染料木黄酮通过SIRT1/p53信号通路对蛛网膜下腔出血后早期脑损伤的作用  

Impact of genistein on early brain injury following subarachnoid hemorrhage in mice via the SIRT1/p53 signaling pathway

作　　者：朱泽超 杨新宇 李侑埕 潘鹏宇 梁国标 Zhu Zechao;Yang Xinyu;Li Youcheng;Pan Pengyu;Liang Guobiao(Department of Neurosurgery,Hospital of the Northern Theater Command,Shenyang 110016,China)

机构地区：[1]北部战区总医院神经外科,沈阳110016

出　　处：《中华神经创伤外科电子杂志》2023年第5期261-269,共9页Chinese Journal Of Neurotraumatic Surgery：Electronic Edition

基　　金：国家自然科学基金(81971133)。

摘　　要：目的探讨染料木黄酮(genistein)对蛛网膜下腔出血(SAH)后早期脑损伤(EBI)的影响和作用机制。方法选取成年雄性C57BL/6J小鼠156只,采用血管内穿刺的方法建立小鼠SAH模型。预实验:选取72只小鼠,设置两个时间点24、72 h,每个时间点36只。将每个时间点的小鼠随机分为6组:假手术组(Sham)、模型组(SAH)、溶剂组(SAH+Vehicle)、低剂量组(genistein 5 mg/kg)、中剂量组(genistein 15 mg/kg)、高剂量组(genistein 30 mg/kg),每组6只,分别对SAH后24、72 h的SAH分级、神经功能、脑水含量进行评估,确定药物最佳注射剂量。先选取54只小鼠随机分为3组:假手术组(Sham)、模型组(SAH)、模型+染料木黄酮组(SAH+genistein),每组18只。采用伊文思蓝检测血脑屏障通透性,Tunel染色观察神经元凋亡的情况,Western blot检测凋亡相关蛋白、血脑屏障相关蛋白的表达。而后选取30只小鼠随机分为5组:假手术组(Sham)、溶剂组(SAH+Vehicle)、模型+染料木黄酮组(SAH+genistein)、模型+染料木黄酮+溶剂组(SAH+genistein+DMSO)、模型+染料木黄酮+抑制剂组(SAH+genistein+Sirtinol),每组6只。采用Western blot检测SIRT1、p53、AC p53的蛋白表达水平。结果(1)SAH后24、72 h,中、高剂量组的出血量评分和脑水含量较溶剂组均明显下降,Garcia评分和平衡木实验评分均明显升高,其中中剂量组的作用最为显著,因此选用染料木黄酮15 mg/kg进行后续实验。(2)SAH后24 h,与模型组比较,模型+染料木黄酮组的伊文思蓝渗出量降低,ZO-1、Occludin和Claudin-5蛋白表达增加,差异均有统计学意义(P<0.05)。Tunel染色和Western blot检测结果显示,与模型组比较,模型+染料木黄酮组神经元凋亡率降低,凋亡蛋白Caspase-3、Cleaved Caspase-3、Bax表达减少,抗凋亡蛋白Bcl-2增加,差异均有统计学意义(P<0.05)。(3)SAH后24 h,与溶剂组比较,模型+染料木黄酮组SIRT1蛋白表达显著增加,p53、AC p53蛋白表达均显著降低,差异�Objective To investigate the effect and mechanism of genistein on early brain injury(EBI)following subarachnoid hemorrhage(SAH).Methods A total of 156 male C57BL/6J mice were selected and the mouse SAH model was established using intravascular puncture.Pre experiment:72 mice were selected and set at two time points of 24 and 72 h,with 36 mice at each time point.Mice at each time point were randomly divided into 6 groups:Sham group,SAH group,solvent group(SAH+Vehicle),low dose group(genistein 5 mg/kg),medium dose group(genistein 15 mg/kg),and high dose group(genistein 30 mg/kg),with 6 mice in each group.The SAH grading,neurological function,and brain water content at 24 and 72 h after SAH were evaluated to determine the optimal injection dose of the drug.After that,54 mice were randomly divided into three groups:Sham group,SAH group and SAH+genistein group,with 18 mice in each group.The permeability of blood-brain barrier was measured by measuring the extravasation of Evans blue.Tunel staining was used to observe cell apoptosis.Western blot was used to verify the expression of apoptosis-related proteins and blood-brain barrier related proteins.Then,30 mice were randomly divided into 5 groups,including Sham group,SAH+Vehicle group,SAH+genistein group,SAH+genistein+DMSO group,SAH+genistein+Sirtinol group,with 6 mice in each group.Western blot was used to verify the protein levels of SIRT1,p53 and AC p53.Results(1)At 24 and 72 h after SAH,the bleeding volume score and brain water content of the medium and high dose groups were significantly reduced compared to the solvent group,while the Garcia score and balance beam experiment score were significantly increased.Among them,the effect of the medium dose group was the most significant.Therefore,15 mg/kg of genistein was selected for subsequent experiments.(2)After 24 h of SAH,compared with the SAH group,the Evans blue exudation in the SAH+genistein group decreased,and the protein expression of ZO-1,Occludin,and Claudin-5 increased,with statistical significance(P<0.05).

关 键 词：蛛网膜下腔出血 染料木黄酮 早期脑损伤 SIRT1/p53信号通路 成年小鼠 

分 类 号：R651.15[医药卫生—外科学]