Iridoid from Eucommia ulmoides Oliv. Exerts Antiarthritis Effects by Inhibiting the JAK2/STAT3 Signaling Pathway In Vivo and In Vitro

The purpose of this study was to investigate the anti-inflammatory effects of EU-Idd both in vivo and in vitro. In vivo, we used the collagen-induced arthritis (CIA) rat model to investigate the efficacy of EU-Idd on rheumatoid arthritis. Hematoxylin-eosin staining and Safranin O-fast green staining were used to evaluate the pathological status of the ankle joints in CIA rats. Micro-CT scanning was used to investigate bone erosion of the ankle joints. In vitro, the effect of EU-Idd on Th17 cell differentiation was identified by flow cytometry. TRAP staining was used to detect osteoclast cells. HFLS-RA model cells, induced by tumor necrosis factor-α(TNF-α), were used to evaluate the anti-inflammatory effects of EU-Idd while the levels of related inflammatory cytokines and JAK2/STAT3 proteins were detected by RT-qPCR and western blotting. EU-Idd alleviated joint inflammation in CIA rats and exerted protective effects on the ankle joints. EU-Idd also prevented the differentiation of CD4+ T cells into Th17 cells, reduced the number of osteoclasts, and improved the expression levels of bone metabolism-related proteins including OPG and RANKL. Moreover, EU-Idd inhibited the invasion and migration of HFLS-RA cells and downregulated the expression of related inflammatory cytokine genes and the protein expression levels of p-JAK2 and p-STAT3, both in vivo and in vitro. EU-Idd exerts anti-inflammatory and osteoprotective effects by regulating the JAK2/STAT3 pathway in rheumatoid arthritis. These results are beneficial to excavate new pharmaceutical ingredients for rheumatoid arthritis from iridoid.


Introduction
Rheumatoid arthritis (RA) is an infammatory autoimmune disease that is mainly characterized by infammation of the synovium, joint destruction, and bone erosion, eventually leading to joint deformity and the loss of joint function [1]. Modern pharmacological studies have found that T helper 17 (T17) cells are the predominant source of interleukin-17 (IL-17) production and are involved in the occurrence and development of various autoimmune diseases, chronic infammatory diseases, and tumors [2]. During the development of RA, interleukin-23 (IL-23) interacts with its receptor to activate the downstream Janus kinase 2 (JAK2) signal transducer and activator of transcription (STAT) signaling pathway and simultaneously induces the diferentiation of CD4 + T cells into T17 cells [3,4], thus increasing the levels of IL-17 cytokines [5]. In RA, IL-17 induces synovial fbroblasts and osteoblasts to activate the receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK) signaling pathway associated with osteoclastogenesis, thus resulting in the destruction of bone [6,7].
It has been found that both ancient and modern prescriptions containing E. ulmoides were mainly used to treat osteoarthritis [8]. Iridoid is isolated from the ethyl acetate fraction of Eucommia ulmoides Oliv. In our previous research, we showed that extracts from diferent parts of E. ulmoides could inhibit infammation and bone destruction in a rat model of collagen-induced arthritis (CIA). For example, an ethyl acetate extract of E. ulmoides was found to reduce arthritis scores and downregulate the expression levels of p-65 and p-IKK proteins in CIA rats [9,10]. Te male fower of E. ulmoides has also been shown to exert antiarthritis efects by acting on the nuclear factor-κB (NF-κB) pathway [11]. A previous study indicated that the iridoids of E. ulmoides may promote the proliferation and diferentiation of human osteoblasts and exert protective efects on bone [12]. However, the precise therapeutic efects of iridoids of E. ulmoides on RA are yet to be investigated. Terefore, in the present study, we comprehensively investigated the efects of EU-Idd on infammation and bone metabolism in a rat model of CIA, with particular emphasis on T17 cells and diferentiation from RAW264.7 cells to osteoclasts and HFLS-RA cells.

Animals.
A total of 32 female rats (120 ± 10 g) were obtained from Beijing CRL Laboratory Animal Co. Ltd. (Beijing, China). All animal protocols were approved by the Laboratory Animal Welfare and Animal Experimental Ethics Committee of Shanghai University of Traditional Chinese Medicine (approval number: PZSHUTCM200717004). Food and water were provided ad libitum. Figure 1 shows a fowchart depicting the animal experiments. In brief, a 2 mg/mL solution of bovine collagen II (CII) (Chondrex, Redmond, WA, USA) was prepared with glacial acetic acid; after refrigeration at 4°C overnight, the solution was mixed with an equal volume of complete Freund's adjuvant (CFA) (Chondrex, Redmond, WA, USA) to prepare a CII/CFA emulsion. Female rats were randomly assigned to four groups: a blank group (Blank, n � 8), a CIA model group (CIA, n � 8), a group treated with iridoids of E. ulmoides (EU-Idd, n � 8), and a group treated with Tripterygium glycosides (TG, n � 8). Iridoids of E. ulmoides were purchased from Daosifu Biotechnology Co. Ltd. (batch number: 180211A, Nanjing, China), High-performance liquid chromatography (HPLC) was used to quantify iridoids of E. ulmoides, and the results are shown in Figure S1. TG was purchased from Fudan Fuhua Pharmaceutical Co. Ltd. (Shanghai, China). Except for the Blank group, the other groups were injected with 0.1 mL of emulsion in the back and the tail. One week later, booster immunization was performed. After successful modeling, the rats in the Blank and CIA groups were intragastrically administered with distilled water while the EU-Idd and TG groups were given 60 mg/kg of EU-Idd and 5.4 mg/kg of TG, respectively, once a day for 7 weeks. Te body weight, paw volume, and arthritis index (AI) of each rat were recorded every 7 days. AI was scored as follows: 0, no redness; 1, red spots or mild swelling of the ankle; 2, moderate swelling of the ankle; 3, severe swelling of the ankle; and 4, deformed ankle with walking difculty. On day 63, the rats were anesthetized for specimen collection. We also used EU-Idd at a dose of 120 mg/kg, but the anti-infammatory efect was not obvious (as shown in Figure S2). Terefore, we did not conduct follow-up infammatory factor detection. After comprehensive literature survey results, we chose the concentration of 60 mg/kg as the fnal dose.

Histological Analysis of the Ankle Joints.
For histological analysis, the left hind ankle joint of rats in each group was fxed in 10% neutral formaldehyde solution (China National Pharmaceutical Group Corporation, Beijing, China) and decalcifed with EDTA Decalcifed Solution (Sangon Biotech, Shanghai, China). Fixed joints were then parafnembedded and cut into sections (5 μm thick). Hematoxylin-eosin (HE) staining was used to explore pannus formation and synovial hyperplasia in the ankle joints. Histological scores were calculated blindly using an established scoring system [13]: 0, normal joint structure, cartilage, and synovial tissue morphology; 1, a small amount of pannus formation, synovial hyperplasia, and no cartilage and bone destruction; 2, a large number of pannus had begun to form, a large amount of synovial hyperplasia, and slight cartilage destruction; 3, massive pannus formation, extensive synovial hyperplasia, and increased cartilage erosion; and 4, destruction of joint structure along with severe erosion of cartilage and bone. Safranin O-fast green (Solarbio, Beijing, China) staining was used to further investigate cartilage destruction; cartilage was scored using an established system [14]: 0, the cartilage structure showed no damage; 1, the cartilage surface was slightly damaged and appeared rough with cracks; 2, the cartilage was slightly to moderately damaged; 3, the local cartilage was severely damaged; and 4, a large area of the cartilage was damaged.

Micro-Computed Tomography (Micro-CT) of the Ankle Joint.
To investigate bone erosion in the CIA rats, the right hind ankle joint was scanned using a high-resolution micro-CT system (SkyScan 1176, Bruker, Germany) after immersion in absolute ethanol. Te scanning layer thickness was 18 μm and system analysis software was used to analyze key microstructural parameters of the bone tissue in a region of interest, such as the bone volume/total volume fraction (BV/ TV) and trabecular separation (Tb.Sp); the analysis range remained the same for all specimens.

Isolation and Magnetic Enrichment of CD4 + T Cells.
Te spleens of rats in the Blank and CIA model groups were cut into pieces in a vertical fow clean bench and then lysed with red blood cell lysing solution (Biosharp, Hefei, China) to obtain splenocytes. Anti-CD4 was then added to the cells and incubated for 2 h. Next, we added sterile immunomagnetic magnetic beads and CD4+T cells were adsorbed by a magnetic rack. Finally, the purity of the CD4 + T cells was detected by fow cytometry.  2.13. ELISA. Te serum levels of TNF-α, IL-17, and IL-23 from CIA rats were measured by ELISA kits in accordance with the manufacturer's instructions. Te same experimental method was also applied to detect the concentrations of IL-1β and IL-6 in the supernatant of HFLS-RA cells. Te cells were treated with concentrations of 40, 80, and 160 μg/mL and stimulated by TNF-α for 24 h. Ten, the cells were centrifuged to obtain a cell supernatant for ELISA. Standard curves were calculated according to the OD value so that we could determine the concentrations of infammatory cytokines. We purchased the following ELISA kits from Multi Sciences (Hangzhou, China): rat TNF-α ELISA Kit (70-EK382/3-96), rat IL-17A ELISA Kit (70-EK317/3-96), human IL-6 ELISA Kit (70-EK106/2-96), and human IL-1β ELISA Kit (70-EK101B-96). An IL-23 ELISA kit (E-EL-R0569c) was obtained from Elabscience (Wuhan, China).
2.14. RT-qPCR. Real-time quantitative PCR (RT-qPCR) was used to detect the levels of mRNA in spleen and cartilage tissue of rats and HFLS-RA cells stimulated by TNF-α. We used Tissue RNA Purifcation Kit PLUS (EZB-RN001-plus) and EZ-press RNA Purifcation Kit (B0004DP) to isolate total RNA in vivo and in vitro. To obtain cDNA, we used a Color Reverse Transcription Kit (A0010CGQ) to reverse transcribe RNA. Te mRNA expression levels were determined by 2 × Color SYBR Green qPCR Master Mix (A0012-R2) on an ABI-7500 qPCR system (Termo Fisher Scientifc, USA) according to the manufacturer's instructions. Te thermocycling conditions were as follows: 95°C for 5 min, 95°C for 10 s, 60°C for 30 s (40 cycles), and then 72°C for 90 s. Te primer sequences are shown in Table 1. Data were normalized to GAPDH expression using the 2 −ΔΔCt method. Te reagents used for RT-qPCR experiments were purchased from EZBioscience (Roseville, MN, USA).

Statistical
Analysis. SPSS 26.0 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis of experimental data. Data are expressed as mean ± standard deviation. One-way ANOVA with Student's t-test and Dunnett's test was used for multiple comparisons. P < 0.05 was considered signifcant.

EU-Idd Inhibited Joint Infammation and Bone
Destruction in CIA Rats

EU-Idd Alleviated Infammatory Symptoms in CIA
Rats. We established a CIA rat model to evaluate the antiarthritic efects of EU-Idd. Changes in body weight of the rats in each group were measured every 7 days from the start of the experiment; the results are shown in Figure 2(a).
Tere was no signifcant diference in body weight between the CIA group and each administration group. However, the fur color of rats in the CIA group was yellow and dull; these rats also ate less and were less active when compared with rats from the EU-Idd group. From the beginning of the experiment, the bilateral paw volume of the rats in each group was measured every 7 days. On the 14 th day of the experiment, the paw volume of the model group was signifcantly greater than that in the blank group (P < 0.05). As shown in Figure 2(b), the paw volume of the CIA group reached a peak on the 21 st day of the experiment. Following treatment with EU-Idd for 7 weeks, the paw volume was signifcantly downregulated. Moreover, the arthritis index decreased from the 21 st day (Figures 2(c) and 2(d)). Tus, iridoid exerts inhibitory efects on joint infammation in CIA rats. Te results arising from HE staining are shown in Figure 2(e). Te joint structure of rats in the blank group was clearly visible. However, we observed synovial tissue hyperplasia in rats from the CIA group and many infammatory cells had infltrated the ankle tissue. Te administration of EU-Idd inhibited synovial hyperplasia of the ankle tissue and improved the pathology of the joint structure ( Figure 2(g)). Te articular cartilage in the ankle was stained by Safranin O-fast green staining; results are shown in Figure 2(f ). After 7 weeks of EU-Idd treatment, the cartilage damage had reduced signifcantly; there was also a reduction in the cartilage score ( Figure 2(g)).

EU-Idd Relieved Bone Erosion in the Ankle Joints.
Micro-CT scanning of the right hind paws of rats from each group provided high-resolution three-dimensional (3D) images and related bone parameters that were able to characterize the level of bone growth and development; results are shown in Figure 3(a). Compared with the CIA group, the EU-Idd treatment group showed less bone erosion, less joint damage, and a clearer joint structure. Bone parameters are shown in Figure 3(b). Compared with the Blank group, the ratio of bone volume per tissue volume (BV/TV) in the CIA group had decreased signifcantly (P < 0.01), thus proving that the bone mass had decreased. However, trabecular separation (Tb.Sp) had increased signifcantly (P < 0.001), thus proving that the mean width of the medullary cavity between the trabecular bone had increased. Conversely, the BV/TV values of the ankle joints from CIA rats increased after EU-Idd treatment while Tb.Sp values decreased.

EU-Idd Reduced the Levels of Infammation and Cytokines Related to Bone Metabolism in CIA Rats.
Te serum concentrations of infammatory cytokines (TNF-α, IL-17, and IL-23) were measured by ELISA; the results are shown in Figure 4(a). Compared with the blank group, the serum levels of TNF-α, IL-17, and IL-23 in the CIA group were signifcantly increased (P < 0.001). Te trend for variation in infammatory cytokines was consistent with the CIA infammation model. Te mRNA expression levels of TNF-α, IL-17, and IL-23 in the spleen of rats from each group were detected by RT-qPCR ( Figure 4(b)). We found that the trend for variation in mRNA expression for the three infammatory cytokines in the spleen was similar to that in the serum. Te administration of EU-Idd signifcantly downregulated the expression levels of TNF-α, IL-17, and IL-23 in the serum and the spleen (P < 0.01, P < 0.001). Collectively, these results suggested that EU-Idd had a certain anti-infammatory efect.
To determine the role of EU-Idd on the expression of factors related to bone metabolism in the joint tissues of rats in each group, we next used RT-qPCR to detect the expression of key genes (Figure 4(c)). Te mRNA expression levels of RANKL, nuclear factor of activated T cells cytoplasmic 1 (NFATc-1), c-Fos, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were signifcantly increased in the CIA rats. Te expression levels of osteoprotegerin (OPG) mRNA, a gene related to osteoblastogenesis, were signifcantly decreased (P < 0.001). Te administration of EU-Idd signifcantly downregulated the mRNA expression levels of RANKL, NFATc-1, c-Fos TRAP, and CTSK in articular cartilage (P < 0.05 or P < 0.01 or P < 0.001) and upregulated the mRNA expression levels of OPG (P < 0.001).  . Te proportions of T17 cells in the peripheral blood and spleens of rats in the CIA group were signifcantly higher than those in the blank group (P < 0.01). Te proportion of T17 cells in the EU-Idd treatment group was signifcantly lower than that in the CIA group (P < 0.05, P < 0.01). Collectively, results suggested that EU-Idd inhibited the expression of T17 cells and might play an important role in inhibiting joint infammation in RA by regulating the production of T17 cells. Te protein expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 in the spleens of rats from each group were detected by western blotting (Figure 5(c)). Next, we investigated the efect of EU-Idd on the JAK2/STAT3 pathway, as shown in Figure 5(d). In the CIA model group, the protein expression levels of p-JAK2/JAK2 and p-STAT3/STAT3 were signifcantly higher than those in the blank group (P < 0.05, P < 0.001). Conversely, compared with the CIA group, the administration of EU-Idd signifcantly downregulated the expression levels of p-JAK2 and p-STAT3 (P < 0.05, P < 0.01, P < 0.001), thus suggesting that EU-Idd efectively inhibited the phosphorylation and activation of the JAK2/STAT3 pathway.

EU-Idd Reduced the Proportion of T17 Cells in CD4 + T Cells.
To obtain more CD4 + T cells, we performed magnetic bead sorting experiments; results are shown in Figures 6(a) and 6(b). Te morphology of CD4 + T cells was observed under an inverted microscope; cell morphology appeared to be relatively uniform, round, and granular, and the cells did not adhere to the wall. After sorting, the cell purity was greater than 80%. After 40, 80, and 160 μg/mL of EU-Idd was applied to the CD4 + T cells for 24 h, we used the All data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, and ### P < 0.001vs Blank; * P < 0.05, * * P < 0.01, and * * * P < 0.001vs CIA. Blank � blank control group. CIA � CIA control group. TG � Tripterygium glycosides. (c) Te mRNA expression levels of cytokines related to bone metabolism in joint tissues of rats from the Blank, CIA, EU-Idd, and TG group were detected by RT-qPCR (n � 6 for each group). All data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, and ### P < 0.001vs Blank; * P < 0.05, * * P < 0.01, and * * * P < 0.001vs CIA. Blank � blank control group. CIA � CIA control group. TG � Tripterygium glycosides.

EU-Idd Inhibited the Phosphorylation of the JAK2/ STAT3 Signaling Pathway during the Diferentiation of T17
Cells. To investigate the mechanism by which EU-Idd inhibited the diferentiation of T17 cells in vitro, we used western blotting to detect the phosphorylated protein expression levels of JAK2 and STAT3 in T17 cells after directional induction for 5 days. Te results are shown in Figure 6(e). Compared with the NC group, the model group showed signifcantly increased protein expression levels of p-JAK2 and p-STAT3 (P < 0.001). Compared with the model group, the 20 μg/mL EU-Idd group showed downregulated expression levels of both JAK2 and STAT3 phosphorylated proteins, although these diferences were not statistically signifcant (P > 0.05). Compared with the model group, the were analyzed and normalized to the total JAK2 and STAT3 proteins (n � 8 for each group). All data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, and ### P < 0.001vs Blank; * P < 0.05, * * P < 0.01, and * * * P < 0.001vs CIA. Blank � blank control group. CIA � CIA control group. TG � Tripterygium glycosides.

10
Evidence-Based Complementary and Alternative Medicine EU-Idd (30, 40 μg/mL) group showed signifcant reductions in the protein expression levels of p-JAK2 and p-STAT3 (P < 0.05 or P < 0.01 or P < 0.001).  Te CD4+T cells used for the CCK-8 assay were isolated from normal rats to investigate the efect on cell viability caused by modeling. Te experiment was repeated three times. Te data are expressed as mean ± SD (n � 4 for each group). * P < 0.05, * * P < 0.01, and * * * P < 0.001vs NC. (d) Efects of diferent concentrations of EU-Idd (20, 30, and 40 μg/mL) on T17 diferentiation. Te CD4 + T cells of CIA rats were induced to diferentiate into T17 cells with stimulatory factors in vitro. After 5 days of induction, the proportion of T17 cells in each group was detected by fow cytometry. (e) Te efects of EU-Idd on the protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 during T17 cell diferentiation. Te data are expressed as mean ± SD (n � 3 for each group). # P < 0.05, ## P < 0.01, and ### P < 0.001vs NC; * P < 0.05, * * P < 0.01, and * * * P < 0.001vs model. NC, the non-induced group. Model � the induced group. Te CD4 + T cells used for assays were isolated from CIA rats.  Figure 8(c). Te model group showed a signifcant increase in the protein expression levels of RANKL, RANK, TRAF6, and CTSK; these proteins are all related to osteoclastogenesis (P < 0.01, P < 0.001). Tere was also a signifcant reduction in the protein expression levels of OPG, a protein related to osteoblastogenesis (P < 0.01, P < 0.001). Conversely, diferent concentrations of EU-Idd signifcantly reduced the protein expression levels of RANKL, RANK, TRAF6, and CTSK and signifcantly increased the expression levels of OPG protein (P < 0.05, P < 0.001). Analysis showed that 40 μg/mL of EU-Idd improved protein levels in the RANKL/OPG axis although these changes were not signifcantly diferent (P > 0.05).  Figure 9(b). After staining, a greater number of stained cells were observed in the model group; in addition, the invasion rate was signifcantly higher than that of the NC group (P < 0.001) (Figure 9(d)). EU-Idd (40, 80, and 160 μg/mL) reduced the rate of invasion and inhibited the invasion of HFLS-RA cells after TNF-α induction. Te efect of EU-Idd group on the migration ability of HFLS-RA cells was judged by scratch assays; the results are shown in Figure 9(c). At 0 h, the size of the scratched area in each group was similar. After 24 h of TNF-α stimulation, the scratched area in the model group was signifcantly smaller (P < 0.01) (Figure 9(e)); the scratched area was signifcantly increased while the scratch healing rate was signifcantly slower (P < 0.001).

EU-Idd Inhibited the Phosphorylation of JAK2 and STAT3 in HFLS-RA Cells.
Total protein was extracted from HFLS-RA cells for western blot assays to explore the mechanisms underlying the anti-infammatory efects of EU-Idd; results are shown in Figure 10(c). Compared with the NC group, the expression levels of p-JAK2 protein were signifcantly increased after stimulation with TNF-α (P < 0.05). Te expression levels of p-STAT3 protein were increased but with no statistical signifcance (P > 0.05). EU-Idd (40, 80, and 160) signifcantly reduced the phosphorylated protein expression level of JAK2 (P < 0.05, P < 0.01, and P < 0.001). EU-Idd signifcantly reduced the expression levels of p-STAT3 protein at concentrations of 40 and 80 μg/ mL, although the downregulation efect of EU-Idd at 160 μg/ mL was not signifcant (P > 0.05).

Discussion
Te iridoid component of E. ulmoides has been shown to exhibit positive anti-infammatory activity and is mainly composed of geniposidic acid, geniposide, and aucubin [15][16][17][18]. Geniposide has been demonstrated to reduce infammation and immunological control, enhance mitochondrial apoptosis in MC3T3-E1 cells, and have boneprotecting property [19,20]. According to the arthritis research of aucubin, it plays an important role in reducing cartilage destruction and preventing apoptosis in chondrocytes in a mouse model of OA, and aucubin has also been proved to exert therapeutic efects on CIA rats and accelerate synovial cell death [21,22]. In CIA model rats, we discovered that EU-Idd had an intervention efect on infammation. Additionally, EU-Idd decreased the paw volume and arthritis index in rats, Densitometry was performed to determine the levels of JAK2 and STAT3 phosphorylation and data were normalized to the total protein levels of JAK2 and STAT3 (n � 4 for each group). Densitometry data for OPG, RANKL, RANK, TRAF6, and CTSK were normalized to GAPDH, respectively (n � 4 for each group). All data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, and ### P < 0.001vs NC; * P < 0.05, * * P < 0.01, and * * * P < 0.001vs model. NC, the non-induced group. Model, the induced group.
improved the pathology of the ankle joints, and had a boneprotective efect. Moreover, it has been shown that EU-Idd negatively regulates the level of expression of associated infammatory cytokines and proteins involved in bone metabolism.
Prior to bone destruction, the principal pathological manifestation is that the synovial tissue is infltrated by many infammatory cells; there is also the secretion of proinfammatory cytokines. Naive CD4 + T cells can diferentiate into Treg and T1, T2, and T17 cells under diferent Western blotting was used to detect JAK2, p-JAK2, STAT3, and p-STAT3 protein expression levels in HFLS-RA cells. (d) Densitometry scans for the phosphorylated levels of JAK2 and STAT3 were analyzed and normalized to the total proteins of JAK2 and STAT3 (n � 4 for each group). All data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, and ### P < 0.001 vs NC; * P < 0.05, * * P < 0.01, and * * * P < 0.001vs model. NC, the non-induced group. Model, the induced group. stimulation conditions. Imbalances in the ratio and function of various T cell subsets and between T17 cells and Treg cells play a crucial role in the pathogenesis of RA [23]. T17 is known to produce a marked efect in RA by producing IL-17, a cytokine that can stimulate synovial fbroblasts to produce a variety of infammatory factors, thus resulting in the induction of synovitis [24]. Moreover, IL-17 activates the expression of RANKL, a protein related to osteoclastogenesis in monocytes, and disrupts bone homeostasis [6]. In this study, we noticed that the ratio of T17 cells in the peripheral blood and spleens of rats in the CIA model was signifcantly higher than that of rats in the blank group. To further verify the efect of EU-Idd on T17 diferentiation in vitro, we directed the diferentiation of CD4 + T cells from CIA rats to T17 cells. TGF-β activates downstream STAT3 together with IL-6; in turn, this leads to high expression levels of RORct, which initiates the diferentiation cascade of T17 cell [25]. According to experimental methods described in the existing literature [26,27], we used cytokine stimulation (TGF-β, IL-6, TNF-α, IL-1β, and IL-23) under coating conditions created by CD3 and CD28 antibodies to induce T17 cells. EU-Idd treatment signifcantly inhibited the diferentiation of T17 cells. Based on these results, it appeared that EU-Idd reduced the production of T17 cells and could regulate the immune system. To better explore the anti-infammatory activity of EU-Idd, we detected changes in related infammatory cytokines. IL-23 is a heterodimeric cytokine composed of p19, IL-12, and IL-23p40 connected by disulfde bonds. Studies have found that IL-23 is a cytokine that maintains T17 expansion and stability [28]. IL-23 exerts its biological efects mainly by binding to its receptor IL-23R to stimulate cells to produce infammatory factors and activate related signaling pathways [29]. A signifcant cytokine in the infammatory response of RA is TNF-α. TNF-α mainly causes cartilage damage in RA by promoting the release of various infammatory mediators, the degradation of cartilage proteoglycan, and the expression of vascular endothelial cell adhesion molecules [30]. In rheumatoid arthritis, the chronic infammation of synovial cells is largely caused by IL-1β overproduction; this directly activates FLS and leads to joint destruction and bone resorption [31,32]. One of the most signifcant pro-infammatory factors in the course of many infammatory reactions is IL-6. Furthermore, IL-6 promotes the recruitment of infammatory cells such as neutrophils into the synovium and promotes the expression of RANKL in synovial cells to stimulate the generation of osteoclasts [33]. We discovered that EU-Idd prevented the infltration of infammatory cells in the ankle joints and the expression of IL-23 and TNF-α of CIA rats and decreased the invasion and migration of HFLS-RA cells. Te protein levels of IL-6 and IL-1β in HFLS-RA and the mRNA expression levels of IL-6, IL-1β, IL-17, and IL-23 in HFLS-RA were downregulated by EU-Idd. Te infammatory reaction was followed by bone destruction. In the early stages of RA, the symptoms of infammation are obvious. We discovered that the paw volume of CIA rats had dramatically risen. Te paw volume stabilized after 42 days. Micro-CT scanning further showed that the bone destruction in CIA rats was severe. Levels of cytokines associated with osteoclasts had also increased signifcantly. RANKL is involved in bone metabolism in regulating the diferentiation of osteoclasts. RANKL acts directly on osteoclast precursors via its receptor RAN which binds to its receptor RANK and recruits TNF-receptorassociated factor 6 (TRAF6) to activate NF-κB, NFATc-1, c-Fos, CTSK, and other major regulators related to osteoclast diferentiation, to induce the diferentiation of precursors into osteoclasts [34,35]. OPG is a soluble decoy receptor of RANKL that can bind to RANKL to inhibit the activation of RANK and has an important role in protecting the bones; it can also negatively regulate the production of osteoclasts [36]. Both c-Fos and NFATc-1 are key transcription factors for the diferentiation of osteoclasts and can initiate the expression of TRAP; CTSK can refect the resorption activity of osteoclasts [37,38]. In vivo, EU-Idd efectively increased the expression of OPG mRNA in CIA rats and reduced the mRNA expression of osteoclast-related cytokines such as RANKL. In vitro, EU-Idd efectively inhibited the diferentiation of RAW264.7 cells into osteoclasts, reduced the protein expression of RANKL, RANK, TRAF6, and CTSK, and increased the protein expression of OPG. Collectively, these data suggested that EU-Idd inhibited the formation of osteoclasts and exerted a protective efect in bone.
Recent studies have shown that the JAK2/STAT3 pathway is a key factor in the infammatory response and bone destruction of RA. Research targeting this pathway could lead to the development of more efective treatments for RA [39]. JAK2 is activated by autophosphorylation; following activation, it can form multiple receptor binding sites and present these sites for STAT3 for phosphorylation and dissociation into the nucleus to regulate the expression of genes related to the transcription factor RORct, and it is essential for the diferentiation of T17 cells [40,41]. Following the activation of the JAK2/STAT3 pathway, memory T lymphocytes can be induced to diferentiate into T17 cells to play a role in maintaining and expanding T17 cells. Research has shown that T17 cells produce a variety of proinfammatory mediators, including IL-17, IL-6, and IFN-c, and IL-6 can further strengthen STAT3 signaling, promote the further diferentiation of T17 cells, and lead to the continuous deterioration of the infammatory response [42]. In our study, phosphorylation of the JAK2/STAT3 protein was shown to be inhibited in CIA rats, while JAK2 and STAT3 were phosphorylated during CD4 + T cell diferentiation into T17 cells. EU-Idd suppresses the protein expression levels of p-JAK2 and p-STAT3 and cellular diferentiation into T17. EU-Idd also inhibited the phosphorylation and activation of the JAK2/STAT3 pathway in HFLS-RA cells stimulated by TNF-α. In terms of bone metabolism, previous researchers found that the protein expression levels of JAK2 and STAT3 were correlated with RANKL levels during osteoclastogenesis. Additionally, inhibiting the JAK2/STAT3 pathway and fostering Tregmediated bone immunity can suppress osteoclast diferentiation and bone resorption [43][44][45][46]. In this study, we observed that EU-Idd reduced the expression levels of p-JAK2 and p-STAT3 during the diferentiation of RAW264.7 cells into osteoclasts induced by RANKL. However, some limitations should be noted. One limitation is that we have not yet explored the optimal therapeutic dose of EU-Idd for rheumatoid arthritis, and another limitation is that our results have not been verifed by using JAK2/STAT3 pathway inhibitor. With the in-depth study of E. ulmoides and rheumatoid arthritis, it could lead to new targets aimed at treatment of rheumatoid arthritis.

Conclusion
EU-Idd could signifcantly inhibit joint infammation and reduce the expression of infammatory cytokines such as IL-17 and IL-23 and relieve bone erosion in the ankle joints in CIA rats. In vitro, EU-Idd inhibited the diferentiation of CD4+ T cells into T17 cells and the diferentiation of RAW264.7 cells into osteoclasts and suppressed the migration and invasion of HFLS-RA cells. Collectively, our results indicate that EU-Idd inhibited the infammatory response of CIA rats and reduced the production of osteoclasts via the JAK2/STAT3 pathway. Consequently, EU-Idd has signifcant potential for the treatment of rheumatoid arthritis.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Li-Dong Tang and Jian-Ying Wang contributed equally. Li-Dong Tang wrote the original draft. Jian-Ying Wang was responsible for methodology. Yan Zhang was responsible for software. Xiao-Yun Chen was responsible for visualization. Lei Zhang was responsible for investigation. Ying Yuan wrote, reviewed, and edited the manuscript.