IL-38-mediated NLRP3/caspase-1 inhibition is a disease-modifying treatment for TMJ inflammation.

Recently, interleukin-38 (IL-38) was identified as an important anti-inflammatory and immunosuppressive factor, but its functional role in temporomandibular joint (TMJ) inflammation remains unknown. This study aimed to elucidate how IL-38 affects chondrocytes and the underlying mechanism that contributes to anti-inflammatory processes in the TMJ. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay, and immunofluorescence analysis were used to verify that IL-38 has anti-inflammatory effects on chondrocytes, and the related key pathways were analyzed by western blotting. SiRNA-IL-38, siRNA-NLRP3, and MCC950 were used to investigate the mechanism underlying the anti-inflammatory effects of IL-38. Inflammation models were induced by injection of complete Freund's adjuvant in TMJ with mouse recombinant IL-38 in in vivo studies. Histological and immunohistochemical analyses were used to investigate histological changes in the cartilage. The results showed that IL-38 inhibited the expression of inflammatory cytokines and MMPs. IL-38 limited inflammation by inhibiting the expression of MAPKs/NF-κB and the NLRP3/caspase-1 pathway. In vivo, IL-38 reduced chondrocyte inflammation and limited cartilage degeneration. This study shows for the first time that IL-38 plays a protective role in TMJ cartilage. IL-38 exerts anti-inflammatory effects through the NLRP3/caspase-1 pathway and may be a promising agent for treating TMJ inflammation.

Luo P ，Zhao T ，He H 《-》

IL-37 inhibits M1-like macrophage activation to ameliorate temporomandibular joint inflammation through the NLRP3 pathway.

IL-37 has been identified as an important anti-inflammatory and immunosuppressive factor. This study was undertaken to explore how IL-37 affects M1/M2-like macrophage polarization and thus contributes to anti-inflammatory processes in the temporomandibular joint. Western blotting, quantitative real-time PCR (qRT-PCR) and immunofluorescence were used to verify the IL-37-induced polarization shift from the M1 phenotype to the M2 phenotype, and the related key pathways were analysed by western blotting. Human chondrocytes were stimulated with M1-conditioned medium (CM) or IL-37-pretreated M1-CM, and inflammatory cytokines were detected. siRNA-IL-1R8 and MCC-950 were used to investigate the mechanism underlying the anti-inflammatory effects of IL-37. Complete Freund's adjuvant-induced and disc perforation-induced inflammation models were used for in vivo studies. Haematoxylin and eosin, immunohistochemical and safranin-O staining protocols were used to analyse histological changes in the synovium and condyle. Western blotting, qRT-PCR and immunofluorescence showed that IL-37 inhibited M1 marker expression and upregulated M2 marker expression. Western blotting and qRT-PCR showed that pretreatment with IL-37 suppressed inflammatory cytokine expression in chondrocytes. IL-37 inhibited the expression of NLRP3 and upregulated the expression of IL-1R8. Si-IL-1R8 and MCC-950 further confirmed that the anti-inflammatory properties of IL-37 were dependent on the presence of IL-1R8 and NLRP3. In vivo, IL-37 reduced synovial M1 marker expression and cartilage degeneration and increased M2 marker expression. IL-37 shifting of the polarization of macrophages from the pro-inflammatory M1 phenotype to the beneficial anti-inflammatory M2 phenotype seems to be a promising therapeutic strategy for treating temporomandibular joint inflammation.

Luo P ，Peng S ，Yan Y ，Ji P ，Xu J ... -  《-》

IL-37b alleviates inflammation in the temporomandibular joint cartilage via IL-1R8 pathway.

Interleukin (IL)-37 is a natural suppressor of innate inflammation. This study was conducted to explore the anti-inflammatory effects of IL-37 in temporomandibular joint (TMJ) inflammation. The expression of IL-37 in the TMJ was measured using ELISA and IHC. Human TMJ chondrocytes were treated with IL-37b and IL-1β, and inflammation-related factors were detected. siRNA-IL-1R8 was transfected into chondrocytes, and the affected pathways were detected. IL-37b was used in disc-perforation-induced TMJ inflammation in SD rats. Micro-CT, IHC, real-time PCR and histological staining were used to quantify the therapeutic effect of IL-37b. IL-37 was expressed in the synovium and the disc of patients with osteoarthritis (OA) and in the articular cartilage of condylar fracture patients. IL-37 was highly expressed in synovial fluid of patients with synovitis than in those with OA and disc displacement and was closely related to visual analogue scale (VAS) score. In vitro, IL-37b suppressed the expression of pro-inflammatory factors. In addition, IL-37b exerted anti-inflammatory effects via IL-1R8 by inhibiting the p38, ERK, JNK and NF-κB activation, while silencing IL-1R8 led to inflammation and upregulation of these signals. In disc-perforation-induced TMJ inflammation in SD rats, IL-37b suppressed inflammation and inhibited osteoclast formation to protect against TMJ. IL-37b may be a novel therapeutic agent for TMJ inflammation.

Luo P ，Feng C ，Jiang C ，Ren X ，Gou L ，Ji P ，Xu J ... -  《-》

Pyroptosis of chondrocytes activated by synovial inflammation accelerates TMJ osteoarthritis cartilage degeneration via ROS/NLRP3 signaling.

Synovial inflammation and chondrocyte death have been widely acknowledged as key contributors to the pathological progression of temporomandibular joint osteoarthritis (TMJ-OA), a degenerative joint disease currently lacking definitive treatments. This study aims to understand the regulatory role of chondrocyte pyroptosis in condylar cartilage degradation during TMJ-OA. The levels of cytokines, cartilage degeneration markers, and pyroptotic biomarkers in the synovium and synovial fluid of temporomandibular disorders (TMD) patients were examined. The synovitis, cartilage degradation, and chondrocyte pyroptosis in wild-type and alpha-kinase 1 (ALPK1)-deficient TMJ-OA mice were then compared following monosodium iodoacetate (MIA) induction. Subsequently, we investigated the downstream mechanisms of cytokines- or macrophage supernatants-induced metabolic disorders and pyroptosis in chondrocytes using primary TMJ chondrocytes and ATDC5 chondrocyte cultures. We found a positive correlation between pyroptotic biomarkers and cartilage degradation mediators and cytokines in the synovial fluid of TMD patients. MIA-induced TMJ-OA mice demonstrated significant synovitis, cartilage degradation, and chondrocyte pyroptosis, which were mitigated in ALPK1-deficient TMJ-OA mice, inflammation-restrained mice. Ex-vivo study revealed the contribution of reactive oxygen species (ROS) to inflammation-irritated macrophage supernatants-induced pyroptosis and metabolic disorders in chondrocytes. Targeting NOD-like receptor protein 3 (NLRP3) alleviated cytokines- or ROS-induced pyroptosis and metabolic disorders in chondrocytes by inhibiting caspase-1 activation and interleukin-1β (IL-1β) secretion. Our findings offer novel insight into the role of synovial inflammation-induced chondrocyte pyroptosis in promoting cartilage degradation during TMJ-OA via the ROS and NLRP3 signaling pathway.

Liu X ，Li Y ，Zhao J ，Hu Z ，Fang W ，Ke J ，Li W ，Long X ... -  《-》

IL-1β mediating high mobility group box protein-1 expression in condylar chondrocyte during temporomandibular joint inflammation.

Temporomandibular joint (TMJ) osteoarthritis(OA)characterized with cartilage degen-eration is associated with inflammation. High mobility group box chromosomal protein-1(HMGB-1)is a potent mediator of inflammation and the trigger of OA. The expression of HMGB-1 in TMJ OA was uncovered, but the role of HMGB-1 in TMJ cartilage degeneration is not fully understood. In this study, the regulation of HMGB-1 in TMJ condylar cartilage was revealed. A complete Freund's adjuvant (CFA)-induced TMJ inflammation animal model was employed and the expression of HMGB-1 was detected at 1st, 2nd, and 6th weeks by immunohistochemistry. TMJ condylar chondrocytes were incubated with IL-1β (10 and 40 ng/ml) at 24, 48, and 72 h, and the translocation and protein level of HMGB-1 were evaluated by immunofluorescence and Western blot. Nuclear HMGB-1 staining was predominantly located in chondrocytes of both the fibrosis and proliferative zones in healthy TMJ. 1st week and 2nd week after CFA injection, immunoreaction could be detected in the cytoplasms of HMGB-1-positive cells and cartilage matrix especially in hypertrophic zone. At 6th week after CFA injection, cartilage matrix expression was disappeared and the cytoplasm expression of HMGB-1 was very weak in hypertrophic zone. HMGB-1 was translocated from the nucleus to the cytoplasm at 48 h after incubated with IL-1β (10 ng/ml and 40 ng/ml). The protein level of HMGB-1 was increased after stimulation and had a peak at 48 h. HMGB-1 might be associated with TMJ inflammation and OA. Insight into the role of HMGB-1 in TMJ inflammation is helpful to add the new knowledge into the pathogenesis of TMJ OA.

Li C ，Cai H ，Meng Q ，Feng Y ，Guo H ，Fang W ，Long X ... -  《-》