渥曼青霉素对大鼠脑外伤诱导的凋亡损害的影响

Effects of wortmannin on traumatic brain injury-induced apoptotic damage in rats

目的探讨渥曼青霉素(wortmannin)对大鼠脑外伤(TBI)模型引起的细胞凋亡的影响及可能机制。方法①健康成年雄性Wistar大鼠100只,随机分为假手术组(sham组)(n=20)、手术对照组(saline组)(n=40)、药物治疗组(wortmannin组)(n=40)。采用改良自由落体法建立大鼠脑外伤模型,sham组暴露硬脑膜而不撞击,wortmannin组打击前左侧脑室注射5μl wortmannin(50 nmol/L),saline组造模前注射5μl生理盐水。②免疫印迹法、Real-time PCR检测脑组织caspase-3、Bax表达情况;DNA凝胶电泳检测脑外伤引起的DNA片段断裂。③图像分析脑体积缺损情况。结果①与sham组相比,saline组脑外伤可明显引起caspase-3、Bax表达增多,Bcl-2表达减少;DNA片段断裂增多,脑体积缺损情况显著。②与saline相比,wortmannin干预能明显抑制caspase-3与Bax的激活(P<0.01),促进Bcl-2表达的增高(P<0.01);抑制脑外伤引起的DNA片段断裂及脑体积缺损(P<0.05)。结论 wortmannin能减轻脑外伤引起的细胞凋亡与脑损害,其机制可能是通过抑制caspase依赖的细胞凋亡途径实现的。

Objective To investigate the effects of wortmannin on traumatic brain injury（TBI）-induced cell apoptosis and its possible mechanisms.Methods ①One hundred male adult Wistar rats were randomly assigned into three groups：sham group（n=20）,saline group（n=40） and wortmannin group（n=40）.The TBI model was performed using a modified free fall method.The rats in sham group were only operated but no impact on cerebral dura mater,while the rats were respectively pretreated with 5 μl of 50 nmol/L wortmannin and 5 μl saline by a single intracerebroventricular（i.v.c.） in wortmannin group and saline group.②The levels of genes and proteins of caspase-3,Bcl-2,Bax were determined by real-time PCR and immunoblotting,respectively.The TBI-induced DNA fragmentation was assessed with DNA fragmentation assay.③TBI-induced damage were also assessed with brain lesion volume.Results ①Compared with sham group,the levels of caspase-3 and Bax activation significantly increased in saline group,whereas the level of Bcl-2 protein decreased and DNA fragmentation increased（P0.01）.Furthermore,TBI resulted in a significant increase in lesion volume.②Compared with saline,wortmannin significantly inhibited the activation of caspase-3 and Bax（P0.01）,promoted Bcl-2 expression（P0.01）.TBI-induced DNA fragmentation and brain lesion volume were attenuated by wortmannin（P0.05）.Conclusion The results suggest that wortmannin could attenuate the TBI-induced brain cell apoptosis and brain damage through the inhibition of the caspase-dependent pathway.Wortmannin may be used as a therapeutic target for treating TBI.