Synedrella nodiflora (Linn.) Gaertn. inhibits inflammatory responses through the regulation of Syk in RAW 264.7 macrophages

Abstract

Synedrella nodiflora (Linn.) Gaertn. (S. nodiflora) has long been used for the treatment of inflammatory diseases, including liver disease, asthma, rheumatism and earache, in tropical countries throughout America, Asia and Africa. However, the biological effects of S. nodiflora have not been extensively studied at the molecular level. Notably, it remains unclear how S. nodiflora exerts anti-inflammatory activity. In the present study, the anti-inflammatory mechanism of a methanol extract of S. nodiflora (MSN) in RAW 264.7 macrophages activated by lipopolysaccharide (LPS) was investigated. Non-cytotoxic concentrations of MSN (≤400 µg/ml) decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which resulted in a decrease in nitric oxide and prostaglandin E₂ (PGE₂) production. The mRNA expression of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α was reduced upon MSN treatment. In addition, the activation of spleen tyrosine kinase (Syk) and Akt was suppressed by MSN. Taken together, these findings recommend the traditional medicinal application of S. nodiflora in the treatment of several inflammation-associated diseases and indicate the possibility of MSN as a novel therapeutic reagent of inflammation-related diseases.

Keywords: inflammatory mediators; lipopolysaccharide; macrophages; nuclear factor-κB; spleen associated tyrosine kinase; synedrella nodiflora.

Figure 1

Effect of MSN on cell viability. (A) RAW 264.7 macrophages were treated with MSN (100, 200, 300, 400 and 600 µg/ml) at 37˚C for 2 h and incubated in the absence or presence of LPS (1 µg/ml) at 37˚C for an additional 24 h. The cell viability was examined using EZ-Cytox assay kit. Cell viability of each group was calculated based on the LPS-treated or LPS-untreated control group. (B) RAW 264.7 macrophages were pre-treated with MSN (100, 200, 300, and 400 µg/ml) at 37˚C for 2 h and then stimulated with LPS (1 µg/ml). After 24 h stimulation, the levels of NO secretion in the culture media were measured using Griess reagent. The amounts of NO secretion were calculated according to a standard curve according to a nitrite standard solution. (C) Total cell lysates were collected after 24 h LPS stimulation, and RT-qPCR analysis was performed. Relative expression levels of iNOS are represented as a bar graph. (D) After 3 h LPS stimulation, iNOS was analyzed by immunoblotting. The expression of iNOS was detected using ECL reagent and quantified by analysis with LabWorks software. All the protein levels were normalized to corresponding tubulin levels. Data are represented as the mean ± SEM. ^*P<0.01 vs. LPS-untreated control group. ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. LPS-treated and MSN-untreated control group. MSN, methanol extract of Synedrella nodiflora (Linn.) Gaertn.; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase.

Figure 2

Inhibitory effects of MSN on pro-inflammatory mediators. RAW 264.7 macrophages were pre-treated with MSN (100, 200, 300, and 400 µg/ml) at 37˚C for 2 h and then stimulated with LPS (1 µg/ml) for an indicated time. (A) After 3 h LPS stimulation, COX-2 was amplified by RT-qPCR, and the expression of each group was compared to that in the LPS-treated group. (B) Total cell lysates were collected after 24 h stimulation, and immunoblot analyses were performed. The protein expression levels of COX-2 were detected using ECL reagent and quantified by analysis with LabWorks software. All the protein levels were normalized to corresponding tubulin levels. Relative expression levels of COX-2 are represented as a bar graph. After stimulation for 24 h, the levels of (C) IL-6 and (D) TNF-α in the cultured media were quantified using ELISA. The secretion of each cytokine was calculated using a standard curve. (E) After 24 h stimulation, IL-1β, IL-6 and TNF-α were amplified by RT-qPCR, and the expression of each group was compared to that in the LPS-treated and MSN-untreated control group. Data are represented as the mean ± SEM. ^*P<0.01 vs. LPS-untreated control group. ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. LPS-treated and MSN-untreated control group. MSN, methanol extract of Synedrella nodiflora (Linn.) Gaertn.; LPS, lipopolysaccharide; COX-2, cyclooxygenase-2; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α.

Figure 3

Inflammatory inhibition of MSN on MAPK phosphorylation and NF-κB activation in RAW 264.7 macrophages. RAW 264.7 macrophages were pre-treated with MSN (100, 200, 300, and 400 µg/ml) for 2 h and stimulated with LPS (1 µg/ml) at 37˚C for indicated times. (A) After stimulation for 3 min, total cell lysates were prepared and immunoblot analyses were performed. Tubulin was used as a loading control. Protein levels were quantified with LabWorks software. Relative expression levels of IκBα and p-IκBα were normalized to tubulin levels and then the ratio of phosphorylated (p)-IκBα vs. total IκBα protein level is shown as a bar graph. (B) Total cell lysates were collected after 15 min of LPS treatment and subjected to immunoblot analysis using appropriate antibodies. Tubulin was used as a loading control. Protein levels were measured using LabWorks software. Levels of phosphorylated MAPKs were normalized to corresponding total MAPK levels. The ratio of phosphorylated vs. total protein level for each MAPK is shown as a bar graph after normalization. (C) Cells were lysed after 3 min stimulation. Samples were subjected to immunoblot analysis. p-TAK1, p-Syk, p-Src, p-Akt, TAK1, Syk, Src, and Akt protein expression levels were determined. The phosphorylation levels of TAK1, Syk, Src, and Akt were normalized to the corresponding total protein levels. The ratios of phosphorylated vs. total protein levels are shown as a bar graph. ^*P<0.01 vs. LPS-untreated control group. ^#P<0.05 and ^###P<0.001 vs. LPS-treated and MSN-untreated control group. MSN, methanol extract of Synedrella nodiflora (Linn.) Gaertn.; LPS, lipopolysaccharide; IκBα, inhibitor of κBα; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; TAK1, transforming growth factor-β-activated kinase 1; Syk, spleen tyrosine kinase; Src, proto-oncogene tyrosine-protein kinase.

Figure 4

The molecular pathway of the inflammatory effects of MSN in LPS-stimulated RAW 264.7 cells. MSN, methanol extract of Synedrella nodiflora (Linn.) Gaertn.; LPS, lipopolysaccharide; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; NO, nitric oxide; PGE₂, prostaglandin E₂; COX-2, cyclooxygenase-2; IκBα, inhibitor of κBα; NF-κB, nuclear factor κB; Akt, protein kinase B; Src, proto-oncogene tyrosine-protein kinase; Syk, spleen tyrosine kinase; TLR4, toll-like receptor 4; TAK1, transforming growth factor-β-activated kinase 1; MAP3K8, mitogen-activated protein kinase kinase kinase 8; MKK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; AP-1, activator protein 1.