Neorogioltriol and Related Diterpenes from the Red Alga Laurencia Inhibit Inflammatory Bowel Disease in Mice by Suppressing M1 and Promoting M2-Like Macrophage Responses

Abstract

Macrophages are central mediators of inflammation, orchestrating the inflammatory response through the production of cytokines and nitric oxide. Macrophages obtain pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, which can be modulated by soluble factors, including natural products. Despite the crucial protective role of inflammation, chronic or deregulated inflammation can lead to pathological states, such as autoimmune diseases, metabolic disorders, cardiovascular diseases, and cancer. In this case, we studied the anti-inflammatory activity of neorogioltriol (1) in depth and identified two structurally related diterpenes, neorogioldiol (2), and O¹¹,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3), with equally potent activity. We investigated the mechanism of action of metabolites 1⁻3 and found that all three suppressed macrophage activation and promoted an M2-like anti-inflammatory phenotype by inducing expression of Arginase1, MRC1, IRAK-M, the transcription factor C/EBPβ, and the miRNA miR-146a. In addition, they suppressed iNOS induction and nitric oxide production. Importantly, treatment of mice with 2 or 3 suppressed DSS-induced colitis by reducing tissue damage and pro-inflammatory cytokine production. Thus, all these three diterpenes are promising lead molecules for the development of anti-inflammatory agents targeting macrophage polarization mechanisms.

Keywords: Laurencia; TNF-alpha; colitis; cytokine; halogenated diterpenes; neorogioltriol; nitric oxide.

Figure 1

Neorogioltriol (1), neorogioldiol (2), and O¹¹,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3) isolated from Laurencia sp. collected from Vatsa bay in Kefalonia island.

Figure 2

Determination of IC₅₀ values and cytostatic potential of metabolites 1–3. (A) Determination of the compound concentration resulting in 50% inhibition of NO production by RAW 264.7 cells. Evaluation of the effect of (B) neorogioltriol (1), (C) neorogioldiol (2), and (D) O¹¹,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3) on the proliferation of RAW 264.7 cells. The cell number was determined using a Neubauer chamber and trypan blue staining, which was normalized to initial cells plated, and compared to cells treated with carbowax 400 0.1% v/v. Statistical analysis was carried out using the Kruskal-Walis non-parametric test in the Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).

Figure 3

Effect of metabolites 1–3 on the expression of pro-inflammatory markers in RAW 264.7 cells following 24 h of incubation with the respective compounds. (A) iNOS mRNA levels measured using real time PCR, (B) TNFα production was measured using ELISA in the supernatant of cell culture, and (C) pre-miR-155 mRNA levels measured using real time PCR. All treatments have been compared to carbowax 400 0.1% v/v treated cells. Statistical analysis was carried out using Kruskal-Walis non-parametric test in Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).

Figure 4

Effect of metabolites 1–3 on the levels of mRNA expression of anti-inflammatory markers in RAW 264.7 cells following 24 h of incubation with the respective compounds. (A) Arginase 1 mRNA levels, (B) MRC1 mRNA levels, (C) IRAK-M mRNA levels, (D) pre-miR-146a mRNA levels, and (E) c/EBPβ mRNA levels were measured using real time PCR. All treatments have been compared to carbowax 400 0.1% v/v treated cells. Statistical analysis was carried out using Kruskal-Walis non-parametric test in Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).

Figure 5

Evaluation of the pro-inflammatory potential of metabolites 1–3 upon LPS activation in RAW 264.7 cells. Cells were pre-incubated for 1 h with the respective compound, then co-incubated with LPS 100 ng/mL for 24 h. The expression profile of pro-inflammatory markers was evaluated. (A) iNOS and (C) pre-miR-155 mRNA levels were evaluated using real time PCR. (B) TNFα production was measured using ELISA in the supernatant of the cell culture. All treatments have been compared to carbowax 400 0.1% v/v treated cells. Statistical analysis was carried out using the Kruskal-Walis non-parametric test in Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).

Figure 6

Evaluation of the anti-inflammatory potential of metabolites 1–3 upon LPS activation in RAW 264.7 cells. Cells were pre-incubated for 1 h with the respective compound and were then co-incubated with LPS 100 ng/mL for 24 h. Expression profile of anti-inflammatory markers was evaluated. (A) Arginase 1 mRNA levels, (B) MRC1 mRNA levels, (C) IRAK-M mRNA levels, (D) pre-miR-146a mRNA levels, and (E) c/EBPβ mRNA were measured using real time PCR. All treatments have been compared to carbowax 400 0.1% v/v treated cells. Statistical analysis was carried out using Kruskal-Walis non-parametric test in Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).

Figure 7

Identification of the potential in vivo anti-inflammatory properties of metabolites 2 and 3 using DSS-induced model of inflammatory bowel disease. (A) Schematic representation of the DSS protocol combined with intraperitoneal treatment of potential anti-inflammatory analogs. Female C57BL/6J mice received 2.5% in their drinking water for five days and were injected intraperitoneally with metabolites 2 and 3 every 48 h starting from day 0. “Control” mice received only drinking water. (B) Photos illustrating the length and the macroscopic view of a representative intestine of each group. (C) Graph with values of colon length of each group. (D) Hematoxylin and eosin staining of colon tissue sections from untreated (left panel) and DSS-treated mice (right panel) showing that treatment with 2 and 3 (lower right panels) ameliorates the disease phenotype (right upper vs. right lower images). Original magnification is x100. (E–G) Quantification of IL-6, IL-1b, and TNFα mRNA expression of groups indicated in the x-axis. Results were normalized to the housekeeping β-actin gene and expressed as RQ values relative to untreated controls, which was given the arbitrary value of ‘1’. Statistical analysis was carried out using Kruskal-Walis non-parametric test in Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).

Figure 7

Identification of the potential in vivo anti-inflammatory properties of metabolites 2 and 3 using DSS-induced model of inflammatory bowel disease. (A) Schematic representation of the DSS protocol combined with intraperitoneal treatment of potential anti-inflammatory analogs. Female C57BL/6J mice received 2.5% in their drinking water for five days and were injected intraperitoneally with metabolites 2 and 3 every 48 h starting from day 0. “Control” mice received only drinking water. (B) Photos illustrating the length and the macroscopic view of a representative intestine of each group. (C) Graph with values of colon length of each group. (D) Hematoxylin and eosin staining of colon tissue sections from untreated (left panel) and DSS-treated mice (right panel) showing that treatment with 2 and 3 (lower right panels) ameliorates the disease phenotype (right upper vs. right lower images). Original magnification is x100. (E–G) Quantification of IL-6, IL-1b, and TNFα mRNA expression of groups indicated in the x-axis. Results were normalized to the housekeeping β-actin gene and expressed as RQ values relative to untreated controls, which was given the arbitrary value of ‘1’. Statistical analysis was carried out using Kruskal-Walis non-parametric test in Graphpad Prism 7.0 and graphs represent mean ± SEM (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001).