A novel modified-curcumin 2.24 resolves inflammation by promoting M2 macrophage polarization

Abstract

To assess resolving-like activity by a novel chemically-modified curcumin (CMC2.24) in a "two-hit" model of diabetes-associated periodontitis. Macrophages from rats were cultured in the presence/absence of either Lipopolysaccharide (LPS, 1st hit); or advanced-glycation-end products (AGE, 2nd hit); or both combined. CMC2.24 was added as treatment. The conditioned media were analyzed for MMP-9, cytokines (IL-1β, IL-6, TNF-α), resolvins (RvD₁, RvE₁, lipoxin A₄), and soluble receptor for AGE (sRAGE). The phenotypes of M1/M2 macrophage were analyzed by flow cytometry. Both LPS/AGE-alone, and two-combined, dramatically increased the secretion of MMP-9 by macrophages. CMC2.24 "normalized" the elevated levels of MMP-9 under all conditions. Moreover, CMC2.24 significantly reduced the secretion of IL-1β and IL-6 with a fewer effects on TNF-α. Importantly, CMC2.24 increased RvD₁ and sRAGE secretion by macrophages exposed to LPS/AGE; and both treatment groups exhibited increased M2 relative to M1 populations. Furthermore, scatter-diagram showed the macrophages gradually shifted from M1 towards M2 with CMC2.24-treated, whereas LPS/AGE-alone groups remained unchanged. CMC2.24 "normalized" cytokines and MMP-9, but also enhanced RvD₁ and sRAGE in macrophages. Crucially, CMC2.24 appears to be a potent inhibitor of the pro-inflammatory M1 phenotype; and a promotor of the pro-resolving M2 phenotype, thus acting like a crucial "switch" to reduce inflammation.

Figure 1

The study design of a “two-hit” cell-culture model of diabetes-associated periodontitis. (a) Resident peritoneal and blood-derived Mφs were harvested from the rat, and separated by density gradient centrifugation, respectively. In an ex-vivo cell culture, Mφs were incubated in the absence (normal control), or presence of either LPS (L, the “1st hit”); or AGE (A, “2nd hit”); or both combined (L + A). CMC2.24 was added to the cultures at 2 or 5 μM. MMP-9 activity was measured by gelatin zymography and scanned densitometrically. Effects of CMC2.24 on MMP-9 in the peritoneal Mφs challenged by LPS-alone (b,c), AGE-alone (d,e), or the “two-hit” (f,g). N, the normal group without any challenge or treatment. L, the LPS-alone challenged group. L + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. A, the AGE-alone challenged group. A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. L + A, the “two-hit” challenged group. L + A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. Each value represents the mean (n = 3/group) ± the standard error. *p < 0.05, **p < 0.005, values were compared between N and L/A/(L + A)-challenged groups at 18-h. ^#p < 0.05, ^##p < 0.005, values were compared between L/A/(L + A)-challenged and L/A/(L + A) + 2.24 (2 or 5)-treated groups at 18-h. The grouping of gels were cropped from different parts of the same gel. The full-length gels were included in the Supplementary Information files (S1-4.)

Figure 2

Effects of CMC2.24 on pro-inflammatory cytokines. The effects of CMC2.24 on the concentrations (pg/mL) of IL-1β (a), IL-6 (b) and TNF-α (c) in rat peritoneal Mφs challenged by LPS-alone, AGE-alone, or the “two-hit”, respectively; and treated with different concentrations (2 or 5 μM) of CMC2.24 at 18-h. N, the normal group without any challenge or treatment. L, the LPS-alone challenged group. L + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. A, the AGE-alone challenged group. A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. L + A, the “two-hit” challenged group. L + A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. Each value represents the mean (n = 3/group) ± the standard error. *p < 0.05, **p < 0.005, ***p < 0.001, values were compared between N and L/A/(L + A)-challenged at 18-h. ^#p < 0.05, values were compared between L/A/(L + A)-challenged and L/A/(L + A) + 2.24 (2 or 5)-treated groups at 18-h.

Figure 3

Effects of CMC2.24 on Resolvin D₁ and soluble RAGE. The effects of CMC2.24 on the concentrations (pg/mL) of RvD₁ (a) and sRAGE (b) in rat peritoneal Mφs challenged by LPS-alone, AGE-alone, or the “two-hit”, respectively; and treated with different concentrations (2 or 5 μM) of CMC2.24 at 18-h. N, the normal group. L, the LPS-alone challenged group. L + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. A, the AGE-alone challenged group. A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. L + A, the “two-hit” challenged group. L + A + 2.24 (2 or 5), the low (2 μM) and high (5 μM) concentrations of CMC2.24-treated groups. Each value represents the mean (n = 3/group) ± the standard error. *p < 0.05, **p < 0.005, ***p < 0.001, values were compared between N and L/A/(L + A)-challenged at 18-h. ^#p < 0.05, ^###p < 0.001, values were compared between L/A/(L + A)-challenged and L/A/(L + A) + 2.24 (2 or 5)-treated groups at 18-h.

Figure 4

Conversion of LPS-challenged Mφs from M1 to M2 phenotype. At 0, 6, and 18 h, the effects of CMC2.24 on the ratio of M2 to M1 phenotype of L-challenged Mφs were indicated in (a) and analyzed by flow cytometry. The effects of CMC2.24 on each phenotype of L-challenged Mφs were indicated in (b) (for the percentage of M1), and (c) (for the percentage of M2), respectively. N, the normal group (grey line). N + 2.24, the CMC2.24 treated N group (black line). L, the LPS-alone challenged group (red line). L + 2.24, the CMC2.24-treated L group (green line). (d) Scatter diagrams of flow cytometry analysis showed the effects of CMC2.24 on the phenotype conversion of L-challenged Mφs, the “shift” from M1 to M2 phenotype after CMC2.24 treatment. Scatter diagrams of four groups (N, N + 2.24, L, L + 2.24) were present together at 0 h, 6 h, and 18 h. Each scatter diagram has 4 quadrants, UL: CD38⁺CD163⁻ (M1-only); UR: CD38⁺CD163⁺ (M1 and M2 mixed); LL: CD38⁻CD163⁻ (M0); LR: CD38⁻CD163⁺ (M2-only). Each value represents the mean (n = 3/group) ± the standard error. *p < 0.05, **p < 0.005, values were compared between L-challenged and L + 2.24-treated groups at 0, 6, and 18 h.

Figure 5

Conversion of AGE-challenged Mφs from M1 to M2 phenotype. At 0, 6, and 18 h, the effects of CMC2.24 on the ratio of M2 to M1 phenotype of A-challenged Mφs were indicated in (a) and analyzed by flow cytometry. The effects of CMC2.24 on each phenotype of A-challenged Mφs were indicated in (b) (for the percentage of M1), and (c) (for the percentage of M2), respectively. N, the normal group (grey line). N + 2.24, the CMC2.24 treated N group (black line). A, the AGE-alone challenged group (red line). A + 2.24, the CMC2.24-treated A group (green line). (d) Scatter diagrams of flow cytometry analysis showed the effects of CMC2.24 on the phenotype conversion of A-challenged Mφs, the “shift” from M1 to M2 phenotype after CMC2.24 treatment. Scatter diagrams of four groups (N, N + 2.24, A, A + 2.24) were present together at 0 h, 6 h, and 18 h. Each scatter diagram has 4 quadrants, UL: CD38⁺CD163⁻ (M1-only); UR: CD38⁺CD163⁺ (M1 and M2 mixed); LL: CD38⁻CD163⁻ (M0); LR: CD38⁻CD163⁺ (M2-only). Each value represents the mean (n = 3/group) ± the standard error. *p < 0.05, **p < 0.005, values were compared between A-challenged and A + 2.24-treated groups at 0, 6, and 18 h.

Figure 6

A scheme to summarize the pleiotropic mechanisms of the novel chemically-modified curcumin 2.24 on resolving inflammation in a “two-hit” model. This “two-hit” model demonstrates a bi-directional manner between diabetes (or other systemic diseases) and periodontitis. Treatment with CMC2.24 can inhibit inflammatory and tissue-destructive mediators including cytokines and MMPs; upregulate resolvin-enhancing (RvD1) activity; and promote the expression of sRAGE; as well as contribute to macrophage polarization from the M1 towards M2 phenotype.