Citri Reticulatae Pericarpium Limits TLR-4-Triggered Inflammatory Response in Raw264.7 Macrophages by Activating RasGRP3

Abstract

Inflammation is an important immune response to pathogen invasion, but excessive inflammation leads to tissue injury and even cytokine storm. Therefore, proper response is needed depending on the intensity of the infection. Ras guanine nucleotide releasing protein 3 (RasGRP3) is a regulator of the TLR-mediated response. In low-intensity inflammation, it negatively regulates production of pro-inflammatory cytokines, especially IL-6. Citri Reticulatae Pericarpium, the peel of Citrus reticulata Blanco, is a major medicinal herb in Korean medicine. The present study aims to investigate whether the Citri Reticulatae Pericarpium extract (CRE) has immunomodulatory activity using the Raw264.7 macrophage. Also, we investigated the effect of CRE on RasGRP3 expression. In the present study, CRE reduced IL-6 production in the low-LPS environment (1 ng/mL) and did not in the high-LPS environment (100 ng/mL). The suppression of IL-6 production in the low-LPS environment (1 ng/mL) was abolished after the pretreatment of RasGRP3 siRNA. The reduced RasGRP3 protein content by 100 ng/mL LPS treatment was increased by CRE treatment. Additionally, nobiletin, a major component of CRE showed a suppressive effect on IL-6 production in the low-LPS environment (1 ng/mL). The present results suggest that CRE alleviates inflammatory response via activating RasGRP3 expression in low-intensity inflammation.

Keywords: Citri Reticulatae Pericarpium; RasGRP3; immunomodulatory effects; macrophage.

Figure 1

Effects of Citri Reticulatae Pericarpium extract (CRE) on the IL-6 production in Raw264.7 cells. Raw264.7 macrophages were treated with CRE (25, 50, 100, 200 μg/mL) 1 h before LPS treatment at a concentration of 1 ng/mL (A) or 100 ng/mL (B). The data are presented as mean ± SD. ^### p < 0.001 compared with the blank control group; and *** p < 0.001 compared with the LPS-treated group via one-way ANOVA (A) F(5, 12) = 3926, (B) F(5, 12) = 9149.

Figure 2

Effects of Citri Reticulatae Pericarpium extract (CRE) on RasGRP3 expression in Raw264.7 cells. Raw264.7 macrophages were treated with 100 μg/mL of CRE 1 h before LPS treatment at a concentration of 1 ng/mL (A) or 100 ng/mL (B). After 18 h post-LPS-treatment, the expression of RasGRP3 was determined using immunoblotting. ^# p < 0.05 ^### p < 0.001 compared with the blank control group; and ** p < 0.01 compared with the LPS-treated group.

Figure 3

Effects of Citri Reticulatae Pericarpium extract (CRE) on the IL-6 production in Raw264.7 cells transfected with RasGRP3 siRNA. Raw264.7 macrophages with or without RasGRP3 siRNA were treated with 100 μg/mL of CRE 1 h before LPS treatment at a concentration of 1 ng/mL (A) or 100 ng/mL (B). The data are presented as mean ± SD. *** p < 0.001 compared with the LPS-treated group by one-way ANOVA (A) F (5, 18) = 491, (B) F(5, 18) = 1663.

Figure 4

Effects of Citri Reticulatae Pericarpium extract (CRE) on the RasGRP3 expression in Raw264.7 cells transfected with RasGRP3 siRNA. Raw264.7 macrophages with or without RasGRP3 siRNA were treated with 100 μg/mL of CRE 1 h before LPS treatment at a concentration of 1 ng/mL (A) or 100 ng/mL (B). After 18 h post-LPS-treatment, the expression of RasGRP3 was determined using immunoblotting. Different letters (a–d) represent statistically significant differences (p < 0.05) (C,D).

Figure 5

Effects of nobiletin on IL-6 production in Raw264.7 macrophages. Raw264.7 macrophages were treated with nobiletin (2.5, 5, 10, 20, 40 μg/mL) 1 h before LPS treatment at the concentration of 1 ng/mL (A) or 100 ng/mL (B). The IL-6 production was determined using a commercial IL-6 assay kit, following manufacturer’s instruction. The data are presented as mean ± SD. ^### p < 0.001 compared with the blank control group; and *** p < 0.001 compared with the LPS-treated group by one-way ANOVA (A) F (6, 21) = 278, (B) (6, 21) = 1335.

Figure 6

Proposed mechanism of immune hypersensitivity suppression activity of Citri Reticulatae Pericarpium extract (CRE) on the inflammatory responses induced by low-level (1 ng/mL) or high-level (100 ng/mL) LPS.