Acrylamide induces human chondrocyte cell death by initiating autophagy­dependent ferroptosis.

Acrylamide (ACR) is formed during heat treatment of foodstuffs and ACR may serve as a probable malignant neoplastic disease agent in all organs and tissues of the human body. However, it is unknown if ACR is associated with ankylosing spondylitis (AS) pathogenesis. Cell viability and proliferation were determined using CCK-8 assay and EdU staining. Flow cytometry was used to determine cell death and cell cycle arrest. Intracellular lipid reactive oxygen species, Fe2+ and mitochondrial membrane potential (MMP) were analyzed using a C11-BODIPY581/591 fluorescent probe, FerroOrange staining and a JC-1 MMP Assay kit, respectively. The present study showed that ACR decreased chondrocyte cell viability in a dose-dependent manner and that ACR significantly promoted chondrocyte senescence. ACR also elevated the expression of cell cycle arrest-associated proteins, including p53, cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor protein, in human chondrocytes. Similarly, DNA damage was also enhanced following ACR treatment in chondrocytes. In addition, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) and the autophagy inhibitor 3-methyladenine abolished ACR-induced cell death in chondrocytes. ACR was shown to activate autophagic flux and induce mitochondrial dysfunction by increasing the MMP. Western blot analysis of ferroptosis-related proteins demonstrated that ACR decreased the expression of glutathione peroxidase 4, solute carrier family 7 member 11, transferrin receptor protein 1 and ferritin heavy chain 1 in chondrocytes whereas Fer-1 abolished these effects. ACR treatment significantly elevated the phosphorylation levels of AMP-activated protein kinase (AMPK) and serine/threonine-protein kinase ULK1 in human chondrocytes. Notably, the effect of ACR was diminished by knockdown of AMPK, as evidenced by reduced lipid reactive oxygen species accumulation and Fe2+ levels. Hence, ACR inhibited cell proliferation and contributed to cell death by inducing autophagy-dependent ferroptosis while promoting autophagy by activating AMPK-ULK1-mTOR signaling in human chondrocytes. It was hypothesized that the presence of ACR in foodstuffs may increase the risk of AS and that decreasing ACR in food products is of importance.

SIX1 attenuates inflammation and rheumatoid arthritis by silencing MyD88-dependent TLR1/2 signaling.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease that severely affects the patients' quality of life. Sine oculis homeobox 1 (SIX1) has been reported as a key regulator of organogenesis and inflammation. This study aimed to explore the effects of SIX1 on RA. METHODS: Wistar rats were immunized with type II collagen to induce an animal model of RA. RA synovial fibroblasts (RASFs) were isolated from the rats. SIX1 expression in RA rats and RASFs was detected by qRT-PCR and western blot. CCK-8, EdU, transwell, flow cytometer, and ELISA were conducted to assay the effects of SIX1 on RASFs. The effects of SIX1 on RA rats were studied by Safranin O staining, H&E staining, and ELISA. Besides, GSEA and KEGG analysis were used to predict the underlying signaling pathways. RESULTS: SIX1 was low expressed in synovial tissue of RA rats and RASFs. SIX1 overexpression inhibited the proliferation, invasion, and levels of TNF-α, IL-6, and IL-8 in RASFs. However, SIX1 overexpression promoted the apoptosis of RASFs. SIX1 overexpression enhanced body weight, and attenuated the cartilage damage, pathological injury, and pro-inflammatory cytokine release of RA rat model. MyD88-dependent TLR1/2 might be a downstream signaling of SIX1. RelA acted as a transcription factor of TLR1/2, and SIX1 inhibited TLR1/2 signaling possibly via interaction with RelA. Adding with Pam3CSK4, a specific agonist of TLR1/2 signaling, attenuated the effects of SIX1 on RASFs. CONCLUSION: SIX1 attenuated inflammation and RA by silencing MyD88-dependent TLR1/2 signaling. SIX1 may be a promising target for RA treatment.

Platelet­rich plasma promotes the migration and invasion of synovial fibroblasts in patients with rheumatoid arthritis.

Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets, and the number of platelets is correlated with rheumatoid activity. PRP is a concentrated source of autologous platelets, and contains several different growth factors and cytokines, including platelet­derived growth factor, transforming growth factor­ß and insulin­like growth factor­1, which stimulate healing of bone and soft tissue. Rheumatoid arthritis (RA) is characterized by synovial hyperplasia, cell activation, articular inflammation and invasion of the synovium into the adjacent bone and cartilage. The adhesion of fibroblast­like synoviocytes (FLSs) onto the extracellular matrix (ECM), migration and invasion are important for the erosion and destruction of the articular cartilage of patients with RA. The aim of the present study was to investigate the effects of PRP on the adhesion, migration and invasion of RA­FLSs. Scratch and Transwell migration assays determined that PRP at a concentration of 2 and 5% significantly enhanced the migration ability of RA­FLSs. Treatment of RA­FLSs with 2 and 5% PRP promoted the adhesion and invasion of the cells. Additionally, the immunofluorescence assay revealed that PRP induced a decrease in the number of centrally located stress fibers and led to an increase in the formation of filopodia and lamellipodia in the detectable leading edge protrusions in RA­FLSs. In addition, reverse transcription­quantitative polymerase chain reaction and western blot analysis determined that PRP upregulated the protein and mRNA expression levels of matrix metalloproteinase­1 (MMP­1). In conclusion, the promotion of RA­FLS cell migration, invasion and adhesion on the ECM by PRP may be modulated through the upregulation of MMP­1 expression and the induction of actin cytoskeletal reorganization.