Extract of Pinus densiflora needles suppresses acute inflammation by regulating inflammatory mediators in RAW264.7 macrophages and mice

Abstract

Context: Pinus densiflora Siebold & Zucc. (Pinaceae) needle extracts ameliorate oxidative stress, but research into their anti-inflammatory effects is limited.

Objective: To investigate antioxidant and anti-inflammatory effects of a Pinus densiflora needles (PINE) ethanol extract in vitro and in vivo.

Materials and methods: We measured levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at various PINE concentrations (25, 50 and 100 μg/mL; but 6.25, 12.5 and 25 μg/mL for interleukin-1β and prostaglandin E₂ (PGE₂)). Thirty ICR mice were randomized to six groups: vehicle, control, PINE pre-treatment (0.1, 0.3 and 1 mg/left ear for 10 min followed by arachidonic acid treatment for 30 min) and dexamethasone. The posttreatment ear thickness and myeloperoxidase (MPO) activity were measured.

Results: PINE 100 μg/mL significantly decreased ROS (IC₅₀, 70.93 μg/mL, p < 0.01), SOD (IC₅₀, 30.99 μg/mL, p < 0.05), malondialdehyde (p < 0.01), nitric oxide (NO) (IC₅₀, 27.44 μg/mL, p < 0.01) and tumour necrosis factor-alpha (p < 0.05) levels. Interleukin-1β (p < 0.05) and PGE₂ (p < 0.01) release decreased significantly with 25 μg/mL PINE. PINE 1 mg/ear inhibited LPS-stimulated expression of cyclooxygenase-2 and inducible NO synthase in RAW264.7 macrophages and significantly inhibited ear oedema (36.73-15.04% compared to the control, p < 0.01) and MPO activity (167.94-105.59%, p < 0.05).

Discussion and conclusions: PINE exerts antioxidant and anti-inflammatory effects by inhibiting the production of inflammatory mediators. Identified flavonoids such as taxifolin and quercetin glucoside can be attributed to effect of PINE.

Keywords: Reactive oxygen species; anti-inflammatory; antioxidant; arachidonic acid; ear oedema; lipopolysaccharide.

Figure 1.

Chromatogram derived from UHPLC-LTQ-Orbitrap analysis of P. densiflora extract.

Figure 2.

Cytotoxicity of PINE towards RAW264.7 macrophages. RAW264.7 macrophages were incubated with various concentrations of PINE for 24 h at 37 °C. The number of viable cells was assessed via MTT assay. The viability of the control group cells was considered to be 100%. Each value represents the mean ± SD of four separate experiments.

Figure 3.

Effects of PINE on oxidative stress and NO production in RAW264.7 cells. (A, B) ROS levels were measured via DCF-DA assay, and fluorescence was detected using a microplate reader (excitation wavelength, 485 nm; emission wavelength, 535 nm). (C) SOD concentration was measured via ELISA. (D) NO level was measured using the Griess reagent. Stimulation was performed by LPS (1 μg/mL) treatment, and the cells were pre-treated with various concentrations of PINE (25, 50 or 100 μg/mL). Dexamethasone (Dexa; 10 μg/mL) was used as the reference compound. Results are expressed as activity percentages, and each value represents the mean ± SD of four separate experiments. Significantly different from the control (^#p < 0.05 and ^###p < 0.001); significantly different from the stimulation group (*p < 0.05, **p < 0.01 and ***p < 0.001); and significant intergroup differences (^$$$p < 0.001).

Figure 4.

Effects of PINE on lipid peroxidation in RAW264.7 macrophages. Cells were pre-treated with PINE (25, 50 or 100 μg/mL) for 30 min and then incubated with 1 μM H₂O₂ and 10 mM FeSO₄ for 15 min to induce lipid membrane peroxidation, followed by sonication. Thiobarbituric acid reacts with MDA to generate a red adduct, which can be quantitated by measuring the absorbance at 532 nm. Caffeic acid (25 μg/mL) was used as the reference compound. Results are expressed as an MDA concentration, and each value represents the mean ± SD of four separate experiments. Significantly different from the control (^###p < 0.001); significantly different from the stimulation group (*p < 0.05 and **p < 0.01).

Figure 5.

Effects of PINE on LPS-induced iNOS and COX-2 protein levels in LPS-stimulated RAW264.7 macrophages. Western blotting analysis for iNOS, COX-2 and β-actin levels. Cells were pre-treated with PINE (25, 50 or 100 μg/mL) for 1 h and then treated with LPS (1 μg/mL) for 12 h. Dexamethasone (Dexa; 10 μg/mL) was used as the reference compound. The results were analysed using Chemidoc XRS and Quantity One. Experiments were repeated four times. Significantly different from the control group (^##p < 0.01 and ^###p < 0.001); significantly different from the stimulation group (*p < 0.05 and **p < 0.01).

Figure 6.

Effects of PINE on arachidonic acid-induced ear oedema in mice. (A) PINE (0.1, 0.3 or 1 mg/ear in ethanol) was applied to the inside of the left ears of ICR mice and given 10 min to absorb. Arachidonic acid (AA; 2 mg/ear dissolved in acetone) was applied for 30 min to the left ears. Dexamethasone (Dexa; 0.1 mg/ear) was applied as the reference compound to the left ear in the Dexa group. Ear thickness was measured using the micro-engineer metre. (B) Representative images of mouse ears and (C) H&E staining for each experimental group (magnification, ×10; scale bar, 100 μm). (D) MPO activity was measured in the ear tissues via colorimetry based on the absorbance at 620 nm. (E) The MDA level was quantified in the ear tissues via the TBARS method. Each value represents the mean ± SD of three separate experiments. Significantly different from the AA treatment group (*p < 0.05, **p < 0.01 and ***p < 0.001). Significantly different from the control (^##p < 0.01 and ^###p < 0.001).