电针镇痛的累积效应与大鼠下丘脑、海马蛋白激酶A表达变化的观察

目的:观察电针(EA)"足三里"-"阳陵泉"镇痛的累积效应,及大鼠下丘脑和海马细胞蛋白激酶A(PKA)活性的变化。方法:Wistar雌鼠68只,随机分为正常对照组、慢性压迫损伤(CCI)组、CCI+EA 2天(d)组、CCI+EA 2周(w)组、去卵巢(OVX)+CCI组、OVX+CCI+EA 2 d组、OVX+CCI+EA 2 w组。Morris水迷宫法检查OVX+D-半乳糖皮下注射动物的学习记忆能力。结扎坐骨神经造成CCI慢性痛模型,电针双侧"足三里"-"阳陵泉"穴(2/15 Hz,1~2 mA,1次/d)。用辐射热照射大鼠双侧足底引起的缩腿反应潜伏期(PWL)的差值作为痛敏分数(HS)判断动物的痛反应;用免疫组化法检测下丘脑和海马组织PKA的活性。结果:CCI术后,与正常组比较,各组HS都明显升高。在单纯CCI动物上,与CCI组比,CCI术后第18天CCI+EA 2 d、CCI+EA 2 w的HS显著减小(P<0.05);CCI+EA 2 w组的HS显著低于CCI+EA 2 d组(P<0.05)。在记忆损害的动物上,OVX+CCI+EA 2 w和OVX+CCI+EA 2 d组HS明显低于OVX+CCI组(P<0.05);而两EA组间HS差异没有显著性意义(P>0.05)。CCI+EA 2 w组下丘脑室旁核(PVN)、弓状核(ARC)和视上核(SON)区PKA的灰度值均明显低于正常对照组(P<0.05),PVN和ARC区也显著低于CCI+EA 2 d组(P<0.05);OVX+CCI+EA 2 w组PVN和SON区的PKA灰度值亦均明显低于OVX+CCI组(P<0.05)。说明EA 2 w可上调PKA的表达,且具有累积性作用。OVX+CCI+EA 2 w组ARC和SON区PKA的灰度值均显著高于CCI+EA 2 w组(P<0.05),提示EA上调PKA表达的累积效应在记忆功能减退的动物上明显减弱。海马PKA的表达与SON等下丘脑核团PKA的表达趋势类似。结论:电针镇痛有累积作用,其累积效应可能与下丘脑、海马细胞PKA的表达上调有关,并且与动物神经记忆有密切关系。

Observation on the Accumulative Analgesic Effect of Electroacupuncture and the Expression of Protein Kinase A in Hypothalamus and Hippocampus in Chronic Pain or/and Ovariectomized Rats

OBJECTIVE: To observe the accumulative analgesic effect of electroacupuncture (EA) and the simultaneous changes of protein kinase A (PKA) expression in hypothalamus and hippocampus in rats with chronic constrictive injury (CCI) or/and learning-memory impairment. METHODS: Sixty-eight female Wistar rats were randomized into normal control, CCI, CCI + EA 2 d, CCI + EA 2 w, OVX + CCI, OVX + CCI + EA 2 d and OVX + CCI + EA 2 w groups, with 10 cases in each except OVX + CCI group (n=8). CCI pain model was established by ligature of the right sciatic nerve with surgical suture. EA (2/15 Hz, 1-2 mA, 30 min) was daily applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 2 days (2 d) and 2 weeks (2 w) respectively. Neuro-memory impairment was induced by OVX plus subcutaneous injection of D-galactose. Hyperalgesic scores (HS) were detected with radiation-heat irradiation and PKA activity was detected by immunohistochemical method. RESULTS: After CCI, HS of all groups increased significantly in comparison with normal control group (P<0.05). In simple CCI rats, compared with CCI group, HS of both CCI + EA 2 d and CCI + EA 2 w groups decreased markedly (P<0.05), and that of CCI + EA 2 w group was evidently lower than that of CCI + EA 2 d group on the 18th day after CCI (P<0.05). In memory impairment rats, HS of both OVX + CCI + EA 2 d and OVX + CCI + EA 2 w groups was significantly lower than that of OVX+ CCI group (P<0.05); and no significant difference was found between OVX + CCI + EA 2 w and OVX + CCI + EA 2 d groups in HS (P>0.05). Results of immunohistochemistry showed that the integral grey values (IGV) of PKA of paraventricular nucleus (PVN), arcuate nucleus (ARC) and supraoptic nucleus (SON) of hypothalamus in CCI + EA 2 w group were significantly lower than those in normal control and CCI + EA 2 d groups (P<0.05). IGV of PVN and SON in OVX + CCI + EA 2 w group were also markedly lower than those in OVX + CCI group (P<0.05), displaying upregulation and cumulative increase of PKA expression by EA 2 w. IGV of PKA, ARC and SON in OVX + CCI + EA 2 w group were markedly higher than those in CCI + EA 2 w group (P<0.05), suggesting an apparent attenuation of the cumulative upregulation effect of PKA expression after learning-memory injury. The changing tendency of PKA expression in hippocampal CA1 region was similar to that of SON after repeated EA of ST36 and GB34. CONCLUSION: Repeated EA has a cumulative analgesic effect in CCI rats, which is probably related to its resultant upregulation of PKA expression in neurons of hypothalamus and hippocampus and the animals' neuro-memory.