醒脑静注射液对大鼠全脑缺血性深昏迷的早期保护作用及其机制

目的：研究醒脑静注射液对大鼠全脑缺血性深昏迷的早期保护作用及其机制。方法：雄性SD大鼠采用四 血管阻塞法建立全脑缺血性深昏迷模型后分组：模型对照组、醒脑静低、中、高剂量组(1.8，3.6，5.4 mL/kg)、醒脑 静(3.6 mL/kg)+PI3K抑制剂组、纳洛酮组(0.04 mL/kg)、醒脑静(3.6 mL/kg)+纳洛酮组(0.04 mL/kg)，每组8只。另设8只 动物为假手术组。给药组手术后静脉给予不同剂量的醒脑静注射液和/或纳洛酮注射液，给药体积10.0 mL/kg，连续 给药3 d；醒脑静+PI3K抑制剂组为造模后立即侧脑室注射10.0 μL PI3K抑制剂LY294002(10 mmol/L)，然后再给予醒脑 静注射液(3.6 mL/kg)。记录首次给药后动物苏醒时间、末次给药后抓力和10 min内自主活动；采用ELISA法检测脑脊 液多巴胺(dopamine，DA)和谷氨酸(glutamate，Glu)的含量；采用HE染色观察脑组织病理改变；采用蛋白质免疫印迹 法检测海马组织蛋白激酶B(Akt)蛋白及其磷酸化蛋白(p-Akt)和cAMP应答元件结合蛋白(cAMP-response element binding protein，CREB)及其磷酸化蛋白(p-CREB)的表达。结果：与模型对照组比较，醒脑静注射液单次给药能明显缩短大 鼠苏醒时间(P<0.05)，连续给药3 d能明显增加大鼠抓力、自主活动总路程、活动次数、活动时间(P<0.05或P<0.01)， 降低DA和Glu的含量(P<0.05或P<0.01)，改善海马组织病理改变。醒脑静注射液能显著诱导Akt和CREB蛋白质的磷酸 化(P<0.05或P<0.01)。PI3K抑制剂LY294002能明显抑制醒脑静注射液对全脑缺血性深昏迷大鼠上述改善作用(P<0.05或 P<0.01)。醒脑静注射液能显著增加纳洛酮注射液对大鼠全脑缺血性深昏迷的上述改善作用(P<0.05或P<0.01)。结论： 醒脑静注射液能明显改善大鼠全脑缺血性深昏迷，其机制与上调PI3K/Akt介导的CREB磷酸化，调节神经递质和保护 海马组织有关。醒脑静注射液与纳洛酮对大鼠全脑缺血性深昏迷有协同增效作用。

Protective effects and mechanisms of Xingnaojing Injection on early global brain ischemic-induced deep coma in rats

Objective: To study the protective effect of Xingnaojing Injection on early global brain ischemiainduced deep coma in rats. Methods: The deep coma model was induced by global brain ischemia by using four-vessel occlusion method in male SD rats. According to the body weight, the rats were randomly divided into 8 groups: a model control group, three different dose of Xingnaojing Injection (1.8, 3.6 and 5.4 mL.kg–1) groups, a Xingnaojing Injection (3.6 mL.kg–1) plus PI3K inhibitor group, a naloxone injection (0.04 mL.kg–1) group and a naloxone injection (0.04 mL.kg–1) plus Xingnaojing Injection (3.6 mL.kg–1) group (n=8 per group). In addition, eight animals served as the sham group were performed same operation with the model group excepting no blockage of the blood vessels. After the operation, three different doses of Xingnaojing Injection and/or naloxone injection were given intravenously once a day for three days. Ten μL PI3K inhibitor (LY294002, 10 mmol/L) was injected via anterior cerebral ventricle at once after global brain ischemia. The awakening time after the first drug treatment, the grasping power and the autonomous activity within 10 min after the last drug treatment were recorded. The levels of both dopamine (DA) and glutamate (Glu) in cerebrospinal fluid were detected by ELISA. The pathological changes were observed in brain tissue slices with HE staining and the protein levels of Akt/p-Akt and cAMP-response element binding protein (CREB)/p-CREB in hippocampus were detected by Western blotting. Results: Comparing with the model group, single administration of Xingnaojing Injection could significantly shorten the waking time (P<0.05) and continuous administration of Xingnaojing Injection for 3 d could increase grasping power, distance, frequency and duration of autonomous activities (P<0.05 or P<0.01) in the deep coma rat. Also, Xingnaojing Injection could inhibit these increases in neurotransmitters DA and Glu contents (P<0.05 or P<0.01), and improve pathological changes of hippocampal tissue. Xingnaojing Injection significantly induced protein phosphorylation of both Akt and CREB (P<0.05 or P<0.01); this effect was inhibited by PI3K inhibitor (P<0.05 or P<0.01). Moreover, the protective effects of naloxone on awakening time, grasping power, the autonomous activity and hippocampus damage in global brain ischemia-induced deep coma could be enhanced by joint use of Xingnaojing Injection (P<0.05 or P<0.01). Conclusion: Xingnaojing Injection could significantly improve deep coma induced by global brain ischemia in rat, which is related to inducing PI3K/Akt-dependent protein phosphorylation of CREB, and reducing hippocampal damage. The protective effect of Xingnaojing Injection is synergistically enhanced by naloxone.