逆灸对阿尔茨海默病大鼠海马CA3区Tau蛋白磷酸化及相关蛋白激酶的影响

Effects of pre-moxibustion on expression of phosphorylated Tau protein and related protein kinases in hippocampal CA3region of AD rats

目的:观察逆灸“百会”“肾俞”“足三里”对阿尔茨海默病(AD)大鼠海马CA3区Tau蛋白及相关蛋白激酶糖原合成酶激酶-3β(GSK-3β)、细胞周期蛋白依赖性激酶-5(CDK5)的影响,探讨逆灸防治AD认知障碍的可能作用机制。方法:SD大鼠随机分为正常组、假手术组、模型组和逆灸组,每组9只。逆灸组取“百会”“肾俞”“足三里”予逆灸疗法,15min/次,1次/d,6d为1个疗程,共干预3个疗程。逆灸完成后,采用双侧海马注射1μLβ-淀粉样蛋白25-35聚集液复制AD大鼠模型。Morris水迷宫检测大鼠空间学习记忆能力,电镜观察海马神经细胞超微结构,苏木精-伊红染色法观察大鼠海马组织病理学变化,Western blot法检测大鼠海马CA3区GSK-3β、CDK5、SynapsinⅠ蛋白表达,免疫组织化学法观察大鼠海马CA3区GSK-3β、CDK5、p-Tau阳性表达。结果:与正常组、假手术组比较,模型组大鼠逃避潜伏期显著延长、平台停留时间显著缩短、穿越原平台次数显著减少(P<0.01);海马超微结构观察显示神经元呈不同程度的水肿,可见突触,突触前囊内突触小泡减少,高尔基体水肿扩张、极性消失;海马组织病理学示神经元数量相对减少,神经元皱缩,部分核仁不显,核膜界限模糊,海马周围神经元见空泡变性;海马CA3区GSK-3β、CDK5、p-Tau蛋白表达升高(P<0.01),SynapsinⅠ表达降低(P<0.01)。与模型组比较,逆灸组大鼠逃避潜伏期显著缩短、平台停留时间显著延长、穿越原平台次数显著增加(P<0.01);海马神经元线粒体空泡变性,管状嵴断裂消失,高尔基体和粗面内质网结构尚正常,髓鞘板层松散,周期间线变大,部分轴索尚存在变性,可见突触,突触前囊内突触小泡减少;神经元结构基本恢复正常;海马CA3区GSK-3β、CDK5、p-Tau蛋白表达降低(P<0.01),SynapsinⅠ表达升高(P<0.05)。结论:逆灸大鼠“百会”“肾俞”“足三里”穴在一定程度上发挥了“未病先防”作用,减缓AD大鼠认知障碍发生发展,其作用机制可能与抑制Tau蛋白过度磷酸化及降低相关蛋白激酶表达有关。

Objective To observe the effect of pre-moxibustion at“Baihui”(GV20),“Shenshu”(BL23)and“Zusanli”(ST36)on expression of Tau protein and related protein kinases as glycogen synthase kinase-3β(GSK-3β),etc.in the hippocampal CA3region of Alzheimer’s disease(AD)rats,so as to explore its mechanism underlying prevention and treatment of AD cognitive impairment.Methods Male SD rats were randomly divided into 4groups:normal control,sham operation,model and pre-moxibustion,with 9rats in each group.Rats of the pre-moxibustion group received moxibustion of GV20,BL23and ST36for 15min,once a day,6days a week for 3weeks.After completion of moxibustion,the AD model was reproduced by injection of amyloid beta-peptide 25-35(Aβ25-35)aggregation solution 1μL(5μg/μL)into the bilateral hippocampus,rats of the sham operation group received injection of the same dose of normal saline into the hippocampus.The spatial learning-memory ability was detected using Morris water maze test,and changes of the ultrastructure of hippocampal neurons were observed using electron microscope,and those of histopathological changes of hippocampus tissue observed using hematoxylin eosin(H.E.)staining.The expression levels of hippocampal GSK-3β,p-Tau,CDK5and Synapsin I proteins were detected by Western blot and immunohistochemistry,respectively.Results No significances were found between the normal control and sham groups in all the indexes(P>0.05).Compared with the control group,the escape latency of place navigation test of Morris water maze test,expression of GSK-3βand CDK5and the immunoactivity of GSK-3β,CDK5and p-Tau were significantly increased(P<0.01),and the residence time in the platform quadrant and the number of platform crossing of spatial prob test and the expression of SynapsinⅠsignificantly reduced in the model group(P<0.01).Following the intervention,the increase of escape latency,expression of GSK-3βand CDK5and the immunoactivity of GSK-3β,CDK5and p-Tau,and the decrease of residence time in the platform quadrant,number of platform crossing and the expression of SynapsinⅠwere reversed in the pre-moxibustion group(P<0.05,P<0.01).Outcomes of ultrastructure and histopathological observations respectively showed edema of hippocampal nerve cells at varying degrees,moderate edema of the cytoplasma,chromatin condensation at the edge of the nucleus,partial mitochondrial vacuolelike degeneration,fracture of tubular crest,edema and expansion of Golgi body,disappearance of polarity,fracture of the rough endoplasmic reticulum,degeneration of ribosome and partial myelin axon and reduced synaptic vesicles in the presynaptic capsule;and reduced number of neurons with shrank body,disappearance of nucleolus and blurred nuclear boundary and vacuole-like degeneration in some of them in the model group,which were relatively milder in the pre-moxibustion group.Conclusion Premoxibustion at GV20,BL23and ST36plays a role in slowing down the occurrence and development of cognitive impairment in AD rats,which may be related to its functions in inhibiting tau protein hyperphosphorylation and reducing the expression of some related protein kinases in the hippocampus.