Anti-inflammatory Effect of Curcuma longa and Allium hookeri Co-treatment via NF-κB and COX-2 Pathways

Abstract

Although inflammation is a host defense mechanism, chronic inflammation mediates several diseases, including cancer, allergy, asthma, and autoimmune diseases, and reportedly, it is associated with a 60% mortality rate. There are several reports on the anti-inflammatory effects of Curcuma longa and Allium hookeri. However, although they can be used as culinary materials and have biological effects, they are not effective anti-inflammatory agents. In this study, we evaluated the synergic effect of C. longa and A. hookeri in order to confirm the possibility of a new anti-inflammatory agent. Based on cell viability and cytokine analyses, the appropriate ratio of C. longa and A. hookeri was confirmed using an air pouch animal model. Then, the anti-inflammatory effect of C. longa and A. hookeri co-treatment was evaluated by measuring the immune cell count and cytokines in the exudate and by comparing the morphological changes and cytokines in inflamed skin samples. Additionally, we evaluated the NF-κB/COX-2 pathway and iNOS levels. The active constituents detected in C. longa were demethoxycurcumin and bisdemethoxycurcumin, and that detected in A. hookeri was methylsulfonylmethane. An in vitro assessment determined the appropriate drug ratio as 3:7. In a carrageenan-induced inflammatory model, co-treatment effectively suppressed inflammatory cytokines, including IFN-γ, IL-1β, IL-6, IL-13, and IL-17, and recovered inflammation-related morphological changes in the skin. The anti-inflammatory effect of the co-treatment was mediated through the NF-κB/COX-2 pathway and iNOS inhibition. We concluded that co-treatment with C. longa and A. hookeri synergistically inhibited inflammation via the NF-κB/COX-2/iNOS pathway.

Figure 1

Identification of active compounds in C. longa extract and 70% ethanol extract of A. hookeri root. (A) At a retention time of 20 to 22 min, the active constituents in C. longa extract such as bisdemethoxycurcumin (BDMC), demethoxycurcumin (DMC), and curcumin, were detected via HPLC analysis. The upper graph is the standard at 450 nm and the lower graph is the sample extract at 450 nm. (B) At a retention time of 10 min, methylsulfonylmethane (MSM) in the 70% ethanolic extract of A. hookeri was confirmed via GC analysis. The upper graph is the standard and the lower graph is the sample extract. DMC, demethoxycurcumin; BDMC, Bisdemethoxycurcumin. MSM, methylsulfonylmethane.

Figure 2

Cell proliferation suppression and pro-inflammatory cytokine regulation depending on the ratio of C. longa to A. hookeri. (A) Including curcumin alone treatment, the ratios of C. longa to A. hookeri were 10:0, 7:3, 5:5, 3:7, and 0:10, and controlled LPS-induced cell proliferation comparable to non-LPS-treated cells. The ratios (7:3, 5:5, 3.7, and 0:10) suppressed cell proliferation and the minimum ratio of cellular proliferation inhibition was 7:3. (B) In most groups, except the curcumin alone treatment group and the 5:5 ratio, IFN-γ levels, increased by LPS treatment, were suppressed by C. longa and A. hookeri co-treatment. Although the 7:3 ratio was most effective in suppressing IFN-γ expression, the ratios 7:3, 3:7, and A. hookeri alone downregulated IFN-γ levels. Unlike A. hookeri, C. longa dose-dependently inhibits IL-6 levels. The C. longa alone treatment most significantly inhibited IL-6 expression, but the 7:3, 5:5, or 3:7 ratios effectively suppressed IL-6 expression. IL-13 levels decreased only by the 3:7 C. longa to A. hookeri. In all groups, TNF-α levels were suppressed, with the lowest level by the 3:7 ratio. All values are presented as mean ± standard deviation. CON vs. ^*p < 0.05; CON vs. ^**p < 0.001 LPS vs. ^$p < 0.05; LPS vs. ^$$p <0.001; C. longa: A. hookeri = 10:0 vs. ^#p < 0.05; C. longa: A. hookeri = 10:0 vs. ^##p < 0.001; C. longa: A. hookeri = 7:3 vs. ^&p < 0.05; C^. longa: A. hookeri = 5:5 vs. ^@p < 0.05; C. longa: A. hookeri = 3:7 vs. ^%p < 0.05.

Figure 3

C. longa and A. hookeri co-treatment suppresses carrageenan-stimulated inflammation. (A) Inhibitory effect of co-treatment against blood cell proliferation in the exudate. Co-treatment with C. longa and A. hookeri dose-dependently inhibits white blood cells and monocyte proliferation, controls eosinophils levels similar to MSM treatment, completely decreases carrageenan treatment-upregulated lymphocyte levels, dose-dependently controls monocyte levels, and only demonstrates a tendency for neutrophil suppression with no statistical comparison. (B) Recovery effect of co-treatment on the air pouch membrane’s morphological changes. (C) Co-treatment suppresses the expression of inflammatory-related cytokines such as IFN-γ, IL-1β, IL-6, IL-13, and IL-17, but not TNF-α. Co-treatment with ≥250 mg/kg C. longa and A. hookeri exerts a stronger inhibitory effect on IFN-γ levels than MSM treatment. Co-treatment dose-dependently suppresses IL-1β, IL-6, and IL-13 levels. However, no significant decrease in TNF-α and IL-10 expression was observed with co-treatment. In the case of IL-17, there is a dose-dependent decrease pattern, which is hard to obtain with co-treatment. (D) Significant inhibitory effects against inflammatory-related cytokines in the dermis such as IFN-γ, TNF-α, IL-1β, IL-6, IL-13, and IL-17. Co-treatment dose-dependently suppresses the expression of cytokines such as IFN-γ, IL-1β, IL-10, and IL-17, and 500 mg/kg treatment completely inhibits the expression of IFN-γ, IL-10, and IL-17. In a dose-dependent manner, the levels of IL-1β, IL-6, and IL-13 are effectively downregulated. Co-treatment effectively inhibits the expression of TNF-α, with complete suppression observed with 250 mg/kg and 500 mg/kg co-treatment. a, control; b, 1% carrageenan treatment; c, 25 mg/kg MSM treatment; d, 50 mg/kg C. longa and A. hookeri co-treatment; e, 250 mg/kg C. longa and A. hookeri co-treatment; f, 500 mg/kg C. longa and A. hookeri co-treatment. Magnification, 200×. Scare bar, 100 μm. All values are presented as mean ± standard deviation. CON vs. ^**p < 0.001; Carrageenan vs. ^$p < 0.05; MSM vs. ^#p < 0.05.

Figure 4

C. longa and A. hookeri co-treatment inhibits NF-κB and COX-2 expression. (A) NF-κB is downregulated in the nucleus and COX-2 in the cytoplasm. NF-κB and COX-2 are not expressed in the control group and only the nucleus is stained with DAPI (blue spots). Carrageenan stimulates the expression of NF-κB in the nucleus and that of COX-2 in the cytoplasm. MSM suppresses the expression of NF-κB and COX-2 induced by carrageenan, but the expression of NF-κB (arrow) and COX-2 (arrowhead) observed is minimal. C. longa and A. hookeri co-treatment significantly inhibits the expression of NF-κB and COX-2. (B) The graph and scores present the immunofluorescent results using the image analyzing program in the K1-Fluo confocal microscope. MSM and C. longa and A. hookeri co-treatment statistically significantly control NF-κB and COX-2 expression effectively. (C) Compared to the density of NF-κB in the nucleus and cytoplasm, the pattern is similar to that observed in the immunofluorescence assay. C. longa and A. hookeri co-treatment blocks the translocation of NF-κB into the nucleus from the cytoplasm and inhibits the expression of COX-2 in the cytoplasm. (D) Depending on the image analysis of the western blotting of NF-κB (in nucleus and in cytoplasm) and COX-2 (in the skin tissue), the density graph based on each band per β-actin was presented using the Image J program. (E) MSM and C. longa and A. hookeri co-treatment significantly inhibited carrageenan-induced iNOS in the serum. The downregulation effect of 500 mg/kg C. longa and A. hookeri co-treatment is similar to that of the control. All values are presented as mean ± standard deviation. Arrow, NF-κB; arrowhead, COX-2. Magnification, 1000×. Scare bar, 50 μm. CON vs. ^*p < 0.05; CON vs. ^**p < 0.001 Carrageenan vs. ^$p < 0.05; Carrageenan vs. ^$$p < 0.001; MSM vs. ^#p < 0.05. Table in Fig. 4. The ratio of NF-κB p65 vs. nucleus or COX-2 vs. nucleus. All values are presented as mean ± standard deviation. CON vs. ^*p < 0.05; CON vs. ^**p < 0.001; Carrageenan vs. ^$p < 0.05; Carrageenan vs. ^$$p < 0.001; MSM vs. ^#p < 0.05.