Abstract:Objective To investigate the effects of silencing the adenosine A3 receptor on the cAMP/Epac signaling pathway and the expression of inflammatory molecules in superficial fascia fibroblasts.Methods The fibroblasts were extracted from the superficial fascia of SD rats by tissue block culture. The expressions of vimentin, CD45, FⅧ and AKA were detected by the immunofluorescence assay on cultured cells. Cells pretreated with adenosine were divided into control group, 10 nmol/L group, 100 nmol/L group, 10 μmol/L group and 100 μmol/L group and cultured for 48 hours. To explore the effects of RNA interference of adenosine, cells were divided into the control group, adenosine group, A3R interference group, and empty vector group. The control group was left untreated, and the adenosine group was treated with 10 μmol/L of adenosine and cultured for 48 hours. Cells in the other two groups were transfected with A3R siRNA via 2 μL of Lipofectamine 2000, incubated in the incubator for 6 h, and cultured for another 48 h after replacement with the maintenance solution. The protein level of the adenosine A3 receptor in the cells was detected by Western blotting. The mRNA expressions of cAMP and Epac were detected by quantitative real-time polymerase chain reaction, while the protein expressions of cAMP and Epac were detected by Western blotting. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay, and the expression of intercellular adhesion molecule 1 (ICAM-1) was determined by immunofluorescence.Results The relative protein expression of the adenosine A3 receptor was different among the control group, 10 nmol/L group, 100 nmol/L group, 10 μmol/L group and 100 μmol/L group (P < 0.05). Pretreatment with adenosine upregulated the protein expression of the adenosine A3 receptor in a dose-dependent manner (P < 0.05). There were differences in the relative protein expression of the adenosine A3 receptor among the control group, adenosine group, A3R interference group and empty vector group (P < 0.05). The interference with the adenosine siRNA but not the empty vector downregulated the protein expression of the adenosine A3 receptor. The relative mRNA expressions of cAMP and Epac in the A3R interference group were higher than those in the control group (P < 0.05), and the relative protein expressions of cAMP and Epac in the A3R interference group were also higher than those in the control group (P < 0.05). The relative expressions of IL-6 and TNF-α in the A3R interference group were higher than those in the control group (P < 0.05).Conclusions In superficial fascia fibroblasts, the adenosine A3 receptor reduces the expressions of inflammatory molecules by downregulating the cAMP/Epac signaling pathway.