电针命门穴对FMR1基因敲除小鼠自主行为学及海马CREB蛋白表达的影响

Effects of Electroacupuncturing Mingmen(GV4) on Autonomous Behavior and Hippocampus CREB Expression of FMR1 Knockout Mice

目的观察针刺督脉命门穴对FMR1基因敲除(KO)小鼠大脑海马区环磷腺苷效应元件结合蛋白(CREB)及其磷酸化CREB(p-CREB)蛋白表达的影响,并探讨其作用机制。方法选取适龄的FMR1基因敲除小鼠,采用聚合酶链式反应(PCR)鉴定小鼠基因表型;采用随机数表法将24只基因型为纯合子的小鼠分为空白组、非经非穴组和命门组,每组各8只。空白组在相同时间、相同条件下仅模拟抓取动作。命门组选取小鼠命门穴,非经非穴组选取小鼠尾根与肛门之间凹陷旁开约1 cm的部位,均于每日固定时间采用由0.5寸毫针自制而成的双极连体针连接电针仪进行干预,电针刺激频率2Hz,强度2mA,连续波,每日30min,连续干预14d;干预后连续两天于同一时间进行自主活动行为学测试,并采用免疫组化法及Western blot法分析海马CREB及其p-CREB蛋白表达量。结果自主行为学测试结果显示:与空白组比较,命门组活动次数和站立次数显著降低(P<0.05);免疫组化结果显示:命门组CREB和p-CREB蛋白的阳性平均光密度表达较空白组显著性升高(P<0.01,P<0.05),命门组p-CREB蛋白的阳性平均光密度表达较非经非穴组显著性升高(P<0.05);Western blot结果显示:各组CREB蛋白表达量比较无统计学意义(P>0.05),非经非穴组与命门组较空白组p-CREB蛋白表达量显著升高(P<0.05,P<0.01)。结论电针命门穴对FMR1基因敲除小鼠自主活动站立行为有影响,且能促进海马CREB、p-CREB蛋白的表达。

Objective: To observe the effects of electroacupuncture(EA) at Mingmen(GV4) on autonomous behavior and CREB and p-CREB expression in hippocampus of fragile X mental retardation 1(FMR1) gene knockout(KO) mice, and explore the mechanism of EA at GV4 on FMR1 KO mice. Methods: Fragile X-1 deficiency mice on appropriate age were chosen and the genotype were identified by polymerase chain reaction(PCR). 24 chose mice were randomly divided into blank group,non-acupoint EA group and GV4 acupoint group, with 8 mice in each group. The GV4 acupoint was selected in the GV4 acupoint EA group, and in the non-acupoints EA group, the area about 1 cm adjacent to the pit between the tail root and the anus was selected. The blank group only simulated grabbing at the same time and under the same conditions, and the rest of the groups took EA intervention by HANS electroacupuncture therapeutic apparatus and chosen the 1 inch acupuncture to make the double pole needle to puncture,the puncture were connected electroacupuncture therapeutic apparatus acupuncturing with continuewave, 2 Hz, 2 m A, 30 min. All groups of mice were treated with daily at fixed time, once a day for 14 days. The experiment mice were tested by autonomic activity during two days after the interesting treatment. And the expression of the related proteins of CREB and p-CREB was detected by immunohistochemistry and western blot. Results: The results of autonomous behavior test showed that compared with the blank group, the activities and standing times of GV4 acupoint EA group decreased significantly(P<0.05). The expression of CREB and p-CREB protein by immunohistochemistry in GV4 acupoint EA group was significantly higher than that of blank group(P<0.01, P<0.05). The expression of p-CREB protein by immunohistochemistry in GV4 acupoint EA group was significantly higher than that of the non-acupoints EA group(P<0.05). There was no statistical significance in the expression of CREB protein in each group by western blot(P>0.05).The expression of p-CREB protein by western blot in GV4 acupoint EA group and the non-acupoints EA group were significantly higher than that of the blank group(P<0.05, P<0.01). Conclusion: EA at the Mingmen(GV4) acupoint could adjust the independent activity of FMR1 gene KO mice and could improve the expression of CREB and p-CREB proteins in hippocampus region of FMR1 gene knockout mice.