-related mRNAs in mouse synovial tissue. n = 6 for all groups, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. DMM: destabilization of the medial meniscus; 4-OI: 4-octyl itaconate. 3.5. 4-OI may regulate the differentiation of Th-17 cells in the synovium and alleviate synovitis The 4-OI drug targets in mice were intersected with PTOA mice synovitis targets from RNA-seq to obtain potential therapeutic targets of 4-OI against synovitis in mice. We identified 18 potential therapeutic targets for treating PTOA mice synovitis with 4-OI ([106]Fig. 6A). GO functional enrichment analysis classified these targets into 4 functional categories: metabolite interconversion enzyme (CD38, PLA2G2E, KMO, PLA2G2D, CES1D, PDE10A, PTGS1, MAOB, PYGL), protein modifying enzyme (MAPK11, MME, ZAP70, GCK, MMP9, MMP13), transmembrane signal receptor (PTGER1, PTGER4), and chaperone (HSP90AA1) ([107]Fig. 6B). The KEGG pathway analysis of these 18 targets indicated that 4-OI primarily affected the IL-17 signaling pathway and the Th17 cell differentiation signaling pathway ([108]Fig. 6C). The KEGG enrichment analysis of mRNA sequencing results from the synovial tissues of WT and DMM group mice revealed that the Th17 cell differentiation signaling pathway was closely related to the pathogenesis of PTOA synovitis ([109]Fig. 3D). Additionally, the major effector molecules of Th17 cells, IL-17 and its downstream cytokines were associated with the progression of PTOA and pain symptoms. These findings suggested that 4-OI may inhibit Th17 cell differentiation, thereby alleviating PTOA synovitis. Fig. 6. [110]Fig. 6 [111]Open in a new tab Exploring the therapeutic pathway of 4-OI based on network pharmacology. (A) The Venn diagram of drug targets for 4-OI and differential expression Genes for RNA-Seq of DMM and WT mice synovium; (B) Functional classification diagram of intersection targets in mice; (C) KEGG pathway enrichment analysis of intersection targets in mice; (D) Expression levels of mRNAs for positive regulators of Th17 cell differentiation in mouse synovial cells; (E) The expression levels of major effector molecules of Th17 cells and downstream cytokines mRNA in mouse synovial cells.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0010. 4-OI: 4-octyl itaconate; DMM: destabilization of the medial meniscus; WT: wild-type; KEGG: Kyoto encyclopedia of genes and genomes. To verify whether 4-OI inhibits the differentiation of Th17 cells in the synovium, we performed qRT-PCR to measure the expression levels of positive regulatory factors for Th17 cell differentiation (IL-1, IL-6) in the mouse synovial cells of negative control (N) group, lipopolysaccharide (LPS) group, and LPS + 4-OI (400 μM) group. LPS referred to treatment with 2.5 μg/mL LPS for 24 h to simulate the inflammatory response following injury ([112]Fig. 6D), as well as the effector molecules of Th17 cells (IL-17A, IL-17F) and the downstream cytokines of IL-17 (CCL2, MMP13) ([113]Fig. 6E). The results indicated that 4-OI significantly inhibited the expression of positive regulatory factors for Th17 cell differentiation, effector molecules of Th17 cells, and IL-17 downstream cytokines in LPS-induced mouse synovial cells. These findings suggested that 4-OI inhibited the differentiation of Th17 cells in the synovium to treat PTOA synovitis, thereby alleviating the progression of PTOA and associated pain. 4. Discussion The mouse PTOA model displays pathological features that closely resemble those observed in human knee joint PTOA. The mouse PTOA model demonstrates significant synovitis, characterized by an accumulation of M1 macrophages in the synovial lining layer, significant fibrosis in the sub-lining layer, and vascular proliferation. M1 macrophages accumulation within the synovium has been shown to exacerbate cartilage degeneration and osteophyte formation in experimental PTOA.[114]^34 Additionally, fibrosis of the synovial sub-lining layer contributed to joint stiffness and pain in PTOA.[115]^35 Synovium acted as a semipermeable membrane controlling molecular traffic into and out of the joint space, which was essential for preserving the homeostasis of articular cartilage.[116]^9^,[117]^36 Therefore, the suppression of synovitis may alleviate the progression of PTOA and the pain symptoms, improve the inflammatory microenvironment within the joint, and restore joint homeostasis. Intra-articular injection is challenging due to the small knee joint cavity in mice and repeated intra-articular injection can lead to damage and fibrosis of the joint capsule and synovium. Therefore, intraperitoneal administration is chosen to deliver 4-OI to the synovium through the circulation. This method avoids the joint capsule and synovial damage associated with intra-articular injections, allowing for a more accurate assessment of the effects of 4-OI on the pathology of synovitis, while also improving the systemic inflammatory state. Our results indicate that 4-OI can effectively suppress the pathological changes in the synovium of PTOA mice, thereby effectively alleviating pain symptoms and slowing down cartilage degeneration. Compared to currently used OA treatment medications, 4-OI as an endogenous metabolite, can effectively avoid gastrointestinal irritation and cardiovascular adverse events associated with the long-term use of NSAIDs, as well as the increased all-cause mortality associated with the prolonged use of weak opioids.[118]15, [119]16, [120]17 Furthermore, our study demonstrates that 4-OI can alleviate the degeneration of OA cartilage to some extent, thereby avoiding the side effects of exacerbated cartilage damage caused by the use of corticosteroids.[121]^37 In vivo, 4-OI acts on the synovium of PTOA mice by reducing the transcription levels of genes associated with monocyte/macrophage chemotaxis, inflammation and pain, and fibrosis in synovial cells. The reduced expression of genes associated with monocyte/macrophage chemotaxis may lead to decreased chemotaxis of M1 macrophages to the joint synovium, resulting in a lower macrophage presence within the synovial lining. Similarly, the downregulation of genes associated with inflammation and pain may contribute to the relief of joint pain and synovitis. Furthermore, the suppression of fibrosis-related gene expression is likely to alleviate joint stiffness and pain. These results indicate that 4-OI has the potential to mitigate synovitis and pain in the mouse PTOA model by inhibiting the expression of pro-inflammatory and pro-fibrotic genes in synovial cells. Through network pharmacology and bioinformatics analysis, this study further explores the mechanisms of 4-OI in the treatment of synovitis in mice and predicts potential therapeutic targets and signaling pathways. The analysis reveals a significant enrichment of the Th17 cell differentiation signaling pathway. In inflammatory arthropathies, Th17 cells and the key effector molecule IL-17 are critical factors in the onset and maintenance of synovitis.[122]^38 As insights into OA advance, the role of synovitis in OA onset and progression has become a focus of increasing interest.[123]8, [124]9, [125]10 The elevated levels of Th17 cells and IL-17 in the peripheral blood, synovial fluid, and synovial tissue of patients with OA result in an imbalance in the Th17/Treg cell ratio.[126]^39 Research indicates that IL-17 and Th17 can induce cellular senescence in damaged joint cells, promote the progression of OA and synovitis, and exacerbate pain symptoms.[127]^40^,[128]^41 Moreover, IL-17 can also upregulate CCL2 expression in the synovium, and promote M1 macrophage polarization and migration to the synovium, which leads to pathological changes characteristic of synovitis, accelerates OA progression, and intensifies pain symptoms.[129]^42 Thus, inhibiting Th17 cell differentiation and IL-17 expression may be advantageous for treating PTOA synovitis and alleviating disease progression and pain.[130]^43 Studies have shown that itaconate can regulate T cell differentiation by modulating metabolism and epigenetic reprogramming, inhibiting Th17 cell differentiation while promoting Treg cell differentiation, thus reducing IL-17 production.[131]^44 Using qRT-PCR, we demonstrate that 4-OI inhibits the expression of positive regulators and effector molecules associated with Th17 cell differentiation in synoviocytes of inflammatory mice, suggesting that 4-OI inhibits Th17 cell differentiation and expression of the effector molecule IL-17, and thus inhibits changes associated with synovitis, disease progression, and pain in PTOA. In conclusion, this study demonstrates that 4-OI can be administered to mice via intraperitoneal injection as a supplement to exogenous itaconate, allowing it to reach the synovium through body fluids circulation and exert therapeutic effects. This treatment can inhibit synovitis in PTOA mice, improve the inflammatory microenvironment of the synovium, effectively alleviate pain symptoms associated with PTOA, and delay the progression of PTOA. CRediT authorship contribution statement Yu-Zhen Tang: Writing–original draft, Software, Data curation, Conceptualization. Wan Chen: Writing–review & editing, Validation, Supervision. Bao-Yun Xu: Investigation, Formal analysis. Gang He: Methodology, Data curation. Xiu-Cheng Fan: Methodology, Investigation. Kang-Lai Tang: Resources, Project administration, Funding acquisition. Ethical statement All experiments conducted in this study were approved by the Animal Research Ethics Committee of the Army Medical University (ethic cord: AMUWEC20232275). Funding This work was supported by the Foundation of Chongqing Talents (grant no. CQYC2021059825). Declaration of competing interests The authors declare no conflicts of interest. Footnotes Peer review under responsibility of Chinese Medical Association. Contributor Information Wan Chen, Email: chenwanfred@foxmail.com. Kang-Lai Tang, Email: tangkanglai@hotmail.com. References