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)-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 antiinflammatory 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 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.
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.