胡黄连苷Ⅱ对脑缺血损伤后神经元特异性烯醇化酶表达的影响

Effect of picroside Ⅱ on neuron specific enolase after cerebral ischemic injury

目的通过正交试验法验证胡黄连苷Ⅱ对大鼠脑缺血损伤的神经保护作用,并优化其治疗的最佳剂量及时间窗。方法应用双侧颈总动脉结扎法建立大鼠前脑缺血模型,按照正交试验设计分组,经腹腔注射胡黄连苷Ⅱ干预治疗。甲苯胺蓝染色法观察神经细胞结构;流式细胞术观察细胞早期凋亡率;免疫组织化学法和蛋白免疫印迹法(Western blot)定性、定量检测神经元特异性烯醇化酶(NSE)表达;反转录聚合酶链式反应(RT-PCR)检测NSE mRNA转录水平。结果胡黄连苷Ⅱ治疗大鼠脑缺血损伤的最佳时间和剂量:根据甲苯胺蓝染色显示为脑缺血2.0 h腹腔注射胡黄连苷Ⅱ10 mg·kg-1;流式细胞术检测显示为脑缺血1.5h腹腔注射胡黄连苷Ⅱ10 mg·kg-1;免疫组织化学法分析为脑缺血2.0h腹腔注射10 mg·kg-1;Western blot分析为脑缺血2.0 h给予10 mg·kg-1;RT-PCR显示为脑缺血1.5 h给予10 mg·kg-1。结论根据用药剂量最小化和治疗时间窗最大化的原则综合评价,胡黄连苷Ⅱ治疗脑缺血损伤的最佳治疗时间窗为脑缺血1.5~2.0 h,腹腔注射胡黄连苷Ⅱ10 mg·kg-1。

Aim To verify the neuroprotective effect and optimize the therapeutic dose and time window of picroside H by orthogonal test in cerebral ischemic in-jury in rats. Methods The forebrain ischemic models were established by bilateral common carotid artery oc-clusion （BCCAO） methods. The successful rat models were randomly divided into sixteen groups according to orthogonal experimental design and treated by injecting picroside H intraperitoneally at different ischemic time with different doses. The structures of neurons wereobserved by toluidine blue staining; the early apoptotic ratio was detected by flow cytometry; the immunohisto- chemical assay and Western blot were used to detect quantitatively and qualitatively the expression of neuron specific enolase （NSE） ; the reverse transcription level of NSE mRNA was determined by reverse transcription polymerase chain reaction （RT-PCR）. Results The best therapeutic time window and dose of picroside II in cerebral ischemic injury were （ 1 ） ischemia 2. Oh with 1 0 mg ~ kg- 1 according to the results of toluidineblue staining; （2） ischemia 1.5h with 10rag ~ kg^1 according to the examination by flow cytometry; （3） ischemia 2. Oh with I0 mg ~ kg-I according to the a- nalysis of immunohistochemical assay ; （4） ischemia 2. Oh with 10 mg . kg-1 according to the analysis of Western blot; and （5） ischemia 1.5h with 10 mg. kg- 1 according to the analysis of RT-PCR. Conclusion Given the principle longest time window, and time window is ally with lOmg . kg-1 bral ischemic injury of lowest therapeutic dose with the optimized therapeutic dose ecting picroside I1 intraperitone-at ischemia 1.5 -2. Oh in cere-bral ischmic injury.