四君子汤对大鼠学习记忆能力的影响机制

Influence mechanism of Sijunzi Decoction on learning and memory ability of rats

目的研究四君子汤对大鼠学习记忆能力、海马组织P物质(SP)、一氧化氮合酶(NOS)含量和C-fos基因和蛋白表达水平的影响。方法将Wistar大鼠用综合因素法复制脾虚模型(中药干预后,继续造模21 d)。按照随机数字表法将成模大鼠分为5组:模型组、空白对照组和高、中、低3个剂量实验组,每组10只;另取10只正常大鼠作为正常组。造模21 d后,高、中、低3个剂量实验组分别给予24,12,6 g·kg^-1四君子汤药剂灌胃。干预21 d后,收集海马组织。用酶联免疫吸附法检测各组大鼠海马组织中SP、NOS含量,实时荧光定量-PCR法和蛋白质印迹法检测海马组织C-fos基因及蛋白表达情况。结果正常组、模型组、空白对照和高、中、低3个剂量实验组的大鼠学习能力(逃避潜伏期)分别为(40.38±4.96),(85.25±5.92),(81.63±8.61),(78.25±5.28),(81.00±4.21)和(82.88±5.69)s;这6组大鼠的记忆能力(穿越平台次数)分别为(6.50±1.60),(1.38±1.06),(1.50±0.93),(5.13±1.13)和(2.75±1.04),(2.00±0.76)次;这6组大鼠的SP含量分别为(2.53±0.16),(2.16±0.09),(2.20±0.07),(2.47±0.11),(2.26±0.09)和(2.23±0.14)ng·g^-1;这6组大鼠的NOS含量分别为(6.75±0.18),(9.30±0.39),(9.21±0.24),(7.57±0.43),(8.87±0.45)和(9.07±0.33)ng·g^-1;这6组大鼠的C-fos基因表达量(2-△△Ct值)分别为1.00±0.00,1.49±0.16,1.34±0.10,1.23±0.05,1.25±0.06和1.26±0.06;这6组大鼠的C-fos蛋白表达量(灰度值)分别为0.45±0.09,0.78±0.18,0.72±0.20,0.48±0.12,0.56±0.05和0.63±0.07。上述指标:模型组与正常组比较,差异均有统计学意义(均P<0.01);高剂量实验组与模型组比较,差异均有统计学意义(P<0.05,P<0.01)。结论四君子汤可通过调控细胞因子SP及NOS异常分泌,进而下调C-fos基因和蛋白表达水平,从而发挥促进脾虚大鼠学习记忆能力的作用。

Objective To study the effect of Sijunzi Decoction on learning and memory ability,substance P(SP),nitric oxide synthetase(NOS)content and C-fos gene and protein expression in hippocampus of rats.Methods Wistar rats were used to duplicate spleen deficiency model by comprehensive factor method(21 d after the intervention of traditional Chinese medicine).Rats were randomly divided into five groups according random number table:model group,blank control group,and high,medium and low dose experimental groups,10 rats in each group;another 10 normal rats were taken as the normal group.While rats in the high,medium and low dose experimental groups were given 2412,6 g·kg^-1 Sijunzi Decoction by gavage.The hippocampus was collected 21 d after intervention.The contents of SP and NOS in hippocampus were detected by enzyme-linked immunosorbent assay.The expression of C-fos gene and protein in hippocampus was detected by real-time fluorescence quantitative PCR and Western blot,respectively.Results The escape latency in normal group,model group,blank control group and high,middle,low dose experimental groups were(40.38±4.96),(85.25±5.92),(81.63±8.61),(78.25±5.28),(81.00±4.21)and(82.88±5.69)s,respectively;the times of crossing platform in the six groups were(6.50±1.60),(1.38±1.06),(1.50±0.93),(5.13±1.13)and(2.75±1.04),(2.00±0.76)sequence,respectively;the contents of SP in the six groups were(2.53±0.16),(2.16±0.09),(2.20±0.07),(2.47±0.11),(2.26±0.09)and(2.23±0.14)ng·g^-1,respectively;the contents of NOS in the six groups were(6.75±0.18),(9.30±0.39),(9.21±0.24),(7.57±0.43),(8.87±0.45)and(9.07±0.33)ng·g^-1,respectively;the expression of C-fos gene(2-△△Ct)in the six groups were 1.00±0.00,1.49±0.16,1.34±0.10,1.23±0.05,1.25±0.06 and 1.26±0.06,respectively;the expression of C-fos protein in the six groups were 0.45±0.09,0.78±0.18,0.72±0.20,0.48±0.12,0.56±0.05 and 0.63±0.07,respectively.Comparison between model group and normal group,the difference of the factors were significantly(all P<0.01).Comparison between high dose experimental group and model group,the difference of the factors were significantly(P<0.05,P<0.01).Conclusion Sijunzi Decoction can regulate the abnormal secretion of SP and NOS,and then down regulate the expression of C-fos gene and protein,so as to promote the learning and memory ability of spleen deficiency rats.