腺苷A3受体对浅筋膜成纤维细胞cAMP/Epac信号通路及炎症因子表达的影响

Role of adenosine A3 receptor in regulating cAMP/Epac signaling pathway and expression of inflammatory molecules in superficial fascia fibroblasts

目的 探讨沉默腺苷A3受体对浅筋膜成纤维细胞cAMP/Epac信号通路及炎症因子表达的影响。方法 选取SD大鼠浅筋膜组织,通过组织块培养法提取成纤维细胞,细胞免疫荧光实验检测Vimentin、CD45、FⅧ、AKA表达,腺苷预处理细胞并分为Control组、10 nmol/L组、100 nmol/L组、10μmol/L组和100μmol/L组,均培养48 h。腺苷siRNA干扰实验将细胞分为对照组、腺苷组、A3R干扰组、空载组。对照组不予任何处理,腺苷组用10μmol/L腺苷培养48 h,其余两组用2μL Lipofectamine 2000转染A3R siRNA,在培养箱孵育6 h后换上维持液继续培养48 h。Western blotting检测细胞腺苷A3受体蛋白的表达,实时荧光定量聚合酶链反应检测cAMP、Epac mRNA表达,Western blotting检测cAMP、Epac蛋白表达,酶联免疫吸附试验检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平,免疫荧光检测细胞间黏附分子-1表达。结果 Control组、10 nmol/L组、100 nmol/L组、10μmol/L组和100μmol/L组A3R蛋白相对表达量比较,差异有统计学意义(P <0.05),腺苷预处理可剂量依赖性地上调A3R蛋白表达。对照组、腺苷组、A3R干扰组、空载组A3R蛋白相对表达量比较,差异有统计学意义(P <0.05),腺苷siRNA干扰可下调A3R蛋白表达,而空载组并无明显影响。A3R干扰组cAMP、Epac m RNA相对表达量高于对照组(P <0.05)。A3R干扰组cAMP、Epac蛋白相对表达量高于对照组(P <0.05)。A3R干扰组IL-6、TNF-α相对表达量高于对照组(P <0.05)。结论 在浅筋膜成纤维细胞中,腺苷A3受体通过下调cAMP/Epac信号通路减少炎症因子的表达。

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,FⅧand 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,10μmol/L group and 100μmol/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 10μmol/L of adenosine and cultured for 48 hours.Cells in the other two groups were transfected with A3R siRNA via 2μL 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,10μmol/L group and 100μmol/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-αin 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.