长期电针刺激百会、足三里对SHR大鼠海马CA1区微血管和神经元损伤的改善作用

【作者】 田贵华；

【导师】 李志刚； 韩晶岩；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2012， 博士

【摘要】 目的：高血压引发的脑血管病变和海马神经元的损伤是高血压发展为血管性痴呆的病理基础。所以,在改善血压的同时,能否改善脑血管病变,从而减轻海马神经元损伤是治疗高血压及其脑血管病变的关键环节。本研究旨在探讨长期电针刺激百会和足三里抑制自发性高血压大鼠血压升高,进而改善海马CA1区微血管和神经元损伤的作用及其可能的机制。材料和方法：8周龄Wistar和自发性高血压大鼠(Spontaneously Hypertensive Rat, SHR)雄性大鼠随机分为Wistar组、Wistar+(?)穴位组、Wistar+百会、足三里穴位组、SHR组、SHR+非穴位组和SHR+百会、足三里穴位组(每组15只)。穴位组,用针灸针(环球牌,0.3×40 mm,苏州)连接于韩式电针仪(LH202H型,北京),刺激百会和足三里(电流强度1 mA,频率2 Hz,时间20 min,隔日一次,共28次)。非穴位组,于尾部距尾根1 cm及2 cm处施以同样的刺激。对照组只捆绑,不行针灸电刺激。在电针刺激前、刺激后第2、4、6、8周末,分别监测血压；在刺激后第8周测定脑灌流量、观察海马神经元细胞形态、测定脑组织匀浆中血管内皮素-1 (endothelin-1, ET-1) and一氧化氮(nitric oxide, NO)的含量,测定脑组织中血管紧张素Ⅱ受体Ⅰ(angiotensionⅡtypeⅠreceptor, ATIR)、血管紧张素Ⅱ受体Ⅱ(angiotensionⅡtype I receptor, AT2R)、内皮素受体(endothelin receptor, ETAR)、内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的表达。结果：SHR大鼠在8周龄入组时,平均动脉压显著高于Wistar,在入组后第8周平均动脉压上升到170mmHg。在入组后第8周,SHR大鼠的脑血流量显著降低,海马CA1区开放的微血管数显著减少,海马CA1区神经元丢失,出现认知功能障碍,脑组织匀浆中的ET-1含量显著升高,与此同时,脑组织中的AT1R、ETAR的蛋白表达显著升高,而脑组织匀浆中的NO含量、脑组织中的AT2R、eNOS和iNOS的蛋白表达没有显著地变化。长期电针刺激百会和足三里可以显著地抑制SHR大鼠的平均动脉压的上升,改善脑血流量,抑制海马CA1区神经元丢失,改善认知功能障碍,抑制脑组织中的AT1R、ETAR的蛋白表达显著升高,而对脑组织中的AT2R、eNOS和iNOS的蛋白表达没有显著地影响。非百会和足三里的长期电针刺激,则没有观察到上述变化。结论：针灸电刺激百会和足三里在抑制SHR大鼠平均动脉压的升高的同时,可以减轻SHR大鼠海马CA1区微血管和神经元的损伤,该作用与其调节ATIR-ET-1-ETAR的通路相关。更多还原

【Abstract】 AimHypertension caused by cerebrovascular disease and hippocampal neurons injury is is the pathological basis of vascular dementia. So inhibit the blood pressure simultaneously,whether ameliorate cerebral vascular disease, reduce hippocampal neuronal damage is important for hypertension and cerebrovascular disease treatment. This study aimed to investigate long-term electrical-stimulation acupoint DU20 and ST36 inhibit blood pressure of spontaneously hypertensive rats, thereby improving the hippocampal CA1 region of microvascular and neuronal damage and its possible mechanism.Materials and MethodsA total of 90 adult male rats at 8 weeks, including 45 Wistar rats and 45 SHR rats, were randomly divided into 6 groups:Wistar group (n=15), Wistar+nonacupoint group (Wistar+NAP group) (n=15), Wistar+electroacupoint group (Wistar+AP group) (n=15), SHR group (n=15), SHR+nonacupoint group (SHR+NAP group) (n=15) and SHR+electroacupoint group (SHR+AP group) (n=15). The animals in Wistar+AP group and SHR+AP group were subjected to stimulation by electroacupuncture at acupoint DU20 (located at the midmost point of parietal bone) and ST36 (5 mm below head of fibula under knee joint, and 2 mm lateral to the anterior tubercle of the tibia). Sterilized disposable stainless steel needles (0.3 mm×40 mm, Global brand, Suzhou, China) were inserted 2 mm deep at DU20 with a slope of 30 degrees. Perpendicular Needling was perfomed with the depth of 5mm at ST36. Both needles were connected to Han’s Acupoint Nerve Stimulator (Model LH 202H, Huawei LTD, Beijing, China). Electric stimulation took place for 20 mins each time, once every other day for a period of 8 weeks, and the stimulation parameters were disperse-dense waves of 2/100 Hz with an intensity of 1 mA,2 Hz (11). In Wistar+NAP group and SHR+NAP group, the animals received similar treatment as electroacupuncture groups but the acupuncture region was 1 cm and 2 cm from the root of the tail, respectively, to replace DU20 and ST36. The animals in Wistar group and SHR group underwent similar procedure but without electroacupuncture.Before EA and on the end of 2,4,6,8 weeks, blood pressure was measured respectively, after eight weeks EA stimulation, Cerebral blood flow and number of open microvessels in hippocampal CA1 were detected by Laser Doppler and immunohistochemistry.The CA1 histological structure and learning and memory capacity were evaluated by Nissl staining and morris water maze (MWM). Furthermore, endothelin (ET) and nitric oxide (NO) in brain were measured by enzyme-linked immunosorbent assay (ELISA). The expression of angiotensin II type 1 receptor (AT1R), AT2R and endothelin receptor (ETAR), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) were measured by western blot.ResultsThe results showed that compared with Wistar rats, the MAP of SHR rats increased linearly during the observation period, which was significantly inhibited by electro-acupuncture. Accordingly, CBF hipocampal neuronal injury and learning and memory capacity were all improved by electro-acupuncture. In addition, the up-regulation of ATIR, ETAR and ET in SHR rats was abrogated by electro-acupuncture. The present study suggested that electro-acupuncture at "GV20" and "ST36" acupoint inhibited the increase of MAP, as well as the decrease of CBF and hipocampal CA1 injury, which was correlated with suppression of AT1R, ET and ETAR expression in brain.ConclusionThe present study revealed that SHR benefits from long-term electroacupuncture stimulation at DU20 and ST36 significantly, including relief of hypertension, increase in the number of opening microvessels and cerebral blood flow, attenuation of neuron injury, and restoration of cognitive impairment. Moreover, the ELISA and Western blot results indicate an implication of AT1R-ETAR-ET-1 signaling pathway in the electroacupuncture effects observed.更多还原