Ca2+/CaN信号通路在双酚A影响巨噬细胞分泌IL-6中作用

Role of Ca2+/CaN signaling pathway in BPA-induced IL-6 secretion in macrophages

目的探讨Ca2+/CaN信号通路在双酚A影响巨噬细胞分泌白细胞介素6(IL-6)中的作用机制。方法取对数生长期Raw264.7细胞分为对照组、激活剂组、3个双酚A组和抑制剂组。对照组细胞予体积分数为0.10%二甲基亚砜处理;激活剂组细胞予质量浓度为2 mg/L脂多糖处理;3个双酚A组细胞分别予终浓度为1、10、100μmol/L的双酚A处理。另设2个异搏定组和2个他克莫司(FK506)组,分别予终浓度为10、30μmol/L的异搏定或终浓度为250、500 nmol/L的FK506刺激细胞,再予终浓度为100μmol/L的双酚A组处理细胞。分别于培养4、12、24 h时间点收获细胞,采用实时荧光定量聚合酶链式反应检测4、12 h时间点细胞中IL-6、钙调神经磷酸酶(CaN)的mRNA相对表达水平,采用酶联免疫吸附实验检测24 h时间点细胞IL-6和CaN蛋白相对表达水平,采用单管型多功能检测仪检测4 h时间点细胞内钙离子(Ca2+)水平。结果在12 h时间点,IL-6 mRNA相对表达水平在100μmol/L双酚A组细胞分别高于对照组、激活剂组和1、10μmol/L双酚A组(P<0.05),均高于10、30μmol/L异搏定组和250、500 nmol/L FK506组(P<0.05)。CaN mRNA相对表达水平在100μmol/L双酚A组细胞分别高于对照组、激活剂组和1、10μmol/L双酚A组(P<0.05),并高于10、30μmol/L异搏定组(P<0.05)。100μmol/L双酚A组细胞IL-6蛋白相对表达水平分别高于对照组、激活剂组和1、10μmol/L双酚A组(P<0.05);CaN蛋白相对表达水平在100μmol/L双酚A组和10、30μmol/L异搏定组细胞均高于对照组、激活剂组(P<0.05),在10、30μmol/L异搏定组均低于100μmol/L双酚A组(P<0.05)。细胞内Ca2+水平在100μmol/L双酚A组分别高于对照组和激活剂组(P<0.05),在10μmol/L异搏定组低于100μmol/L双酚A组(P<0.05)。结论双酚A可能通过激活Ca2+/CaN信号通路而促进巨噬细胞分泌IL-6。

Objective To investigate the mechanism of calcium ion(Ca2+)/calcineurin(CaN)signaling pathway in bisphenol A(BPA)-induced interleukin-6(IL-6)secretion in macrophages.Methods Raw 264.7 cells in logarithmic growth phase were divided into control group,activator group,BPA low-,medium-and high-dose groups and inhibitor group.The cells in control group were treated with 0.10%dimethyl sulfoxide.The activator group was treated with lipopolysaccharide at mass concentration of 2 mg/L.The 3 BPA groups were treated with BPA at final concentrations of 1,10,or 100μmol/L.Two sets of verapamil or tacrolimus(FK506)groups were given verapamil at the final concentration of 10 or 30μmol/L;or final concentration of FK506 at 250 or 500 nmol/L.Then the cells were treated with final concentration of 100μmol/L BPA.The cells were collected at 4,12,and 24 hours.The mRNA expression of IL-6 and CaN at 4 and 12 hours were detected by real-time fluorescence quantitative polymerase chain reaction.The relative expression of IL-6 and CaN protein was detected at 24 hours by enzyme-linked immunosorbent assay,and the intracellular Ca2+level was detected at 4 hours using a single-tube multi-function detector.Results At 12 hours,the mRNA expression of IL-6 in the 100μmol/L BPA group was higher than that in control group,activator group and the 1 and 10μmol/L BPA groups(P<0.05),and higher than that in the 10 and 30μmol/L verapamil groups,and in the 250 and 500 nmol/L FK506 groups(P<0.05).The mRNA expression of CaN in the 100μmol/L BPA group was higher than that in control group,activator group and 1 and 10μmol/L BPA groups(P<0.05),and higher than in 10 and 30μmol/L verapamil groups(P<0.05).The relative expression of IL-6 protein in the 100μmol/L BPA group was higher than that in control group,activator group and 1 and 10μmol/L BPA groups(P<0.05).The relative expression of CaN protein in 100μmol/L BPA group and 10 and 30μmol/L verapamil groups were higher than that in control group and activator group(P<0.05).The relative expression of CaN protein in the 10 and 30μmol/L verapamil groups were lower than that in the 100μmol/L BPA group(P<0.05).The intracellular Ca2+level in the 100μmol/L BPA group was higher than that in control group and activator group(P<0.05).The intracellular Ca2+level in the 10μmol/L verapamil group was lower than that in the 100μmol/L BPA group(P<0.05).Conclusion BPA might promote the secretion of IL-6 through Ca2+/CaN signaling pathway in macrophages.