Korean J Physiol Pharmacol.  2018 Jul;22(4):379-389. 10.4196/kjpp.2018.22.4.379.

Adenine attenuates lipopolysaccharide-induced inflammatory reactions

• Silwal P
• Lim K
• Heo JY
• Park JI
• Namgung U
• Park SK

• KMID: 2414262
• DOI: http://doi.org/10.4196/kjpp.2018.22.4.379

Abstract

A nucleobase adenine is a fundamental component of nucleic acids and adenine nucleotides. Various biological roles of adenine have been discovered. It is not produced from degradation of adenine nucleotides in mammals but produced mainly during polyamine synthesis by dividing cells. Anti-inflammatory roles of adenine have been supported in IgE-mediated allergic reactions, immunological functions of lymphocytes and dextran sodium sulfate-induced colitis. However adenine effects on Toll-like receptor 4 (TLR4)-mediated inflammation by lipopolysaccharide (LPS), a cell wall component of Gram negative bacteria, is not examined. Here we investigated anti-inflammatory roles of adenine in LPS-stimulated immune cells, including a macrophage cell line RAW264.7 and bone marrow derived mast cells (BMMCs) and peritoneal cells in mice. In RAW264.7 cells stimulated with LPS, adenine inhibited production of pro-inflammatory cytokines TNF-α and IL-6 and inflammatory lipid mediators, prostaglandin E₂ and leukotriene B₄. Adenine impeded signaling pathways eliciting production of these inflammatory mediators. It suppressed IκB phosphorylation, nuclear translocation of nuclear factor κB (NF-κB), phosphorylation of Akt and mitogen activated protein kinases (MAPKs) JNK and ERK. Although adenine raised cellular AMP which could activate AMP-dependent protein kinase (AMPK), the enzyme activity was not enhanced. In BMMCs, adenine inhibited the LPS-induced production of TNF-α, IL-6 and IL-13 and also hindered phosphorylation of NF-κB and Akt. In peritoneal cavity, adenine suppressed the LPS-induced production of TNF-α and IL-6 by peritoneal cells in mice. These results show that adenine attenuates the LPS-induced inflammatory reactions.

Keyword

Adenine; Inflammation; Lipopolysaccharide; Macrophages; Mast cells; Toll-like receptor 4

MeSH Terms

Adenine Nucleotides
Adenine*
Animals
Bone Marrow
Cell Line
Cell Wall
Colitis
Cytokines
Dextrans
Gram-Negative Bacteria
Hypersensitivity
Inflammation
Interleukin-13
Interleukin-6
Lymphocytes
Macrophages
Mammals
Mast Cells
Mice
Mitogen-Activated Protein Kinases
Nucleic Acids
Peritoneal Cavity
Phosphorylation
Protein Kinases
Sodium
Toll-Like Receptor 4
Adenine
Adenine Nucleotides
Cytokines
Dextrans
Interleukin-13
Interleukin-6
Mitogen-Activated Protein Kinases
Nucleic Acids
Protein Kinases
Sodium
Toll-Like Receptor 4

Figure

• 

  Fig. 1 Adenine inhibits secretion of TNF-α and IL-6 from LPS-stimulated RAW264.7 cells.(A–D) Secretion of TNF-α (A, C) and IL-6 (B, D) into the culture medium was determined 6 h after LPS (100 ng/ml) stimulation from RAW264.7 cells in complete culture media. Adenine (A, B, E) or hypoxanthine (C, D, E) was treated for 30 min before LPS stimulation. (E) The cell viability was measured with MTT assay. Significant difference against a control group in each time point of LPS-stimulation is indicated as *(p<0.05).

• 

  Fig. 2 Adenine attenuates LPS-induced eicosanoids and COX-2 productions from RAW264.7 cells.(A, B) RAW264.7 cells pretreated with adenine for 30 min were stimulated with LPS (100 ng/ml) for 6 h. Amounts of PGE2 and LTB4 in the culture supernatant were measured. (C) Western blot analysis of COX-2 in RAW264.7 cells pretreated with adenine and simulated with LPS for 6 h. Graph shows densitometric analysis of the blots. Significant difference against a control group is indicated as *(p<0.05).

• 

  Fig. 3 Adenine suppresses LPS-induced phosphorylation and nuclear translocation of NF-κB p65.(A) RAW264.7 cells were pretreated with adenine for 30 min and stimulated with LPS (100 ng/ml) for indicated time. The cell lysates were analyzed by immunoblotting to detect indicated proteins. Graphs show the densitometric analysis of blots represented as phosphorylated-IκB/NF-κB and phosphorylated-NF-κB/NF-κB. (B) Treated cells with LPS and/or adenine as described in section A were fractionated into cytosolic and nuclear parts and Western blotting was performed to detect the indicated proteins. Graphs show the densitometric analysis of blots represented as p65/actin for cytosolic fraction and p65/PARP (poly (ADP-ribose) polymerase) for nuclear fraction. Significant difference against a control group is indicated as *(p<0.05).

• 

  Fig. 4 Adenine suppresses LPS-induced activation of MAPKs in RAW264.7 cells.RAW264.7 cells pre-treated with adenine (1 mM) for 30 min were stimulated with LPS (100 ng/ml) for indicated time. Cell lysates were used for Western blotting to detect the indicated proteins. Graphs show the densitometric analysis of the blots. Significant difference against a control group is indicated as *(p<0.05).

• 

  Fig. 5 Adenine suppresses the activation of Akt in LPS-induced RAW264.7 cells.RAW264.7 cells pre-treated with adenine (1 mM) for 30 min were stimulated with LPS (100 ng/ml) for indicated time. Cell lysates were used for Western blotting to detect the phosphorylation of Akt (Ser473 and Thr308). Graphs show the densitometric analysis of the blots. Significant difference against a control group is indicated as *(p<0.05).

• 

  Fig. 6 Effects of adenine on cellular AMP level and AMPK phosphorylation.(A) RAW264.7 cells treated with indicated doses of adenine for different time period in complete medium were harvested and AMP-Glo assay was performed to measure the cellular AMP level. Data are normalized to equal protein level in each sample. (B) RAW264.7 cells treated as above were subjected to immunoblot analysis to detect indicated proteins. Significant difference against a control group in each time point is indicated as *(p<0.05).

• 

  Fig. 7 Adenine inhibits LPS-induced activation of mast cells.(A–C) BMMCs pre-treated with adenine for 30 min were stimulated with LPS for 6 h to measure the secretion of TNF-α (A), IL-6 (B) and IL-13 (C) into culture media using ELISA. (D) Western blotting was performed with cell lysates from BMMCs treated with adenine and/or LPS for indicated time to detect indicated proteins. Significant difference against a control group is indicated as *(p<0.05).

• 

  Fig. 8 Adenine suppresses LPS-induced production of pro-inflammatory cytokines in the peritoneal cavity.BALB/c mice were injected into peritoneum with LPS (1 mg/kg) 1 h after injection of adenine (10 mg/kg) or PBS. Peritoneal lavages were collected 1 h after LPS administration to measure the levels of TNF-α (A) and IL-6 (B). Significant difference against a control group is indicated as *(p<0.05).

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