Kinase experiment:

RAW264.7 cells are plated in 100-mm dishes and stimulated with lipopolysaccharide (LPS) for 1?h. Cross-linked chromatin are sonicated into DNA fragment of 0.5 to 1.0?kb. For in vitro biochemical experiments, the sonicated cell lysates are incubated with Adenosine 5'-monophosphate monohydrate (5′-AMP) (400?μM) at 37℃ for 30?min. Then chromatin is precleared with 50% salmon sperm DNA-saturated protein A agarose beads and immunoprecipitated with 5?μg of antibody[2].

Cell experiment:

The RAW264.7 cells (a murine macrophage cell line) are maintained in supplemented RPMI 1640 in an atmosphere at 90% humidity containing 5% CO2 at 37℃. At the end of the pre-incubated period, cells are rinsed with PBS and the medium is supplemented with RPMI 1640 without fetal bovine serum. The cells are pretreated with Adenosine 5'-monophosphate monohydrate (5′-AMP) (400?μM) for 30?min[2].

Animal experiment:

Male WT C57BL/6J mice are used at 8 to 10 weeks of age with a body weight of 20 to 25?g in this work. For Adenosine 5'-monophosphate monohydrate (5′-AMP)-pretreated survival experiment, WT mice are randomly divided into two groups (n=15 in each group). Adenosine 5'-monophosphate monohydrate (5mg/20?g body weight i.p.) or PBS is administered to mice. For general experiment, WT mice are randomly divided into different groups (n=5 in each group): (1) vehicle/vehicle group; (2) Adenosine 5'-monophosphate monohydrate/vehicle group. Mice are killed and the blood is collected from the carotid artery. The liver of each mouse is removed immediately and then is kept at -80℃ until analyzed[2].

Adenosine 5'-monophosphate monohydrate is an adenosine A1 receptor agonist. Adenosine A1 receptor[1]

Adenosine 5'-monophosphate monohydrate is an adenosine A1 receptor agonist[1]. Adenosine 5'-monophosphate monohydrate (5′-AMP)-induced cell toxicity is negligible at concentrations of 25 to 400 μM in RAW264.7 cells. Adenosine 5'-monophosphate monohydrate significantly attenuates the mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in RAW264.7 cells. The dose-dependence of TNF-α and IL-6 mRNA show that at concentration of 400 μM, Adenosine 5'-monophosphate monohydrate exhibits the maximum inhibition. Exposure of cells to Adenosine 5'-monophosphate monohydrate significantly reduces recruitment of NF-κB p65 to TNF-α, IL-6, and IL-1β gene promoters[2].

C57BL/6J mice treated with Adenosine 5'-monophosphate monohydrate (5′-AMP) markedly increases hepatic adenosine level. Survival rate in PBS-treated mice (n=15) is 60% (8 h) and 33.3% (24 h), whereas survival rate in Adenosine 5'-monophosphate monohydrate-treated mice (n=15) is 100% (8 h) and 93.3% (24 h). Serum aspartate transaminase (AST) and alanine transaminase (ALT) levels are significantly lowered in the Adenosine 5'-monophosphate monohydrate group than in the vehicle group. The area and extent of necrosis is attenuated and the infiltration of inflammatory cells is reduced in the Adenosine 5'-monophosphate monohydrate group[2].

[1]. Rittiner JE, et al. AMP is an adenosine A1 receptor agonist. J Biol Chem. 2012 Feb 17;287(8):5301-9. [2]. Zhan Y, et al. Adenosine 5'-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice. Cell Death Dis. 2014 Jan 9;5:e985.