不同频率电针对阿尔茨海默病小鼠脑内胰岛素信号转导通路的影响

Effect of electroacupuncture at different frequencies on brain insulin signaling transduction pathway in Alzheimer’s disease mice

目的:观察不同频率电针对阿尔茨海默病(AD)小鼠学习记忆功能和脑内胰岛素信号转导通路相关蛋白的影响,探讨电针治疗AD的作用机制。方法:将72只SPF级雄性昆明小鼠随机分为空白组、假手术组、模型组、2 Hz电针组、15 Hz电针组、30 Hz电针组,每组12只。模型组和各电针组均采用左侧脑室注射链脲佐菌素(STZ)溶液(8 mg/kg)的方法制备AD模型;假手术组于左侧脑室注射等体积0.9%氯化钠溶液。造模成功后,各电针组于“百会”“大椎”“肾俞”行相应频率电针干预,每日1次,7次为一疗程,疗程间休息1 d,共干预2个疗程。造模后和干预后,各组小鼠行Morris水迷宫测试;采用免疫组化法和Westernblot法检测小鼠干预后海马胰岛素受体(IR)、胰岛素受体底物-1(IRS-1)、磷脂酰肌醇-3激酶(PI3K)蛋白表达。结果:与空白组比较,模型组、2 Hz电针组、15 Hz电针组、30 Hz电针组造模后逃避潜伏期、首次跨越平台时间均延长(P<0.01),跨越平台次数均减少(P<0.01)。干预后,与空白组比较,模型组逃避潜伏期、首次跨越平台时间均延长(P<0.01),跨越平台次数减少(P<0.01)。与模型组比较,2 Hz电针组、15 Hz电针组、30 Hz电针组干预后逃避潜伏期、首次跨越平台时间均缩短(P<0.01),跨越平台次数增加(P<0.05,P<0.01)。与2 Hz电针组比较,15 Hz电针组和30 Hz电针组干预后逃避潜伏期、首次跨越平台时间均缩短(P<0.01,P<0.05),跨越平台次数增加(P<0.05)。与空白组比较,模型组IR、IRS-1、PI3K蛋白表达降低(P<0.01,P<0.05);与模型组比较,15Hz电针组和30Hz电针组IR、IRS-1、PI3K蛋白表达升高(P<0.05,P<0.01);与2 Hz电针组比较,15 Hz电针组和30 Hz电针组IR、IRS-1、PI3K蛋白表达增加(P<0.05)。结论:电针可能通过调节脑内胰岛素信号转导通路改善AD小鼠的学习记忆能力,且15 Hz和30 Hz电针频率效果整体优于2 Hz。

Objective To observe the effect of electroacupuncture(EA) at different frequencies on learning and memory functions, as well as the relevant proteins of brain insulin signal transduction pathway in Alzheimer’s disease(AD) mice and explore the effect mechanism of EA in treatment of AD. Methods Seventy-two SPF Kunming male mice were randomized into a blank group, a sham-operation group, a model group, a 2 Hz EA group, a 15 Hz EA group and a 30 Hz EA group, 12mice in each one. In the model group and each EA group, AD model were established by the injection with streptozotocin(ST2) solution(8 mg/kg) into the left lateral ventricles. In the sham-operation group, 0.9% sodium chloride solution of the same volume was injected into the left lateral ventricles. After successful modeling, in each EA group, EA was applied at "Baihui"(GV 20), "Dazhui"(GV 14) and "Shenshu"(BL 23) with corresponding frequencies, once daily. One course of EA intervention consisted of 7 treatments and 2 courses were given totally at interval of 1 day. After modeling and intervention,Morris water maze test was conducted for the mice of each group. Using immunohistochemistry and Western blot method, the protein expression of insulin receptor(IR), insulin receptor substrate-1(IRS-1) and phosphatidylinositol 3-kinase(PI3K) was detected in the hippocampal of the mice after intervention. Results Compared with the blank group, in the model group, the 2 Hz, 15 Hz and 30 Hz EA groups, the escape latency and the first time of crossing the platform were all extended(P<0.01),and the number of crossing the platform was reduced(P<0.01) after modeling. When compared with the blank group, the escape latency and the first time of crossing the platform were all extended(P<0.01), and the number of crossing the platform was reduced(P<0.01) in the model group after intervention. In the 2 Hz, 15 Hz and 30 Hz EA groups, the escape latency and the first time of crossing the platform were all shortened(P<0.01), and the number of crossing the platform was increased(P<0.05, P<0.01) after intervention when compared with the model group. The escape latency and the first time of crossing the platform were all shortened(P<0.01, P<0.05), and the number of crossing the platform was increased(P<0.05) in the 15 Hz and 30 Hz EA groups in comparison with the 2 Hz EA group. The protein expression levels of IR, IRS-1and PI3K were reduced in the model group when compared with those of the blank group(P<0.01, P<0.05);and these protein expression levels were increased in the 15 Hz and 30 Hz EA groups compared with the model group(P<0.05, P<0.01). Compared with the 2 Hz EA group, the protein expression levels of IR, IRS-1 and PI3K were all elevated in the 15 Hz and 30 Hz EA groups(P<0.05). Conclusion The learning and memory function of AD mice may be improved through regulating brain insulin signaling transconduction pathway with electroacupuncture, and electroacupuncture at 15 Hz and 30 Hz obtains the overall better effect compared with the intervention at 2 Hz.