An Alcohol Extract Prepared From the Male Flower of Eucommia Ulmoides Oliv. Promotes Synovial Cell Apoptosis, Inhibits Osteoclast Differentiation and Ameliorates Bone Destruction in Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease with a complex pathogenesis and is 20 dominated by synovial hyperplasia and bone destruction. Previous research has shown that the male flower of Eucommia 21 ulmoides Oliv. (EF) can exert effect on the inflammation caused by rheumatoid arthritis. However, the effect of EF on synovial cell apoptosis and bone destruction on RA have yet to be investigated. In this study, the effects of the synovial cell apoptosis of the male flower of Eucommia ulmoides Oliv.(EF) on human fibroblast-like synoviocyte -RA (HFLS-RA) cells, the osteoclast differentiation of EF on RAW264.7 cells and the bone destruction of effects of EF on collagen-induced arthritis (CIA) rats were explored. Materials and methods: In vitro , we investigated the anti-proliferative and pro-apoptotic effects of EF on HFLS-RA cells by immunofluorescence assays, flow cytometry, RT-qPCR (Real-time quantitative polymerase chain reaction), and western blotting. We investigated the differentiation into osteoclasts effects of EF on RAW264.7 cells by the TRAP staining and western blotting. In vivo , we used a rat model of collagen-induced arthritis (CIA) to investigate the relative effects of EF on anti-arthritis activity, the toe swelling arthritis score, the serum levels of metabolic bone factors, and pathological conditions. Micro-computed tomography (micro-CT) was used to scan ankle joints while the mRNA and protein levels of factors related to the NF- κB pathway were determined by RT-qPCR and western blotting, respectively. Finally, the main chemical components of EF were identified by HPLC (High Performance Liquid Chromatography). Results: EF inhibited the proliferation of synovial cells and promoted apoptosis in a dose-dependent manner, inhibited the differentiation of osteoclast by inhibiting activation of the NF- κB pathway. We also found that EF reduced articular inflammation in CIA rats, inhibited the expression of pro-angiogenic factors, and delayed the destruction of articular cartilage and bone. Our 37 data indicate that EF acts via a mechanism related to bone metabolism that is induced by the NF- κB pathway. Conclusions: Our findings indicate that EF exerts a potential therapeutic effect on rheumatoid arthritis. Our research will help to elucidate the potential pharmacological mechanisms associated with the beneficial effects of EF and provide an experimental basis for the application of EF in future clinical treatments. EF can exert anti-inflammatory, analgesic, anti-bacterial, and other pharmacological immunoregulation EF often used for daily health care, and the prospects are good for developing and utilizing EF in a wide range of clinical applications In previous published articles, we have preliminarily discussed the effects of EB, and EF on improving inflammation in CIA rats. EF and EB have similar functions. We found EF can reduce the expression of TNF- α and NO in synovial cells and improve t he articular inflammation in CIA rats in vivo [11]. Therefore, on this basis, this study further explored the mechanism of EF on promoting synovial cell apoptosis and inhibiting the bone destruction, explored whether the effect of EF on synovial cell apoptosis and bone destruction on CIA rats is related to the activation of NF- κB pathway and provided experimental basis for the further application of EF. Currently, there are few studies on the treatment of RA by EF, however, the effects of EF on RA have yet to be confirmed. In this study, we investigated the effect of EF on the proliferation, migration, and apoptosis in human fibroblast-like synoviocyte cells-RA (HFLS-RA); we investigated the effect of EF on the osteoclast differentiation in RAW264.7 cell; these cells are the main causes of tissue injury and distortion in the joints in RA. We also evaluated the effects of EF on inflammatory bone destruction via the NF- κB pathway in a rat model of CIA. Our goal was to provide an experimental basis for the rational application of EF in future clinical RA treatments and to provide a modern interpretation of its pharmacological mechanism. cytotoxic 24h. We investigated the effects of EF on the proliferation of LPS-stimulated RAW264.7 cells. that EF 2000 μg/mL was able to inhibit the proliferation of LPS-stimulated RAW264.7 (**p 0.01, ***p 0.001). Western we found that EF osteoclast of RAW264.7 cells and the of NF- κB pathway The pathogenesis of RA is the main synovial cell and the of life Joint into two types: synovial and joint structure destruction. Once has degeneration, it is very difficult to stop or reverse this process [1]. Our current data show that EF can significantly reduce the degree of joint swelling in a rat model of CIA. Pathological sections further showed that EF can reduce partial necrosis of the articular cartilage, reduce pannus formation, fibrous tissue changes, and synovium hyperplasia. Micro-CT scans showed significant improvement in bone destruction in the group of CIA rats treated with EF. These results suggest that EF can effectively inhibit the bone destruction experienced by joints in the rat model of CIA and improve pathological changes within the joints. κB oncogene c-FOS, tartaric phosphatase these degrade bone exacerbate bone erosion [18]. RANKL signaling, via its receptor activator, NF- κB (RANK), important osteoclastic role in bone remodeling [19]. In the CIA group of rats, we observed a significant increase in the levels of osteoclast including TRAP, NFATc1, CTSK, c-FOS, and RANKL, significant levels of collectively, these had induced severe in the cartilage and bone significantly reduced the mRNA levels of , NFATc1 , CTSK , c-FOS , RANKL . The mRNA levels of OPG, a marker of osteogenesis, were significantly higher in the EF group, although this showed some variation. The MMP family is involved in tissue remodeling, repair, and angiogenesis, and is regulated by tissue metalloproteinase inhibitor (TIMP). In the event of osteoarthritis and RA, the family of MMP proteins is up-regulated and the balance with TIMP is destroyed [20]. In this study, we observed reduced levels of TIMP1 in the cartilage of rats in the model group. In contrast, levels of TIMP1 in the EF group increased to varying degrees, thus indicating that EF has the ability to improve arthritis. However, whether EF exerts impact on the family of MMP proteins requires further study. This study provides an experimental basis for the application of EF, but there are still many limitations. It is mainly limited to research design, such as clarifying the effect between active single component of TCM and active single component of TCM, the role of active single component of TCM in active single component of TCM, the main target of TCM, improving the method of in vitro study of TCM extract, etc. On the basis of this experiment, this research group will continue to search for active monomer components of Chinese medicine and further study its target mechanism. In summary, the expression levels of p-p65 NF- κB, p - Iκκαβ , and p- IκBα , in the cartilage of the model group were significantly up-regulated. After treatment, the expression levels of p-p65 NF- κB, p - Iκκαβ , and p- IκBα , in the EF group were significantly reduced. These data demonstrate that EF may improve bone destruction in the rat model of CIA by inhibiting the NF- κB signaling pathway. apoptosis and inhibits osteoclast differentiation by activation of the NF- κB pathway . EF also reduces toes swelling in the rat model of CIA, inhibits the expression of pro-angiogenic factors, and delays the destruction of articular cartilage and bone. The mechanism underlying the effects of EF is related to bone metabolism induced by the NF- κB pathway. We hope that the results of our study will help to elucidate the precise pharmacological mechanisms underlying the actions of EF and provide an experimental basis for the application of EF in the clinical treatment of


Introduction 43
Rheumatoid arthritis (RA) is an autoimmune disease that is dominated by chronic multiarticular inflammation and bone 44 destruction. The main pathological features of RA include the proliferation of synovial cells, pannus formation, the destruction of 45 cartilage and bone, and even joint deformity. These changes cause a great deal of inconvenience to the patients affected by this 46 disease and create a significant burden with regards to daily life and work activities. RA is a very common global disease; the 47 World Health Organization (WHO) lists RA as one of the most troublesome global diseases [1]. Although diagnostic methods 48 and treatments have improved significantly over recent years, studies have found that the currently available drugs for RA are 49 mainly disease-relieving anti-rheumatic drugs (such as methotrexate), non-steroidal anti-inflammatory drugs (NSAIDs), 50 glucocorticoids (such as dexamethasone), and other biological agents [2]. With regards to joint inflammation, even if synovial 51 inflammation has been controlled during the late stages of rheumatoid arthritis, the destruction of bone can still cause irreversible 52 damage to the joints. Therefore, inhibiting the early proliferation of synovial cells, and slowing pannus formation, may represent 53 potential treatment methods with which to reduce the subsequent erosion of cartilage and bone. Traditional Chinese medicine 54 (TCM) has achieved certain curative effects in the treatment of RA. Previous studies have found that three different extracts from 55 Eucommia ulmoides Oliv. can significantly inhibit bone destruction, synovial inflammation, and systemic inflammation [1].

56
The NF-κB pathway plays an important role in a variety of immune diseases. When stimulated by interleukin-1 (IL-1), tumor 57 necrosis factor (TNF-α) and cells activate IκB kinase (Iκκs); this kinase phosphorylates, ubiquitinates and then degrades the IκB 58 protein. This leads to the dissociation of IκB from NF-κB in the cytoplasm. Once transferred into the nucleus, NF-κB regulates 59 the transcription of a range of target genes, promotes angiogenesis and the expression of cytokines such as IL-1β and vascular 60 endothelial growth factor (VEGF) , and thus exacerbates attacks on the bone and cartilage in RA [3]. Therefore, inhibition of the 61 NF-κB pathway may represent a potential therapeutic approach with which to control the bone destruction of RA.

62
There is increasing evidence that Chinese herbal medicines are valuable resources for the treatment of some intractable 63 diseases [4][5]. Eucommia ulmoides Oliv. (EU) is a traditional Chinese medicinal plant. The bark and leaves of EU are used 64 widely in TCM clinics. The active ingredients of EU are able to reduce blood pressure, improve immune function, and can 65 exhibit anti-aging, anti-inflammatory, and anti-tumor effects. EU is one of the most commonly prescribed TCM ingredients used 66 for the clinical treatment of RA [6][7]. In a previous study, we found that an alcohol extract prepared from the bark of Eucommia 67 ulmoides Oliv. (EB) was able to inhibit the proliferation of synovial cells in human rheumatoid arthritis (RA-FLS) in vitro and 68 promote their apoptosis. In vivo, the swelling and arthritic scores of a collagen-induced rheumatoid arthritis (CIA) rat model were 69 reduced when treated with EB; pathological analysis further suggested that synovial inflammatory infiltration was improved and 2 pannus formation was alleviated [8]. However, EB grows slowly and resources are incredibly scarce; consequently, there is an 71 urgent need to identify alternatives. The male flower of Eucommia ulmoides Oliv. (EF) is relatively rich in terms of resources.

72
Previous research has shown that EF can exert anti-inflammatory, analgesic, anti-bacterial, and other pharmacological effects, 73 including immunoregulation [9]. EF is often used for daily health care, and the prospects are good for developing and utilizing 74 EF in a wide range of clinical applications [10]. In previous published articles, we have preliminarily discussed the effects of EB, 75 EL and EF on improving inflammation in CIA rats. EF and EB have similar functions. We found that EF can reduce the 76 expression of TNF-α and NO in synovial cells and improve the articular inflammation in CIA rats in vivo [11]. Therefore, on this 77 basis, this study further explored the mechanism of EF on promoting synovial cell apoptosis and inhibiting the bone destruction, 78 explored whether the effect of EF on synovial cell apoptosis and bone destruction on CIA rats is related to the activation of 79 NF-κB pathway and provided experimental basis for the further application of EF. Currently, there are few studies on the 80 treatment of RA by EF, however, the effects of EF on RA have yet to be confirmed.

81
In this study, we investigated the effect of EF on the proliferation, migration, and apoptosis in human fibroblast-like 82 synoviocyte cells-RA (HFLS-RA); we investigated the effect of EF on the osteoclast differentiation in RAW264.7 cell; these 83 cells are the main causes of tissue injury and distortion in the joints in RA. We also evaluated the effects of EF on inflammatory 84 bone destruction via the NF-κB pathway in a rat model of CIA. Our goal was to provide an experimental basis for the rational 85 application of EF in future clinical RA treatments and to provide a modern interpretation of its pharmacological mechanism. 102 h), filtered and vacuum dried to await experimentation. The concentration of the extract was 1 g/mL [11]. Based on the 103 preliminary experiments conducted by our project team in the early stage and the basic research in the early stage, the effec t was 104 obvious when the drug concentration was 1 g/mL. Therefore, in vivo experiment, the concentration of the high dose was set at 1 105 g/mL and the low dose was set at 0.5 g/mL. To qualitatively investigate the main constituents of ethanol extracts from EF, we 106 performed HPLC analysis.

126
Cell colony forming assay 127 Rat tail collagen was diluted to 3% with PBS and added to a 6-well plate. HFLS-RA cells were cultured in serum-free DMEM 128 for 24 h in 6-well plates (in triplicate). After starvation, the cells were either left untreated (as negative controls) or supplemented 129 with various concentrations of EF (0, 100, 200, 400, 800, and 1600 μg/mL). Cells were treated for 2 days and then cultured for 7 130 days. Following culture, the cells were fixed with 4% pre-cooled paraformaldehyde and stained with 0.5% crystal violet. The 131 crystal violet was eluted with 70% ethanol, and the eluent was transferred to a 96-well culture plate. The optical density (OD) 132 was then read at 595 nm. The relative rate of cell viability was then expressed as a proportion (%) of the control group.   143 μg/mL) and TNF-α (10 ng/mL) for 24 h. Following treatment, the cells were fixed with 4% paraformaldehyde and stained with 144 0.1% crystal violet solution. The cells were then placed onto a glass slide and five fields from each group were selected for 145 photography under an inverted microscope. Cells were eluted with 33% acetic acid in 96-well plates and the OD value was read 146 at 570 nm. The relative rate of cell viability was expressed as a proportion (%) of the control group.

147
Flow cytometric analysis of apoptosis 148 HFLS-RA cells were pre-incubated for 24 h with EF at various concentrations (400, 800, 1600 μg/mL) with or without TNF-α 149 (10 ng/mL). As recommended by the manufacturers of the Annexin V-FITC/PI apoptosis detection kit, cells were re-suspended 150 with binding buffer, and mixed with 5 μL of Annexin V-FITC and PI. Cell apoptosis was then detected by flow cytometry 151 analysis.

153
HFLS-RA cells were cultured in 6-well plates and treated with TNF-α (10 ng/mL) and EF (400, 800, and 1600 μg/mL) for 24 154 h. RNA was extracted with EZ-press RNA Purification Kit and cDNA was synthesized with 4 × Reverse Transcription Master 155 Mix. Real-time quantitative polymerase chain reaction (RT-qPCR) was then used to quantify gene expression levels. RT-qPCR 156 was performed using 2 × SYBR Green qPCR Master Mix on an ABI Prism 7500 qPCR system (Thermo Fisher Scientific) with 157 the following cycling conditions: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, 158 and a final extension at 72°C for 90 s. The primers used for RT-qPCR analysis are shown in Table 1. Data were normalized to the 159 expression of GAPDH using the 2 -ΔΔCT method [1]. All the experiments were repeated three times.    Table 2. Data were normalized to the expression of GAPDH using the 2-ΔΔCT method [1]. All the 237 experiments were repeated three times.

EF inhibited cell migration and invasion in TNF-α stimulated HFLS-RA cells 260
Next, we evaluated the effects of EF on the migration and invasion of HFLS-RA cells. As shown in Fig. 3A and C, the scratch 261 area in the EF group was significantly larger than that in the TNF-α group, thus indicating that the administration of EF inhibited 262 the migration of cells (***p < 0.001). We also carried out an invasion assay to evaluate the effect of EF on the invasion of cells 263 ( Fig. 3B and D). We found that EF significantly inhibited cell invasion (**p < 0.01, ***p < 0.001).

265
Our previous results indicated that EF exhibited notable antiproliferative effects against HFLS-RA cells. In order to investigate 266 the inhibitory effect of EF on the proliferation of HFLS-RA cells, we next investigated the ability of EF to induce apoptosis in 267 HFLS-RA cells by performing flow cytometry and RT-qPCR. As shown in Fig. 4A and B, there was a gradual increase in 268 apoptotic ratio as the concentration of EF increased (*p < 0.05, ***p < 0.001). As shown in Fig. 4C and D, the mRNA levels of 269 IL-1β, IL-6, VEGF, and MMP-9, all decreased significantly with increased concentrations of EF (400, 800, 1600 μg/mL) when 270 compared to the TNF-α group (*p < 0.05, **p < 0.01, ***p < 0.001). Compared with the TNF-α group, the mRNA levels of 271 Caspase3 (cleaved) and Bax in the EF group were significantly higher, and the mRNA levels of Bcl-2 in the EF group were 272 significantly lower (**p < 0.01, ***p < 0.001). These data indicate that EF can induce apoptosis in HFLS-RA cells. To further 273 explore the potential mechanism of EF inducing apoptosis of HFLS-RA cells, we measured the protein levels of factors related to 274 the NF-κB pathway and found that (Fig. 5A,B), compared with the TNF-α group, the protein levels of p-Iκκ, p-IκB, and p-p65, 275 were significantly reduced (**p < 0.01, ***p < 0.001). Consistent with RT-qPCR results, the protein levels of Caspase3 (cleaved) 276 and Bax in the EF group were significantly higher, while the mRNA content of Bcl-2 in the EF group were significantly 277 lower (Fig. 5C,D). These results suggested that EF can induce apoptosis in HFLS-RA cells via the NF-κB pathway.

279
Our previous results indicated that EF had a significant anti-proliferation effect on HFLS-RA cells and could induce apoptosis 280 of HFLS cells. In order to investigate the effect of EF on the osteoclast differentiation of RAW264.7 cells, we next investigated 281 the ability of EF on the osteoclast differentiation of RAW264.7 cells induced by RANKL by TRAP staining and Western blot. As 282 shown in Fig. 6A, the influence of EF on RAW264.7 cell viability was investigated with the CCK-8 assay, EF was not cytotoxic 283 at either 24h. We investigated the effects of EF on the proliferation of LPS-stimulated RAW264.7 cells. Fig. 6B shows that EF 284 (1600, 2000 μg/mL) was able to inhibit the proliferation of LPS-stimulated RAW264.7 (**p < 0.01, ***p < 0.001). By TRAP 285 staining and Western blot (Fig. 6C,D), we found that EF can inhibit the osteoclast differentiation of RAW264.7 cells induced by 286 RANKL and inhibit the activation of NF-κB pathway (*p < 0.05, **p < 0.01, ***p < 0.001).

288
To determine whether EF is able to exhibit inflammatory bone destruction effects on RA in vivo, we established a rat model of 289 CIA. We initiated EF treatment on day 14 after successfully modeling. As shown in Fig. 7A, the CIA rats weighed significantly 290 less than the normal group after modeling than the control group. Our data also showed that the CIA rats showed significant joint 291 inflammation and that EF treatment reduced the severity of disease and joint swelling in the CIA rats ( Fig. 7B and C) (**p < 0.01, 292 ***p < 0.001). In addition, histological results showed that the ankle joints in CIA rats exhibited inflammatory cell infiltration in 293 the soft tissue and the lining of the synovial layer. Data also revealed cartilage destruction which led to pannus formation and 294 narrowing in the joint space ( Fig. 7D and E). EF treatment attenuated joint inflammation by reducing synovial inflammatory cell 295 infiltration and reducing pannus formation in the CIA rats. SafraninO-Fast Green Staining also showed that EF reduced cartilage 296 damage in RA (Fig. 7F).

297
The effect of EF on bone destruction 298 Because EF treatment had a positive effect on pathological bone destruction, we next explored the potential of EF to reduce 299 bone loss; we did this by carrying out morphological analysis and creating a three-dimensional reconstruction of the tibia and 300 ankle ( Fig. 8A and B). We found that BMD and BV/TV were significantly reduced (p < 0.001). There was also a reduction in the 301 quantity of bone trabecula, although this was not statistically significant. There was a significant increase in the separating degree 302 of the Tb.Sp (p < 0.001). In contrast, a high dose of EF partially prevented the sharp decline in bone mass and the deterioration of 303 trabecular microstructures in CIA rats. However, there were no significant differences when low-dose EF treatments were 304 compared to controls.

305
The effect of EF on the expression of bone metabolic indices in serum and spleen 306 CTX-1, ICTP, PINP, and BGP, are commonly used as clinical markers for the detection of osteoporosis. Increases and 307 decreases in these factors can reflect the status of bone destruction. The serum levels of CTX-1 and ICTP, and the mRNA levels 308 of TNF-α, TRAF-6, IL-1β, and IL-17, in the spleen of the CIA group were significantly increased than the control group. Serum 309 levels of PINP and BGP in the CIA group were significantly lower in the CIA group than in the controls (Fig. 9) (#p < 0.05, ##p 310 < 0.01, ###p < 0.001). Following EF administration, the levels of CTX-1 and ICTP, and the mRNA levels of TNF-α, TRAF-6, 311 IL-1β, and IL-17, in the spleen were significantly lower than those in the CIA group; serum levels of PINP and BGP were also 312 significantly higher than in the controls (*p < 0.05, **p < 0.01, ***p < 0.001). In addition, EF reduced the levels of inflammatory 313 factors in the spleen and regulated metabolic events in the bone; this was consistent with the micro-CT results and suggested that 314 EF has a certain regulatory effect on damage caused by osteoarthritis.

315
The effect of EF on bone metabolic indices in joint tissue 316 RANKL, c-FOS, NFATc1, CTSK, and TRAP, are all considered as markers for the production of osteoclasts; VEGF and HIF-1 317 are also considered to be related to angiogenesis. Increased levels of these factors are considered to be indicators of bone damage.

318
As shown in Fig. 10, compared to the normal group, the levels of these factors were significantly higher in the CIA group, but 319 significantly lower in the EF group than in the CIA group (*p < 0.05, **p < 0.01, ***p < 0.001). TIMP-1 is the inhibitory factor 320 for MMP-9 (a biomarker for the local inflammation of joints and is known to be related to bone destruction). The levels of 321 TIMP-1 and OPG in the EF group were significantly increased than the CIA group. Collectively, these data showed that EF can 322 improve the osteoarthritic regulation of bone metabolism at the genetic level.

323
The effect of EF on the NF-κB pathway in joint tissue 324 Next, as shown in Fig. 11, we further explored the potential mechanism underlying the effects of EF on the CIA rat. We 325 measured the protein levels of key factors in the NF-κB pathway and found that, compared with the CIA group, the protein levels 326 of p-Iκκ, p-IκB, and p-p65, were significantly lower in the EF group (*p < 0.05, **p < 0.01, ***p < 0.001). These results 327 suggested that EF could improve joint inflammation and bone destruction by inhibiting activation of the NF-κB pathway.   The effects of EF on the proliferation of HFLS-RA cells with and without TNF-α stimulation. (A) The cytotoxicity of EF in cultured cells was tested for 24 h and 48 h. (B) The anti-proliferative effects of EF against TNF-α stimulated HFLS-RA cells exhibited a concentration-dependent relationship). HFLS-RA cells were incubated with TNF-α (10 ng/mL) and EF (25, 50, 100, 200, 400, 800 and 1600 μg/mL) for 24 h. We then used the CCK-8 assay to determine the extent to which cell proliferation was inhibited (%). Data are represented as mean ± SD (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001, vs. TNF-α cells (HFLS-RA cells treated by TNF-α alone). #p < 0.05, ##p < 0.01, ###p < 0.001, vs. Control cells. (C) A cell colony assay was used to investigate the effect of EF on the proliferation of HFLS-RA cells. (D) Data are represented as mean ± SD (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001, vs. control cells. (E) HFLS-RA cells were seeded onto glass coverslips, starved, and then stimulated for 24 h with TNF-α (10 ng/mL) or EF (400, 800 and 1600 μg/mL). Cells were then stained with the EdU Cell Proliferation Kit .Hoechst (blue) and Azide594 (red) and visualized by uorescence microscopy (200×).