Activation of adenosine A3 receptor attenuates progression of osteoarthritis through inhibiting the NLRP3/caspase‐1/GSDMD induced signalling

Abstract The specific adenosine A3 receptor (A3AR) agonist (CF101) has potential for inflammation and pain in various disease, such as arthritis, cancer and neuropathic pain, while the role of A3AR in post‐traumatic OA and the underlying mechanism is largely unknown. CF101 was orally administrated in OA rats induced by anterior cruciate ligament transection (ACLT) surgery, and the rat primary chondrocytes were stimulated by hydrogen peroxide (H2O2, 300 μM). Histologic grading system was performed for detecting cartilage degeneration and immunohistochemistry for determining pyroptosis. The moleculars associated with cartilage homeostasis and inflammatory cytokines were analysed; moreover, the activation of NLRP3 inflammasome was determined. CF101 treatment significantly attenuated OA cartilage damage, OA‐related pain and cartilage pyroptosis. Chondrocytes stimulated by H2O2 evoked ROS release, thereby promoting the activation of NLRP3 inflammasome and facilitating the cleavage of GSDMD, which ultimately resulted in the mass release of pro‐inflammatory cytokines including IL‐1β and IL‐18, and production of matrix hydrolase. The pre‐treatment with CF101 powerfully inhibited the above process both in vivo and in vitro. Our findings demonstrated that activation of A3AR attenuates OA progression and relieves pain perception through suppression of cartilage degradation and inhibition of ROS/NLRP3/GSDMD signalling, indicating pyroptosis is a potential candidate for OA treatment.

In recent years, there have been numerous molecules and signalling pathways that have been found to be involved in cartilage disorders, which include pyroptosis cell death (a type of regulated cell death that is closely related to inflammation). 5 Pyroptosis occurs depending on the cleavage of procaspase-1 to yield active caspase-1.
Inflammasomes are multimeric protein complexes that assemble in the cytosol and act as platforms for caspase activation. 6 Activation of nucleotide-binding and oligomerization domain-like receptor containing protein 3 (NLRP3) promotes the cleavage of caspase-1 and further proteolytically matures proIL-1β and proIL-18 to their active forms, resulting in pyroptosis through gasdermin D (GSDMD) cleavage. 7 IL-1β, IL-18 and TNFα are the main inflammatory mediators that are involved in the pathophysiology of OA, which lead to suppressing expression of type II collagen and aggrecan, 8 and stimulates the release of matrix metalloproteinase (MMP)-3 and MMP-13. 9 This indicates that NLRP3-induced pyroptosis may contribute to OA progression by releasing pro-inflammatory cytokines. Additionally, increasing evidence suggests that the NLRP3 inflammasome is involved OA pathogenesis, which leads to the synovial inflammation 10 and cartilage degradation. In the synovial tissue of OA model rats, it was proved that the specific caspase1 inhibitor Ac-YVAD-CMK inhibited NLRP3 inflammasome-induced pyroptosis and the expression of pro-inflammatory factors IL-1β, IL-18, as well as alleviated OA pathology. 11 Moreover, fibroblast-like synoviocytes that were stimulated with LPS + ATP resulted in cell pyroptosis, while NLRP3 siRNAs attenuated this effect. These results suggested that LPSinduced fibroblast-like synoviocytes pyroptosis may be mediated by NLRP3 inflammsomes. 12 In addition, Li et al. found that the P2X7 receptor antagonist (A740003) ameliorated monosodium iodoacetateinduced cartilage degradation and OA-like pyroptotic inflammation by rescuing the expression of P2X7, MMP13, NF-κB p65, NLRP3, caspase-1 and IL-1β upregulation. 13 In human, nucleus pulposus stimulated by lactate solution showed NLRP3 inflammasome activation and increased levels of pyroptosis, furthermore degeneration of the extracellular matrix. While lactate-induced NLRP3 inflammasome activation was blocked by ASIC inhibitors and NLRP3 siRNA, 14 these studies indicated the role of pyroptosis in OA cartilage.
The purine nucleoside adenosine is a naturally occurring multifunctional signalling molecule that has potential for managing inflammatory joint disease. 15,16 Adenosine mediates its effects through binding and activating one or more of four G proteincoupled receptors expressed on the cell membrane: A1, A2A, A2B and A3. 17 Currently, a large number of studies have focused on the role of adenosine A2A receptor and adenosine A3 receptor in articular cartilage. Evidence indicated that the loss of glycosaminoglycans, collagen and extracellular matrix occurred in A2A knockout mice, which was accompanied by increased expression of MMPs, cartilage cell hypertrophy and osteophyte formation. 18 In addition, Liu Xiuling et al. (2019) found that the expression of IL-6, MMP-13 and type X collagen was significantly reduced after the use of adenosine-coupled nanoparticles, which was partially reversed by A2A receptor antagonist (ZM 241385) in primary chondrocytes of mice stimulated with IL-1β. 19 These studies suggested that adenosine A2A receptor is directly involved in the maintenance of cartilage homeostasis. Furthermore, activation of adenosine A3A receptor (A3AR) has been documented to exhibit powerful anti-inflammatory activity in a variety of diseases, including bowel inflammation, 20 psoriasis 21 and rheumatoid arthritis (RA). 22 Numerous A3AR agonists and antagonists have been tested for disease intervention. It has been reported that oral administration of CF101 (a selective A3AR agonists) downregulated levels of TNF-a, and increased apoptosis in infiltrating macrophages. 23

| Animals
Sprague-Dawley (SD) rats (bought from Liaoning Changsheng Biotechnology Co., Ltd.) were raised at the experimental animal centre on a standard 12 h dark/light cycle. All experimental procedures and protocols were carried out in accordance with the guidelines for Experimental Animal Ethics Committee of Northeast Agricultural University.

| Rat model of surgically induced OA
Anterior cruciate ligament transection (ACLT) model was induced using 10-week-old male rats. Rats accepted ACLT surgery y (n = 48) were randomly divided into 4 groups (12 rats per group), (1) ACLT group (vehicle), (2) ACLT+CF101 treatment group (100 μg/kg given twice daily), (3) ACLT+MRS1523 treatment group (100 μg/kg given twice daily) and (4) ACLT+MRS1523 + CF101 group (MRS1523 was administrated 30 min before CF101 treatment). Sham surgery was performed on the right knee joints from a separated group of rats as control (n = 12, treatment with vehicle). All treatment was started on Week 1 after ACLT surgery by gavage and was continued until the study was terminated on the last day of Week 6. The dose of each drug we used was based on previous study. 23

| Pain assessment
Secondary mechanical allodynia was assessed with Von Frey filaments (Aesthesio) in a blinded fashion. Mechanical allodynia was measured using the simplified up-down method 25 (n = 12 per group) in which Von Frey filaments were pressed against the plantar surface of the paw until the filaments flexed and held for 6-7 s for estimating paw withdrawal threshold (PWT). Filaments numbered 7 through 14 (0.60-10 g) were used and testing always began with filament 10.
If there was a positive response, the next lower strength hair was applied; if no withdrawal was present, the next higher value was applied. A total of 5 stimulus presentations occurred. Each animal went through two test rounds and whole numbers was used to represent the filament value.
After sacrifice, serum and urine samples were collected from 5 groups. After that, six rats in each group were performed for histological section, and another six rats in each group were performed for collecting synovial fluid.

| Histology and Immunohistochemistry
Knee joint specimens were extracted from the rats (n = 6 per group),

| ELISA
Serum samples, synovial fluid lavage fluids and urine samples were collected (n = 6 per group) and concentrations of IL-1β, IL-18, COX-2, CTX-II, COMP and GAG were measured using specific rat enzymelinked immunosorbent assay (ELISA) kits (Jingmei, Co., Ltd.) according to the protocol provided by the manufacturer. All of the samples were assessed in triplicate.

| Rat primary chondrocytes culture and stimulation
Rat cartilage from femoral condyle, tibial plateau and caput femoris were obtained from lactating rats (14-21-day-old). After trypsinization (0.25%, GIBCO, USA) of cartilage for 30 min, the tissues were cut into small pieces and then digested at 37°C with 0.2% collagenase II (BioFroxx, Germany) for 4 h. The primary chondrocytes were cultured with DMEM/F12, including 10% foetal calf serum (Biological Industries).

| LDH assay
Chondrocytes were plated in 96-well plates before exposure to different drugs (the same as 2.7). The levels of LDH in cell culture supernatants released from injured chondrocytes were determined using LDH Release Assay Kit (Beyotime) following the manufacturer's instruction (n = 6). The absorbance was measured at a wavelength of 490 nm.

| Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using the RNAsimple Total RNA Kit (TianGen

| Western blot
Total protein from chondrocytes were extracted, and protein concentration was determined using BCA Protein Assay Kit (Beyotime; Shang Hai, China). Equal amount of protein samples (30 μg) was separated by 10% or 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a BioTrace NT Nitrocellulose transfer membrane (Pall). The membranes were than blocked with 5% non-fat milk in tris buffered saline tween buffer for 1 or 2 h, and then incubated with primary antibodies against NLRP3

| Measurement of ROS production
Production of intracellular ROS was detected by the fluorescent probe DCFH-DA using ROS Assay Kit (Beyotime). After treatment, chondrocytes were incubated with 10 μM DCFH-DA at 37°C for 20 min in the dark. The culture medium was washed three times with PBS, harvested by trypsinization, and the fluorescence intensity was detected using a Spark Multi-mode microplate readers (Tecan, excitation at 488 nm, emission at 525 nm) and images were performed with microscopy fluorescence (n = 6).

| Intracellular potassium or calcium detection
After treatment, the cells were collected and broke through repeated freezing and thawing methods, then centrifuge and collect the supernatant. Intracellular potassium was detected by Potassium Assay Kit (Nanjing Jiancheng Bioengineering Institute), following the manufacturer's instructions (n = 6).
After treatment, cells were loaded with Ca 2+ indicator dye by incubating the culture dish in standard solution supplemented with 5 μM Fluo-3-AM (Beyotime Biotechnology) for 30 min at 37°C; then, the incubation was washed by PBS for two times. Absorbance was tested using Tecan Multi-Mode Microplate Readers (n = 6).

| Statistical analysis
All data were performed using SPSS 22.0 software or GraphPad Prism 8.3.0 software, and results are expressed as the mean ± standard error of mean (SEM). Statistical analyses were one-way analysis of variance (anova) was performed followed by Tukey's honestly significant difference post hoc test to compensate for multiple pairwise comparisons, and p < 0.05 was considered statistically significant.

| Activation of A3AR inhibits ACLT-induced pain and reduces COX-2 expression
In order to determine the role of A3AR in post-traumatic OA, the knee joints were collected from rats that underwent ACLT surgery.
The expression of A3AR increased in cartilage after ACLT induction, while CF101 treatment downregulated the expression of A3AR ( Figure 1A). Moreover, in the group that received ACL transection, the PWT values showed a clear reduction when compared with the vehicle group at one-week post-surgery, and the pain lasted until 6 weeks after surgery. Oral administration of CF101 had no obvious TA B L E 1 Primer sequences for genes tested with primary chondrocytes of rat  Figure 1C).

| Activation of A3AR ameliorates ACLT-induced OA progression in rats
Next, we investigated the effect of CF101 on cartilage degeneration in experimental OA rats. CF101 administration protected the structure of cartilage in OA through HE staining ( Figure 1D,E).

Safranine
O staining showed that ACLT surgery induced an obvious surface discontinuity, matrix disturbance, disordered chondrocytes, hypertrophic chondrocytes, vertical cracks and increased OARSI score (Figure 2A-C). Additionally, the expression of aggrecan and col II was downregulated in ACLT group ( Figure 2D). Compared to the ACLT rats, the OARSI score was significantly decreased, while the expression of aggrecan and col II was upregulated in the CF101 administration group. All of these changes were reversed by MRS1523 pre-treatment, suggesting the ACLT-induced cartilage degeneration and mechanical hyperalgesia were mediated by A3AR.

| Activation of A3AR reduces extracellular degradation in ACLT-induced OA and in H 2 O 2 -treated chondrocytes
To evaluated the role of A3AR in extracellular degradation induced by ACLT, we determined the expression of MMP-13 and ADAMTS-5.
The expression of MMP-13 and ADAMTS-5 in the cartilage was increased in the ACLT group when compared with sham group, while CF101 treatment decreased the expression of MMP-13 and ADAMTS-5 ( Figure 3A). Moreover, COMP and CTX-II, which are potential biomarker for OA diagnosis, were also decreased in the CF101-treated group, both in urine and synovial fluids, compared to the ACLT group. However, the low levels of GAG showed contrary tendency. The changeover occurred in the MRS1523 pre-treated rats ( Figure 3B). In

| Activation of A3AR ameliorate cartilage damage through inhibiting NLRP3-induced pyroptotic cell death
To

| CF101 suppresses NLRP3 inflammasome activation through inhibiting the production ROS in chondrocytes
Several events have been proposed to clarify the activation of the NLRP3 inflammasome. ROS, potassium (K + ) efflux and calcium (Ca + ) afflux were reported to contribute to the activation of NLRP3 inflammasome. We found that H 2 O 2 stimulation led to an increase of ROS and Ca 2+ , but decreasing K + in chondrocytes. CF101 treatment did not have an effect on the intracellular levels of K + and Ca 2+ ( Figure 6A,B), while it did significantly suppress on the levels of ROS ( Figure 6C,D). The results indicated that A3AR activation downregulated the NLRP3 inflammasome and pro-inflammatory cascades by inhibition of production of ROS.

| DISCUSS ION
Currently, there are no curative therapeutics available for OA, and the underlying mechanism needs to be further explored for personalized treatment. 27 Data from several groups agree on the therapeutic potential of activation of A2AR and A3AR in OA. Previous studies have found that osteoclast differentiation and chondrocyte inflammation were inhibited after activation of A2AR. 28  Intra-articular injections of adenosine prevented the development of OA in rats while the effect was abrogated by the co-injection of ZM 241385 (a selective A2A receptor antagonist). 29 These studies suggested that A2AR activation might be available to OA prevention.
Additionally, activation of A3AR through CF101 treatment is believed to protect OA induced by monosodium iodoacetate (MIA) through and NF-κB pathways, and the effect was abolished by selective adenosine antagonists. 30 Fishman et al. also determined that the PI3K-NF-κB pathway is involved in mediating the anti-inflammatory effect of CF101 in adjuvant-induced arthritis. 31 Both adenosine A2A and A3 agonists have been extensively studied in pain management. 32,33 It is worth noting that A2A agonists were effective for chronic pain, however, accompanied with cardiovascular side effects. 32,33 A3AR agonists (CF101) is also effective in models of neuropathic pain induced by diverse chemotherapeutic agents, 33 and demonstrated a good safety profile. 34 Therefore, our study tested the effect of A3AR agonists on OA cartilage injury and pain, but not A2AR. The results presented here demonstrated that activation of A3AR by CF101 ame- is also known to protect human chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. 39 Our present study confirms that activation of A3AR by CF101 inhibits inflammation in OA disease model, and the effects are associated with NLRP3-induced pyroptosis.
NLRP3 is the best-characterized inflammasome which induces an inflammatory, pyroptotic cell death. 40  activation has been demonstrated to attenuate neuroinflammation by inhibition of NLRP3 inflammasome activation in a model of traumatic brain injury (TBI), 64 and A3AR agonists may be useful adjuncts to opioids to manage their unwanted effects, which was associated with reduced dorsal horn of the spinal cord expression of NLRP3 and caspase-1. 65 These evidence supports our results that CF101 could relieve OA pain through inhibiting pyroptotic cell death by blocking NLRP3 inflammasome.
Our present study suggests that adenosine A3 receptor activation exerts a chondroprotective effect by inhibiting the NLRP3 inflammasome-mediated pyroptosis pathway, which not only enriches the mechanism of action of adenosine A3 receptors, but also provides a useful target for the treatment of OA, and offers a potential therapy for OA pain relief. However, the current study has several limitations. First, our study suggests that CF101 could be used to delay ACLT-induced OA development in a rat model. However, one critical question that we were not able to address is whether CF101 is able to delay OA progression when OA is fully developed. A long-term study needs to be carried out to investigate it. Second, in order to further determine the safety effect of CF101 on OA, large animals and large animal numbers are needed. Third, we have chosen male animals in the present study. In the future, effects of CF101 in female animals should be assessed due to the hormones difference.

| CON CLUS IONS
Taken together, the findings of our study demonstrate activation of A3AR by CF101 treatment attenuates OA pain and progression of disease in a post-traumatic animal model. Furthermore, activation of A3AR alleviates inflammatory process through inhibiting ROS initiated NLRP3 inflammasome activation. Therefore, it is reasonable to speculate that activation of A3AR by CF101 and inhibition of NLRP3 inflammasome activation might be a potential therapeutic strategy to prevent OA.