ABSTRACT
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)-1ß, 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-1ß and IL-6 secretion. BA also downregulated the transcription of vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGF-ß) and decreased the expression of the NF-кB pathway proteins (NF-kB-P65, IkBα, and IKKα/ß) 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-1ß, IL-6, VEGF, and TGF-ß.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Arthritis, Experimental/prevention & control , Cytokines/metabolism , Inflammation Mediators/metabolism , Pentacyclic Triterpenes/pharmacology , Synovial Membrane/drug effects , Synovitis/prevention & control , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Cell Proliferation/drug effects , Cells, Cultured , Collagen Type II , Male , NF-kappa B/metabolism , Phosphorylation , Rats, Wistar , Signal Transduction , Synovial Membrane/metabolism , Synovial Membrane/pathology , Synovitis/chemically induced , Synovitis/metabolism , Synovitis/pathology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Betulinic AcidABSTRACT
BACKGROUND: 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. METHODS: 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)-1ß, 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. RESULTS: EF showed no significant cytotoxicity at concentrations from 10 to 60âµg/mL (cell viabilityâ>â80%) in the CCK-8 cell viability assay. EF inhibited the RAW 264.7 cell proliferation (EF 60âµg/mL, 120âµg/mL, and 250âµg/mL vs. negative control: 87.31â±â2.39% vs. 100.00â±â2.50%, Pâ=â0.001; 79.01â±â2.56 vs. 100.00â±â2.50%, Pâ<â0.001; and 64.83â±â2.50 vs. 100.00â±â2.50%, Pâ<â0.001), suppressed NO (EF 20âµg/mL and 30âµg/mL vs. LPS only, 288.81â±â38.01 vs. 447.68â±â19.07âµmol/L, Pâ=â0.004; and 158.80â±â45.14 vs. 447.68â±â19.07âµmol/L, Pâ<â0.001), TNF-α (LPS+EF vs. LPS only, 210.20â±â13.85 vs. 577.70â±â5.35âpg/mL, Pâ<â0.001), IL-1ß (LPS+EF vs. LPS only, 193.30â±â10.80 vs. 411.03â±â42.28âpg/mL, Pâ<â0.001), and IL-6 (LPS+EF vs. LPS only, 149.67â±â11.60 vs. 524.80â±â6.24âpg/mL, Pâ<â0.001) secretion, and downregulated the mRNA expression of IL-17 (LPS+EF vs. LPS only, 0.23â±â0.02 vs. 0.43â±â0.12, Pâ<â0.001), IL-23 (LPS+EF vs. LPS only, 0.29â±â0.01 vs. 0.42â±â0.06, P=0.002), and IL-10 (LPS+EF vs. LPS only, 0.30â±â0.01 vs. 0.47â±â0.01, P=0.008) in LPS-stimulated RAW 264.7 cells. EF inhibited the LPS-induced NF-κB p65 (LPS+EF 20âµg/mL and 30âµg/mL vs. LPS only: 0.78â±â0.06 vs. 1.17â±â0.08, Pâ<â0.001; and 0.90â±â0.06 vs. 1.17â±â0.08, P =0.002) and inhibitor of kappa B (IκBα) phosphorylation (LPS+EF 20âµg/mL and 30âµg/mL vs. LPS only: 0.25â±â0.01 vs. 0.63â±â0.03, Pâ<â0.001; and 0.31â±â0.01 vs. 0.63â±â0.03, Pâ<â0.001), LPS+EF 30âµg/mL inhibited IκB kinase (IKKα/ß) phosphorylation (LPS+EF 30âµg/mL vs. LPS only, 1.12â±â0.14 vs. 1.71â±â0.25, Pâ=â0.002) in RAW 264.7 cells. Furthermore, EF 10âmg/kg and EF 20âmg/kg inhibited lung tissue inflammation in vivo and suppressed the serum TNF-α (LPS+EF 10âmg/kg and 20âmg/kg vs. LPS only, 199.99â±â186.49 vs. 527.90â±â263.93âpg/mL, P=0.001; and 260.56â±â175.83 vs. 527.90â±â263.93âpg/mL, Pâ=â0.005), and IL-6 (LPS+EF 10âmg/kg and 20âmg/kg vs. LPS only, 41.26â±â30.42 vs. 79.45â±â14.16âpg/âml, Pâ=â0.011; and 42.01â±â26.26 vs. 79.45â±â14.16âpg/mL, Pâ=â0.012) levels and MPO (LPS+EF 10âmg/kg and 20âmg/kg vs. LPS only, 3.19â±â1.78 vs. 5.39â±â1.51âU/g, Pâ=â0.004; and 3.32â±â1.57 vs. 5.39â±â1.51âU/g, Pâ=â0.006) activity in lung tissue. CONCLUSIONS: 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.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Eucommiaceae/chemistry , Flowers/chemistry , Inflammation/chemically induced , Inflammation/drug therapy , Lipopolysaccharides/toxicity , Plant Extracts/chemistry , Plant Extracts/therapeutic use , Animals , Anti-Inflammatory Agents/chemistry , Inflammation/blood , Interleukin-1beta/blood , Macrophages/drug effects , Mice , NF-KappaB Inhibitor alpha/blood , NF-kappa B/blood , Nitric Oxide/blood , RAW 264.7 Cells , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/bloodABSTRACT
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.