P2X7 Receptor Induces Pyroptotic Inflammation and Cartilage Degradation in Osteoarthritis via NF- κ B/NLRP3 Crosstalk

Abstract

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.

Figure 1

MIA induces inflammation and increases P2X7 expression. (a) Cell viability after chondrocytes were treated with different concentrations (0, 0.75, 1.5, and 3 μM) of MIA for 12 or 24 h. (b) Histogram displaying LDH concentrations normalized to the control group. Grey, 0 μM MIA; green, 0.75 μM MIA; red, 1.5 μM MIA; blue, 3.0 μM MIA. (c) Representative flow cytometry scatter plots of double-positive staining for active caspase-1 and propidium iodide (PI). (d) Double-positive rate for active caspase-1 and PI after 12 h, assessed by flow cytometry. (e) Extracellular ATP concentration of chondrocytes measured by the luciferase reaction. (f) Representative western blot, (g) relative fold-change of intensity, and (h) mRNA expression measured by qRT-PCR for P2X7, MMP13, collagen II, and IL-1β level normalized to the control. Data are presented as means ± SD of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 2

A740003 and BzATP regulate P2X7 function. Cell viability was measured for chondrocytes treated with (a) A740003 (10, 20, and 50 μM) or (b) BzATP (10, 50, and 100 μM) for 12 or 24 h. (c, d) Representative western blots, (e, f) relative fold-change of intensity, and (g, h) mRNA expression measured by qRT-PCR for P2X7, MMP13, caspase-1, collagen II, NK-κB p65, and IL-1β level for chondrocytes treated for 12 h with A740003 and BzATP. Grey, 0 μM MIA; orange, 1.5 μM MIA; green, 1.5 μM MIA+10/20/50 μM A740003; red, 1.5 μM MIA+20/50/100 μM BzATP. Data are presented as means ± SD of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 3

NF-κB or NLRP3 inhibitors attenuate pyroptotic inflammation. (a) Cell viability of chondrocytes treated with MIA (1.5 μM) with or without A740003 (20 μM), BzATP (50 μM), BzATP+Bay 11-7082 (10 μM), or BzATP+CY-09 (10 μM) for 12 h. (b) Double-positive staining rate for activated caspase-1 and propidium iodide (PI). (c) Scatter plot evaluating cell viability measured by flow cytometry. (d) ELISA analysis of extracellular inflammatory factor IL-1β. (e) Cell damage analyzed by LDH assay. Data are presented as means ± SD of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 4

Immunofluorescence staining of (a) collagen II (green), (b) NF-κB p65 (red), and (c) caspase-1/PI (green/red) immunoreactivity (scale bar: 50 μm). Nuclei were stained with DAPI (blue). Groupings are the same as shown in Figure 3.

Figure 5

Inhibition of NF-κB and NLRP3 changes cellular morphology and reduces ROS production and inflammatory factor release. (a) Representative TEM images of cellular morphology changes (white arrows) such as cell swelling, cell membrane protrusion, and cell nucleus atrophy (scale bar: 5 μm). Groupings are the same as described in Figure 3. (b, c) Reactive oxygen species levels in cells analyzed by DCF fluorescence intensity. (f) qRT-PCR detection of MMP13, collagen II, P2X7, and IL-1β mRNA levels normalized to the control. (d, e) Representative western blots of protein expression levels. GAPDH was used as the control. Data are presented as means ± SD of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 6

MIA-induced OA evaluated by micro-CT. (a) Frontal views of the knee joints 4 weeks after MIA injection, represented as three-dimensional micro-CT images. (b) Sagittal views of medial compartment subchondral bone. (c) Quantification of bone morphological parameters (BV, BV/TV, Tb.Sp, Tb.N, and Tb.Th). (d) ELISA analysis of IL-1β in joint-cavity-lavage fluid. Data are presented as means ± SD of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01. Scale = 1 mm.

Figure 7

Staining evaluations of inhibitor treatments and P2X7 activation in knee-joint cartilage of MIA-induced OA rats. Cartilage and subchondral bone of knee-joints were stained using (a) H&E and (b) Toluidine Blue O (scale bar: 500 μm). (c) Quantitative analysis of OARSI score from (a, b). (d) TUNEL staining of sagittal central sections of the cartilage in each group (scale bar: 50 μm). (f) Quantitative data for (d). (e) IHC staining of P2X7, MMP13, collagen II, NF-κB p65, NLRP3, IL-1β, and caspase-1 (scale bar: 50 μm). (g) Quantitative analysis of immune-positive cells. Data are presented as means ± SD of at least three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 8

Schematic of the working hypothesis of P2X7 in OA.