PDGFR-β/TGF-β/Smad2/3信号通路调控阿尔茨海默病血脑屏障完整性和学习记忆能力的分子机制研究

Regulatory Mechanism of Blood-brain Barrier Integrity and Learning and Memory Ability in Alzheimer's Disease via PDGFR-β/TGF-β/Smad2/3 Signal Pathway

目的:分析PDGFR-β/TGF-β/Smad2/3信号通路调控阿尔茨海默病(AD)血脑屏障(BBB)完整性和学习记忆能力的分子机制。方法:利用APP/PS1转基因AD小鼠模型,通过水迷路及觅食试验分析学习记忆能力;荧光免疫组织化学法检测海马区血管周细胞增殖率(ki67/desmin)、周细胞覆盖率(desmin/lectin);Western blot检测海马区血管周细胞TGF-βR1及其下游信号通路分子、紧密连接(TJs)蛋白的表达水平。经过外源性PDGF-BB脑室内注射和/或TGF-βR1激酶抑制剂SB431542腹腔内注射预处理后,分别进行上述分析。构建AD体外BBB模型,经过PDGF-BB和/或SB431542作用后,进行异硫氰酸荧光素-牛血清白蛋白(FITC-BSA)渗透性和跨细胞电阻检测。结果:与对照组相比,APP/PS1小鼠经过虚拟平台次数明显减少,达到终点所需时间明显延长(水迷路训练试验),投食区正确选择率明显下降(觅食训练试验);海马区desmin/lectin阳性细胞比例明显下降;TGF-βR1、p-Smad2、p-Smad3蛋白表达水平明显升高;TJs蛋白表达水平明显下降。外源性PDGF-BB可使APP/PS1小鼠经过虚拟平台次数明显增加、达到终点所需时间明显缩短(水迷路正式试验第28天)、投食区正确选择率明显提高(觅食正式试验第28天);海马区desmin/lectin阳性细胞比例明显增加;使TGF-βR1、p-Smad2、p-Smad3蛋白表达水平明显升高;使TJs蛋白表达水平明显升高。SB431542则可部分抑制上述作用。体外试验证明:外源性PDGF-BB可明显降低AD模型组的最终渗透系数,提高24 h时相对TEER值;SB431542则可部分抑制上述作用。结论:PDGFR-β/TGF-β/Smad2/3信号通路可通过诱导周细胞分化、覆盖,提高内皮细胞TJs的表达,调控AD血脑屏障完整性,以促进学习记忆能力恢复。

Objective:To investigate the molecular regulatory mechanism of integrity of blood-brain barrier(BBB)and learning and memory ability in Alzheimer's disease(AD)via PDGFR-β/TGF-β/Smad2/3 signaling pathway.Methods:Using the APP/PS1 transgenic mouse model of AD,the learning and memory abilities of mice were analyzed through water maze test and food search test.The proliferation rate and pericyte coverage rate in hippocampus were measured.The level of TGF-βR1 and its downstream signal pathway molecules,and the levels of tight junctions(TJs)were analyzed.After intraventricular microinjection with exogenous PDGF-BB and/or intraperitoneal injection with SB431542,the transgenic mice were analyzed by the above methods.After PDGF-BB and/or SB431542 treatment,permeability and transcellular resistance were tested in the BBB model in vitro.Results:Compared with the control group,APP/PS1 mice showed the learning and memory disability in the water maze test and food search test,the less proportion of desmin/lectin positive cells in the hippocampus,the lower levels of TGF-βR1,p-Smad2 and p-Smad3 protein,and the lower levels of TJs.Furthermore,exogenous PDGF-BB could significantly enhance the learning and memory ability,increase the pericyte coverage rate,raise the expression levels of TGF-βR1,p-Smad2 and p-Smad3 protein,and heighten the expression levels of TJs proteins.In contrast,SB431542 could partially inhibit the above effects of PDGF-BB treatment.In vitro tests showed that exogenous PDGF-BB could significantly reduce the final permeability coefficient of AD model group and increase the relative TEER value at 24 h;SB431542 can partially inhibit the above effects.Conclusion:PDGFR-β/TGF-β/Smad2/3 signal pathway may play a vital role in regulating the integrity of blood-brain barrier and the recovery of learning and memory ability in AD.