Inhibition of Cdc37 Ameliorates Arthritis in Collagen-Induced Arthritis Rats by Inhibiting Synoviocyte Proliferation and Migration Through the ERK Pathway

Rheumatoid arthritis (RA) is a chronic autoimmune disease that can lead to synovial inflammation, pannus formation, cartilage damage, bone destruction, and ultimate disability. Fibroblast-like synoviocytes (FLS) are involved in the pathogenetic mechanism of RA. Cdc37 (cell division cycle protein 37) is regarded as a molecular chaperone involved in various physiological processes such as cell cycle progression, cell proliferation, cell signal transduction, tumorigenesis, and progression. However, the precise role of Cdc37 in the pathogenesis of rheumatoid arthritis (RA) remains uncertain. In our study, we found that Cdc37 expression was upregulated in human rheumatoid synovia in contrast with the normal group. Interestingly, Cdc37 activated the ERK pathway to promote RA-FLS proliferation and migration in vitro. Ultimately, in vivo experiments revealed that silencing of Cdc37 alleviated ankle swelling and cartilage destruction and validated the ERK signaling pathways in vitro findings. Collectively, we demonstrate that Cdc37 promotes the proliferation and migration of RA-FLS by activation of ERK signaling pathways and finally aggravates the progression of RA. These data indicated that Cdc37 may be a novel target for the treatment of RA.


INTRODUCTION
Rheumatoid arthritis (RA) is a progressive systemic autoimmune disease with pathological features of hyperplasia in synovial tissue, pannus formation, and destruction of bone and cartilage, which would lead to joint dysfunction [1,2]. The pathogenesis of RA is complicated because it involves fibroblast-like synoviocytes (FLS), macrophages, B cells, and T cells [3,4]. Previous studies have shown that FLS have proliferative and migration abilities similar to tumor cells [5,6]. Abnormal synovial hyperplasia which is caused by persistent abnormal FLS proliferation in RA joint cavity leads eventually to cartilage and bone destruction and pathogenesis of RA [7][8][9][10]. Therefore, the inhibition of FLS proliferation and migration could be a useful target in the treatment of RA.
Cdc37 is a molecular chaperone that targets multiple protein kinases and recruits several unstable kinases to Hsp90. These interactions can result in proper protein folding, increased protein stability, and kinase activity [11,12]. As a critical component of cell cycle control, Cdc37 is of great importance to cellular signaling pathways and carcinogenesis [13,14]. Emerging evidence suggests that Cdc37 plays a crucial role in cell proliferation, migration, and cell signal transduction [15,16]. The overexpression of Cdc37 and increased immunoreactivity in human prostate tissue were found compared with that in the normal prostate tissue [17]. Cdc37 is also highly expressed in luminal cells [18]. However, while Cdc37 is involved in a variety of diseases, the biological function of Cdc37 in RA is uncertain based on our group's previous investigations on the Cdc37 biological function of Schwann cell proliferation and migration in sciatic nerve crush [19]. Therefore, we hypothesized that Cdc37 may also be involved in RA development and progression.
A growing body of research is showing that the ERK signaling pathway is associated with the occurrence and development of RA. ERK was activated in the FLS of RA synovium and promoted RA-FLS proliferation and migration [20,21]. The expression of MMPs and proinflammatory cytokines in RA FLS is reduced following the inhibition of the ERK pathway [22,23]. Moreover, impairing activation of ERK significantly inhibited paw edema and ameliorated clinical arthritis in the CIA rats [24,25]. These studies demonstrate that the ERK pathway plays a key role in the pathological process of RA.
In this study, we first examined the expression of Cdc37 in RA synoviocytes and RA-FLS. In addition, we used small interfering RNA (siRNA) to explore the significance of Cdc37 in RA-FLS proliferation and migration in vitro. The overexpression of Cdc37 achieved the same results. These results showed that Cdc37 might play a potential effect on cell proliferation and migration via the ERK signaling pathway in RA. Subsequently, the inhibition of Cdc37 in CIA rats exhibited therapeutic potential which suggested that Cdc37 may be a novel target for RA therapy.

Isolation and Culture of FLS
Synovial tissue was obtained from RA patients (n = 10, mean age 58 ± 8.1 years, 2 males and 8 females) who have undergone total knee arthroplasty in the Orthopedic Department of the Affiliated Hospital of Nantong University; all patients met the criteria for the classification of RA of the American College of Rheumatology(ACR)/European League Against Rheumatism (EULAR) published in 2010. Normal synovium was obtained from meniscal knee arthroscopic surgery patients (n = 10, mean age 53.8 ± 5.49 years, 6 males and 4 females) [26,27]. Tissue samples were digested in typeII collagenase (Sigma) in DMEM-F12(Gibco) for 1-2 h and then gently shaken at 37 °C for 2 h. After centrifugation, the cells were cultured in a DMEM-F12(Gibco) medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin at 37 °C, and 5% CO 2 . Cells of passages 3-6 were used in this study. RA-FLS activation model in vitro was generated by treatment with LPS (1 µg/mL) [28,29], IL-1β (10 ng/mL) [30,31], or Tnfα (10 ng/mL) [32,33]. RA-FLS were transfected with Cdc37-siRNA or with Cdc37 plasmids and then incubated for 24 h with LPS. After LPS stimulation, the cells were harvested for subsequent experiments.

Western Blot Analysis
Proteins were loaded on SDS-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. Membranes were immunoblotted with specific antibodies at 4 °C overnight. Samples were rinsed and incubated with (HRP-labeled Goat Anti-Rabbit IgG(H + L) or Anti-Mouse IgG(H + L) (secondary antibody) for 1 h. The samples were washed with PBST, using the electrochemiluminescence (ECL) chemiluminescence method, and visualized by the BioRad imager. The target bands were analyzed for gray values using Image J software. Anti-PCNA, anti-CyclinD1, and anti-βactin are from Beyotime, and anti-Cdc37 is from Proteintech. Anti-ERK1/2 and anti-phospho-ERK1/2 are from Cell Signaling Technology.

Immunohistochemistry
Tissues were placed in 4% paraformaldehyde solution overnight at 4 °C and were subsequently dehydrated, transparentized, and embedded in paraffin blocks. The blocks were then cut into slices of 4-5 µm. Paraffin sections were dewaxed in xylene, gradient ethanol immersion, and then immersed in an endogenous peroxidase blocker for 20 min and then rinsed with PBS. Primary antibodies against Cdc37 (1:50; Proteintech) were applied to the sections at 4 °C overnight. Enzyme-labeled sheep antirabbit IgG polymer was added and incubated at 37 ℃ were applied to the sections at 4 °C overnight. Enzyme-labeled sheep anti-rabbit IgG polymer was added and incubated at 37 em-8 min and hematoxylin staining solution for 20 s (differentiation, rinse back blue; dehydrated, transparent, and sealed). The stained tissue slices were observed under an optical microscope. Brown color or brown granular shapes in the section represented positive staining.

Cdc37-siRNA Transfection of FLS
SiCdc37 and Cdc37 overexpression plasmids were obtained from GenePharma (Suzhou, China). Lipofectamine 2000 (Life Technologies) was used according to the manufacturer's recommendations, and the medium was replaced with DMEM-F12 with 10% FBS 6 h after transfection. The cells were further cultured for 48 h and used for the following experiments.

Enzyme-Linked Immunosorbent Assay
The primary FLS was transfected with Cdc37 siRNA#2 for 48 h and then stimulated with LPS for 24 h. Cell supernatants were harvested and centrifuged at 12,000 × g for 15 min. Using the appropriate ELISA kits according to the manufacturer h, and then stimulated with LPS for 24 h. Cell supernatants (wBiotek Synergy2) were at a 450 nm wavelength.

Wound-Healing Assays
FLS were plated into a six-well plate and transfected for 48 h. Serum-free DMEM-F12 was then incubated for 12 h. A sterile 10-µL tip was used to scratch the cell monolayer. After scraping, immediately switch to a fresh medium and cultured cells for 24 h.

Cell Migration Assay
FLSs were transfected with Cdc37-siRNA and transferred to a migration chamber at a density of 2 × 10 4 cells/well. The lower chamber was filled with a 500 µL complete medium. After 24 h, the cells at the bottom of the film were fixed with 4% paraformaldehyde and stained with crystal violet, and the cells that had not penetrated the upper chamber were wiped with a cotton swab and observed under a microscope. The Image J software was used to calculate the number of the cells.

EdU Assay
FLSs were seeded in 96-well plates at a concentration of 5×10 4 /100 µL in triplicate and incubated overnight after treatment. The EDU analysis was then performed using a Cell-Light™ EdU DNA Cell Proliferation Kit (RiboBio, China).

Cell Cycle Analysis
Cells were washed with PBS after siRNA transfection and fixed with 70% ethanol overnight at −20 °C. Cells were washed twice with pre-cooled PBS, resuspended in 70% ethanol, and fixed overnight at −20 °C. Make up a 1:9 volume of RNase A:PI working solution to make a working staining solution (KEYGEN BioTECH). After washing the cells with PBS, add 500 µL of preprepared PI/RNase A staining working solution and store at room temperature for 30-60 min in the dark; record the red fluorescence at the excitation wavelength of 488 nm.

RNA Isolation and Quantitative Polymerase Chain Reaction
The total RNA was extracted by the Trizol (Thermofisher, USA) method. Use UNlQ-10 Column Trizol Total RNA Isolation Kit(Sangon Biotech, China) to extract total RNA and then measure the concentration. After reverse transcription into cDNA by using the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd, China), the relative expression of target mRNA was normalized to that of β-actin, which served as an endogenous control. The mRNA expression of Cdc37 was analyzed by Q-PCR amplification according to the manufacturer's protocol (Roche, Penzberg, Germany). Using the relative quantitative algorithm 2^-ΔΔCt method (Livak method) to analyze and compare the ratio between the target gene of the experimental group and the housekeeping gene of the control group. The primers used are as follows: Cdc37 (F:TGA AGA CGA GAC GCACC; R:TCA GTT TCC TCT GGC ACT CG) and β-actin (F:TGG CAC CCA GCA CAA TGA A; R:CTA AGT CAT AGT CCG CCT AGA AGC A).

Animals and Induction of CIA
In this experiment, Wistar male rats, aged 6 weeks, were used (each n = 6). These rats were purchased from the Experimental Animal Center of Nantong University, and all animal experiments have been approved by the Animal Ethics Committee of Nantong University. Under SPF conditions with relative humidity and temperature of 22 ± 3 °C, the animals were housed in a 12-h light-dark cycle and fed with a regular diet and water for 2 weeks, and then the CIA model was induced. Incomplete Freund's adjuvant (Chondrex) and bovine type II collagen (Chondrex, WA) were mixed in a ratio of one to one (1:1). Then, injecting 200 µL of the mixture into the bottom of the rat's tail by intradermal injection. Seven days later, 100 µL of immune enhancer was injected at the same location in the same manner. The arthritis index (AI) was used to evaluate: no swelling scored 0 points, 1 or 2 interphalangeal joint involvement scored 1 point, joint and toe mild swelling scored 2 points, toe and ankle joint swelling scored 3 points, and full paw severe arthritis scored 4 points. Each limb has a maximum of 4 points and a total of 16 points. If the total points exceed 6 points, the modeling is successful.

shRNA Treatment Regiment
Fourteen days after secondary immunization, the rats were divided into two groups as follows: an shRNA interference plasmid group (LV-shCdc37, fragments were synthesized by Miaolingbio (Wuhan, China), a PBS group, and normal rats as the control group. In vitro experiments were screened for effective lentiviral vector-mediated with shRNA virus (concentration of 1×10 8 TU/mL) and injected into the anklejoint cavity of the CIA model animal. Two injections each with a dose of 0.1 mL were administered per week for 3 weeks. The PBS group was injected with an equal volume of PBS in the same way. After the administration was finished, five rats in each group were selected for the efficacy index test.

Hematoxylin − Eosin Staining and Safranin-O Assay
All experimental rats were sacrificed 49 days after the primary immunization, and the ankle joints and synovial membrane were taken. Synovial membranes were used to extract tissue proteins for WB experiments. The ankle joints were fixed with 4% paraformaldehyde for 48 h, washed with PBS, and decalcified in 15% (w/v) EDTA solution. After 45 days, the ankle joints were embedded in paraffin and sectioned. The joint morphology and histopathological changes were observed by HE and Safranin-O staining.

Statistical Analysis
The SPSS 13.0 software (SPSS, Inc., Chicago, IL) was used to analyze all statistics. Two-group and multiple-group differences were evaluated using twotailed Student's t-tests and one-way analysis of variance (F) with Bonferroni's post hoc tests, respectively. All values were expressed as mean ± SEM. P < 0.05 was considered statistically significant.

Expression of Cdc37 is Increased in Synovial Tissue and RA-FLS
We first detected the protein expression of Cdc37 in human synovial tissues by western blot. Cdc37 expression was significantly higher in RA synovial tissues compared with healthy synovial tissues (Fig. 1A). To validate western blot results, we employed immunochemistry to investigate the changes of Cdc37. The staining of Cdc37 was noticeably enhanced compared to healthy groups (Figs. 1B and S1). Consistent with this, the qRT-PCR results showed that Cdc37 expression was increased in RA-FLS, which corresponded to the western blot results (Fig. 1C, D).

Cdc37 Contributes to Pro-Inflammatory Cytokines Release and ERK Signaling Pathway Activation
To further explore the effects of Cdc37 in RA-FLS biological behaviors, we performed RNA interference to suppress Cdc37 expression. Because of Cdc37-siRNA#2 highest inhibition efficiency, we chose it for the following studies ( Fig. 2A). ELISA was performed to clarify the effect of Cdc37 on the production of pro-inflammatory cytokines (IL-6 and IL-1β). RA-FLS transfected with Cdc37-siRNA#2 effectively reduced the levels of IL-6 and IL-1β (Fig. 2B, C). To investigate how Cdc37 affects LPStreated changes in RA-FLS, cells were transfected with Cdc37-siRNA#2 and then incubated for 24 h with LPS.
Previous studies had confirmed that Cdc37 could regulate cell cycle progression and cell proliferation. We detected the protein levels of PCNA and CyclinD1 during the process of RA-FLS proliferation via western blot analysis in our study. The expression of Cdc37 significantly increased in proliferating RA-FLS, which was in accord with the expression of PCNA and CyclinD1 (Fig. 2D). Previous research has demonstrated that Cdc37 was involved in the ERK signaling pathway. To further explore whether Cdc37 had an effect on the activation of ERK signaling and thereby affected cell proliferation and migration, we used PD98059, an inhibitor of ERK1/2. Notably, we observed decreased expression of p-ERK after the depletion of Cdc37. In addition, markedly reduced level of p-ERK upon PD98059 Fig. 1 The expression of Cdc37 is increased in synovial tissue and RA-FLS. A In synovial tissue from RA patients and normal people, the expression of Cdc37 was detected by western blot. B Immunochemistry was performed to detect the expression of Cdc37 in the synovial tissue paraffin section. FLS were isolated from synovial tissue, and the expression of Cdc37 was detected by qRT-PCR (C) and western blot (D). Experiments were repeated for three times, *P < 0.05 introduction with Cdc37-siRNA#2 (Fig. 2E). In parallel, Cdc37 knockdown resulted in an obvious reduction in IL-1β or Tnfα induced phosphorylation of ERK1/2 (Fig. S2).

Knockdown of Cdc37 or Inhibition of ERK Signaling Pathway Attenuates Proliferation and Migration of RA-FLS
Further flow cytometry analyses revealed a decrease of the S phase and impeded G1/S-phase transition after the knockdown of Cdc37 with or without PD98059 Fig. 2 Cdc37 contributes to pro-inflammatory cytokines release and ERK signaling pathway activation. A siRNA of Cdc37 were transfected into RA-FLS, and the efficiency of siRNA knockdown was analyzed by western blot. IL-6 (B) and IL-1β (C) levels in the serum of all groups were measured by ELISA assay. D RA-FLS were transfected with siRNA#2 and negative control siRNA, and then stimulated by LPS (1 µg/mL, 24 h); the expression of PCNA and CyclinD1 were detected by western blot. E RA-FLS were transfected with siRNA#2 and/or then stimulated by PD98059 (100 µM, 24 h). The protein expression levels of p-ERK and ERK were measured by western blot. Experiments were repeated for three times, A * compare with Con; B, C * compare with LPS; D, E * compare with Con; & compare with #2, P < 0.05 ◂ Fig. 3 Knockdown of Cdc37 or inhibition of ERK signaling pathway attenuates proliferation and migration of RA-FLS. The proliferation of RA-FLS was measured by cell cycle analysis (A) and EdU assay (B). The migration of RA-FLS were evaluated by wound healing assay (C) and cell migration assay (D). Experiments were repeated for three times, * compare with Con, & compare with #2, P < 0.05 (Figs. 3A and S3A). EdU assays displayed hindering effects of Cdc37-siRNA#2 and PD98059 on cell proliferation of RA-FLS (Fig. 3B). Next, we performed scratch wound test and transwell assay to determine whether Cdc37 had a pivotal role in RA-FLS migration. Our results demonstrated a slower closure in Cdc37-siRNA#2 transfected cells (Fig. 3C). The number of invaded cells significantly decreased, indicating that the interference of Cdc37 markedly suppressed migration of RA-FLS (Fig. 3D). Similarly, the ability of cell migration was inhibited via inactivation of ERK signaling pathway by PD98059 (Fig. 3C, D).

Cdc37 Promotes RA-FLS Proliferation and Migration by Enhancing ERK Signaling
To validate the aforementioned results, we overexpressed Cdc37 expression by the plasmid. Here, we identified that overexpression of Cdc37 enhanced the status of p-ERK. The up-regulation of p-ERK caused by the overexpression of Cdc37 was rescued by PD98059 (Fig. 4A, B). Likewise, in the presence of Cdc37 overexpression, the cells were increased in the S phase (Figs. 5A and S3B). In addition, it was elucidated that the effect of Cdc37 on RA-FLS proliferation and migration was rescued by the inactivation of the ERK signaling pathway (Fig. 5B-D). All the above evidence suggested a constructive role of Cdc37 on cell proliferation and migration, which depended on the activation of the ERK signaling pathway.

Inhibition of Cdc37 Reduced RA Symptoms and Alleviate Cartilage Destruction in CIA Rats
To verify the therapeutic function of Cdc37 in vivo, we made an intra-articular injection of LV-sh-Cdc37 in CIA rats. As shown in Fig. 6A, the CIA rat's paws had severe swelling and redness. However, the paw swelling degree was significantly alleviated in LV-sh-Cdc37-treated groups and resembled normal group paws. Next, we used the paw thickness as a direct indicator extent of paw swelling. The paw thickness in the LV-sh-Cdc37 group showed obvious improvement over that in the CIA group (Fig. 6B). Remarkably, a similar result was observed based on the AI score (Fig. 6C). These data demonstrated that inhibiting Cdc37 expression remarkably improved clinical outcomes, including paw thickness and arthritis index. Furthermore, we performed H&E and Safranin O staining to analyze Fig. 4 Cdc37 mediates the activation of the ERK signaling pathway. A The plasmid of Cdc37 was transfected into RA-FLS, and the expression of Cdc37 was detected by western blot. B RA-FLS were transfected with Cdc37 plasmid and/or then stimulated by PD98059 (100 µM, 24 h). The protein expression levels of p-ERK and ERK were measured by western blot. Experiments were repeated for three times, * compare with Con, & compare with #2, P < 0.05 pathological changes in the ankle joints. LV-sh-Cdc37treated rats exhibit relatively mild synovial hyperplasia and cartilage destruction (Fig. 6D, E). Besides, the levels of p-ERK were reduced, and the ERK signaling pathway was inactivated (Fig. 6F). In addition, to evaluate possible in vivo toxicity of LV-sh-Cdc37, the main organs (heart, liver, spleen, lung, kidney) were removed and examined by H&E staining. No distinct tissue damage was observed among the groups, implying that inhibition of Cdc37 did not exhibit toxicity in vivo, and the use of LV-sh-Cdc37 in Fig. 5 Cdc37 promotes RA-FLS proliferation and migration by enhancing ERK signaling. The proliferation of RA-FLS was measured by cell cycle analysis (A) and EDU assay (B). The migration of RA-FLS were evaluated by wound healing assay (C) and cell migration assay (D). Experiments were repeated for three times, * compare with Con, & compare with Cdc37 plasmid, P < 0.05 CIA rats exhibited a good safety profile (Fig. S4). In brief, our data revealed that blocked Cdc37 could inhibit the ERK signaling pathway, thus slowing the development of RA.

DISCUSSION
Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease with the symptom of hyperplastic synovium and destruction of joints [34]. Excessive proliferation and migration of FLS directly promote cartilage erosion and articular bone destruction, which eventually results in RA development and progression [35]. Currently, the main strategy for RA treatment is non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs), and more [36]. These drugs are mainly focused on relieving pain and improving patients' quality of life to a certain extent [37]. However, the subsequent side effects of the drugs include gastrointestinal reactions, abnormal liver and kidney functions, osteoporosis, and systemic complications that cannot be ignored [38][39][40]. The introduction of targeted biologics has been regarded as a new effective therapy [41,42]. Increasing evidence has suggested that activated fibroblast-like synoviocytes (FLSs) play a critical role in synovitis inflammation, hyperplasia, pannus formation, and joint destruction. The excessive proliferation and enhanced migration capabilities of RA-FLS are considered the major cause of tissue damage [43]. Therefore, searching for novel factors or mechanisms that regulate biological behaviors and inhibit the activation of FLSs is necessary for the treatment of RA.
Interestingly, previous studies have reported that Cdc37 can influence cell cycle progression, cell proliferation, and migration [17,44]. The orderly transformation of the cell cycle among different phases is helpful to prevent uncontrolled cell proliferation. Particularly, Cdc37 knockdown in human colon cancer cells decreased the association with HSP90 and inhibited cell proliferation via the inhibition of G1/S-phase transition, as well as reduced levels of ERBB2, CRAF, CDK4, and CDK6 [13]. In human prostate cancer cells, the inhibition of growth arrest and multiple signaling pathways was also observed after the silencing of Cdc37 [45]. In human HCC, overexpression of Cdc37 was associated with the downregulation of p16 [46]. Indeed, the growth of HepG2 cells was inhibited in vitro within Cdc37 suppression [16]. In addition, Cdc37 could mediate cell growth and migration during Drosophila wound healing [44]. Meanwhile, Cdc37 contributed to Schwann cell proliferation and migration after sciatic nerve crush [19]. Combining the above results, we can speculate that Cdc37 cannot be neglected for cell cycle regulation, cell proliferation, and migration.
In this study, we first found a high expression of Cdc37 in human synovial tissues and RA-FLS. Next, we demonstrated that Cdc37 had a close connection with RA-FLS proliferation. Interestingly, protein levels of PCNA and CyclinD1 were elevated in the LPS-induced RA-FLS proliferation model. Besides, Cdc37 promoted RA-FLS proliferation by facilitating transformation from G0/G1 phase to the S phase. But the molecular mechanism of Cdc37-mediated cell proliferation and migration was still unclear. Various studies have shown that the activation of the ERK signaling pathway is tightly correlated with cell proliferation and migration in RA [47]. We previously found that Cdc37 might participate in Schwann cell proliferation partially via the ERK pathway. This provides good foundations for our present study. Based on these findings, we sought to determine whether Cdc37 contributes to RA-FLS proliferation and migration through ERK signaling. Amazingly, we detected the up-regulation of p-ERK once Cdc37 was overexpressed in our research. Meanwhile, the knockdown of Cdc37 distinctly caused the down-regulation of p-ERK. Moreover, this change caused by the overexpression of Cdc37 can be reversed by PD98059. Similarly, the promotion of cell proliferation and migration resulting from Cdc37 also could be attenuated by the treatment of PD98059, further indicating that the ERK pathway is related to the influence of Cdc37 on the biological behaviors of RA-FLS. Finally, we found that synovial inflammation and cartilage erosion could be effectively alleviated, and the ERK pathway in joint tissues was inhibited by the injection of sh-Cdc37.
In vivo experiments demonstrated that inhibiting Cdc37 mitigated the symptoms of RA rats.
In conclusion, our study demonstrates the interaction between Cdc37 and the ERK signaling pathway in the regulation of RA-FLS proliferation and migration. This presents new evidence that Cdc37 is a critical factor that Fig. 6 The inhibition of Cdc37 reduced RA symptoms and alleviate cartilage destruction in CIA rats. Representative photographs (A), thickness (B), and arthritis index of hind paw (C) in three groups. HE staining (D) and Safranin-O/fast green staining (E) of ankle joint sections in three groups. E The expression of p-ERK and ERK in synovial tissue of three groups was detected by western blot. Con (control group), CIA (collagen induce rheumatoid arthritis), LV-sh-Cdc37 (CIA rats were intra-articular injection of LV-sh-Cdc37). Experiments were repeated for three times, * compare with Con, & compare with CIA, P < 0.05 ◂ can regulate the biological behaviors of RA-FLS and may be a prospective strategy for RA treatment. On the other side, RA-FLS secrete various pro-inflammatory cytokines and chemokines to establish an inflammatory microenvironment typically that is favorable for the occurrence of RA. However, whether there is an interrelation between Cdc37 and inflammatory microenvironment is not known clearly. Therefore, future studies are needed to investigate the anti-inflammatory effects of Cdc37 on RA.

AUTHOR CONTRIBUTION
Weiwei Sun and Xingxing Mao performed the experiments. Weijie Wu and Yunyi Nan collected and analyzed the data. Hua Xu and Youhua Wang performed joint surgeries and provided synovial tissue. Weiwei Sun and Weijie Wu prepared the manuscript. Chunxiang Xu contributed to the conception of the study. All authors have read and approved the manuscript.

FUNDING
This work was supported by A Project of Nantong Science and Technology Program (JC2020015) and the Research Innovation Program for College Graduates of Jiangsu Province (KYCX20-2800).

AVAILABILITY OF DATA AND MATERIALS
Data are available from the corresponding author on reasonable request.

DECLARATIONS
Ethical Approval This study was approved by the institutional medical ethics committee of the Affiliated Hospital of Nantong University. Informed consent was acquired from all patients prior to surgery.