Triptolide Modulates TREM-1 Signal Pathway to Inhibit the Inflammatory Response in Rheumatoid Arthritis

Abstract

Triptolide (TP), an active component isolated from Tripterygiumwilfordii Hook F, has therapeutic potential against rheumatoid arthritis (RA). However, the underlying molecular mechanism has not been fully elucidated. The aim of this study is to investigate the mechanisms of TP acting on RA by combining bioinformatics analysis with experiment validation. The human protein targets of TP and the human genes of RA were found in the PubChem database and NCBI, respectively. These two dataset were then imported into Ingenuity Pathway Analysis (IPA) software online, and then the molecular network of TP on RA could be set up and analyzed. After that, both in vitro and in vivo experiments were done to further verify the prediction. The results indicated that the main canonical signal pathways of TP protein targets networks were mainly centered on cytokine and cellular immune signaling, and triggering receptors expressed on myeloid cells (TREM)-1 signaling was searched to be the top one shared signaling pathway and involved in the cytokine and cellular immune signaling. Further in vitro experiments indicated that TP not only remarkably lowered the levels of TREM-1 and DNAX-associated protein (DAP)12, but also significantly suppressed the activation of janus activating kinase (JAK)2 and signal transducers and activators of transcription (STAT)3. The expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in lipopolysaccharides (LPS)-stimulated U937 cells also decreased after treatment with TP. Furthermore, TREM-1 knockdown was able to interfere with the inhibition effects of TP on these cytokines production. In vivo experiments showed that TP not only significantly inhibited the TREM-1 mRNA and DAP12 mRNA expression, and activation of JAK2 and STAT3 in ankle of rats with collagen-induced arthritis (CIA), but also remarkably decreased production of TNF-α, IL-1β and IL-6 in serum and joint. These findings demonstrated that TP could modulate the TREM1 signal pathway to inhibit the inflammatory response in RA.

Keywords: TREM-1 signal pathway; Triptolide; bioinformatics analysis; rheumatoid arthritis.

Figure 1

The results of bioinformatics analysis. Shared signaling pathways between gene molecular networks related with rheumatoid arthritis (RA) and protein targets molecular network of Triptolide (TP) in cytokine and cellular immune signaling performed using the Ingenuity Pathway Analysis (IPA) compare module. The signaling pathways of TP were represented as dark blue, while signaling pathways of RA were represented as light blue. (A) Cellular immune signaling; and (B) Cytokine signaling. The red boxes showed that TREM-1 signal pathway was the top one shared signaling pathway and participated in cytokine and cellular immune signaling. TREM-1: triggering receptors expressed on myeloid cells-1; IL: interleukin; iNOS: inducible nitric oxide synthase; GM-CSF: granulocyte-macrophage colony-stimulating factor; JAK: janus kinase; TYK: tyrosine kinase; CNTF: ciliary neurotrophic factor.

Figure 2

TREM-1 mRNA expression in U937 cells after lipopolysaccharides (LPS) stimulation. U937 cells were treated with 100 ng/mL of LPS for 0, 2, 4, 6, 12, 24 and 48 h, respectively. TREM-1 mRNA (A) and protein (B) levels were measured by RT-PCR and Western blots, respectively. All results are presented as mean ± standard deviation (SD) of three independent experiments, each performed in triplicate. * p < 0.05, ** p < 0.01 versus 0 h.

Figure 3

Effects of TP on TREM-1 signal pathway and inflammatory cytokines expression in LPS-induced U937 cells. (A) Effect of TP on TREM-1 mRNA levels in U937 cells, as measured with quantitative RT-PCR; (B) Effect of TP on the secretion of TREM-1, as measured with enzyme-linked immunosorbent assay (ELISA); (C) Effect of TP on TREM-1 protein levels in U937 cells, as measured with western blot; (D) Effect of TP on the expression of TREM-1 on U937 cells surface, as measured with flow cytometry; (E–G) Effect of TP on DAP12, p-JAK2, p-STAT3 protein levels in U937 cells, as measured with western blot; and (H–J) Effect of TP on the secretion of TNF-α, IL-1β, IL-6, as measured with ELISA. Data are expressed as mean ± SD from three independent experiments. ^## p < 0.01, compared to the control group. ** p < 0.01, * p < 0.05, compared to LPS group. FL2-A means the fluorescence peak area in flow cytometric analysis.

Figure 3

Effects of TP on TREM-1 signal pathway and inflammatory cytokines expression in LPS-induced U937 cells. (A) Effect of TP on TREM-1 mRNA levels in U937 cells, as measured with quantitative RT-PCR; (B) Effect of TP on the secretion of TREM-1, as measured with enzyme-linked immunosorbent assay (ELISA); (C) Effect of TP on TREM-1 protein levels in U937 cells, as measured with western blot; (D) Effect of TP on the expression of TREM-1 on U937 cells surface, as measured with flow cytometry; (E–G) Effect of TP on DAP12, p-JAK2, p-STAT3 protein levels in U937 cells, as measured with western blot; and (H–J) Effect of TP on the secretion of TNF-α, IL-1β, IL-6, as measured with ELISA. Data are expressed as mean ± SD from three independent experiments. ^## p < 0.01, compared to the control group. ** p < 0.01, * p < 0.05, compared to LPS group. FL2-A means the fluorescence peak area in flow cytometric analysis.

Figure 4

Effects of TP on inflammatory cytokines expression after TREM-1 knockdown. PMA-induced U937 cells were treated with or without 1 μg/mL LPS, and then transfected with TREM-1 siRNA or control siRNA, respectively. After 48 h, cells were treated with or without TP for another 48 h. (A) TREM-1 mRNA protein levels were determined by Western blot; and (B–D) TNF-α, IL-1β and IL-6 production were determined by ELISA. ^## p < 0.01 versus the control group; * p < 0.05 and ** p < 0.01 versus the group with LPS. The data are presented as the means ± standard deviation from three independent experiments.

Figure 5

Effects of TP on collagen-induced arthritis (CIA) rats. (A) Morphological features of representative ankle joints; (B) Histopathological features of representative ankle joints haematoxylin-eosin (HE) staining, ×20, scale bars = 100 μm; (C) Arthritic score in different days after treatment with TP; (D) Inflammation scores in different groups after treatment with TP, as described in methods. TL represented for TP low dose (9.31 µg/kg) group, TH represented for TP high dose (18.62 µg/kg) group, LEF represented for Leflunomide group. Date are represented as the mean ± SD (n = 7), ^## p < 0.01, comparison with the normal group, * p < 0.05, ** p < 0.01, comparison with the model group.

Figure 6

Effects of TP on the expression of TREM-1 and DAP12 in ankle joints of CIA rats. (A,B) TREM-1 and DAP12 mRNA levels in the ankle joints of CIA rats after treatment with TP; (C) Protein expression of TREM-1 and DAP12 in the ankle joints of CIA rats; (D) Expression of p-JAK2 in the ankle joints of CIA rats; and (E) Expression of p-STAT3 in the ankle joints of CIA rats. Date are represented as the mean ± SD (n = 7), ^# p < 0.05, ^## p < 0.01, in comparison with the normal group; * p < 0.05, ** p < 0.01, comparison with the model group.

Figure 7

Effects of TP on proinflammatory cytokines expression in CIA rats. (A–C) TNF-α, IL-1β and IL-6 levels in serum of CIA rats after treatment with TP; and (D–F) TNF-α, IL-1β and IL-6 mRNA levels in ankle joints of CIA rats after treatment with TP. Data are represented as the mean ± SD (n = 7), ^# p < 0.05, ^## p < 0.01 in comparison with the normal group; * p < 0.05, ** p < 0.01, comparison with the model group.