Crosstalk between ferroptosis and NLRP3, a possible therapeutic target in experimentally-induced rheumatoid arthritis: role of P2Y12R inhibition in modulating P53/SLC7A11/ALOX15 signaling

Abstract

Ferroptosis is critical in progressing and exacerbating rheumatoid arthritis (RA) and other inflammatory joint diseases. Inhibition of the P2Y12 receptors reduced iron overload in macrophages displaying an anti-inflammatory response. Herein, the ameliorative effect of ticagrelor, a reversible P2Y12 inhibitor, against adjuvant-induced arthritis (AIA) in rats was investigated, with a special emphasis on the possible modulation of some inflammatory signals linked to ferroptosis. Particularly, correlation analyses were conducted between nod-like receptor protein 3 (NLRP3) and all assessed parameters. Four groups of rats were assigned: Control group, AIA group (0.1 ml intradermal injection of complete Freund's adjuvant), Ticagrelor group (30 mg/kg, p.o.), and Ticagrelor + AIA group. Ticagrelor exhibited an anti-arthritic effect, evidenced by significant improvements in both macroscopic and histopathological alterations. It effectively inhibited ferroptosis, indicated by a marked upregulation of the ferroptotic inhibitors, solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) to reach 9.80, 2.20, and 8.49-folds (p < 0.0001), along with a notable reduction in the ferroptotic promoters, P53, acyl-CoA synthetase long-chain family member 4 (ACSL4) and arachidonic acid 15-lipoxygenase-1 (ALOX15) by 89.46%, 41.45% and 49.85% (p < 0.0001). It reduced TNF-α and various chemokines (RANTES, MIP-1α, eotaxin-3) to suppress matrix metalloproteinases expression. Furthermore, ticagrelor decreased NLRP3 expression by 48.63% (p < 0.0001) to pinpoint its anti-inflammatory effect. Overall, amending the P53/SLC7A11/ALOX15 axis by ticagrelor mediated its anti-inflammatory and anti-ferroptotic effects. These findings provide preliminary experimental evidences for further investigating the potential impacts of ticagrelor as a treatment for RA.

Keywords: ALOX15; Ferroptosis; NLRP3; Rheumatoid arthritis; SLC7A11; Ticagrelor.

Fig. 1

Experimental diagrammatic presentation: The yellow arrow represents FCA injection at day 0. The orange arrow represents the days of receiving ticagrelor (21 days). Blue arrows symbolize the days of measuring hind paw volume and thickness as well as arthritic score. The green arrow represents the end of the experiment, and adjuvant arthritis hind paws were separated for measuring biochemical markers, histopathology and immunohistochemistry

Fig. 2

Effect of ticagrelor on macroscopic arthritis changes in the AIA rat model. A Paw thickness. B Paw volume. C Arthritic score. D Correlation analysis between NLRP3 and macroscopic arthritis changes. Results are provided as means ± SD; p ≤ 0.05. (ns) non-significance, ^%%% p < 0.001, ^%%%% p < 0.0001 significant difference compared to the control group, ^#### p < 0.0001 significant difference compared to AIA group. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). Two-way ANOVA with Tukey–Kramer post hoc testing was employed for statistical analysis. Correlation analysis was implemented based on Pearson’s correlation coefficient

Fig. 3

Effect of ticagrelor on ferroptosis inhibiting factors in the AIA rat model. A SLC7A11. B GPX4. C FTH1 using ELISA analysis. First bar represents the control group, Second Bar represents the AIA group, Third Bar represents the Ticagrelor group and Fourth Bar represents the Ticagrelor + AIA group. D Correlation analysis between NLRP3 and ferroptosis inhibiting factors. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). The left hind paws of rats from each group were separated on (day 22) then kept at - 80°C for SLC7A11, GPX4 and FTH1 ELISA analysis. Two-way ANOVA with Tukey–Kramer post hoc testing was employed for statistical analysis. Data are provided as means ± SD; p ≤ 0.05. (ns) non-significance, * p < 0.05, **** p < 0.0001. Correlation analysis was implemented based on Pearson’s correlation coefficient

Fig. 4

Effect of ticagrelor on ferroptosis-promoting factors in the AIA rat model. A ACSL4 B ALOX15 using ELISA analysis. First Bar represents the control group, Second Bar represents the AIA group, Third Bar represents the Ticagrelor group and Fourth Bar represents the Ticagrelor + AIA group. C Correlation analysis between NLRP3 and ferroptosis-promoting factors. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). The left hind paws of rats from each group were separated on (day 22) and then kept at – 80 °C for ACSL4 and ALOX15 ELISA analysis. Two-way ANOVA with Tukey–Kramer post hoc testing was employed for statistical analysis. Results are provided as means ± SD; p ≤ 0.05. (ns) non-significance, **** p < 0. 0001. Correlation analysis was implemented based on Pearson’s correlation coefficient

Fig. 5

Effect of ticagrelor on P53 and TNF-α in AIA rat model. A photomicrograph of IHC-Peroxidase-DAB immunostaining of P53 in bone (40 ×), (arrow) shows sever positive expression for P53 in bone cells. B photomicrograph of IHC-Peroxidase-DAB immunostaining of TNF-α in bone (40 ×), (arrow) shows sever positive expression for TNF-α in fibrous tissue of bone. C Bar graph represents P53 staining area %. D Bar graph of TNF-α staining area %. First Bar represents the control group, Second Bar represents the AIA group, Third Bar represents Ticagrelor group and Fourth Bar represents the Ticagrelor + AIA group. E Correlation analysis between NLRP3 and P53 as well as TNF-α. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). Specimens of bone underwent fixation in 10% neutral buffered formalin; after that, they were decalcified in 10% EDTA for immunohistochemistry analysis. Two-way ANOVA with Tukey–Kramer post hoc testing was employed for statistical analysis. Results are provided as means ± SD; p ≤ 0.05. (ns) non-significance, ****p < 0.0001. Correlation analysis was implemented based on Pearson’s correlation coefficient

Fig. 6

Effect of ticagrelor on pro-inflammatory chemokines and NLRP3 in AIA rat model. A RANTES/CCL5. B MIP-1α/CCL3, C Eotaxin-3 /CCL26 D NLRP3 using ELISA analysis. First Bar represents the control group, Second Bar represents the AIA group, Third Bar represents the Ticagrelor group and Fourth Bar represents the Ticagrelor + AIA group. E Correlation analysis between NLRP3 and all chemokine markers. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). The left hind paws of rats from each group were separated on (day 22) then kept at -80°C for RANTES/CCL5, MIP-1α/CCL3, Eotaxin-3/CCL26 and NLRP3 ELISA analysis. Two-way ANOVA with Tukey-Kramer post hoc testing was employed for statistical analysis. Data are provided as means ± SD; p ≤ 0.05. (ns) non-significance, *p < 0.05. ****p < 0.0001. Correlation analysis was implemented based on Pearson’s correlation coefficient

Fig. 7

Effect of ticagrelor on MMP13 and MMP3 expression in the AIA rat model. A MMP13. B MMP3, First Bar represents the control group, Second Bar represents the AIA group, Third Bar represents the Ticagrelor group and Fourth Bar represents the Ticagrelor + AIA group. C Bands of western blot for MMP13 and MMP3 expressions within the left hind paw. D Correlation analysis between NLRP3 and matrix metalloproteinases. To verify the equivalent protein loading, β-actin levels were assessed. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). The left hind paws of rats from each group were separated on (day 22) and then kept at – 80 °C for MMP13 and MMP3 western blot analysis. Two-way ANOVA with Tukey–Kramer post hoc testing was employed for statistical analysis. Results are provided as means ± SD; p ≤ 0.05. (ns) non-significance, **** p < 0.0001. Correlation analysis was implemented based on Pearson’s correlation coefficient

Fig. 8

Effect of ticagrelor on the histological structure of bone, articular cartilage, and synovial membrane of paw tissue in AIA rat model. A Descriptive photographs of the ankle joint of rats. B H&E stain of bone, (star) displaying severe bone fibrosis and (arrow) indicating infiltration by a severe amount of mononuclear inflammatory cells in AIA group, on the other hand, (star) shows moderate bone fibrosis and (arrow) shows infiltration by few numbers of mononuclear inflammatory cells in the Ticagrelor + AIA group. C H&E stain of the articular cartilage surface, (arrow) shows a destruction and roughness of articular surface. D H&E stain of the synovial membrane, (arrow) displays infiltration of the synovial membrane by the high number of mononuclear inflammatory cells in AIA group, However, (arrow) expresses infiltration of synovial membrane by few numbers of mononuclear inflammatory cells in the Ticagrelor + AIA group. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). Specimens of bone, articular cartilage, and synovial membrane underwent fixation in 10% neutral buffered formalin; after that, they were decalcified in 10% EDTA for histopathology analysis

Fig. 9

Effect of ticagrelor on histopathological alteration in paw tissue in AIA rat model. A Histopathological changes score of cell infiltration B Histopathological changes score of bone and articular destruction C Correlation analysis between NLRP3 and histopathological alteration in paw tissue. Data are represented as a box plot of the median. Kruskal–Wallis test was performed, followed by a post-hoc Dunn’s test for statistical analysis, (ns) non-significance. *p < 0.05, **p < 0.01, ***p < 0.001. Correlation analysis was implemented based on Pearson’s correlation coefficient