β-淀粉样蛋白对大鼠海马神经细胞钙稳态和内质网钙库的影响

Effect of β-amyloid on calcium homeostasis and endoplasmic reticulum calcium storage of hippocampal neurons in rat

目的研究β淀粉样蛋白(β-amyloid,Aβ)对大鼠海马细胞钙稳态和内质网钙库表达的影响。方法将60只成年清洁级雄性SD大鼠按照体质量相匹配的方法分为正常对照组、假手术组、生理盐水组、Aβ_(25-35)高剂量组(7.5μg/μL)、Aβ中剂量组(5.0μg/μL)和Aβ低剂量组(2.5μg/μL),每组10只。Aβ_(25-35)高、中、低剂量组大鼠按分组分别于海马内微量注射对应剂量的Aβ_(25-35),每只动物左右侧海马各注射2μL;并设立生理盐水组(注射2μL 0.9%氯化钠溶液)、假手术组(动物做开颅处理,海马内不注射任何液体)与正常对照组(正常饲养,不做任何处理)。实验动物饲养至注射后14 d,观察并记录大鼠行为和状态,记录体质量和食物摄入总量。应用光学显微镜观察海马组织病理改变,应用透射电镜观察大鼠海马细胞内质网病理学改变;应用流式细胞仪技术检测大鼠海马内游离Ca^(2+)的浓度;用荧光定量PCR方法和Western blot方法测早老素(PS)、肌质网钙ATP酶(SERCA)和ryanodine受体(RyR)的mRNA和蛋白表达水平。应用SPSS 25.0软件对实验结果进行统计分析,多组间比较采用重复测量方差分析和单因素方差分析,组间多重比较采用Dunnett和Tukey检验。结果(1)各实验组大鼠体质量采用重复测量方差分析,发现时间主效应显著(F=153.15,P<0.001),组间效应和交互效应不显著(F=1.547,P=0.374;F=1.598,P=0.113)。与对照组比较,Aβ_(25-35)高、中、低剂量组大鼠在注射后7 d、14 d体质量差异无统计学意义(均P>0.05)。Aβ_(25-35)高、中、低剂量组大鼠食物利用率分别为(22.9±4.0)%、(23.0±4.2)%和(22.6±3.2)%,与对照组[(23.7±5.0)%]相比均差异无统计学意义(均P>0.05)。注射后14 d内,Aβ_(25-35)高剂量组大鼠出现萎靡和嗜睡的表现。(2)病理观察结果显示,Aβ_(25-35)高剂量组和中剂量组大鼠海马细胞内质网裂隙扩张、肥大,线粒体肿胀变形。(3)Aβ_(25-35)注射后14 d,高、中、低剂量组大鼠海马内游离钙所产生的荧光强度分别为(820.43±6.89)、(720.12±4.30)和(680.50±4.32),均高于对照组(592.17±3.97)(均P<0.001)。(4)RT-PCR和Western blot实验结果显示,与对照组相比,Aβ_(25-35)高剂量组和中剂量组大鼠海马细胞内PS、SERCA mRNA和蛋白的表达上调(均P<0.05),同时海马细胞内RyR mRNA和蛋白的表达下调(均P<0.05)。结论Aβ_(25-35)在海马组织内沉积,可以破坏海马组织内Ca^(2+)稳态,游离钙及相关蛋白上调,从而发挥神经毒性作用。

ObjectiveTo study the effect ofβ-amyloid(Aβ)on calcium homeostasis and endoplasmic reticulum calcium storage of hippocampal neurons in rats.MethodsA total of 60 adult male SD rats were randomly divided into six groups by body mass matching method,with 10 rats in each group.Three groups were injected Aβ_(25-35) into the hippocampus(2μL per side),and divided into low dose group(2.5μg/μL),medium dose group(5.0μg/μL)and high dose group(7.5μg/μL)respectively,and the other 3 groups were set up as the normal saline group(2μL 0.9%sodium chloride solution),sham-operated group(rats craniotomy without injection)and normal control group(normal feeding without any treatment).The rats were fed until 14 days after operation,and the behavior and state of the rats were observed and recorded,as well as the body weight and total food intake ratio.And the rat hippocampal cells endoplasmic reticulum pathological change were observed by using the electron microscope and light microscope,meanwhile the concentration of intracellular free Ca^(2+)ions was detected by the laser scanning confocal microscope and the expression level of PS,SERCA and RyR mRNA and protein by real-time PCR and Western blot methods respectively.The experimental results were analyzed using SPSS 25.0 software for statistical analysis.Repeated measurement ANOVA and one-way ANOVA were used for multi-group comparison,and Dunnett test and Tukey test were used for further pairwise comparison.Results(1)The body weight of rats in each group was analyzed by repeated measurement ANOVA,and the difference of time effect was statistically significant(F=153.15,P<0.001),but there was no significant difference between the intergroup effects and interaction effects(F=1.547,P=0.374;F=1.598,P=0.113).The body weight of high,medium and low dose Aβ_(25-35) groups at 7,14 days after injection had no significant difference compared with the control group(all P>0.05).The food utilization rates of the high,medium and low doses of Aβ_(25-35) groups were(22.9±4.0)%,(23.0±4.2)%and(22.6±3.2)%,respectively,and there was no significant difference compared with the control group((23.7±5.0)%,P>0.05).Within 14 days after injection,listlessness and lethargy were observed in rats in the high dose Aβ_(25-35) group.(2)Pathological observation results showed that the endoplasmic reticulum of rat hippocampal cells in the high dose and medium dose groups of Aβ_(25-35) was expanded and swelled,and the mitochondria were swollen and deformed.(3)14 days after Aβ_(25-35) injection,the fluorescence intensity of free calcium in hippocampus of rats in high,medium and low dose groups were(820.43±6.89),(720.12±4.30)and(680.50±4.32),respectively,which were all higher than that in the control group(592.17±3.97)(all P<0.001).(4)RT-PCR and Western blot results showed that compared with the control group,high dose and medium dose Aβ_(25-35) injection could up-regulate the expression of PS and SERCA mRNA and protein in hippocampal cells(all P<0.05),while down-regulate the expression of RyR mRNA and protein in hippocampal cells(all P<0.05).ConclusionThe deposition of Aβ_(25-35) in hippocampal tissues can disrupt the homeostasis of calcium ions in hippocampal tissues,and then cause the increase of free calcium and its related proteins,thus playing the neurotoxic role.