PPAR- γ Activation Alleviates Osteoarthritis through Both the Nrf2/NLRP3 and PGC-1 α/ Δψ m Pathways by Inhibiting Pyroptosis

Abstract

Osteoarthritis (OA) is a common degenerative joint disease with a gradually increasing morbidity in the aging and obese population. Emerging evidence has implicated pyroptosis in the etiology of OA and it may be recognized as a therapeutic target in OA. We have previously reported regarding another disease that peroxisome proliferator-activated receptor gamma (PPAR-γ) activation exerts an anti-inflammatory effect by suppressing the nucleotide-binding and oligomerization domain-like receptor containing protein (NLRP) 3 inflammasome. However, the relationship between PPAR-γ and NLRP3-mediated pyroptosis in OA cartilage and its underlying mechanisms is fully unclear. In this study, we found that the level of NLRP3-mediated pyroptosis in severe lateral femoral condyle cartilage wear in the knee of an OA patient was significantly higher than that in the mild lateral femoral condyle cartilage wear areas. Moreover, in lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced primary chondrocytes and knee OA rat models, we demonstrated that activation of PPAR-γ by pioglitazone (Piog) attenuated LPS/ATP-induced chondrocyte pyroptosis and arthritis. These effects were partially counteracted by either blocking the nuclear factor erythroid-2-related factor (Nrf2)/NLRP3 or PGC1-α/Δψ _m signaling pathway. Simultaneous depression of these two signaling pathways can completely abrogate the protective effects of Piog on OA and chondrocytes. Taken together, Piog protects OA cartilage against pyroptosis-induced damage by simultaneously activating both the Nrf2/NLRP3 and PGC-1α/Δψ _m pathways, which enhances antioxidative and anti-inflammatory responses as well as mitochondrial biogenesis. Therefore, Piog may be a promising agent for human OA cartilage damage in future clinical treatments.

Figure 1

The upregulated levels of NLRP3, caspase-1 P10, and GSDMD-N in the cartilage of the lateral femoral condyle of the knee of a patient with severe OA. (a and b) MRI shows the lateral femoral condyle of the human knee, which has mild and severe lesions. MRI scoring was used to assess injury severity. (c–h) The levels of caspase-1 P10, GSDMD-N, and NLRP3 in mild and severe knee osteoarthritis are shown using immunohistochemical staining (bar: 200 μm). Quantification of the immunohistochemical staining is located below. (i and j) Representative immunoblots of NLRP3, caspase-1 P10, and GSDMD-N in the mild and severe lesion groups. GAPDH was used as an internal reference. Densitometric analysis of the immunoblots is located on the right. The data represent three independent experiments. ∗P < 0.05 and ∗∗P < 0.01.

Figure 2

Piog alleviates osteoarthritis and reduces NLRP3-mediated pyroptosis in rat experiments. (a) Gross observation of the control, OA, Piog, and OA + Piog groups. (b) The rat femur coronals were sectioned and stained using S-O staining. The cartilage area is stained red and the subchondral bone blue. The sections of the control, OA, Piog, and OA + Piog groups were observed under the microscope (bar: 100 μm). (c–f) Immunohistochemical staining was performed to detect the levels of GSDMD-N and NLRP3 in the lateral femoral condyle of the rat knee (bar: 100 μm). The immunohistochemical staining results were quantified. (g) ELISA was used to measure the concentrations of IL-1β and IL-18 in the cartilage. The data represent three independent experiments and are expressed as mean ± SD. ∗∗P < 0.01.

Figure 3

Piog attenuates LPS/ATP-induced pyroptosis of chondrocytes. (a) MTS assays were performed for cell toxicity after Piog treatment for 24 hours. (b and c) MTS and CCK8 assays were performed for rat chondrocyte viability. Piog pretreatment for 2 hours before LPS treatment for 24 hours and subsequent stimulation with ATP for 30 minutes. (d) CCK8 assays were performed for the effect of Piog on LPS/ATP-treated chondrocyte viability after GW antagonizing PPAR-γ using 20 μM Piog, 1 μM LPS, 5 mM ATP, and 10 μM GW. (e and f) Western blot and densitometric analysis of the immunoblots for NLRP3, caspase-1 P10, and GSDMD-N in the control, LPS/ATP, Piog + LPS/ATP, and GW + Piog + LPS/ATP groups. GAPDH was used as an internal reference. (g) ELISA was performed for IL-1β and IL-18 concentrations in the control, LPS/ATP, Piog + LPS/ATP, and Piog + GW + LPS/ATP groups. The data represent three independent experiments and are expressed as mean ± SD. ∗P < 0.05 and ∗∗P < 0.01.

Figure 4

Piog partially attenuates LPS/ATP-induced chondrocyte pyroptosis related to the NLRP3 inflammasome through Nrf2 signaling. (a and b) Western blot analysis for NLRP3, caspase-1 P10, and GSDMD-N in the control, LPS/ATP, Piog + LPS/ATP, and Piog + MSU + LPS/ATP groups. (b) ELISA was performed for IL-1β and IL-18 concentrations. (c) CCK8 assays were performed to determine the effect of Piog on LPS/ATP-treated chondrocyte viability after MSU activated NLRP3. (d) Western blot analysis for Nrf2 in the control, LPS/ATP, Piog, and Piog + LPS/ATP groups. (e) Western blot analysis was performed to detect NLRP3 level after Nrf2 knockdown by siRNA; (f) ELISA was performed to detect IL-1β and IL-18 concentrations. (g) CCK8 assays were performed to determine the effect of Piog on LPS/ATP-treated chondrocyte viability after Nrf2 knockdown by siRNA. GAPDH was used as an internal reference for western blotting. Densitometric analysis of the immunoblots is located on the right or below. The data represent three independent experiments and are expressed as mean ± SD. ∗P < 0.05 and ∗∗P < 0.01.

Figure 5

Piog partially attenuates LPS/ATP-induced pyroptosis of chondrocytes through the PGC1-α/Δψ_m signaling pathway. (a) PPAR-γ activation by Piog rescued PGC1-α expression in the presence of LPS/ATP. Western blot analysis for PGC1-α in the control, LPS/ATP, Piog + LPS/ATP, and Piog + GW + LPS/ATP groups. (b) Cells were pretreated with Piog in the presence or absence of SR and then stimulated with LPS/ATP. The level of NLRP3 was detected using western blotting. (c) ELISA was performed to measure IL-1β and IL-18 levels in the control, LPS/ATP, Piog + LPS/ATP, and Piog + SR + LPS/ATP groups. (d) CCK8 assays were performed to determine the chondrocyte viability in the control, LPS/ATP, Piog + LPS/ATP, and Piog + SR + LPS/ATP groups. (e) The intracellular ROS content was detected in the same five groups using Δψ_m assays. (f and g) The ratio of red fluorescent emission of cells with intact Δψ_m against green fluorescent emission of cells with impaired Δψ_m was plotted to represent the changes in Δψ_m. Fluorescent spectrophotometry was used to detect the Δψ_m of JC-1-stained cells in the control, LPS/ATP, Piog + LPS/ATP, GW + Piog + LPS/ATP, and SR + Piog + LPS/ATP groups (bar: 100 μm). GAPDH was used as an internal reference for western blotting. The characteristic immunoblot pictures are located under their densitometric analysis. The data represent three independent experiments and are expressed as mean ± SD. ∗∗P < 0.01.

Figure 6

Involvement of Nrf2 and PGC-1α in the effect of Piog on pyroptosis in the OA chondrocytes. (a and b) Western blotting experiments and densitometric analysis. SR was used as a PGC-1α inhibitor. Nrf2-siRNA was used for knocking down the level of Nrf2. GAPDH was used as an internal reference. (c) ELISA was used to measure IL-1β and IL-18 concentrations. (d) CCK8 assays were performed to measure chondrocyte viability. ∗∗P < 0.01, n = 5. (e) The action mode of PPAR-γ under Piog stimulation to protect from pyroptosis-mediated OA cartilage damage.