Sauropus brevipes ethanol extract negatively regulates inflammatory responses in vivo and in vitro by targeting Src, Syk and IRAK1

Abstract

Context: Sauropus brevipes Müll. Arg. (Phyllanthaceae) has been used as an effective ingredient in a decoction for the treatment of diarrhoea. However, there was no report on its modulatory role in inflammation.

Objective: This study investigates anti-inflammatory effect of S. brevipes in various inflammation models.

Materials and methods: The aerial part of S. brevipes was extracted with 95% ethanol to produce Sb-EE. RAW264.7 cells pre-treated with Sb-EE were stimulated by lipopolysaccharide (LPS), and Griess assay and PCR were performed. High-performance liquid chromatography (HPLC) analysis, luciferase assay, Western blotting and kinase assay were employed. C57BL/6 mice (10 mice/group) were orally administered with Sb-EE (200 mg/kg) once a day for five days, and peritonitis was induced by an intraperitoneal injection of LPS (10 mg/kg). ICR mice (four mice/group) were orally administered with Sb-EE (20 or 200 mg/kg) or ranitidine (positive control) twice a day for two days, and EtOH/HCl was orally injected to induce gastritis.

Results: Sb-EE suppressed nitric oxide (NO) release (IC₅₀=34 µg/mL) without cytotoxicity and contained flavonoids (quercetin, luteolin and kaempferol). Sb-EE (200 µg/mL) reduced the mRNA expression of inducible NO synthase (iNOS). Sb-EE blocked the activities of Syk and Src, while inhibiting interleukin-1 receptor associated kinases (IRAK1) by 68%. Similarly, orally administered Sb-EE (200 mg/kg) suppressed NO production by 78% and phosphorylation of Src and Syk in peritonitis mice. Sb-EE also decreased inflammatory lesions in gastritis mice.

Discussion and conclusions: This study demonstrates the inhibitory effect of Sb-EE on the inflammatory response, suggesting that Sb-EE can be developed as a potential anti-inflammatory agent.

Keywords: Natural product medicine; anti-inflammatory remedy; flavonoids; gastritis; peritonitis.

Figure 1.

Suppressive activity of Sb-EE against NO production in RAW264.7 cells. (A) To examine the effect of Sb-EE on NO release, Sb-EE pre-treated-RAW264.7 cells were stimulated with LPS (1 µg/mL) for 24 h, and NO levels in supernatant were assessed by NO assay. (B, C) To test the cytotoxicity of Sb-EE, RAW264.7 cells (B) and HEK 293T cells (C) were dose-dependently treated with Sb-EE for 24 h, and then MTT assay was performed. (D) Active phytochemical elements in Sb-EE were assessed by HPLC. The profiles of Sb-EE were compared with standard profiles containing quercetin, luteolin and kaempferol. All data are presented as the mean ± SD of experiments. ^##p < 0.01 compared to the normal group, and **p < 0.01 compared to the LPS-alone treatment group. Rt (min) (area).

Figure 2.

Effect of Sb-EE on inflammatory gene expression and transcriptional factors. (A) To verify the effect of Sb-EE on inflammatory biomarker expression, RAW264.7 cells were pre-treated with Sb-EE, then stimulated with LPS (1 µg/mL) for 6 h. mRNA expression of iNOS, COX-2 and TNF-α was analysed by semi-quantitative RT-PCR. (B, C) To validate the effect of Sb-EE on the transcriptional activity of NF-κB and AP-1, a luciferase assay was performed. HEK 293 cells were transfected with NF-κB-Luc (B) or AP-1-Luc (C) genes, and Flag-MyD88 or CFP-TRIF were additionally overexpressed to activate transcription of NF-κB-Luc and AP-1-Luc genes. Then, HEK 293 cells were treated with Sb-EE (0–200 μg/mL) for 24 h. Luciferase activity was measured using a luminometer. (D) To analyse nuclear translocation of transcription factor subunits, Sb-EE (200 μg/mL) pre-treated RAW264.7 cells were stimulated with LPS for the indicated times. Then, the levels of AP-1 (c-Fos and c-Jun) and NF-κB subunits (p65 and p50) in nuclear lysate were determined by immunoblotting. Lamin A/C was utilized as a loading control. All data are presented as the mean ± SD of experiments. ^##p < 0.01 compared to the normal group, and *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A, D), and MyD88/TRIF-alone group in (B, C)).

Figure 2.

Effect of Sb-EE on inflammatory gene expression and transcriptional factors. (A) To verify the effect of Sb-EE on inflammatory biomarker expression, RAW264.7 cells were pre-treated with Sb-EE, then stimulated with LPS (1 µg/mL) for 6 h. mRNA expression of iNOS, COX-2 and TNF-α was analysed by semi-quantitative RT-PCR. (B, C) To validate the effect of Sb-EE on the transcriptional activity of NF-κB and AP-1, a luciferase assay was performed. HEK 293 cells were transfected with NF-κB-Luc (B) or AP-1-Luc (C) genes, and Flag-MyD88 or CFP-TRIF were additionally overexpressed to activate transcription of NF-κB-Luc and AP-1-Luc genes. Then, HEK 293 cells were treated with Sb-EE (0–200 μg/mL) for 24 h. Luciferase activity was measured using a luminometer. (D) To analyse nuclear translocation of transcription factor subunits, Sb-EE (200 μg/mL) pre-treated RAW264.7 cells were stimulated with LPS for the indicated times. Then, the levels of AP-1 (c-Fos and c-Jun) and NF-κB subunits (p65 and p50) in nuclear lysate were determined by immunoblotting. Lamin A/C was utilized as a loading control. All data are presented as the mean ± SD of experiments. ^##p < 0.01 compared to the normal group, and *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A, D), and MyD88/TRIF-alone group in (B, C)).

Figure 3.

Inhibitory effect of Sb-EE on NF-κB signalling enzymes. (A, B) RAW264.7 cells were treated with LPS (1 µg/mL) for the indicated time in the presence or absence of Sb-EE (200 μg/mL). The levels of phospho- and total-IκBα (A), Src, Syk, p85 and β-actin (B) were determined by immunoblotting using whole lysates. (C) RAW264.7 cells were pre-treated with Sb-EE in a dose-dependent manner, and were thereafter triggered with LPS for 3 min. Then, p-Src, Src and β-actin levels were detected by immunoblotting. (D) To examine the direct effects of Sb-EE on Src and Syk activity, an in vitro kinase assay was performed with purified Src and Syk. ^##p < 0.01 compared to the normal group, and *p < 0.05 and **p < 0.01 compared to the control group (LPS alone group in (A–C)).

Figure 4.

Inhibitory effect of Sb-EE on AP-1 signalling enzymes. (A–C) RAW264.7 cells were treated with LPS (1 µg/mL) for the indicated time in the presence or absence of Sb-EE (200 μg/mL). Then, an immunoblotting assay was performed with antibodies for phospho- and total-forms of target proteins. The antibodies against p-ERK, ERK, p-JNK, JNK, p-p85 and p85 antibodies were used to detect the MAPKs (A). The antibodies against p-TAK1, TAK-1, p-MEK1/2, MEK1/2, p-MKK4/7, MKK4/7, p-MKK3/6 and MKK3/6 were used to detect the upstream enzymes of MAPKs (B, left and right panel). IRAK-1 and IRAK-4 antibodies were used to detect the initially activated enzymes in the AP-1 signalling pathway (C). (D) To examine the direct effects of Sb-EE on IRAK1 activity, an in vitro kinase assay was performed with purified IRAK1. ^##p < 0.01 compared to the normal group, *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A), (B, left and right panel) and (C)).

Figure 4.

Inhibitory effect of Sb-EE on AP-1 signalling enzymes. (A–C) RAW264.7 cells were treated with LPS (1 µg/mL) for the indicated time in the presence or absence of Sb-EE (200 μg/mL). Then, an immunoblotting assay was performed with antibodies for phospho- and total-forms of target proteins. The antibodies against p-ERK, ERK, p-JNK, JNK, p-p85 and p85 antibodies were used to detect the MAPKs (A). The antibodies against p-TAK1, TAK-1, p-MEK1/2, MEK1/2, p-MKK4/7, MKK4/7, p-MKK3/6 and MKK3/6 were used to detect the upstream enzymes of MAPKs (B, left and right panel). IRAK-1 and IRAK-4 antibodies were used to detect the initially activated enzymes in the AP-1 signalling pathway (C). (D) To examine the direct effects of Sb-EE on IRAK1 activity, an in vitro kinase assay was performed with purified IRAK1. ^##p < 0.01 compared to the normal group, *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A), (B, left and right panel) and (C)).

Figure 5.

Anti-inflammatory ability of Sb-EE in LPS-induced peritonitis and HCl/EtOH-triggered gastritis mice. (A, B) Sb-EE (200 mg/kg) was orally injected into C57BL/6 mice daily for five days. Peritonitis was induced by peritoneal injection of LPS (10 µg/kg) for one day. NO assay was performed using peritoneal macrophages (A). Immunoblotting assay was performed with whole lysates obtained from peritoneal exudates. Phospho- or total-forms of ERK, p38, Src, Syk and β-actin were detected using specific antibodies (B). (C) Sb-EE (200 mg/kg) or ranitidine (40 mg/kg) was orally administered in ICR mice twice a day for two days, and then HCl/EtOH was injected for 1 h to induce gastritis. Stomach inflammatory lesions were photographed with a digital camera and then quantified using ImageJ. ^##p < 0.01 compared to the normal group, *p < 0.05 and **p < 0.01 compared to the control group (LPS-alone group in (A, B), and HCl/EtOH alone group in (C)).

Figure 6.

Schematic diagram of the anti-inflammatory properties of Sb-EE through inhibition of AP-1 and NF-κB signalling.