基于ERK/CREB信号通路探讨艾灸肾俞穴改善去卵巢合D-半乳糖阿尔茨海默病样大鼠神经元丢失的机制研究

Study on Mechanism of Moxibustion at Shenshu(BL23)to Improve Neuron Loss in Ovariectomized D-galactose AD-like Rats Based on ERK/CREB Signaling Pathway

目的研究艾灸肾俞穴通过激活海马区ERK/CREB信号通路改善去卵巢合D-半乳糖腹腔注射诱导阿尔茨海默病(Alzheimer’s disease,AD)样大鼠学习记忆能力及神经元丢失的机制研究。方法选用3月龄SD雌性未孕大鼠40只,随机分为正常组、模型组、肾俞组、抑制剂组,每组10只。除正常组外,其他3组均摘除双侧卵巢,术后3 d,每日按照150 mg·kg^(-1)·d^(-1)剂量腹腔注射D-gal(D-半乳糖)90 d建立阿尔茨海默病样大鼠模型。肾俞组自卵巢切除术后第3天起,采用直径约6 mm定制艾条,分别于双侧"肾俞"上方2~3 cm处悬灸,使局部温度维持在(41±0.5)℃,每穴10 min,每日9:00 am开始治疗,1次/d,5 d为1个疗程,疗程间休息2 d,共治疗12个疗程。抑制剂组是在肾俞组基础上每周皮下注射选择性ERα受体拮抗剂甲基哌啶吡唑methyl-piperidino-pyrazole(MPP),1次/周,共12次。正常组不进行干预,但以相同的方式束缚处理。免疫组化法检测各组大鼠海马区Aβ1-42和雌激素受体α表达;尼氏染色分析各组大鼠海马区神经元数目及形态变化;免疫印迹法检测各组大鼠海马区p-CREB、p-ERK、ERK、CREB的蛋白表达。结果干预3个月后,各组海马组织中ERK和CREB总量没有明显差异,各组逃避潜伏期均呈现随时间推移而下降。与正常组相比,从定位航行实验Day2起,模型组逃避潜伏期明显延长(P<0.001),穿越平台次数明显下降(P<0.01),海马区Aβ1-42表达明显增多(P<0.001),海马CA1、CA3区神经元数目明显下降(P<0.001),P-ERK/T-ERK、P-CREB/T-CREB水平明显下降(P<0.001),海马区ERα表达明显减少(P<0.01);经治疗后,与模型组相比,从定位航行实验Day3起,肾俞组逃避潜伏期明显缩短(P<0.001),穿越平台次数明显增加(P<0.01),海马内Aβ1-42表达明显减少(P<0.001),CA1、CA3区神经元数目明显增加(P<0.01),P-ERK/T-ERK、P-CREB/T-CREB水平明显增加(P<0.001),海马区ERα表达明显升高(P<0.05);与肾俞组比较,抑制剂组逃避潜伏期明显延长(P<0.001),穿越平台次数明显下降(P<0.01),Aβ1-42表达明显升高(P<0.001),CA1、CA3区神经元数目组明显减少(P<0.05,P<0.01),P-ERK/T-ERK、P-CREB/T-CREB水平明显增加(P<0.01,P<0.05),且ERα表达明显降低(P<0.01)。结论激活海马区ERα依赖的ERK/CREB信号通路是艾灸肾俞穴改善去卵巢合D-半乳糖腹腔注射诱导的AD样大鼠学习记忆能力及神经元丢失现象的机制之一。

Objective To study the mechanism of moxibustion at Shenshu(BL23)to improve the ability of learning and memory and neuron loss in AD-like rats induced by activating hippocampal ERK/CREB signaling pathway in AD-like rats.Methods Forty female 3-month-old SD female rats were randomly divided into normal group,model group,Shenshu(BL23)group and inhibitor group,with 10 rats in each group.Except for the normal group,bilateral ovaries were removed.At 3 days after the operation,D-gal(D-galactose)was injected intraperitoneally at a dose of 150 mg/(kg·d)for 90 days to establish an Alzheimer’s disease rat model.The Shenshu(BL23)group was given suspended moxibustion from the 3 rd day after ovariectomy.The experiment used a custom moxa with a diameter of about 6 mm to suspend moxibustion 2~3 cm above Shenshu(BL23)on both sides of the rats to keep the local temperature at(41±0.5)℃.Each acupoint started at 9:00 am every day for 10 minutes one day.One treatment course lasted for 5 days and the interval between courses was 2 days.A total of 12 consecutive courses were performed.In the inhibitor group,the selective ERαreceptor antagonist methyl-piperidino-pyrazole(MPP)was subcutaneously injected weekly on the basis of the Shenshu(BL23)group for a total of 12 weeks.No intervention was performed in the normal group,but the restraint treatment was given in the same way as others.After modeling and intervention,immunohistochemical method was used to detect the expressions of Aβ1-42 and estrogen receptorαin hippocampus of each group.The nissl staining was used to analyze the number and morphological changes of hippocampal neurons in each group.The protein expressions of p-CREB,p-ERK,ERK and CREB in hippocampus of each group were detected by Western blotting.Results After three months of intervention,there was no significant difference in the total amount of ERK and CREB in the hippocampus of each group,and the escape latency of each group decreased with time.Compared with those of the normal group,from day 2 in navigation experiment of MWM,the escape latency of the model group was significantly prolonged(P<0.001),the number of crossing platforms was decreased significantly(P<0.01),and the expression of Aβ1-42 in the hippocampus area was increased significantly(P<0.001).The number of hippocampal CA1 and CA3 neurons was decreased significantly(P<0.001),the levels of P-ERK/T-ERK,P-CREB/T-CREB were decreased significantly(P<0.001),and the expression of ERαin the hippocampus was decreased significantly(P<0.01).After treatment,compared with those of the model group,from day 3 in navigation experiment of MWM,the escape latency of the Shenshu(BL23)group was significantly shortened(P<0.001).The number of crossing platforms was significantly increased(P<0.01),and the expression of Aβ1-42 in the hippocampus was less significant(P<0.001).The number of neurons in CA1 and CA3 areas was increased obviously(P<0.01),levels of P-ERK/T-ERK and P-CREB/T-CREB were increased obviously(P<0.001),and ERαexpression in hippocampus were increased obviously(P<0.05).Compared with Shenshu(BL23)group,the escape latency was prolonged obviously in the inhibitor group(P<0.001),the number of crossing platforms was decreased obviously(P<0.01),and Aβ1-42 expression was increased obviously(P<0.001).The number of neurons in CA1 and CA3 area was reduced obviously(P<0.05,P<0.01).The levels of P-ERK/T-ERK and P-CREB/T-CREB were increased obviously(P<0.01,P<0.05)and the expression of ERαwas decreased obviously(P<0.01).Conclusion Activation of the ERα-dependent ERK/CREB signaling pathway in the hippocampus is one of the mechanisms by which moxibustion at Shenshu(BL23)improving the learning and memory ability and neuronal loss of AD rats induced by ovarian removal and D-galactose intraperitoneal injection.