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中华神经创伤外科电子杂志

中华神经创伤外科电子杂志 ›› 2023, Vol. 09 ›› Issue (05) : 261 -269. doi: 10.3877/cma.j.issn.2095-9141.2023.05.002

基础研究

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
朱泽超, 杨新宇, 李侑埕, 潘鹏宇, 梁国标†()   
  1. 110016　沈阳，北部战区总医院神经外科
  • 收稿日期:2023-03-05 出版日期:2023-10-15
  • 通信作者: 梁国标

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

Zechao Zhu, Xinyu Yang, Youcheng Li, Pengyu Pan, Guobiao Liang†()   

  1. Department of Neurosurgery, Hospital of the Northern Theater Command, Shenyang 110016, China
  • Received:2023-03-05 Published:2023-10-15
  • Corresponding author: Guobiao Liang
  • Supported by:
    National Natural Science Foundation of China(81971133)
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引用本文：

朱泽超, 杨新宇, 李侑埕, 潘鹏宇, 梁国标. 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[J]. 中华神经创伤外科电子杂志, 2023, 09(05): 261-269.

Zechao Zhu, Xinyu Yang, Youcheng Li, Pengyu Pan, Guobiao Liang. Impact of genistein on early brain injury following subarachnoid hemorrhage in mice via the SIRT1/p53 signaling pathway[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2023, 09(05): 261-269.

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，差异均有统计学意义（P<0.05）；与模型+染料木黄酮+溶剂组相比，模型+染料木黄酮+抑制剂组的SIRT1蛋白表达显著下降，p53、AC p53蛋白表达显著增加，差异均有统计学意义（P<0.05）。

结论

染料木黄酮可以通过SIRT1/p53信号通路，减轻脑水肿和神经元凋亡的作用，改善小鼠SAH后的EBI，对SAH具有显著的治疗意义。

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
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). The results of Tunel staining and Western blot analysis showed that compared with the SAH group, the SAH+genistein group had a lower neuronal apoptosis rate, decreased expression of apoptotic proteins Caspase-3, Cleared Caspase-3, and Bax, and increased expression of anti apoptotic protein Bcl-2, with statistical significance (P<0.05). (3) After 24 h of SAH, compared with the SAH+Vehicle group, the SAH+genistein group showed a significant increase in SIRT1 protein expression, while the expression of p53 and AC p53 proteins decreased significantly, with statistical significance (P<0.05); Compared with the SAH+genistein+DMSO group, the SIRT1 protein expression in the SAH+genistein+Sirtinol group significantly decreased, while the expression of p53 and AC p53 proteins significantly increased, with statistical significance (P<0.05).

Conclusion

Genistan can regulate SIRT1/p53 signaling pathway, reduce cerebral edema and neuronal cell apoptosis, and improve EBI after SAH in mice, which has significant therapeutic significance for SAH.

Key words: Subarachnoid hemorrhage, Genistein, Early brain injury, SIRT1/p53 signaling pathway, Adult mice
图1 6组小鼠SAH后24、72 h SAH分级评分、神经功能评分以及脑水含量比较A：SAH后24 h SAH分级评分；B：SAH后24 h Garcia评分；C：SAH后24 h平衡木实验评分；D：SAH后24 h脑水含量；E：SAH后72 h SAH分级评分；F：SAH后72 h Garcia评分；G：SAH后72 h平衡木实验评分；H：SAH后72 h脑水含量；与假手术组比较，aP<0.05；与溶剂组比较，bP<0.05；SAH：蛛网膜下腔出血
Fig.1 Comparison of SAH grading score, neurological function score, and brain water content in 6 groups of mice after SAH at 24 and 72 h
图2 3组小鼠SAH后24 h的伊文思蓝渗出率及血脑屏障相关蛋白表达的比较A：小鼠脑大体图；B：伊文思蓝渗出量；C：ZO-1、Occludin和Claudin-5蛋白表达的Western blot条带；D：ZO-1蛋白表达的定量分析；E：Occludin蛋白表达的定量分析；F：Claudin-5蛋白表达的定量分析；与假手术组比较，aP<0.05；与模型组比较，bP<0.05；SAH：蛛网膜下腔出血
Fig.2 Comparison of Evans blue exudation rate and blood-brain barrier related protein expression in three groups at 24 h after SAH
图3 Tunel染色检验染料木黄酮在SAH后24 h对神经元凋亡的影响A：3组小鼠脑组织神经元凋亡染色图（×400），箭头示凋亡的阳性神经元；B：3组小鼠神经元凋亡率比较；与假手术组比较，aP<0.05；与模型组比较，bP<0.05；SAH：蛛网膜下腔出血
Fig.3 Tunel staining examine the results and quantification of genistein on neuronal apoptosis at 24 h after SAH
图4 Western blot检测3组小鼠SAH后24 h凋亡相关蛋白的表达A：Caspase-3、Cleaved Caspase-3、Bax和Bcl-2蛋白表达的Western blot条带；B：Caspase-3蛋白表达的定量分析；C：Cleaved Caspase-3蛋白表达的定量分析；D：Bax蛋白表达的定量分析；E：Bcl-2蛋白表达的定量分析；与假手术组比较，aP<0.05；与模型组比较，bP<0.05；SAH：蛛网膜下腔出血
Fig.4 Western blot detection of apoptosis-related protein expression in three groups at 24 h after SAH
图5 Western blot检测5组小鼠SAH后24 h SIRT1、p53和AC p53蛋白的表达A：SIRT1、p53和AC p53蛋白表达的Western blot条带；B：SIRT1蛋白表达的定量分析；C：p53蛋白表达的定量分析；D：AC p53蛋白表达的定量分析；与假手术组比较，aP<0.05；与溶剂组比较，bP<0.05；与模型+染料木黄酮+溶剂组比较，cP<0.05；SAH：蛛网膜下腔出血
Fig.5 Western blot detection of SIRT1, p53, and AC p53 protein expression in five groups at 24 h after SAH
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