Hanyu Lu

INTRODUCTION: Quercetin was recently reported to help protect against osteoarthritis (OA) progression, but the molecular mechanism for that protective affect remains unclear. METHODS: Here, OA model rats were intraperitoneally injected with quercetin, and the severity of cartilage damage in the rats was evaluated by H&E, Safranin O, and Toluidine blue, as well as by using the Osteoarthritis Research Society International (OARSI) Scoring System. Additionally, rat chondrocytes were treated with quercetin and then stimulated with IL-1β. The levels of pro-inflammatory cytokines (IL-1β, IL-18, and TNF-α) were detected by ELISA.Cell apoptosis was evaluated by flow cytometry and Hoechst staining. ROS levels were measured using a DCFH-DA probe. Protein expression was evaluated by Western blotting, immunohistochemical staining, and immunofluorescence. RESULTS: Our data showed that quercetin attenuated the degeneration and erosion of articular cartilage, suppressed inflammation and apoptosis, and downregulated the levels of IRAK1, NLRP3, and caspase-3 expression. In vitro data showed that overexpression of NLRP3 could reverse the suppressive effect of quercetin on IL-1β-induced rat chondrocyte injuries. Importantly, rescue experiments confirmed that quercetin inhibited IL-1β-induced rat chondrocyte injuries in vitro by suppressing the IRAK1/NLRP3 signaling pathway. CONCLUSION: Our study indicated that quercetin inhibits IL-1β-induced inflammation and cartilage degradation by suppressing the IRAK1/NLRP3 signaling pathway.

Macrophages polarization plays essential but different roles in most diseases such as atherosclerosis, adipose tissue inflammation, and insulin resistance. Our previous study revealed that protease-activated receptor 2 (PAR2), a G-protein coupled receptor influenced macrophage function, but little is known regarding the regulation of macrophage polarization process and its potential mechanisms. In the present study, bone marrow-derived macrophages (BMDM) isolated from C57/BL6 mice and cultured with L929-conditional medium and murine macrophage cell line RAW264.7 were used to study the function of PAR2 activation in vitro. BMDM was stimulated by the small molecular PAR2 agonist, 2-furoyl-LIGRLO-amide trifluoroacetate salt, followed by transcription factor microarray to screen the significantly activated signaling pathways under PAR2 activation. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression of targeted genes and transcription factors. Immunofluorescence was used to observe the subcellular distribution of transcription factors. Our results demonstrated that M1-like polarization was presented by PAR2 agonist treatment with significant upregulation of interleukin-1β, interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α in BMDM and RAW264.7. Microarray identified forkhead box protein O1 (FOXO1) was significantly increased under PAR2 agonist stimulation, which was confirmed by qPCR and Western blot analysis. Immunofluorescence demonstrated that increased FOXO1 accumulated in the nucleus, which is necessary to promote transcription for targeted genes. We further knocked down FOXO1 expression using small interfering RNA, which alleviated PAR2-induced proinflammatory gene expression. The PAR2/FOXO1 pathway mediated stimulation of proinflammatory genes was further confirmed by tryptase, an endogenous ligand of PAR2. In conclusion, this study demonstrated that PAR2 activation-induced M1 polarization and inflammation through the FOXO1-dependent pathway.

Osteoarthritis (OA) is characterized by an imbalance between M1 and M2 polarized synovial macrophages. Quercetin has shown protective effects against OA by altering M1/M2-polarized macrophages, but the underlying mechanisms remain unclear. In this study, rat chondrocytes were treated with 10 ng/mL of IL-1β. To create M1-polarized macrophages in vitro, rat bone marrow-derived macrophages (rBMDMs) were treated with 100 ng/mL LPS. To mimic OA conditions observed in vivo, a co-culture system of chondrocytes and macrophages was established. ATP release assays, immunofluorescence assays, Fluo-4 AM staining, Transwell assays, ELISA assays, and flow cytometry were performed. Male adult Sprague-Dawley (SD) rats were used to create an OA model. Histological analyses, including H&E, and safranin O-fast green staining were performed. Our data showed a quercetin-mediated suppression of calcium ion influx and ATP release, with concurrent downregulation of TRPV1 and P2X7 in the chondrocytes treated with IL-1β. Activation of TRPV1 abolished the quercetin-mediated effects on calcium ion influx and ATP release in chondrocytes treated with IL-1β. In the co-culture system, overexpression of P2X7 in macrophages attenuated the quercetin-mediated effects on M1 polarization, migration, and inflammation. Either P2X7 or NLRP3 knockdown attenuated IL-1β-induced M1/M2 polarization, migration, and inflammation. Moreover, overexpression of TRPV1 reduced the quercetin-mediated suppressive effects on OA by promoting M1/M2-polarized macrophages in vivo. Collectively, our data showed that quercetin-induced suppression of TRPV1 leads to a delay in OA progression by shifting the macrophage polarization from M1 to M2 subtypes via modulation of the P2X7/NLRP3 pathway.

Tumour necrosis factor-α (TNF-α) is a cytokine involved in systemic inflammation. TNF-α slows down osteogenic differentiation, which may contribute to poor bone development in the inflammatory microenvironment. TNF-α inhibits osteogenic differentiation by activating the JAK-STAT3 pathway, of which Signal transducer and activator of transcription 3 (STAT3)-interacting protein 1 (StIP1, also known as elongator complex protein 2, ELP2) is a key protein in the JAK-STAT3 pathway. We investigated whether and how ELP2 activation mediates the TNF-α-induced pyroptosis during osteoblastic differentiation. Using in vitro cell cultures of preosteoblastic MC3T3-E1 cells, we found that TNF-α exposure causes cell pyroptosis in an inflammatory microenvironment during osteoblastic differentiation. Bioinformatics, protein docking model and co-immunoprecipitation analysis revealed an association between ELP2, STAT3 and NLRP3. Forced ELP2 expression promoted MC3T3-E1 cells pyroptosis, with an increase in the expression of STAT3, NLRP3 inflammasome, GSDMD/GSDME, osteoblast marker genes, and the activity of alkaline phosphatase. In contrast, ELP2 silencing ameliorated MC3T3-E1 cells pyroptosis, and osteogenic differentiation, especially after TNF-α stimulation. The TNF-α-induced cells pyroptosis during osteoblastic differentiation was therefore mediated by ELP2. These results suggest that ELP2 is upregulated at the pyroptosis of MC3T3-E1 cells and inhibits osteogenic differentiation in response to TNF-α through NLRP3-GSDMD/GSDME activation.

Background: Osteoarthritis (OA) is the most common joint disorder, and places a heavy burden on individuals and society. As conventional therapies, such as surgery, rarely cure the disorder, targeted therapies represent a promising alternative. This research sought to explore the potential effect of miR-199a-5p on the development of OA. Methods: Based on the OA rat model, the serum was collected at 6 and 12 weeks, and microRNA (miRNA) sequencing was performed. A bioinformatics analysis was conducted to examine the differentially expressed micro ribonucleic acids, and qRT-PCR (real-time quantitative PCR) was conducted to determine their expression in the joint tissues of rats with OA. Rats articular chondrocytes were collected and treated with a miR-199a-5p antagomir or agomir. Afterwards, cell viability, autophagy was determinated. Dual luciferase was used to verify that miR-199a-5p targets the regulation of mitogen-stimulated protein kinase 4 (MAPK4). Subsequently, in chondrocytes, MAPK was knockdown to rescue the effect of miR-199a-5p inhibition, and cell viability and autophagy were examined. Finally, the OA model was treated with miR-199a-5p antagomir to detect joint pathology, cartilage tissue and inflammatory factor and autophagy was measured. Results: MiR-199a-5p was greatly upregulated in OA, and miRNA was found to be differentially expressed in OA tissues. MAPK4 was identified to be a target gene of miR-199-5p. Inhibiting miR-199a-5p not only decreased the survival of chondrocytes and induced apoptosis, but also relieved inflammation and decreased the content of pro-inflammatory cytokines. Further, the silencing of miR-199a-5p protected the articular cartilage and improved gait abnormalities, but this effect was abrogated by the silencing of MAPK4. Conclusions: The silencing of miR-199a-5p appears to improve gait abnormalities, promote the survival of chondrocytes, and improve the condition of OA. Our findings may lead to the development of miR-199a-5p-based targeted therapy for OA.