二氮嗪预先给药对原代培养大鼠海马神经元缺氧/复氧损伤的保护机制

Diazoxide pretreatment protects primarily cultured hippocampal neurons against anoxia/reoxygenation injury

目的研究ATP敏感性钾通道开放剂二氮嗪预先给药对新生Wistar大鼠原代培养海马神经元缺氧/复氧损伤的保护机制。方法原代培养的新生大鼠海马神经元随机分为2组,二氮嗪组(Dia组),缺氧前给予50μmol/L二氮嗪;对照组(Con组),给予二氮嗪的赋形剂,即含2‰二甲基亚砜的0.01 mol/L磷酸盐缓冲液;分别在缺氧3 h/复氧24 h和缺氧3 h/复氧48 h后,测定神经元存活能力及LDH漏出率;在缺氧3 h/复氧24 h后,测定丙二醛(MDA)和超氧化物歧化酶(SOD)水平;在缺氧3 h、缺氧3 h/复氧24 h和缺氧3 h/复氧48 h时,测定早期神经元凋亡率和死亡率。结果与Con组比较,Dia 组缺氧3 h复氧24 h时神经元存活能力升高,缺氧3 h/复氧48 h时LDH漏出率降低,缺氧3 h/复氧24 h培养液中MDA浓度降低,SOD活性升高(P<0.05或0.01);缺氧复氧各时点Dia组神经元坏死率降低,神经元凋亡率升高(P<0.05或0.01)。结论50 μmol/L二氮嗪预先给药减轻原代培养新生Wistar 大鼠海马神经元缺氧/复氧损伤的机制与增加SOD活性有关。

Objective To investigate the protective effects of pharmacological preconditioning with diazoxide, an ATP-sensitive potassium channel opener, against anoxia/reoxygenation （A/R） injury to primarily cultured hippocampal neurons of newborn Wistar rats and the mechanism involved and the effects of diazoxide preconditioning on MR-induced neuronal apoptosis. Methods Primarily cultured hippocampal neurons prepared by enzymatic digestion of hippocampus isolated from newborn （〈24 h）. Wistar rats were randomly divided into two groups： diazoxide group （Dia） and control group （Con）. In group Dia the hippocampal neurons were incubated with 50 μmol·L^-1 for 30 min before being exposed to 3 h anoxia followed by 24/48h reoxygenation. In Con group the vehicle （2‰ DMSO） was used instead of diazoxide. Viability of neurons and lactate dehydrogenase （LDH） were determined after 24 h and 48h reoxygenation respectively. MDA concentration and SOD activity were checked. Neuronal apoptosis and necrosis after 24 h and 48 h reoxygenation were detected by Annexin in V-PI staining and flow cytometry. Results The viability of hippocampal neurons was significantly higher after 24 h reoxygenation and LDH release was significantly lower after 48 h reoxygenation in group Dia than in control group. Compared with control group the MDA production was significantly reduced and SOD activity was significantly higher in group Dia. The necrosis rate was significantly lower but the early stage apoptosis rate was significantly higher after 3, 24 and 48 h reoxygenation in group Dia than in control group. Conclusion Diazoxide pretreatment can protect the primarily cultured hippocampal neurons of newborn Wistar rats from AIR injury and the increased SOD activity may be involved in the mechanism of neuroprotection. Diazoxide pretreatment reduces A/R-induced neuronal necrosis but increases A/R-induced apoptosis.