Aβ_(1~42)诱导K_(ATP)亚基SUR1/Kir6.2蛋白表达的信号转导通路研究

The effect of signal pathways on the neural K_(ATP) subunits SUR1/Kir6.2 expression induced by Aβ_(1-42)

目的分析探讨β-淀粉样蛋白_(1~42)(Aβ_(1~42))诱导ATP敏感性钾离子通道(K_(ATP))亚基SUR1/Kir6.2蛋白表达上调的潜在信号转导机制。方法采用免疫细胞化学法鉴定原代培养的大鼠皮质和海马神经元,实验组分别加入0.50μmol/L核因子-κB(NF-κB)抑制剂SN50、2μmol/L p38丝裂原激活蛋白激酶(MAPK)抑制剂SB203580或者2μmol/L蛋白激酶C(PKC)抑制剂CTC,于30 min后分别加入2μmol/L Aβ_(1~42),培养72 h后采用Western blotting法检测K_(ATP)亚基SUR1/Kir6.2蛋白的相对表达量。结果(1)NF-κB抑制剂SN50:SN50+Aβ_(1~42)组SUR1/Kir6.2蛋白表达量低于对照组(t=6.237,P=0.010;t=7.136,P=0.004)和Aβ_(1~42)组(t=12.620,P=0.000;t=18.580,P=0.000),SN50组SUR1/Kir6.2蛋白相对表达量低于对照组(t=12.240,P=0.000;t=4.906,P=0.034)、Aβ_(1~42)组(t=18.620,P=0.000;t=16.350,P=0.000)和SN50+Aβ_(1~42)组(t=6.002,P=0.012)。(2)p38 MAPK抑制剂SB203580:SB203580+Aβ_(1~42)组SUR1/Kir6.2蛋白相对表达量低于Aβ_(1~42)组(t=13.010,P=0.000;t=8.506,P=0.001),仅Kir6.2蛋白相对表达量高于对照组(t=7.537,P=0.003);SB203580组SUR1/Kir6.2蛋白相对表达量低于对照组(t=8.089,P=0.002;t=19.380,P=0.000)、Aβ_(1~42)组(t=18.870,P=0.000;t=35.430,P=0.000)和SB203580+Aβ_(1~42)组(t=5.869,P=0.014;t=26.920,P=0.000)。(3)PKC抑制剂CTC:CTC+Aβ_(1~42)组SUR1蛋白相对表达量高于对照组(t=4.756,P=0.040)、但低于Aβ_(1~42)组(t=15.170,P=0.000),CTC组SUR1蛋白相对表达量低于对照组(t=24.000,P=0.000)、Aβ_(1~42)组(t=43.930,P=0.000)和CTC+Aβ_(1~42)组(t=28.760,P=0.000);CTC+Aβ_(1~42)组Kir6.2蛋白相对表达量低于对照组(t=15.000,P=0.000)和Aβ_(1~42)组(t=24.140,P=0.000),CTC组Kir6.2蛋白相对表达量低于对照组(t=9.429,P=0.001)和Aβ_(1~42)组(t=18.570,P=0.000)、但高于CTC+Aβ_(1~42)组(t=5.571,P=0.018)。结论NF-κB、p38 MAPK和PKC信号转导通路参与Aβ_(1~42)上调大鼠皮质和海马神经元K_(ATP)亚基SUR1/Kir6.2蛋白的表达。

Objective To investigate the potential signal transduction mechanism of β-amyloid protein _(1-42) (Aβ_(1-42)) induced up -regulation of ATP sensitive potassium channel K_(ATP) subunit SUR1/Kir6.2 proteins expression. Methods The primary cultured cortical and hippocampal neurons of rats were identified by immunocytochemistry. The experimental groups were added with 0.50 μmol/L nuclear factor-κB (NF-κB) inhibitor SN50, 2 μmol/L p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or 2 μmol/L protein kinease C (PKC) inhibitor CTC, 2 μmol/L Aβ_(1-42) (30 min later), respectively. Western blotting was used to detect the protein expression of K_(ATP) subunit SUR1/Kir6.2 after 72 h of culture. Results 1) NF-κB inhibitor SN50:SUR1/Kir6.2 proteins of K_(ATP) subunit in SN50 + Aβ_(1-42) group were lower than that in control group (t=6.237, P=0.010;t=7.136, P=0.004) and Aβ_(1-42) group (t=12.620, P=0.000;t=18.580, P=0.000), but the SUR1 protein expression of K_(ATP) subunit in SN50 + Aβ_(1-42) group was higher than that in SN50 group (t=6.002, P=0.012). SUR1/Kir6.2 proteins of K_(ATP) subunit in SN50 group were lower than that in control group (t=12.240, P=0.000;t=4.906, P=0.034) and Aβ_(1-42) group (t=18.620, P=0.000;t=16.350, P=0.000). 2) p38 MAPK inhibitor SB203580:the proteins expression of K_(ATP) subunit SUR1/Kir6.2 in SB203580 + Aβ_(1-42) group were lower than that in Aβ_(1-42) group (t=13.010, P=0.000;t=8.506, P=0.001), but higher than that in SB203580 group (t=5.869, P=0.014;t=26.920, P=0.000), but the expression of K_(ATP) subunit Kir6.2 protein in SB203580 + Aβ_(1-42) group was higher than that in control group (t=7.537, P=0.003). The SUR1/Kir6.2 proteins expression of K_(ATP) subunit in SB203580 group were lower than that in control group (t=8.089, P=0.002;t=19.380, P=0.003) and Aβ_(1-42) group (t=18.870, P=0.000;t=35.430, P=0.000). 3) PKC inhibitor CTC:the SUR1 protein expression of K_(ATP) subunit in CTC + Aβ_(1-42) group was higher than that in control group (t=4.756, P=0.040) and in CTC group (t=28.760, P=0.000), but lower than that in Aβ_(1-42) group (t=15.170, P=0.000). The SUR1 protein expression of K_(ATP) subunit in CTC group was lower than that in control group (t=24.000, P=0.000), Aβ_(1-42) group (t=43.930, P=0.000) and CTC + Aβ_(1-42) group. The protein expression of K_(ATP) subunit Kir6.2 in CTC + Aβ_(1-42) group was lower than that in control group (t=15.000, P=0.000), Aβ_(1-42) group (t=24.140, P=0.000) and CTC group (t=5.571, P=0.018). The expression of K_(ATP) subunit Kir6.2 protein in CTC group was lower than that in control group (t=9.429, P=0.001) and in Aβ_(1-42) group (t=18.570, P=0.000). Conclusions NF-κB, p38 MAPK and PKC signaling pathways are involved in Aβ_(1-42) up-regulation of SUR1/Kir6.2 proteins expression of K ATP subunit in cortical and hippocampal neurons of rats.