Siegesbeckia pubescens Makino inhibits Pam3CSK4-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation

Abstract

Background: Siegesbeckia pubescens Makino (SP) is one of the important plant origins for the anti-inflammatory Chinese herbal medicine of Siegesbeckiae Herba. The current investigations indicated that the anti-inflammatory effects of SP were associated with the toll-like receptors (TLRs)-mediated nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathways.

Methods: Raw 264.7 macrophages were pretreated with the 50% ethanol extract of SP (SPE, 50-200 µg/mL) and then co-treated with Pam₃CSK₄ (200 ng/mL) for another 12 h. The inhibitory effect of SPE on Pam₃CSK₄-stimulated NO release and post-inflammatory cytokines secretions were determined using Griess reagent and Elisa kits, respectively. The influence of SPE on NF-κB and MAPKs signaling relevant proteins was measured by Western blotting analysis, while the intracellular nitric oxide (NO) generation and NF-κB/p65 nuclear translocation were determined using Leica TCS SP8 laser scanning confocal microscope. Moreover, the effect of SPE on luciferase reporter gene in NF-κB-luc DNA transfected raw 264.7 cells was determined using the Dual-Glo luciferase assay system kit.

Results: SPE dose-dependently (50-200 µg/mL) attenuated Pam₃CSK₄-induced NO release, post-inflammatory cytokines (IL-6, TNF-α and MCP-1) secretions and intracellular NO generation in raw 264.7 cells. Biologically, SPE suppressed Pam₃CSK₄-induced expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), phosphorylation of NF-κB/p65 and IκBα, but did not significantly show effect on the proteins involved in MAPKs signaling (p38, ERK and JNK). The results were further confirmed by NF-κB-luc reporter gene assay and p65 nuclear translocation assay.

Conclusions: In conclusion, SPE ameliorated Pam₃CSK₄-induced inflammation in raw 264.7 cells through suppressing TLR 1/2-mediated NF-κB activation.

Keywords: Inflammation; NF-κB; Pam3CSK4; Siegesbeckia pubescens Makino; Toll-like receptor 1/2.

Fig. 1

HPLC chromatograms of (a) mixed standards (7.5 μg/mL of rutin, kirenol and darutoside) and (b) SPE (1 mg/mL). 1: rutin; 2: kirenol; 3: darutoside. The contents of rutin, kirenol and darutoside were determined to be 0.27 ± 0.01, 1.81 ± 0.02 and 0.28 ± 0.03%, respectively (n = 3)

Fig. 2

Siegesbeckia pubescens Makino (SP) or SP with Pam₃CSK₄-stimulated did not significantly affect the cell viability and cytotoxicity (n = 3). RAW 264.7 cells were treated with SP with different concentrations for 24 h. a The cell viability was measured by MTT assay and c cell cytotoxicity was measured by LDH assay. RAW 264.7 cells were pretreated with SP for 4 h before Pam₃CSK₄ (200 ng/mL) stimulation for another 12 h. b The cell viability was measured by MTT assay and d cell cytotoxicity was measured by LDH assay

Fig. 3

Siegesbeckia pubescens Makino (SP) exhibited anti-inflammatory effects in Pam₃CSK₄-stimulated RAW 264.7 cells (n = 3). RAW 264.7 cells were pretreated with SP with different concentrations for 4 h before Pam₃CSK₄ (200 ng/mL) stimulation for another 12 h. a Nitric oxide (NO) was determined by Griess assay. TLR1/TLR2 antagonist: CU-CPT22 (CU) was selected as the positive control. We measured the levels of b IL-6, c TNF-α, and d MCP-1 by ELISA assay. *P < 0.05 vs. Pam₃CSK₄-induced, **P < 0.01 vs. Pam₃CSK₄-induced, ***P < 0.001 vs. Pam₃CSK₄-induced

Fig. 4

RAW 264.7 cells were pretreated with Siegesbeckia pubescens Makino (SP) (n = 3) for 4 h before Pam₃CSK₄ (200 ng/mL) stimulation for another 12 h. NO was captured by Leica TCS SP8 laser scanning confocal microscope with 5 μM DAF-FM diacetate (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate). CU-CPT22 (CU) is selected as the positive control

Fig. 5

Effects of Siegesbeckia pubescens Makino (SP) on relevant pathways. RAW 264.7 cells were pretreated with SP (0, 50, 100 and 200 μg/mL) for 4 h and followed with Pam₃CSK₄ (200 ng/mL) addition for 12 h. CU-CPT22 (CU) is selected as the positive control. a Proteins was evaluated by Western blotting assay. b, c Quantification of iNOS and COX-2 protein was detected by densitometric analysis (n = 3). **P < 0.01 vs. Pam₃CSK₄-induced

Fig. 6

Effects of Siegesbeckia pubescens Makino (SP) on NF-κB pathways (n = 3). RAW 264.7 cells were pretreated with SP (0, 50, 100 and 200 μg/mL) for 2 h and followed with Pam₃CSK₄ (200 ng/mL) addition for 4 h. CU-CPT22 (CU) is selected as the positive control. a Proteins was evaluated by Western blotting assay. b, c Quantification was detected by densitometric analysis. d RAW 264.7 cells were transfected with NFκB-luc for 48 h. Cells were pretreated with SP 2 h before Pam₃CSK₄ (200 ng/mL) stimulation for another 4 h. Luciferase activity was determined by Dual-Glo Luciferase Assay. *P < 0.05 vs. Pam₃CSK₄-induced and **P < 0.01 vs. Pam₃CSK₄-induced

Fig. 7

RAW 264.7 cells were pretreated with 200 μg/mL of Siegesbeckia pubescens Makino (SP) (n = 3) for 2 h before Pam₃CSK₄ stimulation for another 4 h. NF-κB/p65 nuclear translocation was determined by immunofluorescence assay