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

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that refers to the aberrant growth of the synovium, which causes inflammation. This excessive growth invades the cartilage and bone, resulting in the progressive deterioration of the joints (McInnes and Schett 2011; Radu and Bungau 2021). RA affects around 3 in every 10⁴ people around the globe and ranks 42^nd highest disability rate among 291 diseases (Nerurkar et al. 2019; Prasad et al. 2023). Due to the obscure pathogenesis of RA and its associated patient suffering, there is an urgent desire for innovative research to develop effective treatments.

Ferroptosis is a newly discovered mode of programmed cell death that is triggered by an excessive iron deposition within cells. Intracellular iron buildup, oxidative degradation of lipids, or perturbations in the system of antioxidants are the primary triggers of ferroptosis, resulting in membrane integrity loss and eventual cell death (Li et al. 2024). Noteworthy, following the entry of ferrous ions into the cytoplasm, a portion of it attaches to ferritin heavy chain 1(FTH1) and undergoes oxidation to produce ferric, which subsequently attaches to ferritin light chain (FTL), forming the ferritin complex, which is then retained within the cell. Excess ferrous ions build up in cells and create unstable iron pools once iron-binding complexes are saturated (Wang et al. 2021). Through the Fenton reaction, excessive ferrous ions can induce substantial reactive oxygen species (ROS) generation, altering the equilibrium of redox processes inside cells, amplifying oxidative damage to lipids, and prompting ferroptosis (Hirayama et al. 2017).

Ferroptosis can only be induced by peroxidase polyunsaturated fatty acids (PUFA) that are integrated into lipids such as phospholipids (Dixon et al. 2012). Acyl-CoA synthetase long-chain family member 4 (ACSL4) is essential for the biosynthesis of membrane phospholipids that contain phosphatidylethanolamines-PUFAs (PUFA-PEs). PUFA-PE is extremely vulnerable to oxidation by lipoxygenases such as arachidonic acid 15-lipoxygenase-1 (ALOX15) and extreme ROS, ultimately resulting in ferroptosis (Li et al. 2024). Furthermore, it can be induced by the impairment of the body's antioxidant defenses (Riegman et al. 2020). The selenium protein, glutathione peroxidase 4 (GPX4) efficiently lowers peroxides and prevents the triggering formation of peroxidized phospholipid throughout the metabolism of arachidonic acid (Friedmann Angeli et al. 2019). Additionally, solute carrier family 7 member 11 (SLC7A11) is the primary subunit of system Xc-, a cystine carrier protein that facilitates the transfer of cystine and glutamate across the cell membranes, promoting the biosynthesis of reduced glutathione (GSH). Thus, disruption of the human body's vital antioxidant system, SLC7A11/GSH/GPX4 accelerates lipid peroxidation, thereby inducing ferroptosis (Chen et al. 2021).

Emerging evidences revealed that ferroptosis promotes the progression and exacerbation of RA and various inflammatory joint disorders. The pathogenic features of RA align with the features of ferroptosis, specifically lipid peroxidation, iron deposition, glutathione loss and GPX4 inactivation (Stockwell 2022; Xie et al. 2021). A growing body of research has reported that the activation of signaling pathways involved in inflammation is very closely associated with ferroptosis. In RA, the alteration of the synovial membrane leads to liberating inflammatory cytokines like tumor necrotic factor alpha (TNF-α) (Telfer and Brock 2004) that induces chondrocytes to release matrix metalloproteinases (MMPs) like MMP13 and MMP3, which are cartilage-breaking enzymes, causing cartilage deterioration in RA (Mengshol et al. 2002; Ostrowska et al. 2018a). Also, the infiltration of synovial tissue induces the production of the nod-like receptor protein 3 (NLRP3) inflammasome, a key precursor of interleukin-1β (IL-1β) and interleukin-18 (IL-18), that augments synovial cell proliferation (Shin et al. 2019).

Interestingly, recent evidences have shown that inhibiting ferroptosis leads to decreased inflammation and defective synovial fibroblast proliferation, indicating a potential role for ferroptosis in the progression of RA (Feng et al. 2024; Liu et al. 2024). These previous investigations have indicated a potential treatment choice for reducing inflammation, joint damage and RA progression in patients by targeting vital ferroptotic pathways.

Ticagrelor is the first approved oral drug as a reversible P2Y12 inhibitor to prevent platelet aggregation in patients who are suffering from coronary artery diseases (Rollini et al. 2016). It elevates tissue adenosine levels, so it acts as an anti-inflammatory and inhibits oxidative stress (Barletta et al. 2012; Nylander et al. 2013). Previous researches indicated that ticagrelor's renoprotective effects were mediated by its ability to exert anti-inflammatory, antioxidant, and anti-apoptotic actions that maintain renal function and structure (El-Mokadem et al. 2021; Mansour et al. 2022). Additionally, ticagrelor showed a neuroprotective effect in Parkinsonism by inhibiting apoptosis (Muneeb et al. 2025). Moreover, ticagrelor mitigated cecal ligation and puncture (CLP)-induced polymicrobial sepsis in mice, likely through modulation of pro-inflammatory and oxidative stress signaling pathways (Mueen et al. 2023). Added to that, prior research proved that it could inhibit osteoclast differentiation and promote bone generation in a mice model (Mediero et al. 2016). Along with prior evidences, its potential effect on arthritis has not been previously discussed. Interestingly, in vitro, P2Y12 inhibition diminished inflammatory response and ferroptosis in iron-overloaded macrophages by outflowing excessive iron from macrophages into the blood circulation (Hu et al. 2024).

In light of this background, the current study aimed to investigate the possible novel anti-arthritic effect of ticagrelor in the adjuvant-induced arthritis (AIA) model in rats with the possible modulation of some inflammatory signals linked to ferroptotic cell death. In particular, the crosstalk between ferroptosis and NLRP3 was highlighted as a potential target for RA therapies.

In our study, ticagrelor modulated P53/SL7A11/ALOX15 axis to attenuate inflammatory response linked to ferroptosis in the AIA rat model.

To date, there is no complete treatment for RA that has been established in humans and the typically administered drugs just help in mitigating its serious symptoms (Aletaha and Smolen 2018). Therefore, there is a critical need for the discovery of future therapy approaches. For the first time, our research focuses on evaluating the anti-inflammatory and anti-ferroptotic effects of ticagrelor against AIA.

The AIA is an experimental paradigm that simulates the pathophysiology of human RA, including chronic joint swelling and pain, pro-inflammatory cytokine release, cartilage erosion, and bone destruction. While it mimics many downstream inflammatory and destructive processes, it does not typically involve the persistent, self-perpetuating loss of self-tolerance or the robust production of specific autoantibodies seen in human RA (Jones et al. 2018). However, it is wieldy used to bio-screen new drugs for RA (El-Sayed et al. 2014; Zhu et al. 2020). We had been used male rats based on three key considerations: First, the historical precedent in scientific research, where male animals have traditionally been used to minimize variability introduced by hormonal fluctuations. Second, and more critically, estrogen has well-documented anti-inflammatory effects (Camporez et al. 2019) which could confound our investigation into the specific role of ticagrelor in AIA-related inflammation. Third, estrogen is an important hormone in the regulation of bone turnover and bone cell activity (Lara-Castillo 2021). By using male rats exclusively, we aimed to reduce this potential hormonal interference and ensure a more controlled assessment of the inflammatory response and the damage in the experimental arthritis model. Additionally, (Schuh et al. 2023) in their investigation of collagen induced arthritis (CIA) in middle-aged mice, observed an earlier onset of arthritis and more severe consequences on joints, bones, and kidneys in male mice compared to females. This finding highlights that sex is a crucial biological variable influencing disease severity and progression in inflammatory arthritis models. While the specific immune mechanisms underlying these sex differences and the limited duration of symptoms require further investigation, the demonstration of more severe disease outcomes in males supports our strategy of utilizing male rats to ensure a consistently robust and uniform disease phenotype for evaluating therapeutic interventions.

Ticagrelor administration showed a significant diminution in paw volume, thickness, and arthritic score in AIA rats that was confirmed in the histopathological examination. It ameliorated bone fibrosis and destruction of cartilage by reducing the number of infiltrated mononuclear inflammatory cells within the bone and synovial membrane. These findings demonstrate ticagrelor's efficacy in attenuating disease activity in the AIA model, suggesting its potential to alleviate pain and improve mobility in affected individuals.

Ferroptosis in RA has been linked to disrupted iron metabolism. The synovial fluid and membrane of RA patients with severe disease activity showed elevated levels of iron and lipid oxidation (Wu et al. 2022). A previous study has indicated decreased FTH expression by erastin that induced ferroptosis in RA (Zhou et al. 2022), which, besides its role in iron storage, has ferroxidase activity inhibiting ROS generation by converting ferrous to ferric iron (Mesquita et al. 2020). This observation is in harmony with our results where AIA rats showed a marked decline in FTH1 expression signifying the contribution of iron metabolism in inducing ferroptosis in RA.

One promising strategy for reducing ferroptosis is to enhance SLC7A11 levels which is indispensable for GSH synthesis, an essential co-activator for GPX4, the most significant anti-lipid peroxidase (Miao et al. 2022). Inactivation of GPX4 induced by GSH depletion secondary to SLC7A11 suppression eventually causes ferroptosis (Imai et al. 2017; Seibt et al. 2019). In this study, decreased expression of SLC7A11 and GPX4 was detected in AIA rats. Coherent with these results, previous studies showed a decrease in SLC7A11 and GPX4 levels enhancing ferroptosis in human synovial cells and AIA rats model (Luo and Zhang 2021; Peng et al. 2022). In a similar fashion, a marked diminution in GPX4 content was detected in the articular cartilage of RA patients (Zhou et al. 2022).

Emerging evidences reported that ACSL4 and ALOX15 are crucial ferroptosis mediators by promoting the peroxidation of lipids (Qin et al. 2021; Sun et al. 2023). Some prior evidences have shown that elevating active ferrous decreased GPX4 and increased ACSL4 and ALOX15. These effects induced dysfunction of GSH and consequently oxidation of different multifunctional phospholipids (Zhang et al. 2021). In myocardial infarction, inhibition of ALOX15 was able to limit ferroptosis and lipid peroxidation while simultaneously elevating GSH levels in the PUFA-enriched cells (Ma et al. 2022). Similarly, an ALOX15 inhibitor reversed ferroptosis-associated decreased expression of the ferroptotic repressors, GPX4 and SLC7A11 in an in vitro model of neurotoxicity (Zhao et al. 2022).

In addition, the P53 gene, a powerful tumor suppressor, could provoke ferroptosis by decreasing the expression of the SLC7A11 protein, hindering the antioxidant effect (Hong et al. 2017). Furthermore, p53 promoted ALOX15 activity by inhibiting SLC7A11, thereby triggering ferroptosis in bladder cancer cells (Li et al. 2023). Previous evidences have shown that P53 is often overproduced in people with RA and animal models (Peng et al. 2022; Sun and Cheung 2002) to coincide with our results, where upregulation of P53 expression was detected in bone cells of AIA rats. In the current investigation, ticagrelor was effective in hampering ferroptotic cell death in AIA rats as revealed by the noticeable induction of the ferroptosis suppressors, FTH1, GPX4, and SLC7A11 along with the prominent diminution in the ferroptosis promoters, ACSL4 and ALOX15. This anti-ferroptotic effect could be partially ascribed to its ability to reduce P53 expression as demonstrated in AIA rats treated with ticagrelor in our study. These findings propose a potential modulatory role for ticagrelor in antioxidant defense mechanisms during AIA. In accordance with our results, the antioxidant effect of ticagrelor has been widely tackled in various investigations through its ability to boost GPX expression. Ticagrelor increased levels of GPX in rat kidneys experiencing ischemia–reperfusion injury and it was observed to attenuate ROS by elevating GPX and SOD within the renal tissue of sepsis mice model (BAĞCIOĞLU et al. 2017; Lv et al. 2022). Similar findings were observed in mice with carrageenan-induced thrombi (Zhang et al. 2022) and in both lung and heart cells of rats exposed to abdominal aorta ischemia and reperfusion (Findik et al. 2016).

Ferroptosis is strongly associated with the activation of signaling pathways driving inflammatory response. Numerous mediators, including chemokines, cytokines, and MMPs, are released in the RA synovium, triggering chronic inflammation as verified herein and previously. Fibroblast-like synoviocytes (FLSs) and chondrocytes secrete MMPs and cytokines such as TNF-α, encouraging the demolition of bone and cartilage (Ostrowska et al. 2018b). TNF-α stimulateds iron buildup in vitro by FLS from RA patients which increases the progression of RA (Chang et al. 2022). Among MMPs, MMP3 can directly break down cartilage and bone tissues (Wang et al. 2022) as well as MMP13 observed in both cartilage and synovial fluid of patients with RA, is a primary enzyme responsible for degrading the extracellular matrix, including type II collagen and proteoglycans. Moreover, MMP13 can contribute to synovial inflammation, stimulate synovial tissue overgrowth, and release cytokines from RA chondrocytes (Hu and Ecker 2021). Preceding studies demonstrated that MMP3 and MMP13 increased in chondrocytes to increase osteoarthritis progression by a ferroptosis promoter like ferric ammonium citrate (Jing et al. 2021a, 2021b). Tchetina et al. reported that by inhibiting TNF-α, iron chelators such as deferoxamine could reverse elevated MMP3 and MMP13 expression (Tchetina et al. 2016).

Several recent studies demonstrated the role of chemokines, including RANTES/CCL5, MIP-α/CCL3, and eotaxin-3/CCL26 in the progression of RA (Murayama et al. 2023; Yang et al. 2023). RANTES/CCL5 promotes the breakdown of bone and articular cartilage in RA by stimulating the production of MMP1 and MMP13 enzymes (Agere et al. 2017). A previous study reported that Met-RANTES, which blocked RANTES/CCL5, mitigated AIA in rats (Shahrara et al. 2005). Likewise, MIP-1α/CCL3 contributed to inflammatory bone loss in the AIA model by the recruitment of osteoclast precursor cells (Toh et al. 2004). Furthermore, previous research has demonstrated that MIP-1α/CCL3 either dose- or time-dependently raised MMP3 in FLS cells in a culture medium (Yang et al. 2023). Prior studies that align with our results had shown that MIP-1α/CCL3 and RANTES/CCL5 increased in AIA and CIA models, suggesting that these chemokines could be therapeutic targets in RA (Szekanecz et al. 2000; Thornton et al. 1999). From the same perspective, Chae et al. reported that eotaxin-3/CCL26 was implicated in RA as a proinflammatory mediator (Chae et al. 2005).

Besides its well-established role in lipid peroxidation during ferroptosis, ALOX15 has pro-inflammatory properties and was shown to be highly expressed in synovial RA membranes (Gheorghe et al. 2009). It was demonstrated that an ALOX15 metabolite upregulated the TNF-α expression in vascular cells and macrophages (Bolick et al. 2005; Dwarakanath et al. 2008). Furthermore, ALOX15 was involved in MMPs upregulation by TNF-α in RA synovial fibroblasts (Wu et al. 2012). In the same context, it has been reported that eotaxin-3/CCL26 was regulated by ALOX15 in the epithelial cells of the lower airways (Li et al. 2019). Similarly, ALOX15 promoted airway inflammation by inducing the expression of chemokines like RANTES/CCL5 and MIP-1α/CCL3 (Xu et al. 2021). These observations highlight the essential role of ALOX15 in promoting inflammatory response through enhancing chemokine-induced MMPs expression as well as inflammatory cells migration in RA.

Our study demonstrates that ticagrelor treatment could alleviate inflammation and promote articular repair in the AIA model. This effect was achieved by reducing TNF-α levels in bone tissue and downregulating MMP13 and MMP3 expressions, which are key enzymes included in cartilage degradation. Through the considerable downregulation of the chemokines RANTES/CCL5, MIP-1α/CCL3, and eotaxin-3/CCL26 in AlA, ticagrelor inhibited the recruitment of inflammatory cells and preserved cartilage and bone integrity, an effect that could be partially ascribed to its ability to attenuate the ALOX15-associated inflammatory response. Coherent with our results, previous studies illustrated that ticagrelor possessed an anti-inflammatory effect via various pathways. Sexton et al. proved the potency of ticagrelor to modulate inflammation by blunting TNF-α in mice that were exposed to lipopolysaccharide (Sexton et al. 2018). Furthermore, in mice model of abdominal aortic aneurysm, ticagrelor in combination with aspirin significantly reduced MMPs expression (Liu et al. 2022). Similarly, ticagrelor could reduce MMPs alone in coronary artery disease patients (Mao et al. 2019).

Furthermore, NLRP3 inflammasome is critically involved in the progression of RA and chondrocyte dysfunction by promoting the formation of pro-inflammatory cytokines contributing to cartilage and bone degradation via inducing MMPs expression (Cheng et al. 2022; Lu et al. 2023). CIA mice and AIA rats models displayed significantly elevated NLRP3 expression in comparison to the normal group (Ding et al. 2016; Zhang et al. 2016), to concur with our results. Moreover, NLRP3 level is positively related to ferroptosis in a rat model of osteoarthritis (Meihe et al. 2021). Preceding evidence proposed a potential relation between the NLRP3 inflammasome, lipid peroxidation and ferroptosis, where a buildup of ferrous iron within cells could activate the NLRP3 inflammasome (Gupta et al. 2023). In the same context, a direct proportion between NLRP3 and ACSL4 has been previously reported (Zhou et al. 2021). Kang et al. also reported that inflammasome activation was negatively regulated by GPX4 (Kang et al. 2018). Also, SLC7A11 impaired NLRP3 inflammasome expression in nonalcoholic steatohepatitis advocating the involvement of ferroptosis in NLRP3 activation (Lv et al. 2024).

Notably, this study showed that ticagrelor significantly diminished NLRP3 expression in the ankle joint of AIA rats. This effect could be attributed to ticagrelor’s capability of modulating the ferropotic markers, SLC7A11, ACSL4, and GPX4 secondary to inhibiting P53. In accordance with our results, ticagrelor demonstrated a distinct therapeutic potential by modulating the NLRP3 inflammasome in the progression of diabetic cardiomyopathy in mice (Chen et al. 2020). In a similar fashion, ticagrelor, beyond its antiplatelet effect, reduced macrophage NLRP3 in vitro in acute coronary syndrome patients (Huang et al. 2021), and following myocardial ischemia reperfusion-induced acute lung injury in rats (Dai et al. 2024). As an effort to interpret our findings concerning the crosstalk throughout the inflammatory response and ferroptosis and their relation with bone damage, the correlation between NLRP3 expression and all evaluated parameters was investigated to assess the role of ticagrelor in alleviating inflammatory burden by downregulating ferroptosis.

Compared to our findings concerning the anti-arthritic effect of ticagrelor through modulating ferroptosis and NLRP3 expression, other approved protective agents against RA have been demonstrated to target ferroptosis-associated oxidative damage and NLRP3 activation. For instance, methotrexate administration resulted in a significant downregulation of malondialdehyde and ACSL4, indicative of reduced lipid peroxidation in joint tissues of the CIA model. This effect was accompanied by upregulation of SLC7A11, GPX4, and FTH1, suggesting a potential protective mechanism against ROS and ferroptosis in RA (Zhou et al. 2023a). Moreover, it mitigated CIA inflammation by reducing NLRP3 expression (Pang et al. 2018). Additionally, hydroxychloroquine treatment, that approved for RA disease, significantly reduced NLRP3 in mouse models of renal injury (Cui et al. 2023; Tang et al. 2018). Similarly, tocilizumab, that targets the interleukin-6 receptor and is approved for RA, reduced kidney damage by preventing ferroptosis through increased levels of GPX4, xCT, and ferritin (Yang et al. 2020). In a similar fashion, recent experimental studies investigated novel protective agents against RA by exploring their ability to hinder ferroptotic cell death (Peng et al. 2022; Zhou et al. 2023a). In addition, an umbrella meta-analysis by (Bideshki et al. 2024) recently affirmed the significant efficacy of curcumin in reducing pain and improving joint function in patients with osteoarthritis which share fundamental destructive processes with RA driven by chronic inflammation and oxidative stress. This effect was previously ascribed to its ability to hinder chondrocyte ferroptosis (Zhou et al. 2023b). The demonstrated benefit of compounds targeting these shared mechanisms further supports the potential of ticagrelor's anti-inflammatory and anti-ferroptotic actions in alleviating pathological changes characteristic of inflammatory arthritis like RA.

Owing to ticagrelor's pharmacological characteristics and growing clinical use, some of its adverse effects such as bleeding tendency, dyspnea, ventricular pause, hyperuricemia and kidney damage are now getting a lot of attention in an effort to make timely changes that will guarantee the medication's safety and maximum clinical benefits (Wei et al. 2024). Notably, ticagrelor at low dosages was reported to be safe for long-term use (Cesaro et al. 2020). Consequently, the implementation of personalized treatment strategies for RA, guided by individual patient factors, remains critical for optimizing therapeutic efficacy while mitigating the risk of dose-related adverse reactions (Rauf et al. 2025; Wei et al. 2024). Moreover, prior research also highlighted hypersensitivity, including angioedema, as an unpredictable type B adverse drug reaction of anti-platelets, independent of dose, time, and frequency, and determined by host factors (Binazon et al. 2013) which is rare with ticagrelor administration (Seecheran et al. 2017).

Taken all together, the present investigation focused on the anti-arthritic abilities of ticagrelor against ferroptosis-associated inflammatory response in the AIA rat model. Ticagrelor abrogated NLRP3 expression and hampered TNF-α and chemokines-induced MMPs activation to inhibit the recruitment of inflammatory cells and preserve cartilage and bone integrity. Modulating the ferroptotic inhibitors, for instance, SLC7A11, GPX4 and FTH1 besides ferroptotic promoters such as ACSL4 and ALOX15 by ticagrelor validates its anti-inflammatory and anti-ferroptotic effects in AIA. Likewise, our data revealed a strong correlation between NLRP3 suppression and modulation of P53/SLC7A11/ALOX15 signaling pathways. The findings of this study may pave the way for future clinical investigations of ticagrelor to help in the management of RA. Furthermore, it also points out the probable mechanism of ferroptosis on cartilage, which could provide further possible targets for future clinical trials for the treatment of RA.

Ferroptosis mechanism should be investigated in other models of arthritis. Further research is needed to explore the functional molecular mechanism of ticagrelor in treated AIA. While AIA model mimics many downstream inflammatory and destructive processes, it does not typically involve the persistent, self-perpetuating loss of self-tolerance or the robust production of specific autoantibodies seen in human RA (Jones et al. 2018). Additionally, clinical researches are required for the safety of ticagrelor. Therefore, future studies will involve detailed acute and chronic toxicity assessments, including preliminary safety assesment such as liver, kidneys, spleen, or lungs weight, and analysis of serum biomarkers for organ function ALT, AST, BUN, and creatinine to establish its full safety profile in this context. These future investigations are essential prerequisites for advancing ticagrelor towards clinical translation for RA. Additionally, our further investigations will explicitly include both male and female rats to systematically investigate sex as a biological variable in the context of ticagrelor's effects on arthritis pathogenesis. This will allow us to assess any sex-specific differences in disease progression, therapeutic response, and underlying mechanisms, fully aligning with contemporary research standards.