One-step TUNEL Apoptosis Assay Kit (Orange Fluorescence)

Osteoarthritis (OA) is an urgent public health problem; however, the underlying causal mechanisms remain unclear, especially in terms of inflammatory mediators in cartilage degradation and chondrocyte imbalance. P2X7 receptor (P2X7R) is a critical inflammation switch, but few studies have examined its function and mechanisms in OA-like pyroptotic inflammation of chondrocytes. In this study, Sprague–Dawley rats were injected in the knee with monosodium iodoacetate (MIA) to induce OA, followed by multiple intra-articular injections with P2X7R antagonist A740003, P2X7R agonist BzATP, NF-κB inhibitor Bay 11-7082, and NLRP3 inhibitor CY-09. Primary rat chondrocytes were harvested and treated similarly. We assessed cell viability, damage, and death via cell viability assay, lactate dehydrogenase (LDH) release, and flow cytometry. Concentrations of adenosine triphosphate (ATP) and interleukin- (IL-) 1β in cell culture supernatant and joint cavity lavage fluid were analyzed by enzyme-linked immunosorbent assay. Changes in expression levels of P2X7 and inflammation-related indicators were analyzed by immunofluorescence, quantitative reverse-transcription polymerase chain reaction, and western blotting. Cell morphology changes and pyroptosis were observed using transmission electron microscopy. Histology, immunohistochemistry, and microcomputed tomography were used to analyze damage to bone and cartilage tissues and assess the severity of OA. Similar to MIA, BzATP reduced cell viability and collagen II expression in a dose-dependent manner. Conversely, A740003 ameliorated MIA-induced cartilage degradation and OA-like pyroptotic inflammation by rescuing P2X7, MMP13, NF-κB p65, NLRP3, caspase-1 (TUNEL-positive and active), and IL-1β upregulation. Additionally, A740003 reduced the caspase-1/propidium iodide double-positive rate, LDH concentration, and reactive oxygen species production. These effects also occurred via coincubation with Bay 11-7082 and CY-09. In conclusion, activated P2X7 promoted extracellular matrix degradation and pyroptotic inflammation in OA chondrocytes through NF-κB/NLRP3 crosstalk, thus, aggravating the symptoms of OA. The study findings suggest P2X7 as a potential target for inflammation treatment, providing new avenues for OA research and therapy.

Moderate-intensity exercise can help delay the development of osteoarthritis (OA). Previous studies have shown that the purinergic receptor P2X ligand gated ion channel 7 (P2X7) is involved in OA development and progression. To investigate the effect of exercise on P2X7 activation and downstream signaling in OA, we used the anterior cruciate ligament transection (ACLT)-induced OA rat model and primary chondrocyte culture system. Our in vivo experiments confirmed that treadmill exercise increased P2X7 expression and that this effect was more pronounced at the later time points. Furthermore, P2X7 activation induced endoplasmic reticulum (ER) stress and increased the expression levels of ER stress markers, such as 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). At the early time points, IRE1 and PERK were activated, and mTOR was inhibited. At the later time points, mTOR was activated, mediating PERK to promote ER stress-apoptosis, whereas IRE1 and autophagy were inhibited. To confirm our observations in vitro, we treated primary chondrocytes with the P2X7 agonist benzoylbenzoyl-ATP (Bz-ATP). Our results confirmed that P2X7-mediated Ca²⁺ influx activated IRE1-mediated autophagic flux and induced PERK-mediated ER stress-apoptosis. To further investigate the role of P2X7 in OA, we injected mTOR antagonist rapamycin or P2X7 antagonist A740003 into the knee joints of ACLT rats. Our results demonstrated that mTOR inhibition induced autophagy, decreased apoptosis, and reduced cartilage loss. However, injection of mTOR agonist MHY1485 or Bz-ATP had the opposite effect. In summary, our results indicated that during the early stages of moderate-intensity exercise, P2X7 was activated and autophagic flux was increased, delaying OA development. At the later stages, P2X7 became over-activated, and the number of apoptotic cells increased, promoting OA development. We propose that the IRE1-mTOR-PERK signaling axis was involved in the regulation of autophagy inhibition and the induction of apoptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA, suggesting that the intensity and duration of exercise play a critical role. We also demonstrated that on a molecular level, P2X7 and its downstream pathways could be potential therapeutic targets for OA.