Plasma samples were all obtained from the China-Japan Friendship Hospital. Twenty-three subjects without RA or other autoimmune diseases were enrolled as control samples (49.21 ± 16.12 years old on average). Forty-two patients were diagnosed with RA according to the 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria, with an average age of 53.52 ± 11.29 years (20). The RA patient group was divided into two groups based on their Disease Activity Score in 28 joints-Erythrocyte Sedimentation Rate (DAS28-ESR) (21, 22), the 5.1≥Activity≥2.6 group (n = 20) or the Activity >5.1 (n = 22) group. This study was reviewed and approved by the Ethics Committee of China-Japan Friendship Hospital with the ethical approval number of 2020-133-K86.

Forty Sprague−Dawley (SD) rats (male, average body weight 200 ± 20 g, 6 weeks old) from the China Institute for Food and Drug Control, Beijing, China were used in this study. The rats were maintained under standard laboratory conditions at 23°C± 2°C with a 12-hour alternating light/dark cycle and were allowed ad libitum access to feed and water. After 3 days of adaptive feeding, arthritis was induced in rats by immunization with bovine type II collagen (Chondrex). Thirty rats were injected subcutaneously with a 50 μL emulsion of bovine collagen II and an equal volume of incomplete Freund’s adjuvant (Chondrex) at the tail root for primary immunization, and the remaining 10 rats were injected with an equal volume of normal saline. Seven days later, CIA rats were constructed by a secondary booster immunization using the same method. Four days after the secondary immunization, rats with different arthritis index (AI) scores (different severities) were equally assigned to each group, and the remaining rats without joint swelling were randomly assigned to each group. The drugs were administered by gavage. The blank control group and CIA group were given 10 mL purified water per kg of weight; the MTX group was given 1.5 mg MTX (Shanghai Sine Pharmaceutical Co) per kg of weight; and the KW2449 group was given 7 mg KW2449 (APExBIO) per kg of weight. All the above gavages were given once daily, and the rats were sacrificed after 28 days of continuous administration. The animal study was reviewed and approved by the Experimental Animal Ethics Committee of China-Japan Friendship Hospital with the ethical approval number of zryhyy21-21-11-02.

The murine macrophage-like cell line RAW 264.7 was obtained from the ATCC. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% (v/v) fetal bovine plasma (Gibco) with 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco) and 100 μg/ml Primocin (In vivoGen) at 37°C in a humid atmosphere with 5% CO₂. Then, RAW 264.7 cells were reseeded in 96-well or 6-well culture plates at densities of 1×10⁵ or 1×10⁶ cells/well and used for follow-up experiments. RAW 264.7 cells were stimulated with LPS (1 μg/ml) to establish a cell inflammation model and LPS (1 μg/ml) plus zVAD (20 μM; APExBIO) to establish a necroptosis model, with or without KW2449 (1 μM) or RIPA56 (1 μM; MedChemExpress LLC). The cell supernatant was collected 24 h after drug addition, and the cells were collected at the corresponding time points for detection.

RAW 264.7 cells seeded in 96-well culture plates were incubated with different concentrations of KW2449 or DMSO for 24 h. Then, 10 µl Cell Counting Kit-8 (CCK-8; Dojindo) reagent was added to each well for an additional 1.5 h at 37° in the dark. The absorbance at 450 nm was measured with a microplate reader (Bio-Tek Instruments), and the cell viability was calculated using the formula in the manufacturer’s protocol.

Total RNA was extracted using TRIzol (Invitrogen) and then reverse transcribed into cDNA. A Quant Studio 5 Real-Time PCR System (Thermo Fisher Scientific) was used for the qRT-PCR assay. The reaction conditions were set as 95 ° for 30 s followed by 40 cycles of 95 ° for 5 s and 60 ° for 34 s. The relative expression of genes was calculated using the 2^-△△Ct formula with β-actin as an internal control. The primers were synthesized by Sangon Biotech (Sangon Biotech).

The NO content in the cell supernatant was detected by an NO assay kit (Beyotime). The reagent was added and incubated at room temperature for 10 to 20 min according to the instructions. The absorbance at 540 nm was immediately measured with a microplate reader (Bio-Tek Instruments).

The levels of TNF-α, IL-6 and IL-1β in the cell supernatant were detected by an ELISA kit (ABclonal) according to the manufacturer’s instructions. RIPK1 and MLKL levels in human plasma and cell supernatant were detected by an ELISA kit (Cloud-Clone Corp) according to the manufacturer’s instructions.

IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, GM-CSF, G-CSF, IFN-γ, IL-17A/CTLA-8 and TNF-α levels in rat plasma were measured using a Th Complete 14-Plex Rat ProcartaPlex Panel (Thermo Fisher Scientific) according to the manufacturer’s instructions. Fluorescence was detected using a Luminex 200 system (Luminex Corporation) and analyzed using ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific). Only the cytokines that were above the detection limit were used in the analysis.

Cells were collected 6 h after drug intervention and lysed for 30 min on ice by RIPA lysis buffer with 1% protease and protein phosphatase inhibitor cocktail (Beyotime Biotechnology). Proteins were loaded onto 12% Super-PAGE Bis-Tris gels, separated by electrophoresis and then transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk for 1 h at room temperature, followed by incubation with antibodies against RIPK1, p-RIPK1, p-RIPK3, p-MLKL (Cell Signaling Technology, Cat# 3493, 31122, 91702, 37333), RIPK3, MLKL (Abcam, Cat# ab62344, ab243142) and β-actin (Santa Cruz Biotechnology Dallas, Cat# sc-47778) at 4 ° overnight. The proteins on the membranes were detected with a ChemiDoc XRS+ Gel Imaging System (Bio-Rad), after 2 hours of incubation with HRP-coupled goat anti-rabbit, goat anti-rat or goat anti-mouse secondary antibodies.

The cells in each group were incubated with fluorescein isothiocyanate-labelled annexin V (Annexin-V-FITC) and propidium iodide (PI) using an apoptosis kit according to the manufacturer’s instructions (BD Biosciences, Cat# 556547). After 15 min of incubation in the dark at room temperature, cell death was analyzed by flow cytometry (FACS; BD Biosciences).

Cells in each group were double-stained with acridine orange/ethidium bromide (AO/EB) according to the instructions. The cell morphology was observed by an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). A minimum of 200 cells per area were observed and counted separately by the following four morphologies: viable, early apoptotic, late-apoptotic and necroptotic cells (25).

Ripk1 siRNA and negative control siRNA were obtained from Gene Pharma (Suzhou, China). The sequences of Ripk1-mus-1633 siRNA were 5′- GCAGAGAGCUCGUGAGAAUTT-3′ (forward) and 5′- AUUCUCACGAGCUCUCUGCTT-3′ (reverse). The cells were stimulated with LPS (1 μg/ml) plus zVAD (20 μM) (LZ) 24 hours after transfection by Lipofectamine 3000 (Invitrogen Life Technologies), and the expression of RIPK1 was measured by qRT−PCR after another 24 hours. Cells were stimulated with LZ 72 hours after transfection, and RIPK1 expression was measured by WB 6 hours later.

GraphPad Prism software (version 9.0) was used for statistical analyses. All data are presented as the mean ± SEM. The two-tailed Student’s t test was used for comparisons between two groups, and Welch’s T test was used for nonnormally distributed data. One-way ANOVA was used to compare the data between three or more groups, and Brown-Forsythe and Welch ANOVA tests were used for nonnormal distributions. Statistical significance was considered when the P value < 0.05.

To determine the efficacy of KW2449 on RA, methotrexate (MTX), which has been considered effective in the clinical treatment of RA for many years, was selected as a positive control drug (26, 27). After 28 days of intragastric administration, we found that both KW2449 and MTX showed obvious therapeutic effects on CIA rats, not only improving the severity of joint swelling and reducing the arthritis score ( Figures 1A, B ) but also reducing the release of inflammatory cytokines in the plasma of CIA rats, such as interleukin-17A (IL-17A), interleukin-1α (IL-1α), interleukin-2 (IL-2) and interleukin-6 (IL-6) (p<0.05; p<0.01) ( Figure 1E ). Histopathological results showed that compared with the blank control group, the ankle joint structure of rats in the CIA group was damaged, and the erosion of cartilage and bone was serious, accompanied by a large amount of pannus formation, synovial hyperplasia and inflammatory cell infiltration. After KW2449 or MTX treatment, these pathological characteristics improved, and the histopathological score decreased significantly (p<0.05) ( Figures 1A, C ). To further evaluate bone destruction, we performed three-dimensional reconstruction of the rat ankle joint by microcomputed tomography (micro-CT), which showed significant bone destruction and increased BS/BV values (p<0.01) in the CIA group, which were improved after treatment with KW2449 or MTX (p<0.01) ( Figures 1A, D ). This showed a protective effect in both treatment groups. These results indicate that KW2449 can inhibit the progression of CIA rats. It can alleviate joint bone destruction and joint tissue damage in CIA rats and reduce the release of plasma inflammatory cytokines. We next explored the mechanism by which KW2449 exerted its therapeutic effects on CIA rats.

It has been reported that RIPK1-mediated necroptosis is associated with autoimmune diseases (4–7). To verify whether RIPK1-mediated necroptosis also has the same effect in RA patients, we detected and compared the plasma levels of RIPK1 and MLKL in RA patients and healthy controls. We found that the plasma levels of RIPK1 and MLKL in RA patients were significantly higher than those in healthy people, and the plasma levels of RIPK1 and MLKL in the severe group were higher than those in the mild-moderate group (p<0.05; p<0.01) ( Figures 2A, B ). These results suggest that the RIPK1-mediated necroptosis pathway plays a role in the pathogenesis of RA and is positively correlated with disease severity.

We detected the plasma levels of RIPK1 and MLKL in each group of rats and found that their levels in the CIA group were significantly higher than those in the control group (p<0.05; p<0.01) ( Figures 2C, D ). This suggests that RIPK1-mediated necroptosis plays an important role in the pathogenesis of CIA. The levels of RIPK1 and MLKL decreased to varying degrees after treatment with KW2449 (p<0.05), but there was no significant change in the MTX treatment group ( Figures 2C, D ), suggesting that KW2449, but not MTX, had an inhibitory effect on RIPK1.

The proteins involved in the RIPK1-mediated necroptosis pathway were examined in the LPS-induced inflammation model and LZ-induced necroptosis model in vitro. To clarify the inhibitory effect of KW2449 on RIPK1, the type III RIPK1 inhibitor RIPA56 was selected as the positive control in the cell experiments (18, 28). The results showed that the levels of proteins involved in the RIPK1-mediated necroptosis pathway were slightly increased after LPS induction, but the difference was not statistically significant, and there was no significant change after treatment with RIPA56 or KW2449 ( Figures 2E, F ). However, after LZ induction, the levels of RIPK1, RIPK3 and MLKL increased significantly (p<0.05; p<0.01), accompanied by increased levels of p-RIPK1, p-RIPK3 and p-MLKL (p<0.05; p<0.01), which were decreased to varying degrees after treatment with RIPA56 or KW2449 (p<0.05; p<0.01) ( Figures 2E, F ). This observation illustrates the inhibitory effect of KW2449 on the RIPK1-mediated necroptosis pathway. Moreover, these results suggest that necroptosis cannot be induced by LPS stimulation alone and that a caspase inhibitor, such as zVAD, must be added. Based on the in vivo and in vitro experiments, it is reasonable to believe that the mechanism by which KW2449 exerts its therapeutic effect on CIA rats is the inhibition of RIPK1-mediated necroptosis.

RIPK1 is involved in the regulation of multiple signaling pathways, such as inflammation, apoptosis and necroptosis (13, 14). Therefore, for the previously detected inhibitory effect of KW2449 on inflammation, it is difficult to tell whether it acts directly on RIPK1-mediated inflammation or by acting on necroptosis to reduce the release of inflammatory cytokines. To this end, we examined the changes in inflammatory cytokines in the LPS-induced inflammation model and LZ-induced necroptosis model after KW2449 treatment. First, we found that KW2449 was non-toxic up to 1 µM, but began to show toxicity to cells at 10 µM by CCK-8 assay, so we selected 1 µM for subsequent cell experiments ( Figure 3A ). Second, the efficacy against LZ-induced necroptosis was detected at 6 h, 12 h, 18 h and 24 h ( Figure 3B ). The results showed that KW2449 had a better inhibitory effect on RIPK1 at 12 h and 18 h and reduced the expression of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6 and interleukin-1β (IL-1β), at the mRNA level ( Figure 3B ). In parallel, we also examined the effects of KW2449 or RIPA56 on LZ-induced necroptosis at 12 h and found that both had similar inhibitory effects on inflammatory cytokines at the mRNA level ( Figure 3D ). Although the changes in RIPK1 at the mRNA level after LZ stimulation were small, they had a clear effect on its protein level and the expression of inflammatory cytokines ( Figures 2E, F , 3C, D ). Finally, by measuring the expression of inflammatory cytokines in the cell supernatant, we found that both RIPA56 and KW2449 showed weak inhibitory effects on inflammatory cytokines in the LPS-induced inflammation model, and only when zVAD was added to induce necroptosis did they show significant inhibitory effects (p<0.01) ( Figure 3C ). In addition, we found that KW2449 or RIPA56 reduced the release of nitric oxide (NO), another inflammation-related marker, in both LPS-induced and LZ-induced models, although the effect was more pronounced in the LZ-induced model (p<0.01) ( Figure 3E ), which may be due to its complex network relationship (29). Collectively, we found that KW2449 exerted a stronger anti-inflammatory effect on LZ-induced necroptosis than on LPS-induced inflammation in RAW 264.7 cells. This suggests that the mechanism by which KW2449 reduces inflammatory cytokines is mainly through the inhibition of RIPK1-mediated necroptosis rather than a direct effect.

At present, many types of regulated cell death have been found (8), and whether KW2449 acts on LZ-induced necroptosis or apoptosis was further verified in our experiments by cell staining and morphological observation. To avoid natural cell apoptosis in the experiment as much as possible, we chose to detect and analyze the cells 24 h rather than 48 h after the addition of the drug. Cells were stained with Annexin V-FITC/PI, and the proportion of different cell death types in each group was detected using flow cytometry ( Figures 4A–E ). We identified AV+PI- cells as apoptotic cells and AV+PI+ cells as necroptotic cells and counted them. We found that the main cause of cell death after LPS stimulation alone was apoptosis, while LZ stimulation produced more necroptosis. After treatment with KW2449 or RIPA56, the proportion of apoptotic cells did not change significantly, but the number of necroptotic cells was significantly reduced (p<0.01) ( Figures 4F–H ).

Because the use of flow cytometry alone cannot distinguish between late-apoptotic and necroptotic cells, our counting of necroptotic cells inevitably includes with the presence of late-apoptotic cells. To distinguish between different types of cell death, AO/EB staining was performed on the cells, and the morphology and staining of the cells were analyzed by a high-content imaging system ( Figure 5A ). The results showed that necroptosis was the main cause of cell death in the LZ-induced cell model, and the proportion of late-apoptotic cells was low. Therefore, the effect of late-apoptotic cells mixed in necroptotic cell count was negligible when detected by flow cytometry, and the proportion of AV+PI+ cells could appropriately represent the proportion of necroptotic cells in this experiment ( Figures 5A, B ). Moreover, treatment with KW2449 or RIPA56 resulted in increased overall cell viability and decreased cell death, with a significant reduction in the number of necroptotic cells (p<0.01), whereas the number of late-apoptotic cells was too low to be discussed here ( Figures 5B, C ). These results clarify that the inhibition of RIPK1 by KW2449 improves necroptosis rather than apoptosis.

After confirming the effect of KW2449 on RIPK1 and necroptosis, the role of RIPK1 in the necroptosis pathway was further clarified. Multiple upstream and downstream proteins are involved in the necroptosis pathway, including RIPK1, RIPK3 and MLKL. To further verify whether RIPK1 is essential in this pathway, we used small interfering RNA (siRNA) to knock down RIPK1 and detected the expression of inflammatory cytokines in each group. The results showed that the levels of inflammatory cytokines were increased in the LZ model compared to the control group, and RIPK1 knockdown significantly reduced these increased inflammatory cytokines at both the mRNA and protein levels (p<0.05; p<0.01) ( Figures 6A-D ). These results suggest that RIPK1 is critical for LZ-induced necroptosis and that the use of the RIPK1 inhibitor KW2449 is an effective way to improve the inflammation caused by necroptosis.