氯化锂对Aβ1-42引起的星形胶质细胞谷氨酸释放的抑制作用及其对海马神经元损伤的保护作用

Inhibitory effect of lithium chloride on Aβ1-42-induced astrocyte glutamate release and its protective effect on hippocampal neuronal injury

目的:研究氯化锂对β淀粉样蛋白1-42(Aβ1-42)引起的星形胶质细胞(AS)谷氨酸释放的抑制作用,并探讨其对海马神经元损伤的保护作用及机制。方法:原代培养AS及海马神经元,AS分为对照组和Aβ1-42+不同剂量氯化锂组(分别加入0.25、0.50和1.00 mmol∙L-1氯化锂作用2周,加入250 nmol∙L-1 Aβ1-42刺激),采用荧光探针技术检测AS中Ca^2+水平,采用高效液相色谱法检测AS谷氨酸的释放量,Western blotting法检测AS中瞬时受体电位通道蛋白1(TRPC1)、钠-钙交换蛋白1(NCX1)和电压门控钙离子通道(Cav1.2)蛋白表达水平。海马神经元成熟化后分为对照组、Aβ1-42组和Aβ1-42+不同剂量氯化锂组,加入Aβ1-42处理的含AS的条件培养基(ACM)培养并以不含AS的条件培养基为对照,倒置相差显微镜下观察海马神经元突起总长度(TDBL)、一级突起数(PDN)和最大分支级数(MBO),Griess法测定培养液中一氧化氮(NO)释放量。结果:与对照组比较,Aβ1-42+不同剂量氯化锂组AS中Ca^2+水平明显降低(P<0.05),Ca^2+再充功能降低(P<0.05),谷氨酸释放量明显减少(P<0.05),TRPC1蛋白表达水平明显降低(P<0.05),NCX1和Cav1.2蛋白表达水平差异无统计学意义(P>0.05)。与对照组比较,Aβ1-42组海马神经元NO释放量明显增加(P<0.05),TDBL、PDN和MBO明显减少(P<0.01);与Aβ1-42组比较,Aβ1-42+不同剂量氯化锂组海马神经元NO释放量明显减少(P<0.01),TDBL、PDN和MBO明显增加(P<0.05或P<0.01)。加入Aβ1-42处理的不含AS的培养基培养,各组海马神经元NO释放量比较差异均无统计学意义(P>0.05)。结论:氯化锂对Aβ1-42引起的AS中Ca^2+依赖的谷氨酸释放具有抑制作用,该作用机制可能与其下调TRPC1受体表达有关,氯化锂可通过抑制神经元中NO的释放减轻海马神经元的损伤。

Objective:To investigate the inhibitory effect of lithium chloride on amyloidβ1-42(Aβ1-42)-induced astrocyte glutamate release,and to explore its protective effect on hippocampal neuronal injury and mechanism.Methods:The astrocytes(AS)and hippocampal neurons were primarily cultured.The AS were divided into control group and Aβ1-42+different doses(0.25,0.50 and 1.00 mmol∙L-1)of lithium chloride groups.After treated with lithium for 2 weeks,the AS were stimulated with 250 nmol∙L-1 Aβ1-42.The Ca^2+level in AS was detected by fluorescence probe technique,the release amount of glutamate of AS was measured by HPLC method,and Western blotting method was performed to detect the expression levels of transient receptor potential channel 1(TRPC1),Na+/Ca^2+exchanger 1(NCX1)and CACNA1C(Cav1.2)proteins in AS.The cultured hippocampal neurons were divided into control group,Aβ1-42 group and Aβ1-42+different doses of lithium chloride groups.The conditioned medium containing AS or not treated with Aβ1-42 were added,respectively.The total dendrite branch length(TDBL),number of primaryorder dendrite(PDN),maximum branch order(MBO)of hippocampal neurons were observed by inverted phase-contrast microscope.The amount of NO release in culture solution was measured by Griess method.Results:Compared with control group,the levels of Ca^2+in AS in Aβ1-42+different doses of lithium chloride groups were significantly decreased(P<0.05),the Ca^2+influx was reduced(P<0.05),the release amounts of glutamate were decreased significantly(P<0.05),and the expression levels of TRPC1 protein were decreased significantly(P<0.05),but the expression levels of NCX1 and Cav1.2 proteins had no significant differences(P>0.05).Compared with control group,the release amount of NO in hippocampal neurons in Aβ1-42 group was significantly increased(P<0.05),and TDBL,PDN and MBO were significantly decreased(P<0.01).Compared with Aβ1-42 group,the release amounts of NO in hippocampal neurons in Aβ1-42+different doses of lithium chloride groups were decreased(P<0.01),and TDBL,PDN and MBO were significantly increased(P<0.01).The release amounts of NO in hippocampal newrons in various groups had significant difference after cultured with Aβ1-42-treated culture medium without AS(P>0.05).Conclusion:Lithium chloride has inhibitory effect on Aβ1-42-induced Ca^2+-dependent glutamate release in AS,and its mechanism may be related to down-regulation of TRPC1 receptor expression;lithium chloride can alleviate the injury of hippocampal neurons by inhibiting the release of NO in the neurons.