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目的研究氯胺酮预先给药对缺氧复氧诱导大鼠小脑颗粒神经元(CGNs)凋亡的影响,及磷脂酰肌醇-3-激酶(P13K)/Akt通路在其中的作用。方法出生7～8 d的清洁级SD大鼠,雌雄不拘,体重15～20g；制备CGNs,培养的8d的CGNs随机分为5组,对照组(A组)、缺氧复氧组(B组)、氯胺酮预先给药组(C组)、PI3K/Akt通路的特异性抑制刺Ly294002-氯胺酮预先给药组(D组)、Ly294002组(E组)。B组将CGNs放入特制缺氧盒中,缺氧3h后复氧；C组缺氧前1h加入200μmol/L氯胺酮；D组加入氯胺酮前30 min加入20μmol/L Ly294002；E组加入20μmol/L Ly294002。复氧16h后进行下述指标的观察。二乙酸荧光素染色法测定CGNs存活率,Hoechst 33258核染色检测CGNs凋亡细胞核,琼脂糖凝胶电泳检测DNA片断化水平,蛋白免疫印迹法(Westem blot法)检测CGNs磷酸化A kt、磷酸化GSK3β及总Akt水平。结果缺氧复氧可诱导CGNs的凋亡,降低CGNs磷酸化Akt和磷酸化GSK3β水平(P＜0．05),氯胺酮预先给药可减轻缺氧复氧诱导的上述改变,氯胺酮对CGNs的这种保护作用可被Ly294002部分抑制。结论氯胺酮预先给药可减轻缺氧复氧诱导大鼠CGNs凋亡,其机制与激活PI3K/Akt通路有关。
Objective To investigate the effects of ketamine preconditioning on apoptosis of cerebellar granule neurons (CGNs) induced by hypoxia-reoxygenation and the role of phosphatidylinositol 3-kinase (P13K) / Akt pathway in them. Methods CGNs were randomly divided into 5 groups, control group (A group), hypoxia-reoxygenation group (B group) and control group ), Ketamine preconditioning group (group C), PI3K / Akt pathway specific inhibition of Ly294002-ketamine preconditioning group (group D) and Ly294002 group (group E). In Group B, CGNs were placed in a special anoxia box and reoxygenated 3h after hypoxia; Group C 200mol / L ketamine 1h before hypoxia; Group D 20μmol / L Ly294002 30 min before adding ketamine; Group E 20μmol / L Ly294002. After reoxygenation for 16h, the following indexes were observed. The survival rate of CGNs was determined by fluorescein diacetate staining, apoptotic nuclei of CGNs were detected by Hoechst 33258 nuclear staining, DNA fragmentation was detected by agarose gel electrophoresis, Western blotting was used to detect phosphorylated Akt, phosphorylation GSK3β and total Akt levels. Results Hypoxia and reoxygenation induced the apoptosis of CGNs, decreased the levels of phosphorylated Akt and phosphorylated GSK3β in CGNs (P <0.05), and pretreatment with ketamine reduced the above-mentioned changes induced by hypoxia-reoxygenation. This effect of ketamine on CGNs The protective effect of Ly294002 can be partially inhibited. Conclusions Ketamine pretreatment can reduce the hypoxia-reoxygenation-induced apoptosis of CGNs in rats, which is related to the activation of PI3K / Akt pathway.