Korean J Physiol Pharmacol.  2019 Nov;23(6):493-499. 10.4196/kjpp.2019.23.6.493.

Rhodanthpyrone A and B play an anti-inflammatory role by suppressing the nuclear factor-κB pathway in macrophages

• Kim KS
• Han CY
• Han YT
• Bae EJ

• KMID: 2461042
• DOI: http://doi.org/10.4196/kjpp.2019.23.6.493

Abstract

Macrophage-associated inflammation is crucial for the pathogenesis of diverse diseases including metabolic disorders. Rhodanthpyrone (Rho) is an active component of Gentiana rhodantha, which has been used in traditional Chinese medicine to treat inflammation. Although synthesis procedures of RhoA and RhoB were reported, the biological effects of the specific compounds have never been explored. In this study, the anti-inflammatory activity and mechanisms of action of RhoA and RhoB were studied in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment with RhoA and RhoB decreased inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW 264.7 cells and in thioglycollate-elicited mouse peritoneal macrophages. In addition, it downregulated transcript levels of several inflammatory genes in LPS-stimulated RAW 264.7 cells, including inflammatory cytokines/chemokines (Tnfa, Il6, and Ccl2) and inflammatory mediators (Nos2 and Ptgs2). Macrophage chemotaxis was also inhibited by treatment with the compounds. Mechanistic studies revealed that RhoA and RhoB suppressed the nuclear factor (NF)-κB pathway, but not the canonical mitogen activated protein kinase pathway, in LPS-stimulated condition. Moreover, the inhibitory effect of RhoA and RhoB on inflammatory gene expressions was attenuated by treatment with an NF-κB inhibitor. Our findings suggest that RhoA and RhoB play an anti-inflammatory role at least in part by suppressing the NF-κB pathway during macrophage-mediated inflammation.

Keyword

Inflammation; Lipopolysaccharide; Macrophages; NF-κB pathway; Rhodanthpyrone

MeSH Terms

Animals
Chemotaxis
Cyclooxygenase 2
Gene Expression
Gentiana
Inflammation
Interleukin-6
Macrophages*
Macrophages, Peritoneal
Medicine, Chinese Traditional
Mice
Nitric Oxide Synthase Type II
Protein Kinases
RAW 264.7 Cells
Cyclooxygenase 2
Interleukin-6
Nitric Oxide Synthase Type II
Protein Kinases

Figure

• 

  Fig. 1 Chemical structures of Rhodanthpyrone (Rho)A and RhoB, and the effects of RhoA and RhoB on the viability of RAW 264.7 cells. (A) Chemical structures of RhoA and RhoB. In (B), the results are expressed as the percentage of surviving cells treated with RhoA or RhoB for 24 h relative to the unstimulated surviving RAW 264.7 cells (n = 3). Values are expressed as mean ± SEM.

• 

  Fig. 2 Prevention of lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression by Rhodanthpyrone (Rho)A and RhoB. (A) RAW 264.7 cells were pretreated with the indicated concentrations of RhoA or RhoB for 1 h, and then 10 ng/ml LPS was added for 24 h. Nitric oxide (NO) levels in the culture supernatant were determined (n = 3). (B) iNOS and COX-2 protein levels were analyzed in RAW 264.7 cells treated with LPS (10 ng/ml) in the presence or absence of RhoA or RhoB as indicated (n = 5). (C, D) RAW 264.7 cells were exposed to LPS (10 ng/ml) with or without RhoA or RhoB as indicated, and the protein (C) and mRNA (D) levels of iNOS and COX-2 were measured (n = 5). (E) Intraperitoneal macrophages were isolated as described in “Methods”. iNOS and COX-2 protein levels after stimulation with LPS (10 ng/ml) in the presence or absence of RhoA or RhoB were determined. Values are expressed as mean ± SEM. **p < 0.01 vs. untreated control; #p < 0.05 and ##p < 0.01 vs. LPS alone.

• 

  Fig. 3 Suppression of lipopolysaccharide (LPS)-stimulated pro-inflammatory cytokines/chemokine production by Rhodanthpyrone (Rho) A and RhoB. RAW 264.7 cells were treated with indicated concentrations of RhoA or RhoB for 1 h and then stimulated with 10 ng/ml LPS for 24 h. (A) mRNA levels of indicated genes were determined using qPCR (n = 3). (B) CCL2 and tumor necrosis factor (TNF)-α protein levels in the culture supernatant were determined using enzyme-linked immunosorbent assays (ELISA) (n = 3). (C) Representative images of the migration assay after staining cells with crystal violet (left). Cells that migrated to the bottom of the inserts were counted, and the quantification results are expressed as the relative ratio (%) (right). CM, conditioned medium. Values are expressed as mean ± SEM. *p < 0.05 and **p < 0.01 vs. LPS alone.

• 

  Fig. 4 Suppression of lipopolysaccharide (LPS)-stimulated nuclear factor (NF)-κB pathway by Rhodanthpyrone (Rho)A and RhoB. (A) RAW 264.7 cells were treated with 40 µM RhoA or RhoB for 1 h and then stimulated with 10 ng/ml LPS for 24 h. Phosphorylated- and total-inhibitory κB kinase (IKK), p65, and inhibitory κB kinase α (IκBα) were determined using western blotting. (B) RAW 264.7 cells transfected with NF-κB luciferase were treated with LPS alone, or LPS with RhoA or RhoB, and then NF-κB luciferase activity in cell lysates were assayed (n = 5–6). (C) The levels of phosphorylated-and total mitogen activated protein kinases (MAPKs) were analyzed in RAW 264.7 cells treated as in panel A. (D) RAW 264.7 cells were treated with RhoA or RhoB (40 µM, 1 h) in the presence or absence of Bay 11-7082 (5 mM), and then stimulated with LPS (10 ng/ml, 24 h). Values are expressed as mean ± SEM. **p < 0.01 vs. untreated control; ##p < 0.01 vs. LPS alone.

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