Eucommia ulmoides Oliv. is a medicinal plant native to China, with its bark (Eucommiae Cortex) traditionally being used for medicinal purposes. Previous research has shown that Eucommia male flowers can exert anti-inflammatory, analgesic, antibacterial, and other pharmacological effects, including immune regulation. This study explored the anti-inflammatory effects of the 70% ethanol extract of male flowers (EF) of E. ulmoides in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-administered mice. Cytotoxicity of EF for RAW 264.7 cells was investigated using Cell Counting Kit-8. The production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 was determined using enzyme-linked immunosorbent assays. IL-17, IL-23, and IL-10 mRNA levels were determined using quantitative real-time polymerase chain reaction. Activation of the nuclear factor (NF)-κB pathway in RAW 264.7 cells was investigated via Western blotting. In vivo anti-inflammatory effects of EF were studied in an LPS-induced acute inflammation mouse model by analyzing lung tissue histopathology, serum TNF-α and IL-6 levels, and myeloperoxidase (MPO) activity in lung tissue. EF showed no significant cytotoxicity at concentrations from 10 to 60 μg/mL (cell viability > 80%) in the CCK-8 cell viability assay. EF inhibited the RAW 264.7 cell proliferation (EF 60 μg/mL, 120 μg/mL, and 250 μg/mL vs. negative control: 87.31 ± 2.39% vs. 100.00 ± 2.50%, P = 0.001; 79.01 ± 2.56 vs. 100.00 ± 2.50%, P < 0.001; and 64.83 ± 2.50 vs. 100.00 ± 2.50%, P < 0.001), suppressed NO (EF 20 μg/mL and 30 μg/mL vs. LPS only, 288.81 ± 38.01 vs. 447.68 ± 19.07 μmol/L, P = 0.004; and 158.80 ± 45.14 vs. 447.68 ± 19.07 μmol/L, P < 0.001), TNF-α (LPS+EF vs. LPS only, 210.20 ± 13.85 vs. 577.70 ± 5.35 pg/mL, P < 0.001), IL-1β (LPS+EF vs. LPS only, 193.30 ± 10.80 vs. 411.03 ± 42.28 pg/mL, P < 0.001), and IL-6 (LPS+EF vs. LPS only, 149.67 ± 11.60 vs. 524.80 ± 6.24 pg/mL, P < 0.001) secretion, and downregulated the mRNA expression of IL-17 (LPS+EF vs. LPS only, 0.23 ± 0.02 vs. 0.43 ± 0.12, P < 0.001), IL-23 (LPS+EF vs. LPS only, 0.29 ± 0.01 vs. 0.42 ± 0.06, P=0.002), and IL-10 (LPS+EF vs. LPS only, 0.30 ± 0.01 vs. 0.47 ± 0.01, P=0.008) in LPS-stimulated RAW 264.7 cells. EF inhibited the LPS-induced NF-κB p65 (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.78 ± 0.06 vs. 1.17 ± 0.08, P < 0.001; and 0.90 ± 0.06 vs. 1.17 ± 0.08, P =0.002) and inhibitor of kappa B (IκBα) phosphorylation (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.25 ± 0.01 vs. 0.63 ± 0.03, P < 0.001; and 0.31 ± 0.01 vs. 0.63 ± 0.03, P < 0.001), LPS+EF 30 μg/mL inhibited IκB kinase (IKKα/β) phosphorylation (LPS+EF 30 μg/mL vs. LPS only, 1.12 ± 0.14 vs. 1.71 ± 0.25, P = 0.002) in RAW 264.7 cells. Furthermore, EF 10 mg/kg and EF 20 mg/kg inhibited lung tissue inflammation in vivo and suppressed the serum TNF-α (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 199.99 ± 186.49 vs. 527.90 ± 263.93 pg/mL, P=0.001; and 260.56 ± 175.83 vs. 527.90 ± 263.93 pg/mL, P = 0.005), and IL-6 (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 41.26 ± 30.42 vs. 79.45 ± 14.16 pg/ ml, P = 0.011; and 42.01 ± 26.26 vs. 79.45 ± 14.16 pg/mL, P = 0.012) levels and MPO (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 3.19 ± 1.78 vs. 5.39 ± 1.51 U/g, P = 0.004; and 3.32 ± 1.57 vs. 5.39 ± 1.51 U/g, P = 0.006) activity in lung tissue. EF could effectively inhibit the expression of inflammatory factors and overactivation of neutrophils. Further investigation is needed to evaluate its potential for anti-inflammation therapy.

Eucommia ulmoides Oliv., a native Chinese plant species, has been used as a traditional Chinese medicine formulation to treat rheumatoid arthritis (RA), strengthen bones and muscles, and lower blood pressure. Various parts of this plant such as the bark, leaves, and flowers have been found to have anti‐inflammatory properties. E. ulmoides has potential applications as a therapeutic agent against bone disorders, which were investigated in this study. In vitro, RA joint fibroblast‐like synoviocytes (RA‐FLS) were treated with different concentrations (0, 25, 50, 100, 200, 400, 800, and 1000 μg/mL) of E. ulmoides bark, leaf, and male flower alcoholic extracts (EB, EL, and EF, respectively) to determine their potential cytotoxicity. Tumor necrosis factor‐ (TNF‐) α and nitric oxide (NO) levels in RA‐FLS were quantified using enzyme‐linked immunosorbent assay (ELISA). Furthermore, collagen‐induced arthritis (CIA) rats were treated with EB, EL, EF, Tripterygium wilfordii polyglycoside (TG) or the normal control (Nor), and then ankle joint pathology, bone morphology, and serum and spleen inflammatory cytokine levels were evaluated. The results showed that, in RA‐FLS, EB, EL, and EF were not cytotoxic; EB and EF reduced TNF‐α supernatant levels; and EB, EL, and EF reduced NO levels. The results of in vivo experiments showed that EB, EL, and EF alleviated ankle swelling and joint inflammation, while all extracts diminished inflammatory cell infiltration, pannus and bone destruction, and bone erosion. All tested extracts inhibited interleukin‐ (IL‐) 6, IL‐17, and TNF‐α mRNA in the spleen of CIA rats, while EB most effectively reduced osteoclasts and inhibited bone erosion. EF showed the most obvious inhibition of inflammatory factors and pannus. Thus, EB, EL, and EF may alleviate bone destruction by inhibiting inflammation.

To compare efficacy of different extracts from Eucommia ulmoides Oliv. with both immune inflammation and joint destruction in collagen induced arthritis (CIA) rat model. Rats were divided into normal group (Nor), control group (CIA), TG group (treated with tripterygium glycoside), E70 group (treated with 70% ethanol extract from Eucommia ulmoides Oliv.), EA group (treated with ethyl acetate fraction from E70), and EN group (treated with n‐butyl alcohol fraction from E70). All extracts from Eucommia ulmoides Oliv. could significantly inhibit ankle swelling, pathological manifestations, and cytokine levels in serum and spleen, by using foot volume measurement, H&E staining, ELISA, and RT‐QPCR methods, respectively. All extracts could significantly inhibit rough joint surface and marginal osteophytes, improve RANKL/OPG ratio, and decrease MMP‐9 expression, by using micro‐CT and immunohistochemical staining. The activation of IKK/NF‐κB signaling pathway was also inhibited by all extracts. In addition, ethyl acetate fraction from E70 presented better effect on RANKL/OPG system. This study identified effective extracts from Eucommia ulmoides Oliv. relieving immune inflammation and maintaining structural integrity of joints in CIA rats.

Background: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease dominated by synovial hyperplasia and bone destruction. The male flower of Eucommia ulmoides Oliv. (EF) has been shown to exert effects on the inflammation caused by RA. However, how EF affects synoviocyte apoptosis and bone destruction on RA have not been investigated thoroughly. The effects of EF on apoptosis of human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) cells, osteoclast differentiation of RAW264.7 cells, and bone destruction in a collagen-induced arthritis (CIA) model in rats were explored. Methods: First, the main components of EF were identified by high-performance liquid chromatography. In vitro, we investigated the anti-proliferative and pro-apoptotic effects of EF on HFLS-RA cells by immunofluorescence assays, flow cytometry, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting; we also investigated how EF influenced the differentiation of RAW264.7 cells into osteoclasts. In vivo, we used a rat model of CIA to investigate the effects of EF on anti-arthritis activity, toe swelling, Arthritis Score, serum levels of metabolic bone factors, and pathologic conditions. Micro-computed tomography was used to scan ankle joints. mRNA and protein expression of factors related to the nuclear factor-kappa B (NF-κB) pathway were determined by RT-qPCR and western blotting, respectively. Results: EF inhibited synoviocyte proliferation and promoted apoptosis in a dose-dependent manner. EF inhibited osteoclast differentiation by inhibiting activation of the NF-κB pathway. EF reduced articular inflammation in CIA rats, inhibited the expression of pro-angiogenic factors, and delayed the destruction of articular cartilage and bone. Our data indicated that EF acted via a mechanism related to bone metabolism induced by the NF-κB pathway. Conclusions: EF exerts a potential therapeutic effect upon RA. Our research will help to elucidate the potential pharmacologic mechanisms associated with the beneficial effects of EF, and provide an experimental basis for EF application in clinical treatments. Graphical

Betulinic acid (BA) inhibits the migration, invasion, and cytoskeletal reorganization of fibroblast-like synoviocytes (RA-FLS) in patients with rheumatoid arthritis. Here, to further explore the mechanism of action of BA in collagen-induced arthritis (CIA) rats, we investigated the pharmacodynamic effects of BA on synovial inflammation in a rat model of type II CIA. After inducing hind paw swelling, the rats were divided into four groups: healthy controls (normal), and rats that underwent CIA and received methotrexate treatment (MTX), BA treatment (BA), or no treatment (CIA). Body weight and hind paw swelling were determined regularly, and arthritis scores were calculated weekly. On day 35, rats were sacrificed and their hind ankle joints sectioned and stained with hematoxylin and eosin for histopathological evaluation. BA significantly reduced CIA-induced hind paw swelling, synovial tissue proliferation, cartilage destruction, and vasospasm. BA treatment also decreased serum interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels in rats with CIA. The CCK-8 assay was used to detect the proliferation of isolated vimentin⁺CD68⁻ RA-FLS; RA-FLS were stimulated with TNF-α in vitro. BA significantly inhibited TNF-α-stimulated RA-FLS proliferation, as well as IL-1β and IL-6 secretion. BA also downregulated the transcription of vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) and decreased the expression of the NF-кB pathway proteins (NF-kB-P65, IkBα, and IKKα/β) in the TNF-α-stimulated RA-FLS. These results indicate that BA alleviated the symptoms of CIA by inhibiting synoviocyte proliferation, modifying TNF-α- and NF-кB-related inflammatory pathways, and downregulating inflammatory mediators and growth factors including IL-1β, IL-6, VEGF, and TGF-β.