14-3-3η Induces a Multidrug-Resistant Phenotype in Rheumatoid Arthritis via p53 Dysregulation: Insights from AIA Models

14-3-3η Induces a Multidrug-Resistant Phenotype in Rheumatoid Arthritis via p53 Dysregulation: Insights from AIA Models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article 14-3-3η Induces a Multidrug-Resistant Phenotype in Rheumatoid Arthritis via p53 Dysregulation: Insights from AIA Models Vincent Kam Wai Wong, Li Jun Yang, Wei Zhang, Yuan Qing Qu, Yuping Wang, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6528370/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rheumatoid arthritis (RA) is a chronic autoimmune condition complicated by drug resistance issues, significantly challenging its treatment. The protein 14-3-3η has been noted for its association with drug resistance in various cancers, and while it is particularly expressed at high levels in RA patients, its role in the disease's pathogenesis is not well understood. This study explores how 14-3-3η contributes to drug resistance in RA, particularly focusing on its interactions with the p53 protein. Through techniques including qPCR, Western blot, ELISA, BLI, immunoprecipitation, and the use of adjuvant-induced arthritis (AIA) transgenic models, we observed elevated levels of 14-3-3η in the peripheral blood mononuclear cells (PBMCs) of RA patients, correlating with both disease activity and duration. Enhanced expression of 14-3-3η was found to induce resistance to methotrexate (MTX) in AIA rats, which could be countered by treatment with arsenic trioxide (ATO). Our mechanistic studies suggest that the overexpression of 14-3-3η promotes a multidrug resistance phenotype in rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) through the downregulation of p53. Furthermore, adenovirus-mediated p53 supplementation in AIA rats was able to mitigate and reverse the MTX resistance caused by 14-3-3η. These findings delineate a regulatory function for 14-3-3η in the pathogenesis and drug resistance of RA via p53 modulation, proposing it as a viable target for treating drug-resistant RA. Biological sciences/Immunology/Autoimmunity Health sciences/Biomarkers/Predictive markers 14-3-3η Methotrexate resistance p53 Adjuvant-induced arthritis RAFLS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Rheumatoid arthritis (RA), the most prevalent chronic inflammatory disease, primarily triggers joint destruction which results in persistent physical disability and constantly developing irreversible dysfunction[ 1 – 3 ]. This complex pathology, affecting an estimated 1% of the global populace, imposes a substantial socioeconomic burden due to enduring synovitis, cartilage breakdown, and bone erosion[ 4 ]. Despite no definitive cure for RA, disease conditions can be ameliorated. Traditional treatments aim to sustain clinical remission with low activity by administering disease-modifying anti-rheumatic drugs (DMARDs), such as methotrexate (MTX) and leflunomide (LEF), biologic DMARDs, nonsteroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids[ 5 ]. For instance, DMARDs and biologic DMARDs achieve disease-modifying ability by obstructing with inflammatory signaling pathways. NSAIDs temper inflammation, pain and swelling, by inhibiting cyclooxygenase activity but are not known to improve joint damage. Glucocorticoids also aid in modulating inflammation but lack disease-modifying activity[ 6 ]. The European League Against Rheumatism (EULAR) recommends the use of DMARDs as first-line treatments, which target proinflammatory mediators such as tumor necrosis factor-a (TNF-α) and interleukin (IL)-6[ 5 ]. However, biologic DMARDs are recommended when the first DMARD regimen fails to achieve treatment outcomes with poor prognostic features, such as persistent moderate or high disease activity, high acute phase reactant levels, and a high swollen joint count[ 5 ]. Nevertheless, long-term drug treatment increases drug resistance due to the chronic nature of RA[ 7 ]. With 20–30% of RA patients refractory to existing antirheumatic drugs[ 5 ], thus necessitates new treatment strategies for drug-resistant RA. Despite significant advancements in RA management over the past thirty years, a notable number of patients do not benefit from multiple DMARDs. This subset of patients often has 'difficult to treat', 'resistant', or 'refractory' RA[ 8 ]. Factors contributing to antirheumatic treatment resistance in these kinds of RA include mutations in the p53 tumor suppressor gene and overexpression of ABCB1/MDR-1/ P-gp transporters. Our previous research emphasized the role of p53 gene mutations, ABC family transporters, and personal factors in antirheumatic drug resistance, which could lead to development of new personalized RA therapies with enhanced drug sensitivity[ 9 ]. We further elucidated the part p53 mutations (such as R202S, R213*, and R248Q) play in promoting MTX resistance, in which mutations have been identified in RA patients[ 10 ]. However, our recent work has unraveled how the p53 R213* mutant affects RA pathogenesis, demonstrating that this mutant can mitigate inflammatory arthritis in AIA rats through inhibition of the TBK1 IRF3 innate immune response[ 11 ]. When conventional DMARDs prove ineffective, biologic DMARDs are often recommended. However, some patients remain resistant, with 40% of RA patients unresponsive to individual biologic therapies, and 5–20% refractory to all such treatments[ 12 ]. The 14-3-3 protein family, which consists of seven isoforms (β, γ, ε, η, τ, ζ, and σ) widely expressed within cellular chaperonin in eukaryotic cells, regulates multiple cellular functions such as cell cycle progression, maintenance of DNA damage checkpoints, apoptosis, and cytoskeletal dynamics, by binding to the Ser-X-pSer or Ser-X-pThr (X represents an arbitrary amino acid) residue[ 13 – 15 ]. Recent research uncovered an important role of the 14-3-3 isoforms (ζ, σ and η), linked with target chemotherapy and drug resistance in cancer, in the multidrug resistance phenomenon in hepatocellular carcinoma[ 16 , 17 ]. Furthermore, 14-3-3η had been reported to be a novel RA-related biomarker that induces the expression of multiple inflammatory factors, thereby facilitating the pathogenesis of RA[ 18 ]. Emerging knowledge regarding the roles of 14-3-3η in RA and its clinical implications as a diagnostic, prognostic and therapeutic response surrogate as well as potential drug target for RA treatment[ 19 ]. Interestingly, this protein isoform was found to be overexpressed in RA, suggesting its role in the disease. This study investigates the pathological contribution of 14-3-3η to drug resistance in autoimmune arthritis. Our study shows that 14-3-3η overexpression via AAV5 nullifies the therapeutic impact of MTX, leading to extreme paw swelling, significant hind paw volume and arthritis score increase, and exacerbated bone erosion in arthritic joints. Consistent with our findings in transgenic knock in (KI) rats, the inducible expression of 14-3-3η in AIA 14 − 3−3η (+/+) rats could also result in the release of proinflammatory cytokines and severe arthritic conditions in AIA rats with MTX treatment. Notably, these effects can be reversed with arsenic trioxide (ATO)-a tool drug known to eliminate 14-3-3η via ubiquitin-degradation. Mechanistic studies revealed that the overexpression of 14-3-3η contribute to apoptosis resistance by reducing the gene expression of p53. AAV-mediated overexpression of p53 markedly reversed the drug-resistant phenotype of AIA 14 − 3−3η (+/+) rats. Collectively, this study thus proposes 14-3-3η as a potential therapeutic target for drug-resistant rheumatoid arthritis or other inflammatory diseases. Results Elevated 14-3-3η levels have been found in the peripheral blood mononuclear cells (PBMCs) of RA patients The relationship between 14-3-3η expression and chronic joint inflammation in RA patients was investigated using qPCR to detect 14-3-3η mRNA levels in PBMCs from RA patients, osteoarthritis (OA) patients, and healthy individuals. As shown in Fig. 1 A, the 14-3-3η mRNA expression level in PBMCs of RA patients ranged from moderate to high, unlike in normal or PBMCs of OA patients where expression levels were marginal. This suggests that 14-3-3η may hold a regulatory role in the pathogenesis of RA but not in OA. Given the chronic nature of RA, drug therapy is often required for many years, which can potentially lead to the development of drug resistance[ 20 ]. Considering that 14-3-3η has been shown to promote drug resistance in hepatocellular carcinoma[ 16 ], we examined the correlation between 14-3-3η expression and disease duration in RA. Our results indicated a positive association between the two, i.e., increased 14-3-3η expressions corresponds to extended disease duration (Fig. 1 B). Furthermore, some RA patients with high disease activity do not respond to treatment with DMARDs or oral corticosteroids[ 21 ], although the mechanism of this drug resistance is not clear. To explore the link between drug resistance and the expression of 14-3-3η, we investigated the correlation between disease activity as rated by DAS28 scores and 14-3-3η expression in PBMCs of RA patients. As shown in Fig. 1 C, a close correlation was identified between higher 14-3-3η expressions with the more severe disease activity. Simultaneously, several RA patients with high disease activity (DAS28>4.7) also exhibited extremely high expression of 14-3-3η in their PBMCs. These findings suggest 14-3-3η maybe a potential target for therapeutic intervention in severe RA, symbolizing an untapped opportunity in the field. Adeno-associated virus (AAV)-mediated overexpression of 14-3-3η contributes to MTX (methotrexate) resistance in adjuvant-induced arthritis (AIA) rats, whereas Arsenic trioxide (ATO) reverses 14-3-3η mediated MTX resistance in AIA rats Our previous studies demonstrated that 14-3-3η plays an important role in inducing and maintaining the anti-oxidation/MDR state in HCC[ 16 ]. Interestingly, gene expression analysis on clinical samples demonstrated a significant increase in 14-3-3η expression in RA patients and also indicated a positive correlation between 14-3-3η expression and disease duration in RA patients (Fig. 1 A-B). To further address the role of 14-3-3η in AIA rats, one of the most commonly used standard arthritis models and reflecting a number of clinical characteristics of RA in humans[ 22 ], we created a treatment timeline for the in vivo study of 14-3-3η in an AIA rat model (Fig. 1 D). To overexpress the 14-3-3η protein in AIA rats, articular injections of an adeno-associated virus 5 (AAV5) encoding 14-3-3η were conducted in the AIA rat model (Fig. 1 E). For AIA animal immunization, 8 week old male SD rats were immunized as described previously[ 23 ]. After that, the knee of each AIA rat was intra-articularly injected with a low dose of 3 × 10 9 PFU of AAV5-14-3-3η, a high dose of 1 × 10 11 PFU of AAV5-14-3-3η, or AAV5-null (a virus vector control) prior to adjuvant mixture injection on the same day. MTX (7.6 mg/kg/week) was orally administered to AIA rats as a positive control. On the other hand, ATO was used as a tool drug to test whether ATO-induced degradation of 14-3-3η[ 16 ] could reverse the MTX resistance phenotype in AIA rats. For this purpose, ATO (6 mg/kg/3 days) was intraperitoneally administered to AIA rats overexpressing 14-3-3η in the presence of MTX. Inflammatory symptoms were assessed every 3 days to monitor hind paw volume and arthritic scoring, and the AIA rats were then sacrificed on day 30. As shown in Fig. 1 F-G, AIA rats treated with AAV5-null (i.e., model group) first displayed the arthritic symptoms characterized by edema and/or erythema in the paws around day 12 after immunization and showed severe paw swelling by day 27, suggesting that the AIA model control group was successfully established. As shown in Fig. 1 F-H, AIA rats were intra-articularly injected with AAV5-null and gavage feeding with the same volume of normal saline as a model control. The paw edema of the rats was significantly greater than that of the healthy controls. AIA rats intra-articularly injected with a high dose of AAV5-14-3-3η were also observed the similar degree of paw edema, hind paw volume and arthritic score compared with those of the model control group, suggesting that the overexpression of 14-3-3η did not aggravate the inflammatory response. As expected, MTX treatment markedly suppressed the paw edema of the AIA rats compared with that of the model control group. However, AAV-mediated overexpression of 14-3-3η at both low and high doses markedly abolished the anti-arthritic effects of MTX, leading to severe paw edema and increased hind paw volume and arthritis score in AIA rats in a dose-dependent manner, suggesting that overexpression of 14-3-3η might contribute to MTX treatment failure. In addition, ATO was used as a tool drug to treat AIA rats overexpressing 14-3-3η in the presence of MTX. Of note, the paw edema, hind paw volume and arthritis score were significantly improved after ATO/MTX combined treatment in comparison with MTX treatment alone, suggesting that ATO might bind and trigger the degradation of 14-3-3η, thereby reversing MTX resistance phenotypes in AIA rats. Meanwhile, the AAV5-mediated expression of 14-3-3η in synovial tissues and PBMCs were also validated by qPCR. As shown in Fig. 1 H, AAV5-14-3-3η injection significantly elevated the expression of the 14-3-3η mRNA in both synovial tissues and PBMCs from AIA rats, with a 3- to 4-fold increase in the 14-3-3η mRNA level compared with the levels in the AIA-AAV5-null group, confirming that AAV5-14-3-3η was also successfully overexpressed in AIA rats. Effects of 14-3-3η overexpression and ATO treatment on bone destruction in the paw articulations of MTX-resistant AIA rats To determine the severity of bone destruction in AIA rats overexpressing 14-3-3η with or without ATO treatment, all treatment groups of AIA rats were subjected to radiological examinations by micro-CT analysis. Representative radiographs of hind paws from the 7 different groups are shown in Fig. 2 A. The severity of bone destruction in the AIA rats were further quantified and compared in the analysis of bone mineral density (BMD), cortical mineral density (TMD), trabecular number (Tb.N), and total porosity among all treatment groups (Fig. 2 B). Overall, the micro-CT data demonstrated that cartilage destruction and bone erosion were severe in the AIA rats either with or without injection of AAV5-14-3-3η. MTX treatment drastically inhibited bone erosion in arthritic joints compared with that in the model group. In contrast, the MTX-treated AIA rats injected with a low dose or high dose of AAV5-14-3-3η exhibited poor therapeutic effects, leading to an aggravated bone erosion in arthritic joints (Fig. 2 A-B), indicating that the overexpression of 14-3-3η in AIA rats contributes to MTX treatment failure. Conversely, MTX combined with ATO treatment successfully reversed 14-3-3η-mediated MTX resistance in AIA rats, as indicated by the recovery of severe bone destruction in the AIA model. Taken together, these results suggested that the overexpression of 14-3-3η contributes to MTX resistance in AIA rats, whereas treatment with ATO could reverse bone erosion in MTX-resistant AIA rats. Accordingly, ATO may show a good protective effect on paw articulation in 14-3-3η-mediated MTX-resistant AIA rats. As shown in Fig. 2 C, stenosis of the articular cavity and synovial hyperplasia appeared in the AIA rats injected with the AAV5-null virus control group compared with those in the healthy control group, which exhibited a normal articular cavity and a smooth surface of cartilage. Notably, the overexpression of 14-3-3η in AIA rats did not cause further severe cartilage destruction or bone erosion, suggesting that the overexpression of 14-3-3η does not increase the severity of inflammation in the AIA model. Apparently, AIA rats treated with MTX alone acquired a normal articular cavity and exhibited lowest synovial hyperplasia and inflammatory cell infiltration. Besides, when the AIA rats treated with either a low dose or high dose of AAV5-14-3-3η, MTX treatment failure was found and followed by cartilage destruction, synovial hyperplasia and immune cell infiltration compared with those in AIA rats treated with MTX alone. Collectively, the overexpression of 14-3-3η can lead to MTX resistance in AIA rats. However, ATO treatment completely reversed these MTX-resistant phenotypes in AIA rats, regardless of the degree of stenosis in articular cavity, synovial hyperplasia or infiltration of immune cells. Gene expression analysis of proinflammatory cytokines in synovial tissues or blood samples from ATO-treated AIA rats overexpressing 14-3-3η To further evaluate the inflammatory potency of 14-3-3η expression in AIA rats, the levels of proinflammatory markers were examined in blood serum, synovial tissues and peripheral blood mononuclear cells (PBMCs) from AIA rats. As shown in Fig. S1 A-C , the gene expression of TNF-α, IL-1β, IL-2, IL-6, IL-8 and MCP-1 was upregulated in the AIA rats injected with the AAV5-null virus control compared with the healthy controls. The gene expression of 14-3-3η was also monitored in all treatment groups to confirm the level of 14-3-3η gene expression after AAV5-14-3-3η injection. Notably, the overexpression of 14-3-3η in AIA rats induced by the injection of a high dose of AAV5-14-3-3η did not further increase the gene expression of inflammatory cytokines, suggesting that the overexpression of 14-3-3η does not increase the severity of inflammation in the AIA model. Apparently, AIA rats treated with MTX alone exhibited markedly suppressed gene expression of the above proinflammatory cytokines in the synovium. However, MTX treatment failed to suppress the expression of these proinflammatory genes in AIA rats injected with either a low dose or a high dose of AAV5-14-3-3η compared with AIA rats treated with MTX alone, suggesting that the overexpression of 14-3-3η can lead to MTX resistance in AIA rats. Consistently, ATO treatment effectively reversed these MTX-resistant in AIA rats in which the gene expression of these inflammatory cytokines in synovial tissue or PBMCs was significantly inhibited. Taken together, 14-3-3η might be an important target leading to MTX treatment failure in AIA rats. Moreover, ATO, a tool drug facilitating the proteasomal degradation of 14-3-3η, abolished 14-3-3η-mediated MTX resistance in AIA rats. Examination of matrix metalloproteinases expression in synovial tissue or PBMCs from ATO-treated AIA rats overexpressing 14-3-3η Many studies have reported the involvement of matrix metalloproteinases (MMPs) in inflammatory diseases, including RA. The cytokines mentioned above can induce the expression of MMPs and proteinases, which are largely responsible for the irreversible destruction of cartilage, bone and tendons in the joints [ 24 ]. In addition, Receptor activator of nuclear factor-kappa B ligand (RANKL) belongs to the TNF superfamily, is an activated T-cell–producing factor that modulates dendritic cell survival and plays an essential role in osteoclast biology [ 25 ]. Of note, the expression of RANKL is increased in the synovial tissues of patients with RA[ 26 ], it plays an essential role of the RANKL–RANK pathway in arthritic bone destruction of a series of animal experiments[ 27 ]. As shown in Fig. S2 , the expression of MMP-1, MMP-3, MMP-9 and RANK in the synovium or PBMCs of the model group was markedly elevated compared with that in the healthy control group. The mRNA expression of the abovementioned genes in the synovium or PBMCs of AIA rats was markedly suppressed by MTX treatment. In contrast, when the AIA rats were treated with either a low dose or a high dose of AAV5-14-3-3η, MTX treatment did not suppress the expression of these genes. Similarly, in MTX-resistant AIA rats treated with ATO, the expression of genes encoding key matrix metalloproteinases in synovial tissue was significantly suppressed. These findings indicated that ATO may protect cartilage and bone tissue by inhibiting the 14-3-3η-mediated mRNA expression of MMP-1, MMP-3 and MMP-9, as well as suppressing the level of RANKL. Conditional knock-in of 14-3-3η contributes to MTX resistance in AIA rats Alternatively, we further elucidated the function of 14-3-3η in AIA model by generation of transgenic SD rats using the Tet-On system. The tet regulatory system in this approach permitted tissue-specific and doxycycline-inducible control of 14-3-3η expression in transgenic rats. As shown in Fig. 3 A-B, the AIA arthritic models were established using 14-3-3η +/+ SD rats. Consistent with the finding that AAV-mediated overexpression of 14-3-3η contributes to MTX resistance in AIA rats, the arthritis score, degree of paw edema and degree of bone destruction were significantly greater in the AIA WT group than in the healthy control WT group. In MTX treated AIA WT rats, the arthritis score, paw edema and bone destruction of the rats were markedly lower than those in the AIA WT group. However, in AIA rats overexpressing 14-3-3η (AIA 14 − 3−3η+/+ ) with MTX treatment, transgenic knock-in of 14-3-3η completely abolished the therapeutic effect of MTX, leading to severe paw edema, severe arthritis and severe bone erosion compared with those in AIA WT+MTX group, demonstrating that knock-in of 14-3-3η could lead to MTX resistance. On the other hand, we observed that the paw edema, hind paw volume, arthritis score and bone destruction were significantly recovered after ATO combined with MTX treatment, compared with the group of AIA 14 − 3−3η+/+ (MTX) (Fig. 3 A-D ). Meanwhile, the erythrocyte sedimentation rate (ESR) also matched very well with the arthritis condition of MTX-treated 14-3-3η knock-in AIA rats with or without ATO combined treatment (Fig. 3 E ) . On the other hand, the release of proinflammatory cytokines and matrix metalloproteinases was higher in the AIA 14 − 3−3η+/+ (MTX) group than in the AIA WT+ (MTX) group. However, when MTX-resistant AIA 14 − 3−3η+/+ (MTX) rats were co-treated with ATO, the gene expression of these inflammatory cytokines in PBMCs was significantly inhibited ( Fig. S3A) . Finally, the gene expression of 14-3-3η were also validated in the PBMCs of the AIA 14 − 3−3η+/+ (MTX) transgenic rats ( Fig. S3B ). ATO treatment enhances the proteasomal degradation of 14-3-3η via ubiquitination in RAFLSs Our previous work demonstrated that ATO targeted 14-3-3η for its degradation in hepatocarcinoma cells[ 16 ]. To investigate whether ATO treatment affects the expression of 14-3-3η in RAFLSs, we determined both the mRNA and protein expression of 14-3-3η in RAFLSs upon ATO treatment. Firstly, cell viability assay demonstrated that ATO inhibited the growth of RAFLSs in a dose-dependent manner, with an IC 50 of 26 ± 0.49 µM (Fig. 4 A). In addition, qPCR analysis indicated that the mRNA expression of 14-3-3η was dose-dependently downregulated upon treatment with different concentrations of ATO (0, 5, 10, 15, 20, or 25 µM) for 24 h (Fig. 4 B). Furthermore, Western blot analysis also indicated that the addition of ATO dose-dependently suppressed the protein expression of 14-3-3η (Fig. 4 C). Here, we further validated the binding affinity of ATO on the 14-3-3η protein. As shown in Fig. 4 D, ATO dose-dependently bound to 14-3-3η, and the binding curves of 14-3-3η suggested that a simple 1:1 binding mode occurred, with R 2 = 0.9964. The results of BLI analysis suggested that 14-3-3η exhibits good binding affinity for ATO. Therefore, these findings revealed that ATO could target 14-3-3η and inhibit its gene and protein expression in RAFLSs. Based on the abovementioned results, ATO treatment suppressed the gene transcription and protein expression of 14-3-3η. To further investigate whether ATO treatment leads to the proteasomal degradation of 14-3-3η, the RAFLSs overexpressed with 14-3-3η were treated with the 26S proteasome inhibitor MG132[ 28 ], in the presence or absence of ATO. The 14-3-3η protein was immunoprecipitated with a specific antibody and then probed with antibody against ubiquitin. Compared with no ATO treatment, ATO treatment markedly enhanced the ubiquitination of 14-3-3η in RAFLSs (Fig. 4 E). These findings suggested that ATO facilitates the proteasomal degradation of 14-3-3η via ubiquitination. The overexpression of 14-3-3η contributes to MDR phenotypes in RAFLSs Apart from animal studies of the drug-resistant potency of 14-3-3η, the multidrug resistance (MDR) effects of overexpressing 14-3-3η in RAFLS cells in response to many anti-arthritic drugs, such as MTX, hydroxychloroquine (HCQ), leflunomide (LEF), tacrolimus, and sulfasalazine (SSZ), were also examined. For this purpose, RAFLSs were transfected with a control vector or plasmid containing 14-3-3η-Flag. As shown in Fig. 5 A, the Western blot results demonstrated that 14-3-3η was successfully overexpressed in RAFLSs. Cell viability assays demonstrated that the RAFLS cells transfected with 14-3-3η-Flag-tagged plasmids were more resistant to anti-arthritic drugs (MTX, HCQ, LEF, SSZ and tacrolimus) than the RAFLS cells transfected with the control vector (Fig. 5 A). The IC50s (µM) and resistance factors of these five drugs for the overexpression of 14-3-3η in RAFLS cells were as follows: MTX (11.59 ± 0.87 µM vs. over 100 µM with resistance factor > 8.63), HCQ (30.9 ± 0.23 µM vs. over 100 µM with resistance factor > 3.24), LEF (16.6 ± 0.17 µM vs. 47.9 ± 0.32 µM with resistance factor 2.89), SSZ (26.9 ± 0.25 µM vs. 33.9 ± 0.47 µM with resistance factor 1.26) and tacrolimus (12 ± 0.24 µM vs. 28.8 ± 0.23 µM with resistance factor 2.40). These results demonstrated that the DMARD drugs MTX, HCQ, LEF, SSZ, and tacrolimus are insensitive to RAFLSs in the presence of 14-3-3η. PCR array analysis identifies p53 tumor suppressor gene as drug resistance-related gene in RAFLSs overexpressing 14-3-3η To explore the target drug resistance genes in 14-3-3η-overexpressing RAFLS cells, a total of 84 genes ( Supplementary Table 2 ) related to drug resistance were compared between the control vector- and 14-3-3η-Flag-tagged plasmid-transfected RAFLS cells via real-time PCR microarray of Human Cancer Drug Resistance PCR Array Targeted RNA Virtual Panels. Western blot analysis demonstrated that 14-3-3η was successfully overexpressed in RAFLSs (Fig. 5 B). Besides, the transcripts detected via a scatter plot indicated that 4 genes (green dots), TP53, ERBB3, ERBB4 and NAT2, were downregulated (> 1.5-fold) after the overexpression of the 14-3-3η gene compared with those in the control vector group. These genes are responsible for diverse functions related to cell proliferation, apoptosis, the cell cycle, lysosomal function and ATP-binding cassette[ 29 – 33 ]. The black dots indicate unchanged gene expression. To further validate the significant changes in gene expression, p53, a mostly downregulated gene, was validated by qPCR and Western blotting. As shown in Fig. 5 C, mRNA and protein expression of p53 were significantly suppressed in RAFLS cells overexpressed with the 14-3-3η compared to those in the vector Ctrl group. However, p53 overexpression did not elevate 14-3-3η expression (Fig. 5 D), suggesting that p53 might act downstream of 14-3-3η. This result is therefore consistent with the reported function of p53, which is commonly responsible for drug resistance in cancer. The overexpression of 14-3-3η may contribute to apoptosis resistance in RAFLSs The p53 tumor suppressor gene regulates apoptosis in cancer and RA[ 34 ]. We therefore investigated whether apoptotic pathways are affected by 14-3-3η-mediated downregulation of p53. We then overexpressed 14-3-3η in RAFLSs and determined the expression of apoptotic markers. As expected, antiapoptotic markers such as Bcl-2 and Bcl-xl were markedly upregulated, whereas proapoptotic markers, including Bax and cytochrome c, were significantly downregulated with p53 protein in RAFLSs transfected with 14-3-3η (Fig. 5 E), whereas the gene and protein expression of p53 were also found to be concomitantly downregulated in PBMC or synovial tissues of MTX-treated AIA rats overexpressing 14-3-3η (AIA 14 − 3−3η+/+ ) (Fig. 5 F), suggesting that RAFLSs may develop apoptosis-resistant phenotypes upon the reduction of p53 expression. Accordingly, 14-3-3η may mediate MTX resistance in RAFLSs through the downregulation of p53, thereby promoting apoptosis resistance pathways. To investigate the mechanism underlying 14-3-3η-mediated p53 protein reduction, we performed co-immunoprecipitation (co-IP) and Western blot assays in 293T cells with or without FLAG-14-3-3η overexpression. As shown in Fig. 5 G, the input panel presented the Western blot analysis of the total cell lysate, while the IP panel showed the Western blot of the cell lysate immunoprecipitated with anti-Flag magnetic beads. Notably, in lanes 3 and 4 of the IP blot, p53 protein was only detected in the FLAG-14-3-3η-immunoprecipitated lysate, indicating that p53 is a 14-3-3η-interacting protein. To assess the mechanism of 14-3-3η-mediated p53 reduction, cells were then treated with MG-132, a known inhibitor for proteasomal degradation. In both input and IP blotting, MG-132 treated-immunoprecipitated cell lysate (lane 4) revealed a significantly higher expression level of interacting p53 proteins, compared with that of lane 3, suggesting that the interaction between 14-3-3η and p53 accelerated p53 degradation by proteasomal degradation (Fig. 5 G). Exogenous p53 supplementation reverses the MTX resistance phenotype in AIA transgenic rats An inextricable link between the 14-3-3η-mediated drug resistance phenotype in RA and p53 has been clearly established. To further address whether the p53 tumor suppressor gene plays an equally critical role in transgenic animal models with 14-3-3η overexpression, we adopted an adeno-associated virus (AAV5-p53) to induce p53 overexpression in methotrexate (MTX)-resistant AIA 14 − 3−3η+/+ transgenic rats via tail vein injection. As shown in Fig. 6 A, the paw swelling and arthritis scores revealed that arthritis symptoms in AIA rats overexpressing 14-3-3η remained comparable to those in AIA rats following MTX treatment. Conversely, AIA rats receiving only AAV5-p53 presented minimal relief of paw swelling, whereas the AIA 14 − 3−3η+/+ group receiving both AAV5-p53 and MTX presented significantly improvement in paw swelling (Fig. 6 A). Quantitative analysis of 14-3-3η and p53 expression levels in PBMCs from all experimental rats indicated that p53 expression was markedly suppressed in the 14-3-3η-overexpressing groups (AIA 14 − 3−3η+/+ and AIA 14 − 3−3η+/+ (MTX) ) than in the control group. In contrast, p53 expression was significantly elevated in the rats after receiving of AAV5-p53 injections (Fig. 6 B). Furthermore, the swollen paws from all experimental groups were captured and collected at the time of sacrifice, and subjected to micro-CT analysis for their joint bone destruction and total porosity. As shown in Fig. 6 C, the paw images with yellow arrows indicated the specific sites of bone destruction, multiple bone lesions were also observed in the AIA 14 − 3−3η+/+ (MTX) group despite MTX treatment. These findings suggest that the therapeutic efficacy of MTX in mitigating bone destruction was compromised in AIA 14 − 3−3η+/+ transgenic rats. In contrast, AIA 14 − 3−3η+/+ transgenic rats receiving exogenous p53 intervention, especially those treated concurrently with MTX, exhibited significant decrease in both joint swelling and bone damaging (Fig. 6 C). In addition, the positive bone improvement-related indicators, including bone mineral density (BMD), tissue mineral density (TMD), bone volume fraction (BV/TV), and trabecular number (Tb.N), were markedly increased, whereas the negative indicator, total porosity, was decreased. Collectively, the corresponding MicroCT score was recovered to a range of 0.6–0.8, indicating the mild bone destruction (Fig. 6 D). Furthermore, erythrocyte sedimentation rate (ESR) analysis also indicated that MTX-treated 14-3-3η-overexpressing AIA rats presented a significant reduction in the ESR following exogenous p53 intervention (Fig. 6 E). Additionally, rats subjected to continuous doxycycline (Dox) induction presented slightly lower body weights than non-induced rats did ( Fig. S4A ). Splenomegaly, a characteristic pathological feature of AIA rats and an indicator of inflammatory response intensity, was generally observed in 14-3-3η-overexpressing rats even after MTX treatment. However, the spleen organ index was significantly reduced in the rats treated with both AAV5-p53 and MTX ( Fig. S4B ). Adenovirus–mediated p53 expression sensitizes the immunomodulatory effect of MTX in the AIA 14−3−3η+/+ transgenic rat To assess the level of autoimmune activation and disease progression in AIA rats, we analyzed the populations of Th17 (IL-17A) and Treg (Foxp3) cells in the peripheral blood via flow cytometry. The results indicated that AIA 14 − 3−3η+/+ rats treated with AAV5-p53 combined with MTX would markedly shift the immune balance toward an immunosuppressive status (Fig. 7 A-B), as evidenced by an increase of Foxp3/IL-17A ratio. In contrast, MTX treatment alone did not alter the Foxp3/IL-17A balance under the same conditions (Fig. 7 C). Additionally, we evaluated the CD4/CD8 T cells ratio, which is typically elevated in AIA rats due to heightened immune activity. Our findings revealed that exogenous p53 significantly reduced the CD4 + /CD8 + T cells ratio in peripheral blood, suggesting that p53 suppresses the overactive autoimmune response, facilitating the effective treatment of AIA rats ( Fig. S4C-E ). Quantitative PCR analysis revealed that exogenous p53 significantly enhanced the therapeutic effect of MTX by downregulation of IL-6, IL-1β and TNF-α expression in AIA 14 − 3− 3η+/+ rats (Fig. 7 D). To assess immune homeostasis comprehensively, we further performed multiplex flow cytometry analysis (LEGENDplex™) on rat serum to quantify multiple cytokines. Consistently, our results demonstrated that combined treatment of AAV5-p53 and MTX significantly decreased the serum levels of TNF-α, IFN-γ, IL-6, IL-1α, IL-17A, GM-CSF, IL-18, IL-12p70, IL-33, and IL-1β in the AIA 14−3− 3η+/+ transgenic rat (Fig. 7 E). Discussion RA is a chronic inflammatory autoimmune disease that primarily affects joints and has a wide range of extra-articular and systemic manifestations. Its pathological features include synovial hyperplasia, inflammatory cell infiltration, pannus formation and bone tissue injury, resulting in joint swelling and pain and eventually bone destruction and dysfunction[ 35 ]. Refractory RA seriously affects patients’ quality of life. However, the specific mechanism of drug resistance in RA remains unclear. Most of the drugs commonly used in clinical practice have limited efficacy and many side effects. Two members (ζ and σ) of the 14-3-3 proteins have been confirmed to be associated with chemotherapy resistance and resistance to molecular targeted drugs in cancer[ 36 , 37 ]. It has been shown that 14-3-3η plays an important role in inducing/maintaining the multidrug resistance phenotype in hepatocellular carcinoma [ 16 ]. Interestingly, we also found that 14-3-3η is aberrantly highly expressed in RA. However, whether 14-3-3η contributes to drug resistance in RA is not known. In this study, we report that 14-3-3η is a new therapeutic target for drug-resistant RA or other inflammatory diseases. We demonstrated that 14-3-3η overexpression via AAV5 negates the therapeutic impact of MTX, leading to extreme paw swelling, a significant increase in hind paw volume and arthritis score, and exacerbated bone erosion in arthritic joints. Our data also shown that in transgenic knock-in (KI) rats, the inducible expression of 14-3-3η in AIA 14 − 3−3η+/+ rats could also result in the release of proinflammatory cytokines and severe arthritic conditions in AIA rats treated with MTX. Notably, these effects can be reversed with arsenic trioxide (ATO), a tool drug known to eliminate 14-3-3η via ubiquitin-mediated degradation[ 16 ]. Mechanistic studies revealed that the overexpression of 14-3-3η contributes to apoptosis resistance by reducing the gene expression of p53. AAV-mediated overexpression of p53 markedly reversed the drug resistance of AIA 14 − 3−3η+/+ rats. The AIA model has the same immunological and pathological characteristics as human RA and is considered an ideal animal model for RA[ 38 ]. An AIA rat model was established to evaluate the drug resistance of 14-3-3η. The results showed that AAV-mediated overexpression of 14-3-3η contributed to MTX resistance in AIA rats, which aggravated basic signs such as paw swelling and the arthritis index. It also increases joint inflammation and synovial tissue destruction, suggesting a potential therapeutic target for 14-3-3η. Indeed, we confirmed that ATO, a tool drug that inhibits 14-3-3η, reverses 14-3-3η-mediated MTX resistance in AIA rats. Cytokines regulate a wide range of inflammatory processes in the pathogenesis of RA, especially proinflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and MCP1[ 39 ]. The cytokines mentioned above can induce the expression of matrix metalloproteinases (MMPs) and proteinases, which are largely responsible for the irreversible destruction of cartilage, bone and tendons in joints[ 40 ]. Although the inflammatory response is not the only characteristic of RA, it is a major problem for patients. Therefore, controlling the inflammatory response may be a viable way to treat RA. We found that ATO treatment significantly inhibited the levels of TNF-α, IL-1β, IL-6, IL-8 and MCP1 in serum and synovial tissues, as well as the expression of MMP-1, MMP-3, MMP-9 and RANK, suggesting that ATO can improve 14-3-3η-mediated MTX resistance in the systemic inflammatory response of AIA rats by regulating inflammatory cytokines. We also confirmed that the inducible expression of 14-3-3η in AIA 14 − 3−3η+/+ rats could also result in the release of proinflammatory cytokines and severe arthritic conditions in AIA rats treated with MTX. Notably, these effects can be reversed with ATO, which is known to eliminate 14-3-3η via ubiquitin-mediated degradation. In vitro experimental results confirmed that ATO treatment enhances the proteasomal degradation of 14-3-3η via ubiquitination in RAFLSs. These results are in line with other research groups’ reports, which show that ATO could be considered a potential molecular targeted agent for the treatment of HCC[ 16 ]. During the course of RA, the synovium transforms into hyperplastic invasive tissue, leading to cartilage and bone destruction. RAFLSs in the synovial lining develop aggressive phenotypes and produce pathogenic mediators that lead to the occurrence and progression of disease, playing a major role in RA pathophysiology[ 41 ]. Therefore, there has been recent interest in RAFLSs as therapeutic targets in RA to avoid side effects associated with many existing drug-resistant RA treatments. To examine the drug resistance potency of 14-3-3η in vitro, the multidrug resistance (MDR) effects of overexpressing 14-3-3η in RAFLSs were examined in many antiarthritic drugs, such as methotrexate, hydroxychloroquine, leflunomide, tacrolimus, and sulfasalazine. The RAFLSs were transfected with a control vector or a 14-3-3η-Flag-tagged plasmid. We found that the overexpression of 14-3-3η contributes to MDR phenotypes in RAFLSs. Furthermore, to explore the clear target drug resistance genes in 14-3-3η-overexpressing RAFLSs, whole-cell mRNA expression analysis was carried out by using a human cancer drug resistance PCR array targeting RNA virtual panels. The results indicated that the expression of 4 genes (green dots), p53, ERBB3, ERBB4 and NAT2, was downregulated (> 1.5-fold) after the 14-3-3η gene was overexpressed compared with that in the control vector group. Since 14-3-3η overexpression results in the greatest increase in p53 expression, we further investigated the mechanism by which it promotes drug resistance in RAFLSs. We then confirmed that p53 overexpression cannot increase 14-3-3η expression, suggesting that p53 might be downstream of 14-3-3η. The p53 tumor suppressor protein regulates apoptosis in cancer and RA. We therefore investigated whether apoptotic pathways are affected by 14-3-3η-mediated downregulation of p53. We further investigated the downstream p53-related pathway and found that 14-3-3η-mediated MTX resistance in RAFLSs may occur through the downregulation of p53, thereby upregulating apoptosis resistance pathways. This result is consistent with the reported function of p53, which is responsible for drug resistance in cancer. Numerous studies have demonstrated that various isoforms of 14-3-3 (such as γ, ε, ζ, τ, and σ) regulate p53 through direct interactions. However, the interaction between 14-3-3 isoforms (β and η) and p53 remains unexplored. Additionally, isoforms such as 14-3-3 α, γ, ε, ζ, η, and τ tend to promote cell survival, potentially facilitating p53 degradation by positively regulating MDM2[ 14 ]. More intriguingly, we employed AAV5-p53 as an intervention approach to effectively reverse the drug-resistant phenotype of rats in the 14-3-3η (+/+) transgenic AIA model. Various pathological alterations of arthritis were significantly ameliorated, encompassing paw swelling, bone destruction, Foxp3/IL-17A immune equilibrium and serum inflammatory factor levels. Therefore, our results elaborated that 14-3-3η mediates the progression of drug resistance in RA via elimination of p53. The supplementation of exogenous p53 markedly enhances the sensitivity of MTX-resistant AIA rats due to 14-3-3η overexpression. Indeed, the role of p53 in the development of drug resistance has been extensively studied. Notably, gene mutations that impair or abolish p53 function is strongly associated with resistance to several widely used therapeutic agents, including cisplatin, doxorubicin, paclitaxel, gemcitabine, and tamoxifen[ 42 , 43 ]. These mutations, predominantly located in the DNA-binding domain of p53, often result in either a loss of p53 expression or the production of dysfunctional mutant proteins. Consequently, p53 can paradoxically serve as a protector of cancer cells rather than a tumor suppressor [ 43 ].Furthermore, drug resistance mechanisms frequently involve disruptions in the balance between pro-apoptotic and anti-apoptotic pathways, with p53 playing a central regulatory role in apoptosis [ 42 ]. Our study is the first to report the relationship between 14-3-3η and p53, which fills a gap in the research on the 14-3-3 family and p53. On the other hand, dysfunction of p53 in RA fibroblast-like synoviocytes (RA-FLS) leads to apoptosis resistance and increased production of inflammatory mediators like IL-6 and MMP-1, contributing to chronic synovial inflammation. p53 inhibition reduces FOXP3 expression, resulting in decreased regulatory T cells (Tregs) and increased polarization of CD4 + T cells toward Th17 cells, which produce high levels of IL-17, promoting osteoclastogenesis. This p53 dysfunction activates NF-κB and MAPK signaling pathways, escalating the expression of pro-inflammatory cytokines and matrix metalloproteinases. IL-6 plays a pivotal role in shifting the balance toward Th17 cells, enhancing the inflammatory response. Deficient p53 in T cells contributes to reduced Treg proportions and heightened Th17 differentiation in RA patients, exacerbating autoimmune arthritis. Overall, impaired p53-dependent apoptosis and proliferation in synovial cells are crucial mechanisms driving chronic inflammation and cartilage destruction in RA[ 44 – 46 ]. Besides, previous studies demonstrated that ATO may target the 14-3-3η protein for its ubiquitination and degradation. Here, we further validated the binding affinity of ATO for the 14-3-3η protein. We confirmed that ATO exhibited good binding affinity for 14-3-3η and that it inhibited the mRNA and protein expression of 14-3-3η in a dose-dependent manner. However, ATO could also facilitate the proteasomal degradation of 14-3-3η via ubiquitination, which is coincided to our previous studies, as addressed above. In conclusion, our research underscores the pivotal role of 14-3-3η in fostering drug resistance in RA by modulating p53 expression. We used gene transfer with AAV5-14-3-3η to effectively demonstrate MTX resistance in AIA rats. Furthermore, we developed doxycycline-inducible 14-3-3η knock-in transgenic SD rats using CRISPR/Cas-mediated genome engineering, which successfully confirmed these MTX-resistant effects in AIA rats. Our findings confirm that 14-3-3η induces MTX resistance in RAFLSs/AIA rats by suppressing p53, thereby activating signaling pathways that confer resistance to apoptosis. Remarkably, supplementing with p53 significantly reversed this MTX resistance phenotype. Additionally, our study is the first to report that arsenic trioxide (ATO), used here as an experimental drug, effectively counters 14-3-3η-mediated MTX resistance in AIA rats. These discoveries open avenues for exploring new compounds targeting this novel resistance mechanism, potentially leading to innovative treatments for MTX-resistant RA. Methods Chemicals and reagents Inactivated Mycobacterium tuberculosis (BD#231141) was purchased from BD Company (New Jersey, USA). Mineral oil and arsenic trioxide (ATO, As2O3, > 99% purity) were obtained from Sigma‒Aldrich (Shanghai, China). Methotrexate (MTX), hydroxychloroquine (HCQ), leflunomide (LEF), sulfasalazine (SSZ) and tacromus with purities above 99.0% were obtained from the China National Institute for Food and Drug Control (Beijing, China). Doxycycline was purchased from Beyotime (Shanghai, China). Lipofectamine 3000 reagent and an MTT kit were purchased from Invitrogen (Shanghai, China). MG-132 was purchased from MedChemExpress (New Jersey, USA). All the compounds were dissolved in dimethyl sulfoxide (DMSO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL (Gaithersburg, USA). Cell culture and transfection Immortalized RAFLSs were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in a 5% CO 2 , 37°C humidified incubator in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, UK). The control vector and pcDNA-3.1-14-3-3η-Flag were synthesized by Generay Biotech (Shanghai, China). RAFLS cells were transiently transfected using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol (Note: plasmids DNA used: 5 ng/ml). After transfection, the cells were cultured in fresh medium supplemented with 10% FBS for another 48 h before being used for other experiments. Cytotoxicity assay s Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 5.0 mg·ml − 1 ) assay. In brief, RAFLSs (5×10 3 cells per well) were seeded in 96-well plates for 24 hrs. Cells were then treated with indicated concentrations of compounds dissolved in DMSO, whereas DMSO alone was used as controls. Subsequently, MTT (10 µL) was added to each well for 4 hr, followed by the addition of 100 µL solubilization buffer (10% SDS in 0.01 mol·L − 1 HCl) and overnight incubation. The optical density was read at a wavelength of 570 nm with a microplate reader. The percentage of cell viability was calculated by the following formula: Cell viability (%) = A treated /A control × 100. The data were obtained in triplicate from three independent experiments. Quantitative reverse-transcription PCR (qRT‒PCR) Total RNA was extracted and reverse-transcribed into cDNA using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. The expression of mRNAs was determined using SYBR Premix Ex Taq Master Mix (2×) (Takara). The relative expression level of the target gene was calculated using the comparative Ct method. β-Actin was used as an internal control to normalize sample differences. The sequences of the primers used for qRT‒PCR analysis are presented in Supplementary Table 1. Western blotting Total proteins were harvested from tissues or RAFLS cells using 1×RIPA lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS and protease inhibitors; Wheaton Science) on ice for 30 min and analyzed via SDS‒PAGE. The PVDF membrane was milk blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, followed by incubation with anti-14-3-3η (CST, 1:1000, #940), anti-Flag (CST, 1:1000, #14793S), anti-ubiquitin (CST, 1:1000, #20326S) and anti-β-actin (Abcam, 1:3000, ab134175) antibodies overnight at 4°C. Immunocomplexes were visualized through chemiluminescence using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, USA). Co-immunoprecipitation (Co-IP) After RAFLSs were pretreated with or without 20 µM MG-132 for 2 h, the cells were then exposed to 10 µM ATO for 6 h. Then, the cells were extracted for 30 min with immunoprecipitation lysis buffer (Beyotime Co., Ltd.). After centrifugation of the preparations, 100 µg of total protein was incubated with 14-3-3η antibody(dilution,1:100) at 4°C overnight. Then, the protein-antibody complexes were incubated with IgG Sepharose beads (Beyotime Co., Ltd.) at 4°C for another 12 h. Afterwards, the supernatants were removed (positive control), and the beads were washed three times (residual supernatants served as a negative control), boiled to remove protein from the beads. Positive control, negative control and protein interactions were analyzed by Western Blot. RT 2 profiler human cancer drug-resistance PCR array analysis Total RNA was obtained from 14-3-3η-overexpressing RAFLS cells via a Qiagen RNeasy® Mini Kit (Qiagen). A human cancer drug resistance-specific RT‒PCR array (PAHS-012ZA, Qiagen) was used to evaluate the potential differential genes expression in RAFLSs after 14-3-3η overexpression. The drug resistance array comprised 87 genes (Supplementary Table 2) selected based on their involvement in drug resistance. Five housekeeping genes served as positive controls. Total RNA was reverse transcribed using the RT 2 First Strand Kit. Real-time PCR was carried out on a ViiA™ 7 Real Time PCR System (Applied Biosystems, USA) using the RT 2 SYBR® Green qPCR Mastermix (Qiagen, Germany) according to the manufacturer’s instructions. Data analysis was performed using Qiagen’s integrated web-based software package for the PCR Array System, which automatically performs all the ΔΔCt-based fold-change calculations from the raw threshold cycle data. Patient recruitment and specimens A blood sample was collected from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy volunteers at the Affiliated Hospital of Southwest Medical University, with the collection process being approved by both the hospital and the research ethics committee under the reference number (KY 2021010). After obtaining informed consent, the researchers proceeded to conduct an epidemiological survey and classification of all volunteers, strictly following the standards set by the American College of Rheumatology. The survey included detailed clinically relevant information and data, which are comprehensively outlined in Supplementary Table 3. Peripheral blood specimens from patients and healthy donors were obtained in blood collection tubes. Peripheral blood mononuclear cells were extracted in red cell lysis buffer (Beyotime Biotechnology Inc., Shanghai) for further analysis. Experimental animals Wild-type male Sprague‒Dawley (SD) rats (6 weeks, 80–120 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. All rats were housed in clean plastic cages and maintained at 23 ± 1°C with 55% relative humidity, a 12-h light/dark cycle (8:00–20:00) and ad libitum access to pure water and standard rat pellets. The study protocol was reviewed and approved by Macau University of Science and Technology (Protocol approval code: MUSTARE-003-2020). All experiments, including animal breeding, experimental operations, and animal euthanasia, were performed in accordance with the guidelines established by the committee. Generation of a rat AAV5 vector and intra-articular administration Adeno-associated virus 5 (AAV5) was manufactured by Hanbio Biotechnology (Shanghai, China). The AAV5 vector was generated after cloning the full-length coding sequence of 14-3-3η into the adeno-associated virus vector pHBAAV5-CMV-3×flag-ZsGreen. AAV5 packaging was performed by cotransfecting AAV5-293 cells with the recombinant AAV5 vector, pAAV-RC vector, or pHelper vector. AAV5 was collected from the AAV5-293 cell supernatant, condensed, and purified for further animal experiments. The virus titer was 2.0×10 12 vg/mL. Viral titers were determined by quantitative PCR of the CMV (cytomegalovirus) sequence, and the viral stock in this study was diluted with phosphate-buffered saline (PBS) and adjusted to final concentrations of 3 × 10 9 and 1 × 10 11 viral genomes (vg)/µl. Rats received an intra-articular injection of AAV5 expressing 14-3-3η. The rats were caught alive, and the skin above the articular joint was shaved. Finally, the rats were injected intra-articularly with AAV5-14-3-3η in PBS using 25-gauge needles (TERUMO Company, Philippines) and 25 µl CASTIGHT syringes (TERUMO Company, Philippines). Establishment of a rat adjuvant-induced arthritis (AIA) model and drug administration Male SD rats were randomly divided into 7 groups (n = 8): (1) healthy control, (2) AIA model + AAV5-null, (3) AIA model + AAV5-14-3-3η high dose (HD, 1×10 11 PFU), (4) AIA model + methotrexate (MTX, 7.6 mg/kg/week, gavage) + AAV5-null, (5) AIA model + MTX (7.6 mg/kg/week) + AAV5-14-3-3η low dose (LD, 3×10 9 PFU), (6) AIA model + MTX (7.6 mg/kg/week) + AAV5-14-3-3η high dose (HD, 1×10 11 PFU), an equal volume of AAV virus was injected on day 0, and (7) arsenic trioxide (ATO, 6 mg/kg/3 days, intraperitoneal injection) + AIA model + MTX (7.6 mg/kg/week) + AAV5-14-3-3η (1×10 11 PFU). The HBAAV5/CMV/EGFP r14-3-3η vector (AAV5-14-3-3η) was used. Mineral oil containing 5.0 mg/ml M. tuberculosis was ground and rolled intensively until the mixture turned white. Rats were injected with adeno-associated virus into the knee joints, and 0.1 ml of the mixture was subsequently injected subcutaneously at the base of the tail on day 0. The rats were sacrificed on day 30. Finally, the blood, organs and joint tissue were harvested for biological assays, paw volume assessment and micro-CT analysis. The ankle joints were collected for H&E staining, followed by microscopic observation. Generation of inducible 14-3-3η (gene name: YWHAH) knock-in (KI) rats To generate inducible YWHAH KI rats, we performed targeted knock-in in rat embryos using the CRISPR/Cas9 system as described below: potential Cas9 cleavage sites were identified by screening genomic regions of interest using online software. The sequence of the sgRNA used was GGCCGAGTCGCGAGCGACATGGG. The homology arms for targeted knock-in were amplified by PCR using a BAC clone as a template. In addition to the homologous arms, the targeting construct also contained the Kozak-tTS/rtTA-BGH pA-anti[TRE promoter-Kozak-Rat YWHAH CDS-3×GGGGS-EGFP-rBG pA] fragment. The aim was to replace the ATG start codon. tTS/rtTA is driven by the rat YWHAH endogenous promoter, and the Rat YWHAH CDS-3xGGGGS-EGFP is driven by the TRE promoter. When the rats were treated with doxycycline (Dox), the YWHAH CDS-3xGGGGS-EGFP was expressed. One-cell-stage zygotes were obtained by mating SD males with SD females superovulated by the injection of pregnant mare serum gonadotropin and human chorionic gonadotropin. The Cas9 protein, synthesized sgRNA and targeting vector were coinjected into the cytoplasm of pronuclear-stage embryos. The injected embryos were cultured in KSOM medium overnight, and those that developed to the two-cell stage were transferred into the oviduct of pseudopregnant females. Pups were genotyped, and correct integration of the fragment carrying the Kozak-tTS/rtTA-BGH pA-anti[TRE promoter-Kozak-Rat YWHAH CDS-3×GGGGS-EGFP-rBG pA] construct was confirmed by Sanger sequencing. The correctly integrated mutant founder rats were further back-crossed with wild-type (WT) SD rats for the F1 generation. The F1 animals were genotyped by PCR, and the positive animals were confirmed by Sanger sequencing. All experimental procedures were performed at the AAALAC-accredited facilities at Cyagen Biosciences, Inc. (Guangzhou, China). Male SD rats were randomly divided into 5 groups (n = 8) as follows: (1) healthy control WT , (2) AIA WT , (3) AIA WT+MTX , (4) AIA 14 − 3−3η+/+ (MTX) , and (5) AIA 14 − 3−3η+/+ (MTX+ATO) . The rats were sacrificed on day 30. Finally, the blood, organs and joint tissue were harvested for biological assays, paw volume assessment and micro-CT analysis. Doxycycline treatment Doxycycline hyclate (Beyotime Biotechnology, China) was prepared in phosphate-buffered saline (PBS). Doxycycline hyclate (8 mg/kg) was intraperitoneally administered to transgenic rats for 7 consecutive days to induce 14-3-3η overexpression in YWHAH-KI rats before the AIA animal model was established. Doxycycline hyclate (8 mg/kg) was intraperitoneally administered to YWHAH-KI rats every 5 days to maintain high 14-3-3η expression. Arthritis scoring assessment From day 0 to day 30, the volumes and arthritis status of the hind paws were measured every 3 days. The arthritis score (0 to 4) was blindly evaluated by unsuspecting researchers according to the following criteria: 0, normal; 1, mild redness of the ankle or tarsal joint; 2, mild redness and swelling of the ankle to the tarsal bone; 3, moderate swelling from the ankle to the metatarsal joints; and 4, severe swelling of the ankles, paws, and fingers. At the end of the treatment, the paw volume was measured along with the toe volume. Histopathological analysis In the AIA models, the ankle joints were dissected from anesthetized rats by chloral hydrate, fixed in a buffered 4% paraformaldehyde solution for 24 h, and embedded in paraffin. Finally, paraffin sections (3 µm) were cut and stained with hematoxylin and eosin (H&E). Micro-CT analysis Rat hind paws were harvested, and soft tissues, including muscles and skins, were dissected from all treatment groups. The remaining tissues, including the whole original rat knee joints, were fixed in formaldehyde and then stored in 70% ethanol. CT scanning was performed using high-resolution µCT (Skyscan 1172). Images were captured and reconstructed with CTAn v1.9 and NRecon v1.6., and then analyzed by 3-D model visualization software (CTVol v2.0). A voltage of 50 kVp, a resolution of 5.7 µm per pixel and a current of 200 µA were used for the scanner. After scanning, each sample was assigned a random number for blinded assessment and then processed for image processing. Transverse, coronal and sagittal images of the knee joints were used for images analysis. The region of interest covering the surface area of the tibia was collected. ELISA Enzyme-linked immunosorbent assay (ELISA) was conducted as described previously [ 23 ]. Briefly, blood was obtained from the orbital sinus of mice. Serum levels of IgG antibodies were measured using a commercially available ELISA kit (Bethyl Laboratories, USA). Horseradish peroxidase (HRP) activity was measured using tetramethyl benzidine as a substrate (eBioscience, USA). Biolayer interferometry analysis (BLI) All BLI binding assays were performed using an Octet RED96 (ForteìBIO, China). A shake speed of 1000 rpm and plate temperature of 30°C were conducted for all the experiments. PBS was used as the kinetics buffer. To prepare 14-3-3η-bound test probes, super streptavidin (SSA) optic fiber probes (ForteìBIO, USA) were run at baseline in PBS for 60 s, loaded in 200 µL of biotinylated 14-3-3η solution at 125 µg/mL for 600 s, run at baseline again in PBS for 60 s, and stored at 4°C dipped in PBS. For the binding kinetics assays, a series of dilutions of six concentrations of ATO dissolved in PBS were added to a black polypropylene 96-well microplate (Greiner Bio-One, Germany) with PBS filling the remaining wells. One row was left as a PBS-only negative control. Each well contained a total volume of 200 µL. An assay cycle consisted of 120 s of baseline incubation in PBS followed by 120–180 s of association in compound solution followed by 120–180 s of dissociation in PBS, and this cycle was repeated for every concentration and with both a 14-3-3η-loaded probe and a blank probe. Analysis of the BLI results was performed using ForteìBIO Data Analysis software version 9.0. The curves were aligned to dissociation, the Y axis was aligned to the last 5 s of baseline steps, and the last 5 s of the association step were considered the steady state. Specific binding to 14-3-3η was subtracted from the blank probe control and PBS negative control by selecting the “Double References” mode. A 1:1 binding model was assumed for the binding kinetics analysis. The KD, Kon, Koff and R2 values are recorded. Statistical analysis GraphPad Prism V8.4.3 software (San Diego, USA) was used for the statistical analysis and data visualization. Multiple comparisons of data between groups were performed by one-way ANOVA or two-way ANOVA. The data are presented as the means ± SEMs, and p < 0.05 was considered to indicate statistical significance. Declarations Data availability The data that support the findings of the study are available from the corresponding author upon reasonable request. Source data are pro-vided with this paper. Acknowledgments This work was supported by grants from the Macao Science and Technology Development Fund, grant codes: 0033/2019/AFJ, 0113/2023/RIA2, and 002/2023/ALC. Author information These authors contributed equally: Li Jun Yang, David Wei Zhang. Authors and Affiliations Dr. Neher’s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine & Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, China Li Jun Yang, David Wei Zhang, Yuan Qing Qu, Yu Ping Wang, Xiong Fei Xu, Wei Dan Luo, Betty Yuen Kwan Law, Zhi-Hong Jiang, Rui Hong Chen, Cong Ling Qiu, Xi Chen, Lin Na Wang, Jiu Jie Yang, Vincent Kam Wai Wong Macau University of Science and Technology Zhuhai MUST Science and Technology Research Institute Betty Yuen Kwan Law, Zhi-Hong Jiang, Vincent Kam Wai Wong Department of Gastroenterology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, Jiangsu Province, China Yuan Li The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China Yuan Li The Affiliated Hospital of Southwest Medical University, Southwest Medical University, Luzhou, 646000, China Wei Dan Luo Author contributions V.K.W.W., Y.L. conceived, conceptualization, sought funding and oversaw the project; B.Y.K.L. Writing - review and editing; L.-J.Y. and D.W.Z. are co-first authors, as they conducted the experiments, interpreted all the results and wrote the manuscript; Y.-Q.Q. performed the Co-IP experiments of p53 and 14-3-3η; Y.-Q.Q., Y.-P.W., X.-F.X., W.-D.L. and R.-H.C. formal analysis, data curation; Z.-H.J. resources and sought funding; C.-L.Q., X.C., L.-N.W. and J.-J.Y. data curation; All authors approved the manuscript. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Consent for publication Consent to publish has been obtained from all authors. Additional information Supplementary information Full supplementary information is listed in the Supplementary Materials document. Correspondence and requests for materials should be addressed to Yuan Li or Vincent Kam Wai Wong. References Firestein GS, McInnes IB. Immunopathogenesis of Rheumatoid Arthritis. Immunity. 2017;46(2):183-96. Imboden JB. The immunopathogenesis of rheumatoid arthritis. Annu Rev Pathol. 2009;4:417-34. Ostrowska M, Maśliński W, Prochorec-Sobieszek M, Nieciecki M, Sudoł-Szopińska I. Cartilage and bone damage in rheumatoid arthritis. Reumatologia. 2018;56(2):111-20. Nemtsova MV, Zaletaev DV, Bure IV, Mikhaylenko DS, Kuznetsova EB, Alekseeva EA, et al. Epigenetic Changes in the Pathogenesis of Rheumatoid Arthritis. Front Genet. 2019;10:570. Smolen JS, Landewé RBM, Bergstra SA, Kerschbaumer A, Sepriano A, Aletaha D, et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2022 update. Ann Rheum Dis. 2023;82(1):3-18. Smolen JS, Aletaha D, Barton A, Burmester GR, Emery P, Firestein GS, et al. Rheumatoid arthritis. Nat Rev Dis Primers. 2018;4:18001. van der Heijden JW, Dijkmans BA, Scheper RJ, Jansen G. Drug Insight: resistance to methotrexate and other disease-modifying antirheumatic drugs--from bench to bedside. Nat Clin Pract Rheumatol. 2007;3(1):26-34. Buch MH, Eyre S, McGonagle D. Persistent inflammatory and non-inflammatory mechanisms in refractory rheumatoid arthritis. Nat Rev Rheumatol. 2021;17(1):17-33. Zhang KX, Ip CK, Chung SK, Lei KK, Zhang YQ, Liu L, et al. Drug-resistance in rheumatoid arthritis: the role of p53 gene mutations, ABC family transporters and personal factors. Curr Opin Pharmacol. 2020;54:59-71. Qiu C, Chan JTW, Zhang DW, Wong IN, Zeng Y, Law BYK, et al. The potential development of drug resistance in rheumatoid arthritis patients identified with p53 mutations. Genes Dis. 2023;10(6):2252-5. Zeng Y, Ng JPL, Wang L, Xu X, Law BYK, Chen G, et al. Mutant p53(R211*) ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response. Inflamm Res. 2023;72(12):2199-219. Rivellese F, Surace AEA, Goldmann K, Sciacca E, Çubuk C, Giorli G, et al. Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial. Nat Med. 2022;28(6):1256-68. Lu M, Dai Y, Xu M, Zhang C, Ma Y, Gao P, et al. The Attenuation of 14-3-3ζ is Involved in the Caffeic Acid-Blocked Lipopolysaccharide-Stimulated Inflammatory Response in RAW264.7 Macrophages. Inflammation. 2017;40(5):1753-60. Falcicchio M, Ward JA, Macip S, Doveston RG. Regulation of p53 by the 14-3-3 protein interaction network: new opportunities for drug discovery in cancer. Cell Death Discov. 2020;6(1):126. Lee YK, Hur W, Lee SW, Hong SW, Kim SW, Choi JE, et al. Knockdown of 14-3-3ζ enhances radiosensitivity and radio-induced apoptosis in CD133(+) liver cancer stem cells. Exp Mol Med. 2014;46(2):e77. Qiu Y, Dai Y, Zhang C, Yang Y, Jin M, Shan W, et al. Arsenic trioxide reverses the chemoresistance in hepatocellular carcinoma: a targeted intervention of 14-3-3η/NF-κB feedback loop. J Exp Clin Cancer Res. 2018;37(1):321. Qiu Y, Shan W, Yang Y, Jin M, Dai Y, Yang H, et al. Reversal of sorafenib resistance in hepatocellular carcinoma: epigenetically regulated disruption of 14-3-3η/hypoxia-inducible factor-1α. Cell Death Discov. 2019;5:120. Zeng T, Tan L. 14-3-3η protein: a promising biomarker for rheumatoid arthritis. Biomark Med. 2018;12(8):917-25. Maksymowych WP, Marotta A. 14-3-3η: a novel biomarker platform for rheumatoid arthritis. Clin Exp Rheumatol. 2014;32(5 Suppl 85):S-35-9. Quan LD, Thiele GM, Tian J, Wang D. The Development of Novel Therapies for Rheumatoid Arthritis. Expert Opin Ther Pat. 2008;18(7):723-38. Tsujimura S, Saito K, Nawata M, Nakayamada S, Tanaka Y. Overcoming drug resistance induced by P-glycoprotein on lymphocytes in patients with refractory rheumatoid arthritis. Ann Rheum Dis. 2008;67(3):380-8. Kabir I, Ansari I. A review on in vivo and in vitro experimental models to investigate the anti-inflammatory activity of herbal extracts. Asian J Pharm Clin Res. 2018;11(11):29. Zhang N, Zhang H, Law BYK, Dias I, Qiu CL, Zeng W, et al. Sirtuin 5 deficiency increases disease severity in rats with adjuvant-induced arthritis. Cell Mol Immunol. 2020;17(11):1190-2. Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front Biosci. 2006;11:529-43. O'Gradaigh D, Compston JE. T-cell involvement in osteoclast biology: implications for rheumatoid bone erosion. Rheumatology (Oxford). 2004;43(2):122-30. Crotti TN, Smith MD, Weedon H, Ahern MJ, Findlay DM, Kraan M, et al. Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis. Ann Rheum Dis. 2002;61(12):1047-54. Feng X, Shi Y, Xu L, Peng Q, Wang F, Wang X, et al. Modulation of IL-6 induced RANKL expression in arthritic synovium by a transcription factor SOX5. Sci Rep. 2016;6:32001. Crawford LJ, Walker B, Ovaa H, Chauhan D, Anderson KC, Morris TC, et al. Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132. Cancer Res. 2006;66(12):6379-86. Pucci B, Kasten M, Giordano A. Cell cycle and apoptosis. Neoplasia. 2000;2(4):291-9. Lee H, Lee H, Chin H, Kim K, Lee D. ERBB3 knockdown induces cell cycle arrest and activation of Bak and Bax-dependent apoptosis in colon cancer cells. Oncotarget. 2014;5(13):5138-52. Wali VB, Haskins JW, Gilmore-Hebert M, Platt JT, Liu Z, Stern DF. Convergent and divergent cellular responses by ErbB4 isoforms in mammary epithelial cells. Mol Cancer Res. 2014;12(8):1140-55. Rösch L, Herter S, Najafi S, Ridinger J, Peterziel H, Cinatl J, et al. ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P-glycoprotein. Mol Oncol. 2023;17(1):37-58. Wang CL, Liu ZP, Guo L. NAT2 knockdown inhibits the development of colorectal cancer and its clinical significance. Eur Rev Med Pharmacol Sci. 2021;25(9):3460-9. Aubrey BJ, Kelly GL, Janic A, Herold MJ, Strasser A. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ. 2018;25(1):104-13. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365(23):2205-19. Huang XY, Ke AW, Shi GM, Zhang X, Zhang C, Shi YH, et al. αB-crystallin complexes with 14-3-3ζ to induce epithelial-mesenchymal transition and resistance to sorafenib in hepatocellular carcinoma. Hepatology. 2013;57(6):2235-47. Reis H, Pütter C, Megger DA, Bracht T, Weber F, Hoffmann AC, et al. A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors. Biochim Biophys Acta. 2015;1854(6):641-50. Wang S, Zhou Y, Huang J, Li H, Pang H, Niu D, et al. Advances in experimental models of rheumatoid arthritis. Eur J Immunol. 2023;53(1):e2249962. McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7(6):429-42. Khokha R, Murthy A, Weiss A. Metalloproteinases and their natural inhibitors in inflammation and immunity. Nat Rev Immunol. 2013;13(9):649-65. Nygaard G, Firestein GS. Restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes. Nat Rev Rheumatol. 2020;16(6):316-33. Hientz K, Mohr A, Bhakta-Guha D, Efferth T. The role of p53 in cancer drug resistance and targeted chemotherapy. Oncotarget. 2017;8(5):8921-46. Cao X, Hou J, An Q, Assaraf YG, Wang X. Towards the overcoming of anticancer drug resistance mediated by p53 mutations. Drug Resist Updat. 2020;49:100671. van Hamburg JP, Asmawidjaja PS, Davelaar N, Mus AM, Colin EM, Hazes JM, et al. Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17A production. Arthritis Rheum. 2011;63(1):73-83. Kimura A, Kishimoto T. IL-6: regulator of Treg/Th17 balance. Eur J Immunol. 2010;40(7):1830-5. Taghadosi M, Adib M, Jamshidi A, Mahmoudi M, Farhadi E. The p53 status in rheumatoid arthritis with focus on fibroblast-like synoviocytes. Immunol Res. 2021;69(3):225-38. Additional Declarations (Not answered) Supplementary Files Supplementarymaterials.docx Supplementary materials WesternBlotsoriginalimages.docx Western Blots original images Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6528370","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449683468,"identity":"b44faba7-c45b-40b6-9b3d-dca5d9255ef5","order_by":0,"name":"Vincent Kam Wai Wong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACCSgtB6WZiddiDMSMDSRpSWwgWotk+xkziY87atPXzkh//oChwjqxQezwAbxapHlyzCRnnjmeu+1GjmEDw5n0xAbptAS8WuQYcrfd5m07BtLC2MDYdhioJccAvxb+t2At6WY30h82MP4jQou0BNiWmgSzGwmGDYwNRGiRnPH++8+ZbQcMt515Yzgj4Vi6cRshv0icT0s2+NhWJ292PP3Bhw811rL90skH8GqBgsMQCmQ8GzHqgaCOSHWjYBSMglEwIgEA1Q5LW42MSegAAAAASUVORK5CYII=","orcid":"","institution":"Macau University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Vincent","middleName":"Kam Wai","lastName":"Wong","suffix":""},{"id":449683469,"identity":"c2772eb9-3b2a-434f-88b8-74473a4048d4","order_by":1,"name":"Li Jun Yang","email":"","orcid":"","institution":"State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"Jun","lastName":"Yang","suffix":""},{"id":449683470,"identity":"e884d08a-3795-4dd7-a495-34ae77952b80","order_by":2,"name":"Wei Zhang","email":"","orcid":"","institution":"Dr. Neher's Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine \u0026 Faculty of Chinese Medicine, Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":449683471,"identity":"f79f2e93-224e-48ac-82d7-2fe396dac47a","order_by":3,"name":"Yuan Qing Qu","email":"","orcid":"https://orcid.org/0000-0003-3733-3661","institution":"State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"Qing","lastName":"Qu","suffix":""},{"id":449683472,"identity":"90822957-d974-4c10-a700-ca2f3ec06210","order_by":4,"name":"Yuping Wang","email":"","orcid":"","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuping","middleName":"","lastName":"Wang","suffix":""},{"id":449683473,"identity":"ac09a13b-d617-4256-8525-c5b21a03e709","order_by":5,"name":"Xiong Fei Xu","email":"","orcid":"","institution":"Dr. Neher’s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine \u0026 Faculty of Chinese Medicine, Macau University of Science and Te","correspondingAuthor":false,"prefix":"","firstName":"Xiong","middleName":"Fei","lastName":"Xu","suffix":""},{"id":449683474,"identity":"9df6eb7e-8a0d-4082-9a6d-c57e7a4820b1","order_by":6,"name":"Wei Dan Luo","email":"","orcid":"https://orcid.org/0000-0002-5432-2481","institution":"State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"Dan","lastName":"Luo","suffix":""},{"id":449683475,"identity":"226064c5-fb90-459c-b3c6-5cbb1d3addd6","order_by":7,"name":"Betty Yuen-Kwan Law","email":"","orcid":"https://orcid.org/0000-0002-8926-3960","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Betty","middleName":"Yuen-Kwan","lastName":"Law","suffix":""},{"id":449683476,"identity":"f6321a4e-cf86-49df-a0fe-d073c51ec19f","order_by":8,"name":"Zhi-Hong Jiang","email":"","orcid":"https://orcid.org/0000-0002-7956-2481","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhi-Hong","middleName":"","lastName":"Jiang","suffix":""},{"id":449683477,"identity":"88a05604-7f36-4295-ac1a-6b815581d22e","order_by":9,"name":"Ruihong Chen","email":"","orcid":"","institution":"The College of Pharmacy in Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruihong","middleName":"","lastName":"Chen","suffix":""},{"id":449683480,"identity":"c90995c5-2833-4b17-b4da-3421c2605d8c","order_by":10,"name":"Cong Ling Qiu","email":"","orcid":"","institution":"Dr. Neher’s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine \u0026 Faculty of Chinese Medicine, Macau University of Science and Te","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"Ling","lastName":"Qiu","suffix":""},{"id":449683481,"identity":"dafb3274-b5e3-44d3-a2e9-27109d5ff534","order_by":11,"name":"Xi Chen","email":"","orcid":"","institution":"State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Chen","suffix":""},{"id":449683482,"identity":"44632a3e-b11f-4e31-9950-8d88c3ab1400","order_by":12,"name":"Linna Wang","email":"","orcid":"","institution":"Dr. Neher’s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Linna","middleName":"","lastName":"Wang","suffix":""},{"id":449683483,"identity":"05697164-5de3-43eb-babf-c5ec756ebd58","order_by":13,"name":"Jiujie Yang","email":"","orcid":"","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiujie","middleName":"","lastName":"Yang","suffix":""},{"id":449683484,"identity":"1544b472-6f1c-4f35-bf8e-c6b6dbb4323f","order_by":14,"name":"Yuan Li","email":"","orcid":"","institution":"The Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-04-25 11:10:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6528370/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6528370/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82203906,"identity":"4a052851-9bb9-410c-8d25-a5ff1c97adf9","added_by":"auto","created_at":"2025-05-07 16:50:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1556048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of the RA biomarker 14-3-3η induces the methotrexate (MTX) resistant phenotype in AIA rats.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) qPCR analysis of 14-3-3η mRNA expression in human peripheral blood mononuclear cell (PBMC) samples. The expression of 14-3-3η in RA patients (n=51) was significantly higher than that in healthy donors (n=29) and osteoarthritis (OA) patients (n=7). P values were determined via an unpaired two-tailed \u003cem\u003et\u003c/em\u003e test. (\u003cstrong\u003eB\u003c/strong\u003e) qPCR detection of 14-3-3η expression in PBMC samples from RA patients revealed a positive correlation between 14-3-3η expression and disease duration. (\u003cstrong\u003eC\u003c/strong\u003e) Correlation between 14-3-3η expression and disease activity in PBMCs of RA patients (assessed by DAS28 score). Y-axis: relative expression level of 14-3-3η mRNA. X-axis: disease activity of individual RA patient estimated based on the DAS28. Pearson correlation analysis was used to determine the statistical significance (n=21). (\u003cstrong\u003eD\u003c/strong\u003e) Treatment timeline of\u003cem\u003e in vivo\u003c/em\u003eexperiments for the study of 14-3-3η in the AIA rat model.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eE\u003c/strong\u003e) Establishment of the AIA model with adeno-associated virus injection in the keen joint region and sample processing after animal sacrifice. (\u003cstrong\u003eF\u003c/strong\u003e) The measurement of hind paw volume and clinical arthritic score in every 3 days post-immunization until day 30. (\u003cstrong\u003eG\u003c/strong\u003e) Images of hind paw swelling were captured on day 30. (\u003cstrong\u003eH\u003c/strong\u003e) mRNA expression of 14-3-3η in the synovial tissue and PBMCs of AIA rats. The data are presented as the means ± SEMs. One-way ANOVA, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the AIA group treated with MTX + AAV5-null; △\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the AIA group treated with MTX + AAV5-14-3-3η high-dose; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the healthy control group.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/fde54ab1e3dc87fbc001a548.png"},{"id":82203913,"identity":"f341a1d1-ac2b-4666-a575-add7bfe3c83f","added_by":"auto","created_at":"2025-05-07 16:50:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3018119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative micro-CT images of hind joints and H\u0026amp;E staining of animal ankle joints harvested from MTX-resistant AIA rats.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative micro-CT images of hind paws of AIA rats with 14-3-3η overexpression in the presence or absence of MTX or MTX/ATO. Red arrows indicate bone erosion. \u003cstrong\u003e(B)\u003c/strong\u003e Micro-CT analysis of MTX-resistant AIA rats before and after treatment. Five disease‐related indices of micro-CT analysis, namely,the calcaneus bone mineral density (BMD), bone volume fraction (BV/TV), cortical mineral density (TMD), trabecular number (Tb.N), and total porosity percentage, were used to evaluate bone damage. Micro-CT scores were obtained from the above five disease‐related indices of micro-CT analysis. \u003cstrong\u003e(C) \u003c/strong\u003eH\u0026amp;E staining images of ankle joint sections from the 7 different groups after 4 weeks treatment with or without AAV5-14-3-3η infection. The animalankle joints were harvested, dehydrated and then stained with H\u0026amp;E (with a sagittal section thickness of 1000 μm). The red arrow indicates stenosis of the articular cavity, destruction of cartilage and synovial hyperplasia. The data are expressed as the mean ± SEM from n=8 rats per group. One-way ANOVA, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the MTX + AAV5-null treated AIA group; △\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the AAV5-14-3-3η HD + MTX-treated AIA group; # \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, significantly different from the healthy control group.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/7285e4e9052a03208ddd2d76.png"},{"id":82204724,"identity":"5487deef-be1f-47d2-b2dd-b9d380945842","added_by":"auto","created_at":"2025-05-07 16:58:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1362708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe drug-resistant role of inducible 14-3-3η in AIA transgenic rats. (A) \u003c/strong\u003eThe measurement of the hind paw volume and clinical arthritic score in every 3 days post-immunization until day 30. \u003cstrong\u003e(B) \u003c/strong\u003eImages of hind paw swelling were captured on day 30. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative micro-CT images of the hind paws of AIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e rats in the presence or absence of MTX or MTX/ATO. Red arrows indicate bone erosion. \u003cstrong\u003e(D)\u003c/strong\u003e Micro-CT analysis of MTX-resistant AIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e rats before and after treatment. Five disease‐related indices of micro-CT analysis, namely, the calcaneus bone mineral density (BMD), bone volume fraction (BV/TV), cortical mineral density (TMD), trabecular number (Tb.N), and total porosity percentage, were used to evaluate bone damage. \u003cstrong\u003e(E) \u003c/strong\u003eMeasurement of the ESR in PBMCs from different groups on day 30. The data are presented as the means ± SEMs. One-way ANOVA, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the AIA \u003csup\u003eWT+MTX\u003c/sup\u003e group; △\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the AIA\u003csup\u003e14-3-3η+/+(MTX) \u003c/sup\u003egroup; #\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, significantly different from the healthy control group.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/78c15f01d59b5c948c054483.png"},{"id":82204726,"identity":"bce00b86-d9d9-4eba-8177-e4cdca1493c5","added_by":"auto","created_at":"2025-05-07 16:58:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":514803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy of the arsenic trioxide (ATO)-mediated protein degradation of 14-3-3η. (A)\u003c/strong\u003e The viability of ATO-treated RAFLSs were measured by MTT assay. \u003cstrong\u003e(B) \u003c/strong\u003emRNA expression analysis of 14-3-3η in ATO-treated RAFLSs. \u003cstrong\u003e(C)\u003c/strong\u003e Protein expression of 14-3-3η in ATO-treated RAFLSs. RAFLSswere treated with ATO for 24 h, and the protein expression of 14-3-3η was detected by Western blot using an anti-14-3-3η antibody, whereas β-actin was used as a loading control. \u003cstrong\u003e(D) \u003c/strong\u003eBLI assay demonstrating the binding affinity of the interaction between immobilized 14-3-3η and different concentrations of ATO. Right panel, steady-state analysis of the interaction between immobilized 14-3-3η and ATO. \u003cstrong\u003e(E) \u003c/strong\u003eWestern blot analysis of ubiquitination in the 14-3-3η-immunoprecipitation complex. RAFLS cells overexpressing 14-3-3η protein were pretreated with 20 μMMG132 for 2 h, and then the cells were further exposed to 20 μMATO for 24 h. 14-3-3η was immunoprecipitated with aspecific antibody against 14-3-3η. The results are presented asthe mean ± SEM of 3 independent experiments (n=3, one-way ANOVA, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, significantly different from the RAFLS cells treated with 0μM ATO).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/75f4c2c05d30c0fbd3b2905d.png"},{"id":82203912,"identity":"06b7a20b-7dad-46b9-b218-f1f52cc62f1e","added_by":"auto","created_at":"2025-05-07 16:50:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1139546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe overexpression of 14-3-3η contributes to apoptosis resistance \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e suppression of p53 in RAFLSs.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Western blot image indicating the overexpression of 14-3-3η in RAFLSs. The flag-tagged 14-3-3η protein was probed with an anti-flag antibody, and b-actin was used as a loading control. MTT cytotoxicity assay of MTX, HCQ, LEF, SSZ and tacrolimus in RAFLSswith or without 14-3-3η overexpression. \u003cstrong\u003e(B) \u003c/strong\u003eComparison of drug resistance gene expression profiles in RAFLS cells transfected with or without 14-3-3η plasmid by PCR array analysis. Green dots represent downregulated genes with at least a 1.5-fold change,and black dots indicate unchanged gene expression. RAFLSs were validated to have 14-3-3η overexpression by Western blot detection using an anti-Flag antibody. \u003cstrong\u003e(C)\u003c/strong\u003e Downregulation of gene and protein expression of p53 were validated by qPCR and Western blot in 14-3-3η-overexpressing RAFLSs. \u003cstrong\u003e(D) \u003c/strong\u003e14-3-3η protein expression did not change in p53-overexpressing RAFLSs.\u003cstrong\u003e (E)\u003c/strong\u003e The protein expression of p53 and the pro-apoptotic markers Bax and cytochrome c were significantly downregulated in 14-3-3η-overexpressing RAFLSs. The levels of the anti-apoptotic markers Bcl-2 and Bcl-xl were significantly increased in RAFLSs overexpressing 14-3-3η.\u003cstrong\u003e (F)\u003c/strong\u003e The gene and protein expression of p53 were markedly downregulated in 14-3-3η-overexpressing transgenic rats. Quantitative bar graph showing the expression levels of the p53 gene in the PBMCs of WT rats and 14-3-3η-overexpressing transgenic rats. Western blot images of p53 protein expression in 14-3-3η-overexpressing transgenic rats compared with WT rats. All samples were biologically independent. Data are presented as the mean ± SEM, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 between two groups. In (A-E), the results of 3 independent experiments are presented as the mean ± SEM (unpaired two-tailed t test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(G)\u003c/strong\u003e Immunoprecipitation (IP) analysis of 293T cells with or without 14-3-3η overexpression in the presence of proteasomal inhibitor, MG-132, the protein expression of 14-3-3η and p53 were detected from the IP protein lysates with specific antibodies respectively. **\u003cem\u003eP\u003c/em\u003e≤ 0.01, ***\u003cem\u003eP\u003c/em\u003e ≤ 0.001 compared with control sample (lane 1). ##\u003cem\u003eP\u003c/em\u003e ≤ 0.001 compared with sample in lane 3, one-way ANOVA analysis.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/8cd806a33a93aee94f4a77c9.png"},{"id":82204725,"identity":"31c760fb-21ff-41d5-aa1c-8bb2fdbc632d","added_by":"auto","created_at":"2025-05-07 16:58:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1561298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTail vein injection of AAV5-p53 alleviates the exacerbation of inflammation in the AIA \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e14-3-3η+/+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e transgenic rat. (A)\u003c/strong\u003e Hind paw swelling and arthritis scores of AIA\u003csup\u003e14-3-3η+/+\u003c/sup\u003e transgenic rat intra-articularly injected with AAV5-p53. Dox was added to the drinking water (0.5 mg/ml) of transgenic\u003csup\u003e \u003c/sup\u003erats, and the rats were fed continuously for 5 days. During the subsequent 30 days, Dox was used to induce 14-3-3η expression in\u003csup\u003e \u003c/sup\u003eAIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003etransgenic rats at intervals of one week. AAV5-p53 was injected into the tail vein of\u003csup\u003e \u003c/sup\u003etransgenic rats as needed (one week before modeling). After AIA induction, the arthritis score and hind paw volume were measured every three days for 30 consecutive days. **P \u0026lt; 0.01 for the AIA model group compared with the healthy control group; ##P \u0026lt; 0.01 for the AIA+MTX group compared with the AIA model group; ++P \u0026lt; 0.01 for the AIA\u003csup\u003e14-3-3η+/+ (AAV5-p53 + MTX) \u003c/sup\u003egroup compared with the AIA\u003csup\u003e14-3-3η+/+ (AAV5-p53)\u003c/sup\u003e group. \u003cstrong\u003e(B)\u003c/strong\u003e Gene expression levels of the 14-3-3η and p53 in rat PBMCs. Blood samples were collected on day 30. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative photographs of rat paws and 3D micro-CT images of damaged swollen articular bone reconstructed using the Inveon Research Workplace at 19 μm resolution. Yellow arrows indicate typical areas of bone destruction. \u003cstrong\u003e(D)\u003c/strong\u003e The micro-CT score of the degree of bone destruction in rat joints was calculated by normalizing five disease-related indicators: bone mineral density (BMD), trabecular number (mm-1) (Tb. N), cortical bone tissue mineral density (g/cm3) (TMD), bone volume fraction (BV/TV), and total porosity (as a percentage of total area). \u003cstrong\u003e(E)\u003c/strong\u003e Determination of rat erythrocyte sedimentation rate (ESR) in the AIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e transgenic rat. On day 30, blood was collected using a 1.6 ml sodium citrate erythrocyte sedimentation assay tube. All samples were biologically independent, and statistical significance was calculated by t tests. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n.s.: no significant difference. The data are expressed as the mean ± SEM (all groups: n = 6).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/0fc1a5081ab9c017b543e844.png"},{"id":82203911,"identity":"579c7440-6847-466d-bec9-d92a367a3af7","added_by":"auto","created_at":"2025-05-07 16:50:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1399236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunomodulatory effect of intravenous injection of the AAV5-p53 in the AIA \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e14-3-3η+/+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e transgenic rat. (A)\u003c/strong\u003e Flow cytometry images showing the purification of Foxp3 and IL-17A cells gated on CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes. Four groups of AIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e transgenic rats were induced with Dox (0.5 mg/ml) every other week for 5 consecutive days. Two groups of rats were intravenously injected with the AAV5-p53 adenoviral vector (1×10\u003csup\u003e11\u003c/sup\u003e PFU) via the tail vein and monitored for 30 days. Blood lymphocytes were collected from these animals and analyzed for T-cell activation using fluorescent antibodies against CD45, CD3, CD4, CD8, and Foxp3 \u003cem\u003evia \u003c/em\u003eflow cytometry. \u003cstrong\u003e(B)\u003c/strong\u003e Quantitative bar graph showing the percentages of Foxp3\u003csup\u003e+ \u003c/sup\u003eT and IL-17A\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells. \u003cstrong\u003e(C)\u003c/strong\u003e Bar graph showing the percentages of Foxp3\u003csup\u003e+\u003c/sup\u003e Tregs over IL17A\u003csup\u003e+\u003c/sup\u003e Th17 cells among CD4\u003csup\u003e+\u003c/sup\u003e T cells. \u003cstrong\u003e(D)\u003c/strong\u003e Gene expression levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in the PBMCs of AIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e transgenic rats. PBMC samples were collected on the last day of experiment (day 30). \u003cstrong\u003e(E)\u003c/strong\u003e Assay of various cytokine concentrations in the serum of AIA \u003csup\u003e14-3-3η+/+\u003c/sup\u003e transgenic rats. Blood serum was collected from rats before sacrifice (day 30) and analyzed using a multiplex flow assay kit. All samples were biologically independent, and statistical significance was calculated using t tests. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, n.s.: no significant difference. The data are presented as the means ± SEMs (all groups: n = 6).\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/2ae26f8b2d3646fcee75c807.png"},{"id":89854397,"identity":"9240e0be-2268-469c-b00a-404389a4cfdf","added_by":"auto","created_at":"2025-08-25 18:24:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13357568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/45652a6e-8721-4dca-b2ee-a5462671fdaf.pdf"},{"id":82203934,"identity":"d36adc4b-c83f-4397-b1c6-4ac8fa60705b","added_by":"auto","created_at":"2025-05-07 16:50:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":63986062,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/8617327e0a535e80244668c8.docx"},{"id":82203924,"identity":"0b46e635-3df9-46d0-8709-e29fb7dd1b74","added_by":"auto","created_at":"2025-05-07 16:50:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25917348,"visible":true,"origin":"","legend":"Western Blots original images","description":"","filename":"WesternBlotsoriginalimages.docx","url":"https://assets-eu.researchsquare.com/files/rs-6528370/v1/6a0d0cf0c4a7c4a430914bab.docx"}],"financialInterests":"(Not answered)","formattedTitle":"\u003cp\u003e14-3-3η Induces a Multidrug-Resistant Phenotype in Rheumatoid Arthritis \u003cem\u003evia\u003c/em\u003e p53 Dysregulation: Insights from AIA Models\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA), the most prevalent chronic inflammatory disease, primarily triggers joint destruction which results in persistent physical disability and constantly developing irreversible dysfunction[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This complex pathology, affecting an estimated 1% of the global populace, imposes a substantial socioeconomic burden due to enduring synovitis, cartilage breakdown, and bone erosion[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite no definitive cure for RA, disease conditions can be ameliorated. Traditional treatments aim to sustain clinical remission with low activity by administering disease-modifying anti-rheumatic drugs (DMARDs), such as methotrexate (MTX) and leflunomide (LEF), biologic DMARDs, nonsteroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For instance, DMARDs and biologic DMARDs achieve disease-modifying ability by obstructing with inflammatory signaling pathways. NSAIDs temper inflammation, pain and swelling, by inhibiting cyclooxygenase activity but are not known to improve joint damage. Glucocorticoids also aid in modulating inflammation but lack disease-modifying activity[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The European League Against Rheumatism (EULAR) recommends the use of DMARDs as first-line treatments, which target proinflammatory mediators such as tumor necrosis factor-a (TNF-α) and interleukin (IL)-6[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, biologic DMARDs are recommended when the first DMARD regimen fails to achieve treatment outcomes with poor prognostic features, such as persistent moderate or high disease activity, high acute phase reactant levels, and a high swollen joint count[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Nevertheless, long-term drug treatment increases drug resistance due to the chronic nature of RA[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. With 20\u0026ndash;30% of RA patients refractory to existing antirheumatic drugs[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], thus necessitates new treatment strategies for drug-resistant RA.\u003c/p\u003e \u003cp\u003eDespite significant advancements in RA management over the past thirty years, a notable number of patients do not benefit from multiple DMARDs. This subset of patients often has 'difficult to treat', 'resistant', or 'refractory' RA[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Factors contributing to antirheumatic treatment resistance in these kinds of RA include mutations in the p53 tumor suppressor gene and overexpression of ABCB1/MDR-1/ P-gp transporters. Our previous research emphasized the role of p53 gene mutations, ABC family transporters, and personal factors in antirheumatic drug resistance, which could lead to development of new personalized RA therapies with enhanced drug sensitivity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. We further elucidated the part p53 mutations (such as R202S, R213*, and R248Q) play in promoting MTX resistance, in which mutations have been identified in RA patients[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, our recent work has unraveled how the p53\u003csup\u003eR213*\u003c/sup\u003e mutant affects RA pathogenesis, demonstrating that this mutant can mitigate inflammatory arthritis in AIA rats through inhibition of the TBK1 IRF3 innate immune response[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. When conventional DMARDs prove ineffective, biologic DMARDs are often recommended. However, some patients remain resistant, with 40% of RA patients unresponsive to individual biologic therapies, and 5\u0026ndash;20% refractory to all such treatments[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 14-3-3 protein family, which consists of seven isoforms (β, γ, ε, η, τ, ζ, and σ) widely expressed within cellular chaperonin in eukaryotic cells, regulates multiple cellular functions such as cell cycle progression, maintenance of DNA damage checkpoints, apoptosis, and cytoskeletal dynamics, by binding to the Ser-X-pSer or Ser-X-pThr (X represents an arbitrary amino acid) residue[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recent research uncovered an important role of the 14-3-3 isoforms (ζ, σ and η), linked with target chemotherapy and drug resistance in cancer, in the multidrug resistance phenomenon in hepatocellular carcinoma[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, 14-3-3η had been reported to be a novel RA-related biomarker that induces the expression of multiple inflammatory factors, thereby facilitating the pathogenesis of RA[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Emerging knowledge regarding the roles of 14-3-3η in RA and its clinical implications as a diagnostic, prognostic and therapeutic response surrogate as well as potential drug target for RA treatment[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Interestingly, this protein isoform was found to be overexpressed in RA, suggesting its role in the disease.\u003c/p\u003e \u003cp\u003eThis study investigates the pathological contribution of 14-3-3η to drug resistance in autoimmune arthritis. Our study shows that 14-3-3η overexpression via AAV5 nullifies the therapeutic impact of MTX, leading to extreme paw swelling, significant hind paw volume and arthritis score increase, and exacerbated bone erosion in arthritic joints. Consistent with our findings in transgenic knock in (KI) rats, the inducible expression of 14-3-3η in AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η (+/+)\u003c/sup\u003e rats could also result in the release of proinflammatory cytokines and severe arthritic conditions in AIA rats with MTX treatment. Notably, these effects can be reversed with arsenic trioxide (ATO)-a tool drug known to eliminate 14-3-3η via ubiquitin-degradation. Mechanistic studies revealed that the overexpression of 14-3-3η contribute to apoptosis resistance by reducing the gene expression of p53. AAV-mediated overexpression of p53 markedly reversed the drug-resistant phenotype of AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η (+/+)\u003c/sup\u003e rats. Collectively, this study thus proposes 14-3-3η as a potential therapeutic target for drug-resistant rheumatoid arthritis or other inflammatory diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eElevated 14-3-3η levels have been found in the peripheral blood mononuclear cells (PBMCs) of RA patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe relationship between 14-3-3η expression and chronic joint inflammation in RA patients was investigated using qPCR to detect 14-3-3η mRNA levels in PBMCs from RA patients, osteoarthritis (OA) patients, and healthy individuals. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the 14-3-3η mRNA expression level in PBMCs of RA patients ranged from moderate to high, unlike in normal or PBMCs of OA patients where expression levels were marginal. This suggests that 14-3-3η may hold a regulatory role in the pathogenesis of RA but not in OA. Given the chronic nature of RA, drug therapy is often required for many years, which can potentially lead to the development of drug resistance[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Considering that 14-3-3η has been shown to promote drug resistance in hepatocellular carcinoma[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we examined the correlation between 14-3-3η expression and disease duration in RA. Our results indicated a positive association between the two, i.e., increased 14-3-3η expressions corresponds to extended disease duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, some RA patients with high disease activity do not respond to treatment with DMARDs or oral corticosteroids[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], although the mechanism of this drug resistance is not clear. To explore the link between drug resistance and the expression of 14-3-3η, we investigated the correlation between disease activity as rated by DAS28 scores and 14-3-3η expression in PBMCs of RA patients. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, a close correlation was identified between higher 14-3-3η expressions with the more severe disease activity. Simultaneously, several RA patients with high disease activity (DAS28\u0026gt;4.7) also exhibited extremely high expression of 14-3-3η in their PBMCs. These findings suggest 14-3-3η maybe a potential target for therapeutic intervention in severe RA, symbolizing an untapped opportunity in the field.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdeno-associated virus (AAV)-mediated overexpression of 14-3-3η contributes to MTX (methotrexate) resistance in adjuvant-induced arthritis (AIA) rats, whereas Arsenic trioxide (ATO) reverses 14-3-3η mediated MTX resistance in AIA rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous studies demonstrated that 14-3-3η plays an important role in inducing and maintaining the anti-oxidation/MDR state in HCC[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Interestingly, gene expression analysis on clinical samples demonstrated a significant increase in 14-3-3η expression in RA patients and also indicated a positive correlation between 14-3-3η expression and disease duration in RA patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To further address the role of 14-3-3η in AIA rats, one of the most commonly used standard arthritis models and reflecting a number of clinical characteristics of RA in humans[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], we created a treatment timeline for the in vivo study of 14-3-3η in an AIA rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To overexpress the 14-3-3η protein in AIA rats, articular injections of an adeno-associated virus 5 (AAV5) encoding 14-3-3η were conducted in the AIA rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). For AIA animal immunization, 8 week old male SD rats were immunized as described previously[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After that, the knee of each AIA rat was intra-articularly injected with a low dose of 3 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e PFU of AAV5-14-3-3η, a high dose of 1 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e PFU of AAV5-14-3-3η, or AAV5-null (a virus vector control) prior to adjuvant mixture injection on the same day. MTX (7.6 mg/kg/week) was orally administered to AIA rats as a positive control. On the other hand, ATO was used as a tool drug to test whether ATO-induced degradation of 14-3-3η[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] could reverse the MTX resistance phenotype in AIA rats. For this purpose, ATO (6 mg/kg/3 days) was intraperitoneally administered to AIA rats overexpressing 14-3-3η in the presence of MTX. Inflammatory symptoms were assessed every 3 days to monitor hind paw volume and arthritic scoring, and the AIA rats were then sacrificed on day 30. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G, AIA rats treated with AAV5-null (i.e., model group) first displayed the arthritic symptoms characterized by edema and/or erythema in the paws around day 12 after immunization and showed severe paw swelling by day 27, suggesting that the AIA model control group was successfully established.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H, AIA rats were intra-articularly injected with AAV5-null and gavage feeding with the same volume of normal saline as a model control. The paw edema of the rats was significantly greater than that of the healthy controls. AIA rats intra-articularly injected with a high dose of AAV5-14-3-3η were also observed the similar degree of paw edema, hind paw volume and arthritic score compared with those of the model control group, suggesting that the overexpression of 14-3-3η did not aggravate the inflammatory response. As expected, MTX treatment markedly suppressed the paw edema of the AIA rats compared with that of the model control group. However, AAV-mediated overexpression of 14-3-3η at both low and high doses markedly abolished the anti-arthritic effects of MTX, leading to severe paw edema and increased hind paw volume and arthritis score in AIA rats in a dose-dependent manner, suggesting that overexpression of 14-3-3η might contribute to MTX treatment failure. In addition, ATO was used as a tool drug to treat AIA rats overexpressing 14-3-3η in the presence of MTX. Of note, the paw edema, hind paw volume and arthritis score were significantly improved after ATO/MTX combined treatment in comparison with MTX treatment alone, suggesting that ATO might bind and trigger the degradation of 14-3-3η, thereby reversing MTX resistance phenotypes in AIA rats. Meanwhile, the AAV5-mediated expression of 14-3-3η in synovial tissues and PBMCs were also validated by qPCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, AAV5-14-3-3η injection significantly elevated the expression of the 14-3-3η mRNA in both synovial tissues and PBMCs from AIA rats, with a 3- to 4-fold increase in the 14-3-3η mRNA level compared with the levels in the AIA-AAV5-null group, confirming that AAV5-14-3-3η was also successfully overexpressed in AIA rats.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of 14-3-3η overexpression and ATO treatment on bone destruction in the paw articulations of MTX-resistant AIA rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine the severity of bone destruction in AIA rats overexpressing 14-3-3η with or without ATO treatment, all treatment groups of AIA rats were subjected to radiological examinations by micro-CT analysis. Representative radiographs of hind paws from the 7 different groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The severity of bone destruction in the AIA rats were further quantified and compared in the analysis of bone mineral density (BMD), cortical mineral density (TMD), trabecular number (Tb.N), and total porosity among all treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Overall, the micro-CT data demonstrated that cartilage destruction and bone erosion were severe in the AIA rats either with or without injection of AAV5-14-3-3η. MTX treatment drastically inhibited bone erosion in arthritic joints compared with that in the model group. In contrast, the MTX-treated AIA rats injected with a low dose or high dose of AAV5-14-3-3η exhibited poor therapeutic effects, leading to an aggravated bone erosion in arthritic joints (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B), indicating that the overexpression of 14-3-3η in AIA rats contributes to MTX treatment failure. Conversely, MTX combined with ATO treatment successfully reversed 14-3-3η-mediated MTX resistance in AIA rats, as indicated by the recovery of severe bone destruction in the AIA model. Taken together, these results suggested that the overexpression of 14-3-3η contributes to MTX resistance in AIA rats, whereas treatment with ATO could reverse bone erosion in MTX-resistant AIA rats. Accordingly, ATO may show a good protective effect on paw articulation in 14-3-3η-mediated MTX-resistant AIA rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, stenosis of the articular cavity and synovial hyperplasia appeared in the AIA rats injected with the AAV5-null virus control group compared with those in the healthy control group, which exhibited a normal articular cavity and a smooth surface of cartilage. Notably, the overexpression of 14-3-3η in AIA rats did not cause further severe cartilage destruction or bone erosion, suggesting that the overexpression of 14-3-3η does not increase the severity of inflammation in the AIA model. Apparently, AIA rats treated with MTX alone acquired a normal articular cavity and exhibited lowest synovial hyperplasia and inflammatory cell infiltration. Besides, when the AIA rats treated with either a low dose or high dose of AAV5-14-3-3η, MTX treatment failure was found and followed by cartilage destruction, synovial hyperplasia and immune cell infiltration compared with those in AIA rats treated with MTX alone. Collectively, the overexpression of 14-3-3η can lead to MTX resistance in AIA rats. However, ATO treatment completely reversed these MTX-resistant phenotypes in AIA rats, regardless of the degree of stenosis in articular cavity, synovial hyperplasia or infiltration of immune cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene expression analysis of proinflammatory cytokines in synovial tissues or blood samples from ATO-treated AIA rats overexpressing 14-3-3η\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the inflammatory potency of 14-3-3η expression in AIA rats, the levels of proinflammatory markers were examined in blood serum, synovial tissues and peripheral blood mononuclear cells (PBMCs) from AIA rats. As shown in \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C\u003c/b\u003e, the gene expression of TNF-α, IL-1β, IL-2, IL-6, IL-8 and MCP-1 was upregulated in the AIA rats injected with the AAV5-null virus control compared with the healthy controls. The gene expression of 14-3-3η was also monitored in all treatment groups to confirm the level of 14-3-3η gene expression after AAV5-14-3-3η injection. Notably, the overexpression of 14-3-3η in AIA rats induced by the injection of a high dose of AAV5-14-3-3η did not further increase the gene expression of inflammatory cytokines, suggesting that the overexpression of 14-3-3η does not increase the severity of inflammation in the AIA model. Apparently, AIA rats treated with MTX alone exhibited markedly suppressed gene expression of the above proinflammatory cytokines in the synovium. However, MTX treatment failed to suppress the expression of these proinflammatory genes in AIA rats injected with either a low dose or a high dose of AAV5-14-3-3η compared with AIA rats treated with MTX alone, suggesting that the overexpression of 14-3-3η can lead to MTX resistance in AIA rats. Consistently, ATO treatment effectively reversed these MTX-resistant in AIA rats in which the gene expression of these inflammatory cytokines in synovial tissue or PBMCs was significantly inhibited. Taken together, 14-3-3η might be an important target leading to MTX treatment failure in AIA rats. Moreover, ATO, a tool drug facilitating the proteasomal degradation of 14-3-3η, abolished 14-3-3η-mediated MTX resistance in AIA rats.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExamination of matrix metalloproteinases expression in synovial tissue or PBMCs from ATO-treated AIA rats overexpressing 14-3-3η\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMany studies have reported the involvement of matrix metalloproteinases (MMPs) in inflammatory diseases, including RA. The cytokines mentioned above can induce the expression of MMPs and proteinases, which are largely responsible for the irreversible destruction of cartilage, bone and tendons in the joints [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, Receptor activator of nuclear factor-kappa B ligand (RANKL) belongs to the TNF superfamily, is an activated T-cell\u0026ndash;producing factor that modulates dendritic cell survival and plays an essential role in osteoclast biology [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Of note, the expression of RANKL is increased in the synovial tissues of patients with RA[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], it plays an essential role of the RANKL\u0026ndash;RANK pathway in arthritic bone destruction of a series of animal experiments[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs shown in \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e, the expression of MMP-1, MMP-3, MMP-9 and RANK in the synovium or PBMCs of the model group was markedly elevated compared with that in the healthy control group. The mRNA expression of the abovementioned genes in the synovium or PBMCs of AIA rats was markedly suppressed by MTX treatment. In contrast, when the AIA rats were treated with either a low dose or a high dose of AAV5-14-3-3η, MTX treatment did not suppress the expression of these genes. Similarly, in MTX-resistant AIA rats treated with ATO, the expression of genes encoding key matrix metalloproteinases in synovial tissue was significantly suppressed. These findings indicated that ATO may protect cartilage and bone tissue by inhibiting the 14-3-3η-mediated mRNA expression of MMP-1, MMP-3 and MMP-9, as well as suppressing the level of RANKL.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConditional knock-in of 14-3-3η contributes to MTX resistance in AIA rats\u003c/h2\u003e \u003cp\u003eAlternatively, we further elucidated the function of 14-3-3η in AIA model by generation of transgenic SD rats using the Tet-On system. The tet regulatory system in this approach permitted tissue-specific and doxycycline-inducible control of 14-3-3η expression in transgenic rats. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B, the AIA arthritic models were established using 14-3-3η\u003csup\u003e+/+\u003c/sup\u003e SD rats. Consistent with the finding that AAV-mediated overexpression of 14-3-3η contributes to MTX resistance in AIA rats, the arthritis score, degree of paw edema and degree of bone destruction were significantly greater in the AIA\u003csup\u003eWT\u003c/sup\u003e group than in the healthy control\u003csup\u003eWT\u003c/sup\u003e group. In MTX treated AIA\u003csup\u003eWT\u003c/sup\u003e rats, the arthritis score, paw edema and bone destruction of the rats were markedly lower than those in the AIA\u003csup\u003eWT\u003c/sup\u003e group. However, in AIA rats overexpressing 14-3-3η (AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e) with MTX treatment, transgenic knock-in of 14-3-3η completely abolished the therapeutic effect of MTX, leading to severe paw edema, severe arthritis and severe bone erosion compared with those in AIA\u003csup\u003eWT+MTX\u003c/sup\u003e group, demonstrating that knock-in of 14-3-3η could lead to MTX resistance. On the other hand, we observed that the paw edema, hind paw volume, arthritis score and bone destruction were significantly recovered after ATO combined with MTX treatment, compared with the group of AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+ (MTX)\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D\u003cb\u003e).\u003c/b\u003e Meanwhile, the erythrocyte sedimentation rate (ESR) also matched very well with the arthritis condition of MTX-treated 14-3-3η knock-in AIA rats with or without ATO combined treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. On the other hand, the release of proinflammatory cytokines and matrix metalloproteinases was higher in the AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+ (MTX)\u003c/sup\u003e group than in the AIA\u003csup\u003eWT+ (MTX)\u003c/sup\u003e group. However, when MTX-resistant AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+ (MTX)\u003c/sup\u003e rats were co-treated with ATO, the gene expression of these inflammatory cytokines in PBMCs was significantly inhibited (\u003cb\u003eFig. S3A)\u003c/b\u003e. Finally, the gene expression of 14-3-3η were also validated in the PBMCs of the AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+ (MTX)\u003c/sup\u003e transgenic rats (\u003cb\u003eFig. S3B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eATO treatment enhances the proteasomal degradation of 14-3-3η via ubiquitination in RAFLSs\u003c/h3\u003e\n\u003cp\u003eOur previous work demonstrated that ATO targeted 14-3-3η for its degradation in hepatocarcinoma cells[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To investigate whether ATO treatment affects the expression of 14-3-3η in RAFLSs, we determined both the mRNA and protein expression of 14-3-3η in RAFLSs upon ATO treatment. Firstly, cell viability assay demonstrated that ATO inhibited the growth of RAFLSs in a dose-dependent manner, with an IC\u003csub\u003e50\u003c/sub\u003e of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, qPCR analysis indicated that the mRNA expression of 14-3-3η was dose-dependently downregulated upon treatment with different concentrations of ATO (0, 5, 10, 15, 20, or 25 \u0026micro;M) for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Furthermore, Western blot analysis also indicated that the addition of ATO dose-dependently suppressed the protein expression of 14-3-3η (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Here, we further validated the binding affinity of ATO on the 14-3-3η protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, ATO dose-dependently bound to 14-3-3η, and the binding curves of 14-3-3η suggested that a simple 1:1 binding mode occurred, with R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9964. The results of BLI analysis suggested that 14-3-3η exhibits good binding affinity for ATO. Therefore, these findings revealed that ATO could target 14-3-3η and inhibit its gene and protein expression in RAFLSs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the abovementioned results, ATO treatment suppressed the gene transcription and protein expression of 14-3-3η. To further investigate whether ATO treatment leads to the proteasomal degradation of 14-3-3η, the RAFLSs overexpressed with 14-3-3η were treated with the 26S proteasome inhibitor MG132[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], in the presence or absence of ATO. The 14-3-3η protein was immunoprecipitated with a specific antibody and then probed with antibody against ubiquitin. Compared with no ATO treatment, ATO treatment markedly enhanced the ubiquitination of 14-3-3η in RAFLSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These findings suggested that ATO facilitates the proteasomal degradation of 14-3-3η \u003cem\u003evia\u003c/em\u003e ubiquitination.\u003c/p\u003e\n\u003ch3\u003eThe overexpression of 14-3-3η contributes to MDR phenotypes in RAFLSs\u003c/h3\u003e\n\u003cp\u003eApart from animal studies of the drug-resistant potency of 14-3-3η, the multidrug resistance (MDR) effects of overexpressing 14-3-3η in RAFLS cells in response to many anti-arthritic drugs, such as MTX, hydroxychloroquine (HCQ), leflunomide (LEF), tacrolimus, and sulfasalazine (SSZ), were also examined. For this purpose, RAFLSs were transfected with a control vector or plasmid containing 14-3-3η-Flag. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the Western blot results demonstrated that 14-3-3η was successfully overexpressed in RAFLSs. Cell viability assays demonstrated that the RAFLS cells transfected with 14-3-3η-Flag-tagged plasmids were more resistant to anti-arthritic drugs (MTX, HCQ, LEF, SSZ and tacrolimus) than the RAFLS cells transfected with the control vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The IC50s (\u0026micro;M) and resistance factors of these five drugs for the overexpression of 14-3-3η in RAFLS cells were as follows: MTX (11.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 \u0026micro;M vs. over 100 \u0026micro;M with resistance factor\u0026thinsp;\u0026gt;\u0026thinsp;8.63), HCQ (30.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u0026micro;M vs. over 100 \u0026micro;M with resistance factor\u0026thinsp;\u0026gt;\u0026thinsp;3.24), LEF (16.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u0026micro;M vs. 47.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 \u0026micro;M with resistance factor 2.89), SSZ (26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u0026micro;M vs. 33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 \u0026micro;M with resistance factor 1.26) and tacrolimus (12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 \u0026micro;M vs. 28.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u0026micro;M with resistance factor 2.40). These results demonstrated that the DMARD drugs MTX, HCQ, LEF, SSZ, and tacrolimus are insensitive to RAFLSs in the presence of 14-3-3η.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR array analysis identifies p53 tumor suppressor gene as drug resistance-related gene in RAFLSs overexpressing 14-3-3η\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the target drug resistance genes in 14-3-3η-overexpressing RAFLS cells, a total of 84 genes (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e) related to drug resistance were compared between the control vector- and 14-3-3η-Flag-tagged plasmid-transfected RAFLS cells via real-time PCR microarray of Human Cancer Drug Resistance PCR Array Targeted RNA Virtual Panels. Western blot analysis demonstrated that 14-3-3η was successfully overexpressed in RAFLSs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Besides, the transcripts detected via a scatter plot indicated that 4 genes (green dots), TP53, ERBB3, ERBB4 and NAT2, were downregulated (\u0026gt;\u0026thinsp;1.5-fold) after the overexpression of the 14-3-3η gene compared with those in the control vector group. These genes are responsible for diverse functions related to cell proliferation, apoptosis, the cell cycle, lysosomal function and ATP-binding cassette[\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The black dots indicate unchanged gene expression.\u003c/p\u003e \u003cp\u003eTo further validate the significant changes in gene expression, p53, a mostly downregulated gene, was validated by qPCR and Western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, mRNA and protein expression of p53 were significantly suppressed in RAFLS cells overexpressed with the 14-3-3η compared to those in the vector Ctrl group. However, p53 overexpression did not elevate 14-3-3η expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting that p53 might act downstream of 14-3-3η. This result is therefore consistent with the reported function of p53, which is commonly responsible for drug resistance in cancer.\u003c/p\u003e\n\u003ch3\u003eThe overexpression of 14-3-3η may contribute to apoptosis resistance in RAFLSs\u003c/h3\u003e\n\u003cp\u003eThe p53 tumor suppressor gene regulates apoptosis in cancer and RA[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We therefore investigated whether apoptotic pathways are affected by 14-3-3η-mediated downregulation of p53. We then overexpressed 14-3-3η in RAFLSs and determined the expression of apoptotic markers. As expected, antiapoptotic markers such as Bcl-2 and Bcl-xl were markedly upregulated, whereas proapoptotic markers, including Bax and cytochrome c, were significantly downregulated with p53 protein in RAFLSs transfected with 14-3-3η (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), whereas the gene and protein expression of p53 were also found to be concomitantly downregulated in PBMC or synovial tissues of MTX-treated AIA rats overexpressing 14-3-3η (AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), suggesting that RAFLSs may develop apoptosis-resistant phenotypes upon the reduction of p53 expression. Accordingly, 14-3-3η may mediate MTX resistance in RAFLSs through the downregulation of p53, thereby promoting apoptosis resistance pathways.\u003c/p\u003e \u003cp\u003eTo investigate the mechanism underlying 14-3-3η-mediated p53 protein reduction, we performed co-immunoprecipitation (co-IP) and Western blot assays in 293T cells with or without FLAG-14-3-3η overexpression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, the input panel presented the Western blot analysis of the total cell lysate, while the IP panel showed the Western blot of the cell lysate immunoprecipitated with anti-Flag magnetic beads. Notably, in lanes 3 and 4 of the IP blot, p53 protein was only detected in the FLAG-14-3-3η-immunoprecipitated lysate, indicating that p53 is a 14-3-3η-interacting protein. To assess the mechanism of 14-3-3η-mediated p53 reduction, cells were then treated with MG-132, a known inhibitor for proteasomal degradation. In both input and IP blotting, MG-132 treated-immunoprecipitated cell lysate (lane 4) revealed a significantly higher expression level of interacting p53 proteins, compared with that of lane 3, suggesting that the interaction between 14-3-3η and p53 accelerated p53 degradation by proteasomal degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\n\u003ch3\u003eExogenous p53 supplementation reverses the MTX resistance phenotype in AIA transgenic rats\u003c/h3\u003e\n\u003cp\u003eAn inextricable link between the 14-3-3η-mediated drug resistance phenotype in RA and p53 has been clearly established. To further address whether the p53 tumor suppressor gene plays an equally critical role in transgenic animal models with 14-3-3η overexpression, we adopted an adeno-associated virus (AAV5-p53) to induce p53 overexpression in methotrexate (MTX)-resistant AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e transgenic rats via tail vein injection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the paw swelling and arthritis scores revealed that arthritis symptoms in AIA rats overexpressing 14-3-3η remained comparable to those in AIA rats following MTX treatment. Conversely, AIA rats receiving only AAV5-p53 presented minimal relief of paw swelling, whereas the AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e group receiving both AAV5-p53 and MTX presented significantly improvement in paw swelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Quantitative analysis of 14-3-3η and p53 expression levels in PBMCs from all experimental rats indicated that p53 expression was markedly suppressed in the 14-3-3η-overexpressing groups (AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e and AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+ (MTX)\u003c/sup\u003e) than in the control group. In contrast, p53 expression was significantly elevated in the rats after receiving of AAV5-p53 injections (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, the swollen paws from all experimental groups were captured and collected at the time of sacrifice, and subjected to micro-CT analysis for their joint bone destruction and total porosity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, the paw images with yellow arrows indicated the specific sites of bone destruction, multiple bone lesions were also observed in the AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+ (MTX)\u003c/sup\u003e group despite MTX treatment. These findings suggest that the therapeutic efficacy of MTX in mitigating bone destruction was compromised in AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e transgenic rats. In contrast, AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e transgenic rats receiving exogenous p53 intervention, especially those treated concurrently with MTX, exhibited significant decrease in both joint swelling and bone damaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In addition, the positive bone improvement-related indicators, including bone mineral density (BMD), tissue mineral density (TMD), bone volume fraction (BV/TV), and trabecular number (Tb.N), were markedly increased, whereas the negative indicator, total porosity, was decreased. Collectively, the corresponding MicroCT score was recovered to a range of 0.6\u0026ndash;0.8, indicating the mild bone destruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Furthermore, erythrocyte sedimentation rate (ESR) analysis also indicated that MTX-treated 14-3-3η-overexpressing AIA rats presented a significant reduction in the ESR following exogenous p53 intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Additionally, rats subjected to continuous doxycycline (Dox) induction presented slightly lower body weights than non-induced rats did (\u003cb\u003eFig. S4A\u003c/b\u003e). Splenomegaly, a characteristic pathological feature of AIA rats and an indicator of inflammatory response intensity, was generally observed in 14-3-3η-overexpressing rats even after MTX treatment. However, the spleen organ index was significantly reduced in the rats treated with both AAV5-p53 and MTX (\u003cb\u003eFig. S4B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAdenovirus\u0026ndash;mediated p53 expression sensitizes the immunomodulatory effect of MTX in the AIA \u003csup\u003e14\u0026minus;3\u0026minus;3η+/+\u003c/sup\u003e transgenic rat\u003c/h2\u003e \u003cp\u003eTo assess the level of autoimmune activation and disease progression in AIA rats, we analyzed the populations of Th17 (IL-17A) and Treg (Foxp3) cells in the peripheral blood via flow cytometry. The results indicated that AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e rats treated with AAV5-p53 combined with MTX would markedly shift the immune balance toward an immunosuppressive status (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B), as evidenced by an increase of Foxp3/IL-17A ratio. In contrast, MTX treatment alone did not alter the Foxp3/IL-17A balance under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Additionally, we evaluated the CD4/CD8 T cells ratio, which is typically elevated in AIA rats due to heightened immune activity. Our findings revealed that exogenous p53 significantly reduced the CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T cells ratio in peripheral blood, suggesting that p53 suppresses the overactive autoimmune response, facilitating the effective treatment of AIA rats (\u003cb\u003eFig. S4C-E\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative PCR analysis revealed that exogenous p53 significantly enhanced the therapeutic effect of MTX by downregulation of IL-6, IL-1β and TNF-α expression in AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;\u0026thinsp;3η+/+\u003c/sup\u003e rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). To assess immune homeostasis comprehensively, we further performed multiplex flow cytometry analysis (LEGENDplex\u0026trade;) on rat serum to quantify multiple cytokines. Consistently, our results demonstrated that combined treatment of AAV5-p53 and MTX significantly decreased the serum levels of TNF-α, IFN-γ, IL-6, IL-1α, IL-17A, GM-CSF, IL-18, IL-12p70, IL-33, and IL-1β in the AIA \u003csup\u003e14\u0026minus;3\u0026minus;\u0026thinsp;3η+/+\u003c/sup\u003e transgenic rat (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRA is a chronic inflammatory autoimmune disease that primarily affects joints and has a wide range of extra-articular and systemic manifestations. Its pathological features include synovial hyperplasia, inflammatory cell infiltration, pannus formation and bone tissue injury, resulting in joint swelling and pain and eventually bone destruction and dysfunction[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Refractory RA seriously affects patients\u0026rsquo; quality of life. However, the specific mechanism of drug resistance in RA remains unclear. Most of the drugs commonly used in clinical practice have limited efficacy and many side effects. Two members (ζ and σ) of the 14-3-3 proteins have been confirmed to be associated with chemotherapy resistance and resistance to molecular targeted drugs in cancer[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It has been shown that 14-3-3η plays an important role in inducing/maintaining the multidrug resistance phenotype in hepatocellular carcinoma [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Interestingly, we also found that 14-3-3η is aberrantly highly expressed in RA. However, whether 14-3-3η contributes to drug resistance in RA is not known. In this study, we report that 14-3-3η is a new therapeutic target for drug-resistant RA or other inflammatory diseases. We demonstrated that 14-3-3η overexpression via AAV5 negates the therapeutic impact of MTX, leading to extreme paw swelling, a significant increase in hind paw volume and arthritis score, and exacerbated bone erosion in arthritic joints. Our data also shown that in transgenic knock-in (KI) rats, the inducible expression of 14-3-3η in AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e rats could also result in the release of proinflammatory cytokines and severe arthritic conditions in AIA rats treated with MTX. Notably, these effects can be reversed with arsenic trioxide (ATO), a tool drug known to eliminate 14-3-3η via ubiquitin-mediated degradation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mechanistic studies revealed that the overexpression of 14-3-3η contributes to apoptosis resistance by reducing the gene expression of p53. AAV-mediated overexpression of p53 markedly reversed the drug resistance of AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e rats.\u003c/p\u003e \u003cp\u003eThe AIA model has the same immunological and pathological characteristics as human RA and is considered an ideal animal model for RA[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. An AIA rat model was established to evaluate the drug resistance of 14-3-3η. The results showed that AAV-mediated overexpression of 14-3-3η contributed to MTX resistance in AIA rats, which aggravated basic signs such as paw swelling and the arthritis index. It also increases joint inflammation and synovial tissue destruction, suggesting a potential therapeutic target for 14-3-3η. Indeed, we confirmed that ATO, a tool drug that inhibits 14-3-3η, reverses 14-3-3η-mediated MTX resistance in AIA rats. Cytokines regulate a wide range of inflammatory processes in the pathogenesis of RA, especially proinflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and MCP1[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The cytokines mentioned above can induce the expression of matrix metalloproteinases (MMPs) and proteinases, which are largely responsible for the irreversible destruction of cartilage, bone and tendons in joints[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Although the inflammatory response is not the only characteristic of RA, it is a major problem for patients. Therefore, controlling the inflammatory response may be a viable way to treat RA. We found that ATO treatment significantly inhibited the levels of TNF-α, IL-1β, IL-6, IL-8 and MCP1 in serum and synovial tissues, as well as the expression of MMP-1, MMP-3, MMP-9 and RANK, suggesting that ATO can improve 14-3-3η-mediated MTX resistance in the systemic inflammatory response of AIA rats by regulating inflammatory cytokines. We also confirmed that the inducible expression of 14-3-3η in AIA\u003csup\u003e14\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026minus;3η+/+\u003c/sup\u003e rats could also result in the release of proinflammatory cytokines and severe arthritic conditions in AIA rats treated with MTX. Notably, these effects can be reversed with ATO, which is known to eliminate 14-3-3η via ubiquitin-mediated degradation. In vitro experimental results confirmed that ATO treatment enhances the proteasomal degradation of 14-3-3η via ubiquitination in RAFLSs. These results are in line with other research groups\u0026rsquo; reports, which show that ATO could be considered a potential molecular targeted agent for the treatment of HCC[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring the course of RA, the synovium transforms into hyperplastic invasive tissue, leading to cartilage and bone destruction. RAFLSs in the synovial lining develop aggressive phenotypes and produce pathogenic mediators that lead to the occurrence and progression of disease, playing a major role in RA pathophysiology[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, there has been recent interest in RAFLSs as therapeutic targets in RA to avoid side effects associated with many existing drug-resistant RA treatments. To examine the drug resistance potency of 14-3-3η in vitro, the multidrug resistance (MDR) effects of overexpressing 14-3-3η in RAFLSs were examined in many antiarthritic drugs, such as methotrexate, hydroxychloroquine, leflunomide, tacrolimus, and sulfasalazine. The RAFLSs were transfected with a control vector or a 14-3-3η-Flag-tagged plasmid. We found that the overexpression of 14-3-3η contributes to MDR phenotypes in RAFLSs. Furthermore, to explore the clear target drug resistance genes in 14-3-3η-overexpressing RAFLSs, whole-cell mRNA expression analysis was carried out by using a human cancer drug resistance PCR array targeting RNA virtual panels. The results indicated that the expression of 4 genes (green dots), p53, ERBB3, ERBB4 and NAT2, was downregulated (\u0026gt;\u0026thinsp;1.5-fold) after the 14-3-3η gene was overexpressed compared with that in the control vector group. Since 14-3-3η overexpression results in the greatest increase in p53 expression, we further investigated the mechanism by which it promotes drug resistance in RAFLSs. We then confirmed that p53 overexpression cannot increase 14-3-3η expression, suggesting that p53 might be downstream of 14-3-3η. The p53 tumor suppressor protein regulates apoptosis in cancer and RA. We therefore investigated whether apoptotic pathways are affected by 14-3-3η-mediated downregulation of p53. We further investigated the downstream p53-related pathway and found that 14-3-3η-mediated MTX resistance in RAFLSs may occur through the downregulation of p53, thereby upregulating apoptosis resistance pathways. This result is consistent with the reported function of p53, which is responsible for drug resistance in cancer. Numerous studies have demonstrated that various isoforms of 14-3-3 (such as γ, ε, ζ, τ, and σ) regulate p53 through direct interactions. However, the interaction between 14-3-3 isoforms (β and η) and p53 remains unexplored. Additionally, isoforms such as 14-3-3 α, γ, ε, ζ, η, and τ tend to promote cell survival, potentially facilitating p53 degradation by positively regulating MDM2[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. More intriguingly, we employed AAV5-p53 as an intervention approach to effectively reverse the drug-resistant phenotype of rats in the 14-3-3η\u003csup\u003e(+/+)\u003c/sup\u003e transgenic AIA model. Various pathological alterations of arthritis were significantly ameliorated, encompassing paw swelling, bone destruction, Foxp3/IL-17A immune equilibrium and serum inflammatory factor levels. Therefore, our results elaborated that 14-3-3η mediates the progression of drug resistance in RA via elimination of p53. The supplementation of exogenous p53 markedly enhances the sensitivity of MTX-resistant AIA rats due to 14-3-3η overexpression. Indeed, the role of p53 in the development of drug resistance has been extensively studied. Notably, gene mutations that impair or abolish p53 function is strongly associated with resistance to several widely used therapeutic agents, including cisplatin, doxorubicin, paclitaxel, gemcitabine, and tamoxifen[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These mutations, predominantly located in the DNA-binding domain of p53, often result in either a loss of p53 expression or the production of dysfunctional mutant proteins. Consequently, p53 can paradoxically serve as a protector of cancer cells rather than a tumor suppressor [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].Furthermore, drug resistance mechanisms frequently involve disruptions in the balance between pro-apoptotic and anti-apoptotic pathways, with p53 playing a central regulatory role in apoptosis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our study is the first to report the relationship between 14-3-3η and p53, which fills a gap in the research on the 14-3-3 family and p53.\u003c/p\u003e \u003cp\u003eOn the other hand, dysfunction of p53 in RA fibroblast-like synoviocytes (RA-FLS) leads to apoptosis resistance and increased production of inflammatory mediators like IL-6 and MMP-1, contributing to chronic synovial inflammation. p53 inhibition reduces FOXP3 expression, resulting in decreased regulatory T cells (Tregs) and increased polarization of CD4\u003csup\u003e+\u003c/sup\u003e T cells toward Th17 cells, which produce high levels of IL-17, promoting osteoclastogenesis. This p53 dysfunction activates NF-κB and MAPK signaling pathways, escalating the expression of pro-inflammatory cytokines and matrix metalloproteinases. IL-6 plays a pivotal role in shifting the balance toward Th17 cells, enhancing the inflammatory response. Deficient p53 in T cells contributes to reduced Treg proportions and heightened Th17 differentiation in RA patients, exacerbating autoimmune arthritis. Overall, impaired p53-dependent apoptosis and proliferation in synovial cells are crucial mechanisms driving chronic inflammation and cartilage destruction in RA[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBesides, previous studies demonstrated that ATO may target the 14-3-3η protein for its ubiquitination and degradation. Here, we further validated the binding affinity of ATO for the 14-3-3η protein. We confirmed that ATO exhibited good binding affinity for 14-3-3η and that it inhibited the mRNA and protein expression of 14-3-3η in a dose-dependent manner. However, ATO could also facilitate the proteasomal degradation of 14-3-3η via ubiquitination, which is coincided to our previous studies, as addressed above.\u003c/p\u003e \u003cp\u003eIn conclusion, our research underscores the pivotal role of 14-3-3η in fostering drug resistance in RA by modulating p53 expression. We used gene transfer with AAV5-14-3-3η to effectively demonstrate MTX resistance in AIA rats. Furthermore, we developed doxycycline-inducible 14-3-3η knock-in transgenic SD rats using CRISPR/Cas-mediated genome engineering, which successfully confirmed these MTX-resistant effects in AIA rats. Our findings confirm that 14-3-3η induces MTX resistance in RAFLSs/AIA rats by suppressing p53, thereby activating signaling pathways that confer resistance to apoptosis. Remarkably, supplementing with p53 significantly reversed this MTX resistance phenotype. Additionally, our study is the first to report that arsenic trioxide (ATO), used here as an experimental drug, effectively counters 14-3-3η-mediated MTX resistance in AIA rats. These discoveries open avenues for exploring new compounds targeting this novel resistance mechanism, potentially leading to innovative treatments for MTX-resistant RA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eInactivated Mycobacterium tuberculosis (BD#231141) was purchased from BD Company (New Jersey, USA). Mineral oil and arsenic trioxide (ATO, As2O3, \u0026gt; 99% purity) were obtained from Sigma‒Aldrich (Shanghai, China). Methotrexate (MTX), hydroxychloroquine (HCQ), leflunomide (LEF), sulfasalazine (SSZ) and tacromus with purities above 99.0% were obtained from the China National Institute for Food and Drug Control (Beijing, China). Doxycycline was purchased from Beyotime (Shanghai, China). Lipofectamine 3000 reagent and an MTT kit were purchased from Invitrogen (Shanghai, China). MG-132 was purchased from MedChemExpress (New Jersey, USA). All the compounds were dissolved in dimethyl sulfoxide (DMSO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL (Gaithersburg, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eImmortalized RAFLSs were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in a 5% CO\u003csub\u003e2\u003c/sub\u003e, 37°C humidified incubator in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, UK). The control vector and pcDNA-3.1-14-3-3η-Flag were synthesized by Generay Biotech (Shanghai, China). RAFLS cells were transiently transfected using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol (Note: plasmids DNA used: 5 ng/ml). After transfection, the cells were cultured in fresh medium supplemented with 10% FBS for another 48 h before being used for other experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytotoxicity assay\u003c/b\u003e \u003cem\u003es\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 5.0 mg·ml\u003csup\u003e− 1\u003c/sup\u003e) assay. In brief, RAFLSs (5×10\u003csup\u003e3\u003c/sup\u003e cells per well) were seeded in 96-well plates for 24 hrs. Cells were then treated with indicated concentrations of compounds dissolved in DMSO, whereas DMSO alone was used as controls. Subsequently, MTT (10 µL) was added to each well for 4 hr, followed by the addition of 100 µL solubilization buffer (10% SDS in 0.01 mol·L\u003csup\u003e− 1\u003c/sup\u003e HCl) and overnight incubation. The optical density was read at a wavelength of 570 nm with a microplate reader. The percentage of cell viability was calculated by the following formula: Cell viability (%) = A\u003csub\u003etreated\u003c/sub\u003e/A\u003csub\u003econtrol\u003c/sub\u003e × 100. The data were obtained in triplicate from three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse-transcription PCR (qRT‒PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted and reverse-transcribed into cDNA using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. The expression of mRNAs was determined using SYBR Premix Ex Taq Master Mix (2×) (Takara). The relative expression level of the target gene was calculated using the comparative Ct method. β-Actin was used as an internal control to normalize sample differences. The sequences of the primers used for qRT‒PCR analysis are presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal proteins were harvested from tissues or RAFLS cells using 1×RIPA lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS and protease inhibitors; Wheaton Science) on ice for 30 min and analyzed via SDS‒PAGE. The PVDF membrane was milk blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, followed by incubation with anti-14-3-3η (CST, 1:1000, #940), anti-Flag (CST, 1:1000, #14793S), anti-ubiquitin (CST, 1:1000, #20326S) and anti-β-actin (Abcam, 1:3000, ab134175) antibodies overnight at 4°C. Immunocomplexes were visualized through chemiluminescence using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eAfter RAFLSs were pretreated with or without 20 µM MG-132 for 2 h, the cells were then exposed to 10 µM ATO for 6 h. Then, the cells were extracted for 30 min with immunoprecipitation lysis buffer (Beyotime Co., Ltd.). After centrifugation of the preparations, 100 µg of total protein was incubated with 14-3-3η antibody(dilution,1:100) at 4°C overnight. Then, the protein-antibody complexes were incubated with IgG Sepharose beads (Beyotime Co., Ltd.) at 4°C for another 12 h. Afterwards, the supernatants were removed (positive control), and the beads were washed three times (residual supernatants served as a negative control), boiled to remove protein from the beads. Positive control, negative control and protein interactions were analyzed by Western Blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRT\u003csup\u003e2\u003c/sup\u003e profiler human cancer drug-resistance PCR array analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was obtained from 14-3-3η-overexpressing RAFLS cells via a Qiagen RNeasy® Mini Kit (Qiagen). A human cancer drug resistance-specific RT‒PCR array (PAHS-012ZA, Qiagen) was used to evaluate the potential differential genes expression in RAFLSs after 14-3-3η overexpression. The drug resistance array comprised 87 genes (Supplementary Table\u0026nbsp;2) selected based on their involvement in drug resistance. Five housekeeping genes served as positive controls. Total RNA was reverse transcribed using the RT\u003csup\u003e2\u003c/sup\u003e First Strand Kit. Real-time PCR was carried out on a ViiA™ 7 Real Time PCR System (Applied Biosystems, USA) using the RT\u003csup\u003e2\u003c/sup\u003e SYBR® Green qPCR Mastermix (Qiagen, Germany) according to the manufacturer’s instructions. Data analysis was performed using Qiagen’s integrated web-based software package for the PCR Array System, which automatically performs all the ΔΔCt-based fold-change calculations from the raw threshold cycle data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePatient recruitment and specimens\u003c/h2\u003e \u003cp\u003eA blood sample was collected from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy volunteers at the Affiliated Hospital of Southwest Medical University, with the collection process being approved by both the hospital and the research ethics committee under the reference number (KY 2021010). After obtaining informed consent, the researchers proceeded to conduct an epidemiological survey and classification of all volunteers, strictly following the standards set by the American College of Rheumatology. The survey included detailed clinically relevant information and data, which are comprehensively outlined in Supplementary Table\u0026nbsp;3. Peripheral blood specimens from patients and healthy donors were obtained in blood collection tubes. Peripheral blood mononuclear cells were extracted in red cell lysis buffer (Beyotime Biotechnology Inc., Shanghai) for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eWild-type male Sprague‒Dawley (SD) rats (6 weeks, 80–120 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. All rats were housed in clean plastic cages and maintained at 23 ± 1°C with 55% relative humidity, a 12-h light/dark cycle (8:00–20:00) and ad libitum access to pure water and standard rat pellets. The study protocol was reviewed and approved by Macau University of Science and Technology (Protocol approval code: MUSTARE-003-2020). All experiments, including animal breeding, experimental operations, and animal euthanasia, were performed in accordance with the guidelines established by the committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of a rat AAV5 vector and intra-articular administration\u003c/h2\u003e \u003cp\u003eAdeno-associated virus 5 (AAV5) was manufactured by Hanbio Biotechnology (Shanghai, China). The AAV5 vector was generated after cloning the full-length coding sequence of 14-3-3η into the adeno-associated virus vector pHBAAV5-CMV-3×flag-ZsGreen. AAV5 packaging was performed by cotransfecting AAV5-293 cells with the recombinant AAV5 vector, pAAV-RC vector, or pHelper vector. AAV5 was collected from the AAV5-293 cell supernatant, condensed, and purified for further animal experiments. The virus titer was 2.0×10\u003csup\u003e12\u003c/sup\u003e vg/mL. Viral titers were determined by quantitative PCR of the CMV (cytomegalovirus) sequence, and the viral stock in this study was diluted with phosphate-buffered saline (PBS) and adjusted to final concentrations of 3 × 10\u003csup\u003e9\u003c/sup\u003e and 1 × 10\u003csup\u003e11\u003c/sup\u003e viral genomes (vg)/µl. Rats received an intra-articular injection of AAV5 expressing 14-3-3η. The rats were caught alive, and the skin above the articular joint was shaved. Finally, the rats were injected intra-articularly with AAV5-14-3-3η in PBS using 25-gauge needles (TERUMO Company, Philippines) and 25 µl CASTIGHT syringes (TERUMO Company, Philippines).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of a rat adjuvant-induced arthritis (AIA) model and drug administration\u003c/h2\u003e \u003cp\u003eMale SD rats were randomly divided into 7 groups (n = 8): (1) healthy control, (2) AIA model + AAV5-null, (3) AIA model + AAV5-14-3-3η high dose (HD, 1×10\u003csup\u003e11\u003c/sup\u003e PFU), (4) AIA model + methotrexate (MTX, 7.6 mg/kg/week, gavage) + AAV5-null, (5) AIA model + MTX (7.6 mg/kg/week) + AAV5-14-3-3η low dose (LD, 3×10\u003csup\u003e9\u003c/sup\u003e PFU), (6) AIA model + MTX (7.6 mg/kg/week) + AAV5-14-3-3η high dose (HD, 1×10\u003csup\u003e11\u003c/sup\u003e PFU), an equal volume of AAV virus was injected on day 0, and (7) arsenic trioxide (ATO, 6 mg/kg/3 days, intraperitoneal injection) + AIA model + MTX (7.6 mg/kg/week) + AAV5-14-3-3η (1×10\u003csup\u003e11\u003c/sup\u003e PFU). The HBAAV5/CMV/EGFP r14-3-3η vector (AAV5-14-3-3η) was used. Mineral oil containing 5.0 mg/ml \u003cem\u003eM. tuberculosis\u003c/em\u003e was ground and rolled intensively until the mixture turned white. Rats were injected with adeno-associated virus into the knee joints, and 0.1 ml of the mixture was subsequently injected subcutaneously at the base of the tail on day 0. The rats were sacrificed on day 30. Finally, the blood, organs and joint tissue were harvested for biological assays, paw volume assessment and micro-CT analysis. The ankle joints were collected for H\u0026amp;E staining, followed by microscopic observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of inducible 14-3-3η (gene name: YWHAH) knock-in (KI) rats\u003c/h2\u003e \u003cp\u003eTo generate inducible YWHAH KI rats, we performed targeted knock-in in rat embryos using the CRISPR/Cas9 system as described below: potential Cas9 cleavage sites were identified by screening genomic regions of interest using online software. The sequence of the sgRNA used was GGCCGAGTCGCGAGCGACATGGG. The homology arms for targeted knock-in were amplified by PCR using a BAC clone as a template. In addition to the homologous arms, the targeting construct also contained the Kozak-tTS/rtTA-BGH pA-anti[TRE promoter-Kozak-Rat YWHAH CDS-3×GGGGS-EGFP-rBG pA] fragment. The aim was to replace the ATG start codon. tTS/rtTA is driven by the rat YWHAH endogenous promoter, and the Rat YWHAH CDS-3xGGGGS-EGFP is driven by the TRE promoter. When the rats were treated with doxycycline (Dox), the YWHAH CDS-3xGGGGS-EGFP was expressed. One-cell-stage zygotes were obtained by mating SD males with SD females superovulated by the injection of pregnant mare serum gonadotropin and human chorionic gonadotropin. The Cas9 protein, synthesized sgRNA and targeting vector were coinjected into the cytoplasm of pronuclear-stage embryos. The injected embryos were cultured in KSOM medium overnight, and those that developed to the two-cell stage were transferred into the oviduct of pseudopregnant females. Pups were genotyped, and correct integration of the fragment carrying the Kozak-tTS/rtTA-BGH pA-anti[TRE promoter-Kozak-Rat YWHAH CDS-3×GGGGS-EGFP-rBG pA] construct was confirmed by Sanger sequencing. The correctly integrated mutant founder rats were further back-crossed with wild-type (WT) SD rats for the F1 generation. The F1 animals were genotyped by PCR, and the positive animals were confirmed by Sanger sequencing. All experimental procedures were performed at the AAALAC-accredited facilities at Cyagen Biosciences, Inc. (Guangzhou, China). Male SD rats were randomly divided into 5 groups (n = 8) as follows: (1) healthy control \u003csup\u003eWT\u003c/sup\u003e, (2) AIA\u003csup\u003eWT\u003c/sup\u003e, (3) AIA\u003csup\u003eWT+MTX\u003c/sup\u003e, (4) AIA\u003csup\u003e14 − 3−3η+/+ (MTX)\u003c/sup\u003e, and (5) AIA\u003csup\u003e14 − 3−3η+/+ (MTX+ATO)\u003c/sup\u003e. The rats were sacrificed on day 30. Finally, the blood, organs and joint tissue were harvested for biological assays, paw volume assessment and micro-CT analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDoxycycline treatment\u003c/h2\u003e \u003cp\u003eDoxycycline hyclate (Beyotime Biotechnology, China) was prepared in phosphate-buffered saline (PBS). Doxycycline hyclate (8 mg/kg) was intraperitoneally administered to transgenic rats for 7 consecutive days to induce 14-3-3η overexpression in YWHAH-KI rats before the AIA animal model was established. Doxycycline hyclate (8 mg/kg) was intraperitoneally administered to YWHAH-KI rats every 5 days to maintain high 14-3-3η expression.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eArthritis scoring assessment\u003c/h2\u003e \u003cp\u003eFrom day 0 to day 30, the volumes and arthritis status of the hind paws were measured every 3 days. The arthritis score (0 to 4) was blindly evaluated by unsuspecting researchers according to the following criteria: 0, normal; 1, mild redness of the ankle or tarsal joint; 2, mild redness and swelling of the ankle to the tarsal bone; 3, moderate swelling from the ankle to the metatarsal joints; and 4, severe swelling of the ankles, paws, and fingers. At the end of the treatment, the paw volume was measured along with the toe volume.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological analysis\u003c/h2\u003e \u003cp\u003eIn the AIA models, the ankle joints were dissected from anesthetized rats by chloral hydrate, fixed in a buffered 4% paraformaldehyde solution for 24 h, and embedded in paraffin. Finally, paraffin sections (3 µm) were cut and stained with hematoxylin and eosin (H\u0026amp;E).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMicro-CT analysis\u003c/h2\u003e \u003cp\u003eRat hind paws were harvested, and soft tissues, including muscles and skins, were dissected from all treatment groups. The remaining tissues, including the whole original rat knee joints, were fixed in formaldehyde and then stored in 70% ethanol. CT scanning was performed using high-resolution µCT (Skyscan 1172). Images were captured and reconstructed with CTAn v1.9 and NRecon v1.6., and then analyzed by 3-D model visualization software (CTVol v2.0). A voltage of 50 kVp, a resolution of 5.7 µm per pixel and a current of 200 µA were used for the scanner. After scanning, each sample was assigned a random number for blinded assessment and then processed for image processing. Transverse, coronal and sagittal images of the knee joints were used for images analysis. The region of interest covering the surface area of the tibia was collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eEnzyme-linked immunosorbent assay (ELISA) was conducted as described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, blood was obtained from the orbital sinus of mice. Serum levels of IgG antibodies were measured using a commercially available ELISA kit (Bethyl Laboratories, USA). Horseradish peroxidase (HRP) activity was measured using tetramethyl benzidine as a substrate (eBioscience, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eBiolayer interferometry analysis (BLI)\u003c/h2\u003e \u003cp\u003eAll BLI binding assays were performed using an Octet RED96 (ForteìBIO, China). A shake speed of 1000 rpm and plate temperature of 30°C were conducted for all the experiments. PBS was used as the kinetics buffer. To prepare 14-3-3η-bound test probes, super streptavidin (SSA) optic fiber probes (ForteìBIO, USA) were run at baseline in PBS for 60 s, loaded in 200 µL of biotinylated 14-3-3η solution at 125 µg/mL for 600 s, run at baseline again in PBS for 60 s, and stored at 4°C dipped in PBS. For the binding kinetics assays, a series of dilutions of six concentrations of ATO dissolved in PBS were added to a black polypropylene 96-well microplate (Greiner Bio-One, Germany) with PBS filling the remaining wells. One row was left as a PBS-only negative control. Each well contained a total volume of 200 µL. An assay cycle consisted of 120 s of baseline incubation in PBS followed by 120–180 s of association in compound solution followed by 120–180 s of dissociation in PBS, and this cycle was repeated for every concentration and with both a 14-3-3η-loaded probe and a blank probe. Analysis of the BLI results was performed using ForteìBIO Data Analysis software version 9.0. The curves were aligned to dissociation, the Y axis was aligned to the last 5 s of baseline steps, and the last 5 s of the association step were considered the steady state. Specific binding to 14-3-3η was subtracted from the blank probe control and PBS negative control by selecting the “Double References” mode. A 1:1 binding model was assumed for the binding kinetics analysis. The KD, Kon, Koff and R2 values are recorded.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism V8.4.3 software (San Diego, USA) was used for the statistical analysis and data visualization. Multiple comparisons of data between groups were performed by one-way ANOVA or two-way ANOVA. The data are presented as the means ± SEMs, and p \u0026lt; 0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of the study are available from the corresponding author upon reasonable request. Source data are pro-vided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Macao Science and Technology Development Fund, grant codes: 0033/2019/AFJ, 0113/2023/RIA2, and 002/2023/ALC.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally:\u0026nbsp;Li Jun Yang, David Wei Zhang.\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDr. Neher\u0026rsquo;s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine \u0026amp; Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi Jun Yang, David Wei Zhang, Yuan Qing Qu, Yu Ping Wang, Xiong Fei Xu, Wei Dan Luo, Betty Yuen Kwan Law, Zhi-Hong Jiang, Rui Hong Chen, Cong Ling Qiu, Xi Chen, Lin Na Wang, Jiu Jie Yang, Vincent Kam Wai Wong\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eMacau University of Science and Technology Zhuhai MUST Science and Technology Research Institute\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetty Yuen Kwan Law, Zhi-Hong Jiang, Vincent Kam Wai Wong\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eDepartment of Gastroenterology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, Jiangsu Province, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuan Li\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eThe Key Laboratory of Modern Toxicology, Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuan Li\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe Affiliated Hospital of Southwest Medical University, Southwest Medical University, Luzhou, 646000, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWei Dan Luo\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eV.K.W.W., Y.L. conceived, conceptualization, sought funding and oversaw the project; B.Y.K.L. Writing - review and editing; L.-J.Y. and D.W.Z. are co-first authors, as they conducted the experiments, interpreted all the results and wrote the manuscript; Y.-Q.Q. performed the Co-IP experiments of p53 and 14-3-3\u0026eta;; Y.-Q.Q., Y.-P.W., X.-F.X., W.-D.L. and R.-H.C. formal analysis, data curation; Z.-H.J. resources and sought funding; C.-L.Q., X.C., L.-N.W. and J.-J.Y. data curation; All authors approved the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent to publish has been obtained from all authors.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003eFull supplementary information is listed in the Supplementary Materials document.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Yuan Li or Vincent Kam Wai Wong.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFirestein GS, McInnes IB. Immunopathogenesis of Rheumatoid Arthritis. Immunity. 2017;46(2):183-96.\u003c/li\u003e\n\u003cli\u003eImboden JB. The immunopathogenesis of rheumatoid arthritis. Annu Rev Pathol. 2009;4:417-34.\u003c/li\u003e\n\u003cli\u003eOstrowska M, Maśliński W, Prochorec-Sobieszek M, Nieciecki M, Sudoł-Szopińska I. Cartilage and bone damage in rheumatoid arthritis. Reumatologia. 2018;56(2):111-20.\u003c/li\u003e\n\u003cli\u003eNemtsova MV, Zaletaev DV, Bure IV, Mikhaylenko DS, Kuznetsova EB, Alekseeva EA, et al. Epigenetic Changes in the Pathogenesis of Rheumatoid Arthritis. Front Genet. 2019;10:570.\u003c/li\u003e\n\u003cli\u003eSmolen JS, Landew\u0026eacute; RBM, Bergstra SA, Kerschbaumer A, Sepriano A, Aletaha D, et al. EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2022 update. Ann Rheum Dis. 2023;82(1):3-18.\u003c/li\u003e\n\u003cli\u003eSmolen JS, Aletaha D, Barton A, Burmester GR, Emery P, Firestein GS, et al. Rheumatoid arthritis. Nat Rev Dis Primers. 2018;4:18001.\u003c/li\u003e\n\u003cli\u003evan der Heijden JW, Dijkmans BA, Scheper RJ, Jansen G. Drug Insight: resistance to methotrexate and other disease-modifying antirheumatic drugs--from bench to bedside. Nat Clin Pract Rheumatol. 2007;3(1):26-34.\u003c/li\u003e\n\u003cli\u003eBuch MH, Eyre S, McGonagle D. Persistent inflammatory and non-inflammatory mechanisms in refractory rheumatoid arthritis. Nat Rev Rheumatol. 2021;17(1):17-33.\u003c/li\u003e\n\u003cli\u003eZhang KX, Ip CK, Chung SK, Lei KK, Zhang YQ, Liu L, et al. Drug-resistance in rheumatoid arthritis: the role of p53 gene mutations, ABC family transporters and personal factors. Curr Opin Pharmacol. 2020;54:59-71.\u003c/li\u003e\n\u003cli\u003eQiu C, Chan JTW, Zhang DW, Wong IN, Zeng Y, Law BYK, et al. The potential development of drug resistance in rheumatoid arthritis patients identified with p53 mutations. Genes Dis. 2023;10(6):2252-5.\u003c/li\u003e\n\u003cli\u003eZeng Y, Ng JPL, Wang L, Xu X, Law BYK, Chen G, et al. Mutant p53(R211*) ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response. Inflamm Res. 2023;72(12):2199-219.\u003c/li\u003e\n\u003cli\u003eRivellese F, Surace AEA, Goldmann K, Sciacca E, \u0026Ccedil;ubuk C, Giorli G, et al. Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial. Nat Med. 2022;28(6):1256-68.\u003c/li\u003e\n\u003cli\u003eLu M, Dai Y, Xu M, Zhang C, Ma Y, Gao P, et al. The Attenuation of 14-3-3\u0026zeta; is Involved in the Caffeic Acid-Blocked Lipopolysaccharide-Stimulated Inflammatory Response in RAW264.7 Macrophages. Inflammation. 2017;40(5):1753-60.\u003c/li\u003e\n\u003cli\u003eFalcicchio M, Ward JA, Macip S, Doveston RG. Regulation of p53 by the 14-3-3 protein interaction network: new opportunities for drug discovery in cancer. Cell Death Discov. 2020;6(1):126.\u003c/li\u003e\n\u003cli\u003eLee YK, Hur W, Lee SW, Hong SW, Kim SW, Choi JE, et al. Knockdown of 14-3-3\u0026zeta; enhances radiosensitivity and radio-induced apoptosis in CD133(+) liver cancer stem cells. Exp Mol Med. 2014;46(2):e77.\u003c/li\u003e\n\u003cli\u003eQiu Y, Dai Y, Zhang C, Yang Y, Jin M, Shan W, et al. Arsenic trioxide reverses the chemoresistance in hepatocellular carcinoma: a targeted intervention of 14-3-3\u0026eta;/NF-\u0026kappa;B feedback loop. J Exp Clin Cancer Res. 2018;37(1):321.\u003c/li\u003e\n\u003cli\u003eQiu Y, Shan W, Yang Y, Jin M, Dai Y, Yang H, et al. Reversal of sorafenib resistance in hepatocellular carcinoma: epigenetically regulated disruption of 14-3-3\u0026eta;/hypoxia-inducible factor-1\u0026alpha;. Cell Death Discov. 2019;5:120.\u003c/li\u003e\n\u003cli\u003eZeng T, Tan L. 14-3-3\u0026eta; protein: a promising biomarker for rheumatoid arthritis. Biomark Med. 2018;12(8):917-25.\u003c/li\u003e\n\u003cli\u003eMaksymowych WP, Marotta A. 14-3-3\u0026eta;: a novel biomarker platform for rheumatoid arthritis. Clin Exp Rheumatol. 2014;32(5 Suppl 85):S-35-9.\u003c/li\u003e\n\u003cli\u003eQuan LD, Thiele GM, Tian J, Wang D. The Development of Novel Therapies for Rheumatoid Arthritis. Expert Opin Ther Pat. 2008;18(7):723-38.\u003c/li\u003e\n\u003cli\u003eTsujimura S, Saito K, Nawata M, Nakayamada S, Tanaka Y. Overcoming drug resistance induced by P-glycoprotein on lymphocytes in patients with refractory rheumatoid arthritis. Ann Rheum Dis. 2008;67(3):380-8.\u003c/li\u003e\n\u003cli\u003eKabir I, Ansari I. A review on in vivo and in vitro experimental models to investigate the anti-inflammatory activity of herbal extracts. Asian J Pharm Clin Res. 2018;11(11):29.\u003c/li\u003e\n\u003cli\u003eZhang N, Zhang H, Law BYK, Dias I, Qiu CL, Zeng W, et al. Sirtuin 5 deficiency increases disease severity in rats with adjuvant-induced arthritis. Cell Mol Immunol. 2020;17(11):1190-2.\u003c/li\u003e\n\u003cli\u003eBurrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis. Front Biosci. 2006;11:529-43.\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Gradaigh D, Compston JE. T-cell involvement in osteoclast biology: implications for rheumatoid bone erosion. Rheumatology (Oxford). 2004;43(2):122-30.\u003c/li\u003e\n\u003cli\u003eCrotti TN, Smith MD, Weedon H, Ahern MJ, Findlay DM, Kraan M, et al. Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients: semiquantitative and quantitative analysis. Ann Rheum Dis. 2002;61(12):1047-54.\u003c/li\u003e\n\u003cli\u003eFeng X, Shi Y, Xu L, Peng Q, Wang F, Wang X, et al. Modulation of IL-6 induced RANKL expression in arthritic synovium by a transcription factor SOX5. Sci Rep. 2016;6:32001.\u003c/li\u003e\n\u003cli\u003eCrawford LJ, Walker B, Ovaa H, Chauhan D, Anderson KC, Morris TC, et al. Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132. Cancer Res. 2006;66(12):6379-86.\u003c/li\u003e\n\u003cli\u003ePucci B, Kasten M, Giordano A. Cell cycle and apoptosis. Neoplasia. 2000;2(4):291-9.\u003c/li\u003e\n\u003cli\u003eLee H, Lee H, Chin H, Kim K, Lee D. ERBB3 knockdown induces cell cycle arrest and activation of Bak and Bax-dependent apoptosis in colon cancer cells. Oncotarget. 2014;5(13):5138-52.\u003c/li\u003e\n\u003cli\u003eWali VB, Haskins JW, Gilmore-Hebert M, Platt JT, Liu Z, Stern DF. Convergent and divergent cellular responses by ErbB4 isoforms in mammary epithelial cells. Mol Cancer Res. 2014;12(8):1140-55.\u003c/li\u003e\n\u003cli\u003eR\u0026ouml;sch L, Herter S, Najafi S, Ridinger J, Peterziel H, Cinatl J, et al. ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P-glycoprotein. Mol Oncol. 2023;17(1):37-58.\u003c/li\u003e\n\u003cli\u003eWang CL, Liu ZP, Guo L. NAT2 knockdown inhibits the development of colorectal cancer and its clinical significance. Eur Rev Med Pharmacol Sci. 2021;25(9):3460-9.\u003c/li\u003e\n\u003cli\u003eAubrey BJ, Kelly GL, Janic A, Herold MJ, Strasser A. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ. 2018;25(1):104-13.\u003c/li\u003e\n\u003cli\u003eMcInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365(23):2205-19.\u003c/li\u003e\n\u003cli\u003eHuang XY, Ke AW, Shi GM, Zhang X, Zhang C, Shi YH, et al. \u0026alpha;B-crystallin complexes with 14-3-3\u0026zeta; to induce epithelial-mesenchymal transition and resistance to sorafenib in hepatocellular carcinoma. Hepatology. 2013;57(6):2235-47.\u003c/li\u003e\n\u003cli\u003eReis H, P\u0026uuml;tter C, Megger DA, Bracht T, Weber F, Hoffmann AC, et al. A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors. Biochim Biophys Acta. 2015;1854(6):641-50.\u003c/li\u003e\n\u003cli\u003eWang S, Zhou Y, Huang J, Li H, Pang H, Niu D, et al. Advances in experimental models of rheumatoid arthritis. Eur J Immunol. 2023;53(1):e2249962.\u003c/li\u003e\n\u003cli\u003eMcInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7(6):429-42.\u003c/li\u003e\n\u003cli\u003eKhokha R, Murthy A, Weiss A. Metalloproteinases and their natural inhibitors in inflammation and immunity. Nat Rev Immunol. 2013;13(9):649-65.\u003c/li\u003e\n\u003cli\u003eNygaard G, Firestein GS. Restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes. Nat Rev Rheumatol. 2020;16(6):316-33.\u003c/li\u003e\n\u003cli\u003eHientz K, Mohr A, Bhakta-Guha D, Efferth T. The role of p53 in cancer drug resistance and targeted chemotherapy. Oncotarget. 2017;8(5):8921-46.\u003c/li\u003e\n\u003cli\u003eCao X, Hou J, An Q, Assaraf YG, Wang X. Towards the overcoming of anticancer drug resistance mediated by p53 mutations. Drug Resist Updat. 2020;49:100671.\u003c/li\u003e\n\u003cli\u003evan Hamburg JP, Asmawidjaja PS, Davelaar N, Mus AM, Colin EM, Hazes JM, et al. Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17A production. Arthritis Rheum. 2011;63(1):73-83.\u003c/li\u003e\n\u003cli\u003eKimura A, Kishimoto T. IL-6: regulator of Treg/Th17 balance. Eur J Immunol. 2010;40(7):1830-5.\u003c/li\u003e\n\u003cli\u003eTaghadosi M, Adib M, Jamshidi A, Mahmoudi M, Farhadi E. The p53 status in rheumatoid arthritis with focus on fibroblast-like synoviocytes. Immunol Res. 2021;69(3):225-38.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"14-3-3η, Methotrexate resistance, p53, Adjuvant-induced arthritis, RAFLS","lastPublishedDoi":"10.21203/rs.3.rs-6528370/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6528370/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRheumatoid arthritis (RA) is a chronic autoimmune condition complicated by drug resistance issues, significantly challenging its treatment. The protein 14-3-3η has been noted for its association with drug resistance in various cancers, and while it is particularly expressed at high levels in RA patients, its role in the disease's pathogenesis is not well understood. This study explores how 14-3-3η contributes to drug resistance in RA, particularly focusing on its interactions with the p53 protein. Through techniques including qPCR, Western blot, ELISA, BLI, immunoprecipitation, and the use of adjuvant-induced arthritis (AIA) transgenic models, we observed elevated levels of 14-3-3η in the peripheral blood mononuclear cells (PBMCs) of RA patients, correlating with both disease activity and duration. Enhanced expression of 14-3-3η was found to induce resistance to methotrexate (MTX) in AIA rats, which could be countered by treatment with arsenic trioxide (ATO). Our mechanistic studies suggest that the overexpression of 14-3-3η promotes a multidrug resistance phenotype in rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) through the downregulation of p53. Furthermore, adenovirus-mediated p53 supplementation in AIA rats was able to mitigate and reverse the MTX resistance caused by 14-3-3η. These findings delineate a regulatory function for 14-3-3η in the pathogenesis and drug resistance of RA via p53 modulation, proposing it as a viable target for treating drug-resistant RA.\u003c/p\u003e","manuscriptTitle":"14-3-3η Induces a Multidrug-Resistant Phenotype in Rheumatoid Arthritis via p53 Dysregulation: Insights from AIA Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 16:50:14","doi":"10.21203/rs.3.rs-6528370/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2fb334d-c36e-48c8-84e8-4f57e68c4e70","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47917005,"name":"Biological sciences/Immunology/Autoimmunity"},{"id":47917006,"name":"Health sciences/Biomarkers/Predictive markers"}],"tags":[],"updatedAt":"2025-08-25T18:00:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 16:50:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6528370","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6528370","identity":"rs-6528370","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}