Decursin negatively regulates LPS-induced upregulation of the TLR4 and JNK signaling stimulated by the expression of PRP4 in vitro

Abstract

The current investigation was carried out to analyze the correlation of bacterial lipopolysaccharide (LPS) and pre-mRNA processing factor 4B (PRP4) in inducing inflammatory response and cell actin cytoskeleton rearrangement in macrophages (Raw 264.7) and colorectal (HCT116) as well as skin cancer (B16-F10) cells. Cell lines were stimulated with LPS, and the expression of PRP4 as well as pro-inflammatory cytokines and proteins like IL-6, IL-1β, TLR4, and NF-κB were assayed. The results demonstrated that LPS markedly increased the expression of PRP4, IL-6, IL-1β, TLR4, and NF-κB in the cells. LPS and PRP4 concomitantly altered the morphology of cells from an aggregated, flattened shape to a round shape. Decursin, a pyranocoumarin from Angelica gigas, inhibited the LPS and PRP4-induced inflammatory response, and reversed the induction of morphological changes. Finally, we established a possible link of LPS with TLR4 and JNK signaling, through which it activated PRP4. Our study provides molecular insights for LPS and PRP4-related pathogenesis and a basis for developing new strategies against metastasis in colorectal cancer and skin melanoma. Our study emphasizes that decursin may be an effective treatment strategy for various cancers in which LPS and PRP4 perform a critical role in inducing inflammatory response and morphological changes leading to cell survival and protection against anti-cancer drugs.

Keywords: JNK; LPS; PRP4; TLR4; decursin; inflammation.

Figure 1.

LPS and PRP4 induces cytokines expression. (A) mRNA and protein levels of PRP4 in control and PRP4-transfected cells. GAPDH and actin were used as the loading control. (B) Western blot and PCR analysis of PRP4 and PRP8 in Raw264.7, HCT116, B16-F10 cells after stimulation with 100 ng/ml LPS. (C) Cells were pre-treated with LPS, followed by transfection with PRP4, and then incubated for 24 h. RT-PCR was performed to examine the mRNA levels of IL-6 and IL-1β. GAPDH was used as internal control. (D) HCT116 cells were transfected with si-RNA-PRP4 using Xfect RNA transfection reagent from Takara as described by the manufacturer. Cells were then stimulated with 100 ng/ml LPS. RT-PCT and western blots were performed on control transfected cells. GAPDH was used as the loading control.

Figure 2.

Decursin inhibits LPS-induced PRP4 expressions and cell morphological alterations. (A) Western blot analysis of PRP4 and PRP8 in Raw264.7, HCT116, B16-F10 cells after stimulation with 100 ng/ml LPS and treating with 10 µM decursin. β-actin was used as a loading control. (B) Cells were pre-treated with LPS and incubated with 10 µM decursin and/or 10 µg/ml polymyxin B for 24 h. Western blot was performed to analyze the expressions of PRP4 and PRP8. β-actin was used as a loading control. (C) Cells were stimulated with 100 ng/ml LPS, followed by transfection with PRP4, and treatment with 10 µM decursin. Cells were stained with phalloidin and observed under a ZEISS LSM 800 confocal microscope at 1000 × magnification.

Figure 3.

LPS and PRP4 induces inflammatory pathways proteins. (A) Cells were pre-treated with LPS and then transfected with PRP4. Cellular proteins were extracted using cell lysis buffer. Proteins were quantified by Bradford assay, and equal amount of proteins were separated on 10% SDS-PAGE. Proteins were then transferred onto nitrocellulose (NC) membranes. NC membranes were then incubated with specific antibodies for NF-κB, and I-κBα overnight at 4°C. Chemiluminescent signals were developed with Clarity™ ECL Western Blotting Substrate. (B) Western blot analysis of TLR4, NF-κB, and I-κBα after stimulating cells with LPS, followed by transfection with PRP4 and treatment with decursin. β-actin served as a loading control.

Figure 4.

LPS stimulates PRP4 by activating TLR4 and JNK. (A) Protein levels of Akt, JNK, Erk, and p-Erk (B) PRP4 and PRP8 protein expressions were analyzed through western blot after incubating the cells with LPS, decursin and SP600125. (C) Cells were stimulated with 100 µg/ml LPS and incubated with or without 10 µM Decursin, and CLI-095 for 24 h. Protein levels of PRP4 and PRP8 were analyzed through western blotting. β-actin was used as a loading control.