【作者】 金建祥；

【导师】 陈高；

【作者基本信息】 浙江大学 ， 外科学（神经外科）， 2018， 硕士

【摘要】 目的蛛网膜下腔出血是一种致死性的脑血管疾病。研究表明,丹参的活性萃取物丹酚酸B镁(magnesium lithospermate B,MLB)在缺血性卒中、阿尔兹海默症等中枢神经系统疾病中均可发挥神经保护作用。然而,MLB在蛛网膜下腔出血(subarachnoid hemorrhage,SAH)早期脑损伤(early brain injury,EBI)病理过程中的作用仍不清楚。因此,本研究旨在探究MLB在SAH后早期脑损伤中的作用及其相关机制。方法采用血管内穿刺法构建大鼠SAH模型。将健康的雄性Sprague-Dawley大鼠随机分为以下4组:假手术+溶剂组、SAH+溶剂组、SAH+MLB组以及SAH+MLB+H89(特异性PKA抑制剂)组。手术后24小时统计比较各组大鼠的死亡率,SAH评分,脑组织含水量以及神经功能评分。通过蛋白质印迹检测不同组别大鼠皮层脑组织内 Bax、Bcl-2、cleaved capase-3、PKA、CREB、Sirtl 的表达情况。通过免疫荧光染色评估各组大鼠脑组织中神经细胞凋亡情况。结果在建模后24小时,与假手术+溶剂组相比,SAH+溶剂组大鼠脑组织的含水量显著增加,并伴有明显的神经功能缺失。此外,SAH+溶剂组大鼠脑组织中Bcl-2/Bax比值下降,cleaved capase-3的表达增多,同时皮层内TUNEL阳性细胞数目明显增加。与SAH+溶剂组相比,MLB治疗显著地降低了 SAH大鼠脑组织内的含水量,改善了大鼠的神经功能,提高Bcl-2/Bax比值,减少cleaved capase-3的表达及皮层内TUNEL阳性细胞的数目。值得注意的是,MLB显著提高了脑组织内PKA的表达、上调了磷酸化的CREB(p-CREB)/CREB的比值,并提高了Sirt1的表达。与SAH+MLB组相比,PKA抑制剂H89预处理组大鼠脑组织内PKA的表达下降,p-CREB/CREB的比值降低,同时Sirt1的表达减少。更为重要的是,H89逆转了 MLB在SAH后介导的抗凋亡的神经保护作用。结论以上研究表明MLB能够显著减轻SAH后的早期脑损伤。MLB在SAH后发挥神经保护作用可能与其调控PKA/CREB/Sirt1信号通路,进而抑制神经细胞凋亡有关。MLB对SAH的临床治疗具有潜在的应用价值。更多还原

【Abstract】 PurposeSubarachnoid hemorrhage(SAH)is a fatal cerebrovascular disease.Previous studies have shown that magnesium lithospermate B(MLB),an effective component of salvia miltiorrhiza,has beneficial effects against neurological diseases such as ischemic stroke,Alzheimer disease.However,the role of MLB in early brain injury(EBI)post SAH is still unclear.The present study was to determine the effects of MLB on EBI following SAH and the underlying molecular mechanisms.MethodsSAH was induced by endovascular perforation in rats.Male Sprague-Dawley rats were divided into four groups at random:sham+vehicle group,SAH+vehicle group,SAH+MLB group and SAH+MLB+H89(a specific PKA inhibitor)group.After 24 h of SAH induction,mortality rate,SAH grade,neurological scores and brain water content of each group were measured and analyzed.The level of Bax,Bcl-2,cleaved capase-3,PKA,CREB and Sirtl in the cortex from each group was examined by western blot.The apoptosis of neurons in the brain was evaluated through immunofluorescence.ResultsAt 24 h after SAH,the brain water content in SAH+vehicle group increase significantly with obvious neurological deficits compared with sham+vehicle grThe Bcl-2/Bax ratio in SAH+vehicle group decreased while the expression of cleaved capase-3 and the number of TUNEL positive cells in the cortex increased.Contrary to the SAH+vehicle group,the treatment of MLB significantly decreased the brain water content in the cortex,improved the neurological functions,increased the Bcl-2/Bax ratio while decreased the expression of cleaved capase-3 and the number of TUNEL-positive cells in the cortex.Notably,MLB significantly upregulated the PKA level,the p-CREB/CREB ratio and the Sirtl expression in the brain.The preconditioning of specific PKA inhibitor H89 downregulated the levels of PKA,p-CREB/CREB ratio and sirtl expression.More importantly,H89 blocked the neuroprotective effects of MLB against SAH.ConclusionMLB treatment significantly attenuated SAH-induced EBI.These neuroprotective effects was possibly related to its regulation of the PKA/CREB/Sirtl signaling pathway and further inhibition of the neural apoptosis.MLB has become a promising method for the clinical treatment of SAH.更多还原