电针对脑缺血再灌注损伤大鼠一氧化氮合酶的影响

Effect of Electroacupuncture on Nitric Oxide Synthase in Rats with Cerebral Ischemia-Reperfusion Injury

目的探讨电针对脑缺血再灌注损伤大鼠一氧化氮合酶(NOS)的调节作用。方法用改良血管内栓线技术建立大鼠局灶性脑缺血再灌注模型,应用亚硝酸盐还原法测定脑组织中一氧化氮(NO)的含量、免疫组织化学技术检测脑组织中神经元型NOS(nNOS)及诱导型NOS(iNOS)的表达。80只SD大鼠分为正常组、脑缺血再灌注组(模型组)、脑缺血再灌注加电针组(针刺组)、电针加磷脂酰肌醇3激酶(PI3-K)抑制剂组(抑制剂组),每组20只。抑制剂组在第一次电针后,经脑立体定位仪按Konig Klippel图谱用微量注射器将5μL的PI3-K的拮抗剂LY294002(400μmol/L)缓慢恒速注入侧脑室。电针穴位选择"水沟"、"承浆"穴,测定大鼠脑组织NO、NOS表达。结果大鼠脑缺血再灌注24 h,海马及大脑皮层NO水平异常增高,nNOS、iNOS蛋白表达增加,与正常组比较,差异有统计学意义(P<0.05);针刺大鼠"水沟"、"承浆"穴,在减少脑缺血再灌注神经元丢失同时,明显抑制了海马、皮层NO水平,nNOS、iNOS蛋白表达异常增加的现象被逆转,与模型组大鼠比较,差异有统计学意义(P<0.05),与正常组比较,差异无统计学意义(P>0.05)。以LY294002拮抗PI3K阻断TrkA受体通路后,针刺对海马及皮层NO、nNOS、iNOS异常增加的抑制效应再次逆转,海马及皮层NO水平、nNOS、iNOS蛋白表达再次增加,与模型组比较,差异有统计学意义(P<0.05)。结论针刺在抗脑缺血再灌注神经元丢失同时,对NOS的异常上调有逆转作用,其逆转作用部分通过TrkA-PI3K信号转导通路介导。

Objective To study the effect of electroacupuncture on nitric oxide synthase（NOS） in rats with cerebral ischemia-reperfusion injury.Methods Focal cerebral ischemia-reperfusion model was established using modified intravascular suture technique.The NO content in the brain tissue was detected by nitrite reduction and the expressions of nNOS and iNOS were detected by immunohistochemistry.Eighty rats in this experiment were divided into the normal group,the cerebral ischemia-reperfusion injury model group（as the model group）,the cerebral ischemia-reperfusion injury＋electroacupuncture group（as the acupuncture group）,and the cerebral ischemia-reperfusion injury＋phosphatidylinositol 3 kinase（PI3-K） inhibitor group（as the inhibitor group）.Each group consisted of twenty rats.Five μL PI3-K inhibitor LY294002（400 μL） was slowly injected at the lateral cerebral ventricle of rats in the inhibitor group at a constant speed using microinjector according to Konig Klippel atlas of the stereotaxis instrument.Shuigou（DU26） and Chengjiang（RN24） were selected to determine levels of NO and NOS.Results After 24-h ischemia-reperfusion,the NO levels of the hippocampus and the cerebral cortex increased abnormally,and the expressions of nNOS and iNOS increased,showing significant difference when compared with those of the normal group（P0.05）.By electroacupucture at Shuigou（DU26） and Chengjiang（RN24）,the ischemic cerebral ischemia-reperfusion injury neuron loss was inhibited.Meanwhile,the high levels of NO,nNOS and iNOS in the cerebral cortex and the hippocampus were significantly inhibited（P0.05）.The abnormally increased expressions of nNOS and iNOS were reversed,showing significant difference when compared with the model group（P0.05）.But when compared with the normal group,there was no significant difference（P0.05）.The effects of electroacupuncture reversed the abnormally increased NO levels of the hippocampus and the cerebral cortex and expressions of nNOS and iNOS after LY294002 oppresses anti-PI3K to block the TrkA acceptor circuit.The NO levels of the hippocampus and the cerebral cortex and expressions of nNOS and iNOS increased again,showing significant difference when compared with the acupuncture group（P0.05）.Conclusions Acupuncture fought against cerebral ischemia and reperfusion in the loss of neurons,at the same time,the abnormal regulation of NOS had reverse effect partly through TrkA/PI3K mediated signal transduction pathway.