SMAD6在Aβ诱导的神经毒性病变中的表达变化

The change of SMAD6 expression in Aβ-induced neurotoxicity

目的探讨母抗Dpp小同源物6(small mothers against decapentaplegic homolog 6,SMAD6)在β淀粉样蛋白(amyloidβ,Aβ)诱导的神经毒性病变中的表达变化。方法取孕15~17d的SD大鼠的胎鼠,进行海马神经元原代培养,经不同浓度(10、15、20μmol/L)Aβ25-35毒性片段处理后,通过定量PCR技术检测SMAD6mRNA表达水平。取2月龄SD雄性大鼠12只,随机分为Aβ注射组和空白对照组,每组6只。采用双侧基底前脑注射Aβ1-40建立大鼠痴呆模型,通过免疫组化及免疫印迹检测基底前脑及海马区SMAD6蛋白表达水平。结果不同浓度Aβ25-35处理组海马神经元内SMAD6mRNA表达均较对照组减少(F=602.1,P<0.01)。免疫组化结果显示,与对照组比较,Aβ1-40注射组大鼠基底前脑区SMAD6蛋白表达减少(68103±1520比96036±1804;t=20.51,P<0.01),而海马区其表达增多(96441±1852比55039±1528;t=29.87,P<0.01);免疫印迹结果显示,与对照组比较,Aβ1-40注射组大鼠基底前脑区SMAD6蛋白表达减少(0.1042±0.0067比1.000±0.0986;t=15.69,P<0.01),而海马区其表达增多(12.5525±2.6803比1.000±0.0135;t=7.465,P<0.01)。结论 Aβ可直接下调神经元内SMAD6mRNA转录及表达,不同脑区内SMAD6蛋白表达可能受负反馈调节机制影响。

Objective To investigate the change of small mothers against decapentaplegic homolog 6（SMAD6）expression in amyloidβ（Aβ）-induced neurotoxicity.Methods 15-17 days of pregnant SD mice were used for the experiment.Hippocampal neurons from the brain of fetal rats were treated with different concentrations of Aβ25-35（10,15,20μmol/L）for 24 h.The mRNA level of SMAD6 in rat hippocampal neurons after Aβ25-35 treatment was assayed through Realtime PCR.Twelve SD rats with the age of 2months old and the weight of 200~220g,were divided into an Aβinjection group and a control group.In vivo,Aβ1-40 was bilaterally injected into the basal forebrain to simulate neuropathological processes of Alzheimer’s disease（AD）,and the levels of SMAD6 protein were explored in the regions of basal forebrain and hippocampus.Results Result of Realtime PCR showed that the levels of SMAD6 mRNA in the Aβgroup significantly decreased compared with the control group（F=602.1,P〈0.01）.Immunohistochemisty results showed that SMAD6 protein was significantly decreased in the Aβgroup relative to the control group in the basal forebrain（68103±1520 vs.96036±1804;t=20.51,P〈0.01）,while there was an opposite change in the hippocampus（96441±1852 vs.55039±1528;t=29.87,P 〈0.01）.Western blot results showed that SMAD6 protein was significantly decreased in the Aβgroup relative to the control group in the basal forebrain（0.1042±0.0067 vs.1.000±0.0986;t=15.69,P〈0.01）,while there was an opposite change in hippocampus（12.5525±2.6803 vs.1.000±0.0135;t=7.465,P〈0.01）.Conclusions Aβdirectly down-regulates SMAD6 expression,and there may be negative feedback regulation of SMAD6 among different brain regions.