核转录因子Κb涉及水通道蛋白-4抗体阳性血清对星形胶质细胞的毒性作用

摘要

Objective To investigate whether NF-κB signal pathway is involved in the toxic effects of AQP4 antibody positive sera to astrocytes in vitro.Methods The purified cerebral cortical astrocytes from newborn Sprague-Dawley rats were used for the experiments. The cultured cells were divided randomly into four groups :a control group ,a pyrrolidinedithiocarbamic acid (PDTC) group ,AQP4 antibody group and an AQP4 antibody + PDTC group. The cells in the control group were given 10% volume ratio of healthy human sera ; cells in the PDTC group were preincubated with PDTC (10 μmol/L ) for 1 h and then treated with healthy human sera ;cells in the AQP4 antibody group were added the same volume of AQP4 antibody-positive sera ; the AQP4 antibody + PDTC group were exposed to AQP4 antibody positive sera after PDTC preincubating. After 12 h of cultivation ,astrocyte viability was tested by M TT assay , the nuclear translocation of NF-κB p65 was detected by immunofluorescense staining ,and the protein expression of NF-κB p65 and phosphorylated p65 (p-p65) were assayed by western blot. Results The translocation of NF-κB p65 from the cytosol into the nucleus were found in the AQP4 antibody group , which was obviously reduced in the AQP4 antibody + PDTC group. The optical density value in the AQP4 antibody + PDTC group and the AQP4 antibody group were 0.4380 ± 0.005 and 0.3897 ± 0.045 (F= 133.355 , P< 0.05) . No statistical difference of the expression levels of NF-κB p65 existed among the groups (P > 0.05) . The AQP4 antibody + PDTC group showed decreased the expression of p-p65 compared with the AQP4 antibody group (F= 10.794 , P < 0.05) . Conclusions AQP4 antibody-positive sera could induce astrocyte injury by activating NF-κB signaling pathway and inhibiting the activity of NF-κB can protected astrocytes against the cytotoxic effect of AQP 4 antibody positive sera. NF-κB signal pathway may be involved in the cytotoxic effect of anti-aquaporin-4 antibody positive sera on astrocytes.%目的 探讨核转录因子 κB(nuclear factor kappa B,NF-κB)在水通道蛋白4(aquaporin-4,AQP4)抗体诱导的星形胶质细胞损伤中的作用.方法 纯化培养新生SD大鼠大脑皮质星形胶质细胞,按随机数字表法分为对照组 、吡咯醛二硫氨基甲酸(PDTC)组 、AQP4抗体阳性血清组(AQP4抗体组)和AQP4抗体+PDTC组,其中对照组加入10%(体积分数)健康人血清,PDTC组给予10μmol/L PDTC预处理1 h后加入等量健康人血清;抗体组加入AQP4抗体阳性血清;AQP4抗体+PDTC组先用PDTC预处理1 h后再加入AQP4抗体阳性血清.细胞培养12 h后采用免疫细胞化学荧光法观察各组NF-κB p65入核情况,MTS比色法检测细胞存活度,免疫印迹法检测细胞内p65蛋白 、磷酸化p65(p-p65)蛋白的表达.结果 AQP4抗体组可见明显NF-κB p65入核,其余三组均未见明显入核;AQP4抗体+PDTC组细胞存活度高于AQP4抗体组(吸光度值分别为0.4380±0.005、0.3897±0.045,F=133.355,P<0.05);各组间NF-κB p65蛋白表达水平比较差异无统计学意义(P>0.05);AQP4抗体+PDTC组p-p65蛋白表达水平较AQP4抗体组明显减少(分别为0.7027±0.020,0.8197±0.027,F=10.794,P<0.05).结论 AQP4抗体阳性血清可能通过激活NF-κB途径促进星形胶质细胞损伤,抑制NF-κB活性对星形胶质细胞具有保护作用.