Cordycepin Suppresses Expression of Diabetes Regulating Genes by Inhibition of Lipopolysaccharide-induced Inflammation in Macrophages

Abstract

Background: It has been recently noticed that type 2 diabetes (T2D), one of the most common metabolic diseases, causes a chronic low-grade inflammation and activation of the innate immune system that are closely involved in the pathogenesis of T2D. Cordyceps militaris, a traditional medicinal mushroom, produces a component compound, cordycepin (3'-deoxyadenosine). Cordycepin has been known to have many pharmacological activities including immunological stimulating, anti-cancer, and anti-infection activities. The molecular mechanisms of cordycepin in T2D are not clear. In the present study, we tested the role of cordycepin on the anti-diabetic effect and anti-inflammatory cascades in LPS-stimulated RAW 264.7 cells.

Methods: We confirmed the levels of diabetes regulating genes mRNA and protein of cytokines through RT-PCR and western blot analysis and followed by FACS analysis for the surface molecules.

Results: Cordycepin inhibited the production of NO and pro-inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha in LPS-activated macrophages via suppressing protein expression of pro-inflammatory mediators. T2D regulating genes such as 11beta-HSD1 and PPARgamma were decreased as well as expression of co-stimulatory molecules such as ICAM-1 and B7-1/-2 were also decreased with the increment of its concentration. In accordance with suppressed pro-inflammatory cytokine production lead to inhibition of diabetic regulating genes in activated macrophages. Cordycepin suppressed NF-kappaB activation in LPS-activated macrophages.

Conclusion: Based on these observations, cordycepin suppressed T2D regulating genes through the inactivation of NF-kappaB dependent inflammatory responses and suggesting that cordycepin will provide potential use as an immunomodulatory agent for treating immunological diseases.

Keywords: cordycepin; immunomodulator; pro-inflammatory cytokines; type 2 diabetes.

Figure 1

Chemical structure of cordycepin.

Figure 2

Effect of cordycepin on NO production in RAW 264.7 cells. Cells were treated with different concentrations of cordycepin; nitrite concentrations in the culture media were determined using Griess reagent assay. The results are reported as mean±S.D. of 3 independent experiments. ^##p<0.01 vs. cells only based on Student's t-test. ^**p<0.01 vs. cells only based on Student's t-test.

Figure 3

Effect of cordycepin on the expression of T2D regulating genes in RAW 264.7 cells. Levels of 11β-HSD1, RANTES, and PPARγ mRNA in RAW 264.7. Cells were incubated with various concentrations of cordycepin in the presence of LPS (100 ng/mL) for 24 hrs. The mRNA levels of T2D regulating genes were determined by RT-PCR analysis. β-actin was used as a control.

Figure 4

Effect of cordycepin on the expression of pro-inflammatory cytokines (A) and related proteins (B) in RAW 264.7 cells. Levels of IL-1β, IL-6, and TNF-α (A) and i-NOS and COX-2 (B) in RAW 264.7 cells. Cells were incubated with various concentrations of cordycepin in the presence of LPS (100 ng/ml) for 24 hrs. Protein (20µg) from each sample was resolved in 8~12% SDS-PAGE and then analyzed by Western blotting. β-actin was used as a control.

Figure 5

Effects of cordycepin on the expression costimulatory molecule. RAW 264.7 cells were cultured with various concentrations of cordycepin (10, 20, 40µg/ml) in the presence of LPS (100 ng/ml) for 24 hours. The surface ICAM-1 (A), B7-1 (B), and B7-2 (C) molecules were labeled with either anti-ICAM-1, anti-B7-1/-2.

Figure 6

Effect of cordycepin on NF-κB activation. Levels of NF-κB protein in RAW 264.7 cells. Cells were incubated with various concentrations of cordycepin in the presence of LPS (100 ng/ml) overnight. Protein from each sample was resolved in 12% SDS-PAGE and then analyzed by Western blotting. β-actin was used in as a control.