Skip to main content
NCBI home page
Journal of Central South University Medical Sciences logoLink to Journal of Central South University Medical Sciences
. 2023 Feb 28;48(2):172–181. [Article in Chinese] doi: 10.11817/j.issn.1672-7347.2023.220167

组蛋白脱乙酰酶6特异性抑制剂tubastatin A对大鼠蛛网膜下腔出血的保护作用及其机制

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms

ZHU Yuwei 1,2, ZHENG Haiping 1, CHEN Chunli 1,✉
Editor: 郭 征
PMCID: PMC10930345  PMID: 36999463

Abstract

Objective

Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms.

Methods

Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery.

Results

The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) .

Conclusion

HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

Keywords: histone deacetylase 6, subarachnoid hemorrhage, early brain injury, cerebral vasospasm, tubastatin A, brain edema, apoptosis, nitric oxide synthase

蛛网膜下腔出血(subarachnoid hemorrhage，SAH)是出血性脑卒中的一种类型，约占所有卒中的5%，年发病率为每10万人2~22.5人，致死率约为30%[1]。作为一种严重的脑血管疾病，SAH患者预后较差，即使保全了生命，也可能会留下残疾或者精神障碍，给社会、家庭及患者本人造成沉重的负担。尽管血管内介入治疗和显微外科手术技术近年来有所进步，SAH患者的预后仍不能令人满意。因此，需要开发治疗SAH的新药物。SAH的损伤机制尚未完全阐明，早期脑损伤(early brain injury，EBI)和脑血管痉挛作为导致SAH患者预后不良的主要原因得到了普遍认可[2-4]。EBI的病理改变主要包括颅内压升高、氧化应激、炎症、血脑屏障的破坏、脑水肿、细胞凋亡等[5-7]。另外，越来越多的证据[8-9]表明内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)表达的下调和诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)表达的上调是导致SAH后脑血管痉挛的重要因素。

组蛋白脱乙酰酶(histone deacetylase，HDAC)通过调节细胞内发挥重要作用的组蛋白和非组蛋白的乙酰化来调控细胞的生命活动[10]。HDAC主要分为I类(HDAC 2/3/8)、II类(HDAC 4/5/6/7/9/10)、III类(Sirt 1~7)、IV类(HDAC 11)，它们分别调控不同的底物[11]。其中，HDAC6是IIb类中的一个比较独特的成员，主要定位于细胞质，与乙酰化微管蛋白α-tubulin、热激蛋白(heat shock protein，HSP)90、β-catenin等蛋白质相互作用，并通过调控这些蛋白质的乙酰化水平参与细胞的迁移、存活、炎症以及氧化应激等过程[12-13]。Rivieccio等[14]研究发现使用HDAC6选择性抑制剂tubastatin A(TubA)或短发夹RNA(shRNA)靶向抑制HDAC6可用于治疗氧化应激造成的神经元损伤和促进神经元的修复。此外，TubA在多种急慢性中枢神经系统疾病的动物模型中被证实有明确的神经保护作用[15-16]。然而，HDAC6在SAH中所扮演的角色及其抑制剂TubA对SAH的神经保护作用和相关机制尚未明确。因此，本研究旨在探讨HDAC6在SAH后EBI中的表达，及TubA对SAH后EBI和脑血管痉挛的保护作用和其机制。

1. 材料与方法

1.1. 动物和主要试剂

成年(8~9周)雄性SD大鼠(280~330 g)均购自湖南斯莱克景达实验动物有限公司。大鼠饲养在实验动物专用饲养房内，温度控制在22 ℃左右，保持通风，明暗循环时间为12 h，并让动物自由饮水和摄食。涉及动物的所有程序严格遵守美国国立卫生研究院实验动物护理和使用指南，所有实验程序均经湘雅二医院实验动物伦理委员会批准(审批号：2020594)。研究过程中尽量减轻动物的痛苦。

TubA购自美国Selleck生物科技有限公司；兔抗HDAC6多克隆抗体购自美国Proteintech公司；兔抗eNOS单克隆抗体、兔抗iNOS多克隆抗体、小鼠抗 β-actin单克隆抗体、小鼠抗神经元细胞核(neuronal nuclei，NeuN)单克隆抗体、小鼠抗胶质纤维酸性蛋白(glial fibrillary acidic protein，GFAP)单克隆抗体和山羊抗离子钙结合适配器分子1(calcium-binding adaptor molecule 1，IBA-1)多克隆抗体均购自英国Abcam公司；增强型化学发光(enhanced chemi-luminescence，ECL)发光液、含吐温的磷酸盐缓冲液(phosphate buffered saline-Tween，PBST)、4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)购自武汉谷歌生物科技有限公司；末端脱氧核苷酸转移酶介导的dUTP缺口标记(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling，TUNEL)染色试剂盒购自瑞士罗氏公司。

1.2. SAH模型的建立

采用改良的颈内动脉穿刺法建立大鼠SAH模型。用戊巴比妥钠(50 mg/kg)腹腔注射麻醉大鼠后，从组织中分离右侧颈总动脉、右侧颈外动脉和右侧颈内动脉。将锐化的4-0尼龙缝线从右侧颈外动脉残端推入右侧颈内动脉，感觉到阻力后再向前推进4 mm，刺破大脑中动脉分叉处。假手术(sham)组大鼠也进行相同的操作，只是不穿破血管。分层缝合颈部切口。术中监测并维持肛温37 ℃。术后单笼饲养大鼠，注意保暖。SAH建模成功的标准是在小鼠颅底有明确的血块而无实质性脑损害。

1.3. 分组及处理

第1部分，为确定SAH早期HDAC6的表达变化，将大鼠随机分为6组：sham组、SAH-3 h组、SAH-6 h组、SAH-12 h组、SAH-24 h组、SAH-48 h组。分别在SAH建模后3、6、12、24 h，取各组大鼠的损伤侧大脑皮质样本行蛋白质印迹法分析。另取2只SAH-24 h组的大鼠，采用免疫荧光双染测定HDAC6在损伤侧大脑皮质的分布。

第2部分，为探讨TubA对SAH大鼠的神经保护作用，将大鼠随机分为4组：sham组、SAH组、SAH+TubAL组(给予TubA 25 mg/kg)、SAH+TubAH组(给予TubA 40 mg/kg)。在建模后24 h时，对各组大鼠行神经功能评分，检测各组大鼠脑组织含水量。

第3部分，为探讨TubA对SAH大鼠神经保护作用的机制，将大鼠随机分为SAH组和SAH+TubA组(给予TubA 40 mg/kg)。于建模24 h后迅速断头，取损伤侧大脑皮质组织行蛋白质印迹法检测HDAC6、eNOS和iNOS的表达水平。在同一时间点，2组各取4只大鼠，行灌注取脑，制作石蜡切片，行苏木精-伊红(hematoxylin and eosin，HE)染色和TUNEL染色。

如果在实验过程中各组大鼠出现死亡或建模失败，则随机使用备用大鼠补充。将TubA溶解在少量的二甲基亚砜(dimethyl sulfoxide，DMSO)中，然后用含30%聚乙二醇300(polyethylene glycol 300，PEG300)的PBS稀释，稀释后DMSO的最终浓度为4%。SAH建模成功后立即腹腔注射TubA。Sham组和SAH模型组在相应时间点给予等量的溶剂。TubA的给药剂量及给药时间参考文献[15-16]。

1.4. 观察指标

1.4.1. 神经功能评分

参照改良后的Garcia评分[17]，评估大鼠SAH后24 h的神经功能评分。评分人对大鼠的分组不知情。分6个方面评价，具体评分标准如下：1)自由活动。让大鼠在全新的鼠笼中自由活动5 min，记录大鼠在笼内的活动及5 min内接触笼壁的情况。大鼠接触3侧或4侧笼壁记3分，接触少于3侧笼壁记2分，仅能少量活动记1分，不能活动记0分。2)四肢活动。抓住鼠尾并将大鼠提起置于空中，观察大鼠四肢自由活动情况。四肢活动如术前记3分；四肢均有活动，但较正常鼠慢记2分；肢体仅有轻微活动记1分；不活动记0分。3)前肢伸展。抓住鼠尾并将鼠提起置于空中，慢慢接近桌沿，观察大鼠双侧前肢伸展的情况。双侧前肢伸展如术前记3分，双侧前肢较术前伸展轻微受限记2分，前肢仅有轻度伸展记1分，前肢无伸展记0分。4)攀爬能力。将大鼠放置于倾斜的金属网格笼盖底部，引诱大鼠向上攀爬，观察其攀爬能力。正常攀爬如术前记3分，攀爬能力较术前减弱记2分，不能攀爬记1分。5)双侧躯干的触碰反应。将大鼠置于笼内，用木棒轻轻接触双侧躯干，观察大鼠的反应。反应正常如术前记3分，反应较前减弱记2分，无反应记1分。6)双侧触须的触碰反应。将大鼠置于笼内，用木棒从后往前轻轻接触双侧触须，观察大鼠的反应。反应正常如术前记3分，反应较前减弱记2分，无反应记1分。总分最低分为3，最高分为18。

1.4.2. 脑组织含水量

在用戊巴比妥钠深度麻醉后断头处死大鼠，快速取出脑组织。立即称右半球质量(湿重)，在105 ℃的烤箱中烘干24 h，再次测量右半球质量(干重)。脑组织含水量=(湿重-干重)/湿重×100%。

1.4.3. 蛋白质的提取及蛋白质印迹法

断头取脑后，在冰上快速分离出损伤侧大脑皮质组织100 mg，用眼科剪充分剪碎，加入含有终浓度为0.1 mol/L苯甲基磺酰氟(phenylmethylsulfonyl fluoride，PMSF)的放射免疫沉淀法(radio- immunoprecipitation assay，RIPA)裂解缓冲液300 μL，经超声充分裂解；在4 ℃下以12 000 r/min离心15 min，收集上清液，行BCA法测定蛋白质浓度。

每组样品取50 μg蛋白质进行聚丙烯酰胺凝胶电泳，采用湿转法将蛋白质转至聚偏二氟乙烯(polyvinylidenefluoride，PVDF)膜，将膜放入含有5%脱脂奶粉的封闭液中在室温下封闭1 h；在4 ℃下孵育一抗(兔抗HDAC6多克隆抗体、兔抗eNOS单克隆抗体、兔抗iNOS多克隆抗体、小鼠抗β-actin单克隆抗体)过夜；用PBST洗膜3次，每次5 min，加入辣根过氧化物酶标记的羊抗兔或羊抗小鼠二抗，在室温下孵育2 h，用PBST洗膜3次，每次5 min，用ECL显影，使用扫描仪扫描胶片，Image J软件进行灰度分析。

1.4.4. 石蜡包埋

将大鼠用戊巴比妥钠深度麻醉后，先灌注生理盐水以排出体内血液，再灌注4%的多聚甲醛完成固定。取完整大鼠脑组织，放入4%的多聚甲醛中浸泡过夜。经常规梯度乙醇脱水后，再以二甲苯透明；经浸蜡与包埋后，制成厚度为4 μm的石蜡切片。

1.4.5. 免疫荧光双染

切片脱蜡和再水化后，用PBS冲洗玻片，在切片上滴加5%的BSA，于25 ℃孵育2 h；在切片上分别加入配制好的一抗(兔抗HDAC6多克隆抗体、小鼠抗NeuN单克隆抗体，小鼠抗GFAP单克隆抗体和山羊抗IBA-1多克隆抗体)，于4 ℃湿盒中孵育过夜；次日，在室温下复温30 min，在切片上分别加入相应的荧光二抗，于4 ℃湿盒中避光孵育2 h；用PBS洗3次，每次5 min；用吸水纸吸干多余水分，使用含DAPI的抗淬灭封片剂封片后，置于荧光显微镜下观察。红色荧光标记HDAC6的表达，绿色荧光分别标记GFAP、IBA-1、NeuN的表达，蓝色荧光标记细胞核DAPI的表达。

1.4.6. TUNEL染色

切片脱蜡和再水化后，用PBS冲洗2次，将冲洗后的切片放置于4%的多聚甲醛溶液中浸泡10 min后，用PBS再冲洗3次；将切片置于蛋白酶K工作溶液中于37 ℃孵育15 min，再次在PBS中洗涤，用含有10%末端脱氧核苷酸转移酶(terminal deoxynucleotidyl transferase，TdT)的绿色荧光素标记的脱氧尿苷三磷酸(deoxyuridine triphosphate，dUTP)溶液处理；最后，用DAPI复染石蜡切片。显示绿色荧光颗粒的为TUNEL阳性细胞。在显微镜下观察5个视野(×400)，取同一视野TUNEL阳性细胞的数目与总细胞的百分比作为凋亡细胞的百分比。

1.4.7. HE染色

切片脱蜡、水化后，先用苏木精染色，再用伊红复染。依次浸入梯度乙醇中脱水5 min；然后浸入二甲苯I 5 min、二甲苯II 5 min透明；取出切片，滴加适量中性树脂，加盖玻片封片，在显微镜下进行观察。参考Wang等[18]描述的方法测量大鼠大脑中动脉的直径，每只大鼠选3张切片用Case viewer软件录入组织切片全扫图，选择大脑中动脉横截面最长内径的起始点，软件根据标尺自动读取的该两点之间数值即为大脑中动脉直径，记录并计算平均值。

1.5. 统计学处理

所有数据采用SPSS 19.0统计软件进行分析，定量数据以均数±标准误( x¯ ±SEM)表示，柱状图用GraphPad Prism 5.0制作。各组间的数据差异比较采用单因素方差分析和Tukey多重比较检验。P<0.05为差异有统计学意义。

2. 结 果

2.1. SAH后48 h内HDAC6的表达变化

在SAH建模后24 h时，SAH模型大鼠脑组织右侧基底池及脑干表面可见血凝块(图1A)。蛋白质印迹法结果显示：损伤侧大脑皮质中HDAC6的蛋白质表达水平在SAH后6 h开始升高(P<0.05)，24 h时达到峰值(P<0.001)，48 h时出现下降，但仍明显高于sham组(P<0.05，图1B)。

图1.

图1

Open in a new tab

SAH后24 h模型大鼠脑组织(A)和SAH早期不同时间点大鼠损伤侧大脑皮质HDAC6的蛋白质表达水平(B)

Figure 1 Brain tissue of model rats at 24 h after SAH (A) and protein expression level of HDAC6 in the injured cerebral cortex of rats at different time points in the early stage of SAH (B)

Values are presented as the mean±standard error of mean (SEM). *P<0.05, ***P<0.001 vs the sham group. SAH: Subarachnoid hemorrhage; HDAC6: Histone deacetylase 6.

2.2. HDAC6的细胞定位

免疫荧光双染结果显示：标记HDAC6的红色荧光与标记NeuN的绿色荧光明显重叠，且HDAC6阳性红色区域主要分布于蓝色荧光标记的DAPI周围。标记HDAC6的红色荧光与标记GFAP和IBA-1的绿色荧光区域只有极少部分重叠(图2)。

图2.

图2

Open in a new tab

免疫荧光双染示SAH后24 h HDAC6的定位

Figure 2 Double immunofluorescence staining showing the localization of HDAC6 at 24 h after SAH

The green arrows shows that HDAC6 (red fluorescence labeling) is mainly colocalization with NeuN (green fluorescence labeling). Scale bar=50 μm. SAH: Subarachnoid hemorrhage; HDAC6: Histone deacetylase 6; NeuN: Neuronal nuclei; GFAP: Glial fibrillary acidic protein; IBA-1: Calcium-binding adaptor molecule 1; DAPI: 4',6-diamidino-2-phenylindole.

2.3. TubA改善SAH大鼠的神经功能并减轻脑水肿

与sham组大鼠比较，SAH组大鼠神经功能评分明显降低(P<0.01，图3A)，脑组织含水量增加(P<0.01，图3B)。与SAH组大鼠比较，SAH+TubAH组神经功能评分明显升高且损伤侧脑组织含水量明显下降，差异均有统计学意义(均P<0.05，图3)；而SAH+TubAL组神经功能改善和脑组织含水量下降均不明显，差异均无统计学意义(均P>0.05，图3)。高剂量(40 mg/kg)TubA能改善SAH大鼠的神经功能并减轻脑水肿。因此，在后续研究中选择40 mg/kg的TubA。

图3.

图3

Open in a new tab

不同剂量的TubA对SAH后24 h神经功能评分(A)和脑组织含水量(B)的影响

Figure 3 Effects of different doses of TubA on score of neurological function (A) and brain edema (B) at 24 h after SAH

Values are presented as the mean±standard error of mean (SEM). **P<0.01 vs the sham group; †P<0.05 vs the SAH group. TubA: Tubastatin A; SAH: Subarachnoid hemorrhage; HDAC6: Histone deacetylase 6.

2.4. HDAC6、eNOS和iNOS的蛋白质表达水平

与sham组大鼠比较，SAH组eNOS表达显著降低(P<0.01)，iNOS和HDAC6表达均明显上调(分别 P<0.05和P<0.01)；与SAH组相比，SAH+TubA组eNOS表达明显升高，iNOS和HDAC6均明显下调(均P<0.05，图4)。

图4.

图4

Open in a new tab

TubA对SAH后24 h HDAC6、eNOS和iNOS表达的影响

Figure 4 Effects of TubA treatment on the expression of HDAC6, eNOS, and iNOS at 24 h after SAH

Values are presented as the mean±standard error of mean (SEM). *P<0.05, **P<0.01 vs the sham group; †P<0.05 vs the SAH group. TubA: Tubastatin A; SAH: Subarachnoid hemorrhage; HDAC6: Histone deacetylase 6; eNOS: Endothelial nitric oxide synthase; iNOS: Inducible nitric oxide synthase.

2.5. TubA减轻SAH诱导的细胞凋亡和脑血管痉挛

与SAH组大鼠比较，SAH+TubA组TUNEL阳性细胞数明显减少，且大脑中动脉直径明显增加，差异均有统计学意义(均P<0.05，图5)。

图5.

图5

Open in a new tab

TubA对SAH后24 h损伤侧大脑皮质细胞凋亡(A)及脑血管痉挛(B)的影响

Figure 5 Effects of TubA treatment on cell apoptosis (A) and cerebral vasospasm (B) in the ipsilateral cortex at 24 h after SAH

Scale bar=20 μm. Values are presented as the mean±standard error of mean (SEM). *P<0.05 vs the SAH group. TubA: Tubastatin A; SAH: Subarachnoid hemorrhage; TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; MCA: Middle cerebral artery.

3. 讨 论

多项研究[19-21]表明HDAC抑制剂在表观遗传学领域治疗中风和神经退行性疾病中具有广泛的应用前景，它分为广谱性HDAC抑制剂和选择性HDAC抑制剂。HDAC6具有2个催化域和1个泛素相互作用域。近年来因其在卒中等神经科疾病中发挥重要作用而受到关注[14]，靶向抑制HDAC6成为开发治疗中枢神经系统疾病新药的热点。TubA是一种强效和高选择性的HDAC6抑制剂，半抑制浓度(half maximal inhibitory concentration，IC50)为15 nmol/L，对除HDAC8外的其他异构体的选择性均超过1 000倍[22]，已在多种中枢神经疾病的动物模型中被证实具有神经保护作用。近年来国内外学者也开始探索其保护机制，Wang等[16]证实TubA能通过乙酰化微管蛋白增加成纤维细胞生长因子21(fibroblast growth factor-21，FGF21)的释放和改善线粒体运输功能障碍；通过与其底物乙酰化微管蛋白的相互作用调节轴突生长及皮质神经元的迁移和分化[23]；通过乙酰化介导的蛋白酶体降解起到抗炎、抗氧化作用[24-25]。尽管HDAC6在许多疾病中所扮演的角色已被深入挖掘，但HDAC6对SAH的影响及其抑制剂对SAH的保护作用尚未有报道。

研究[26]发现：在缺血性脑梗死的细胞模型和动物模型中，HDAC6的表达在脑梗死后3 h开始明显上调。提示其可能参与脑梗死早期的病理过程。Zhang等[15]在阿尔茨海默病转基因小鼠模型中也发现HDAC6的表达相比野生型小鼠显著上调。本研究结果显示：受损大脑皮质中HDAC6的蛋白质表达水平在SAH后6 h开始升高，24 h时达到峰值，48 h时出现下降，但仍明显高于sham组。另外，本研究还发现：在SAH后24 h，HDAC6主要与神经元共定位，很少在星形胶质细胞或小胶质细胞中表达。提示HDAC6可能对大鼠SAH后早期神经元的存活和凋亡发挥重要作用。

基于上述结果，本研究进一步探讨HDAC6特异性抑制剂TubA对SAH后EBI的作用及其机制。结果显示：40 mg/kg的TubA能明显减轻SAH后24 h时的脑水肿及神经功能损伤，而25 mg/kg的TubA没有明显效果。此结果首次表明适宜剂量的TubA对SAH EBI的神经保护作用。Yuan等[27]在大鼠大脑皮质神经元模型中发现，另一种HDAC6特异性抑制剂Tubacin可通过调控活性氧和乙酰化微管蛋白的表达减少神经细胞坏死，提高神经细胞的存活率。本课题组前期的研究[28]发现：在N2a细胞氧糖剥夺再灌注的体外模型中，利用shRNA干扰HDAC6的表达可以抑制胱天蛋白酶(caspase)-3的激活和p115的裂解，从而减少氧糖剥夺再灌注诱导的神经细胞凋亡。与这些结果相符，本研究结果证实TubA能减轻大鼠SAH早期大脑皮质细胞凋亡。

脑血管痉挛与SAH患者的不良预后密切相关，本研究进一步检测各组大鼠大脑中动脉直径以评估TubA对SAH后脑血管痉挛的改善作用。结果发现TubA对SAH导致的脑血管痉挛具有明显的改善作用。在外伤性失血性休克模型中发现TubA可减轻缺氧诱导的内皮细胞高通透性，改善内皮细胞屏障功能[29]。利用shRNA干扰HDAC6的表达能通过降低内皮细胞通透性，改善肺微血管内皮细胞屏障功能[30]。这些研究均初步证实TubA具有潜在的血管保护作用。虽然发生SAH后脑血管痉挛的机制尚未完全阐明，但eNOS和iNOS的表达已被证实在SAH血管痉挛的发病机制中发挥重要调控作用[31-32]。Hino等[33]在SAH猴模型中发现病灶侧大脑中动脉eNOS mRNA的表达明显下调，较对侧约下降56%。在大鼠SAH模型中观察到eNOS的蛋白质表达明显下调，且eNOS表达的下调是SAH后脑血管痉挛的重要原因[32, 34]。近年来，以eNOS为靶点的药物等治疗手段在SAH后的脑血管痉挛中显示出广泛的应用前景[35-37]。另外，研究[38]发现iNOS在机体受外界刺激时产生非特异反应，其诱导产生的NO可加重血管内皮细胞的氧化应激反应，是导致脑血管痉挛的重要炎性介质。Zheng等[39]研究发现静脉注射iNOS抑制剂氨基胍可以通过上调eNOS的表达显著减轻SAH引起的脑血管痉挛。本研究结果表明：SAH建模后24 h，eNOS的表达相对于sham组明显下降，iNOS的表达较sham组明显升高，腹腔注射TubA能上调eNOS的表达，下调iNOS的表达。提示Tub可能是通过调节eNOS与iNOS的平衡改善SAH早期的脑血管痉挛。

综上，本研究结果发现HDAC6在SAH后EBI的病理过程中发挥重要作用。HDAC6的高选择性抑制剂TubA通过减轻脑水肿和减少细胞凋亡，对SAH大鼠EBI和脑血管痉挛起保护作用。此外，TubA减轻脑血管痉挛的作用可能是通过调节eNOS和iNOS的蛋白质表达水平来发挥的。深入研究HDAC6将有望为提高SAH患者生存率，减少SAH患者后遗症提供新的思路。

基金资助

湖南省自然科学基金(2021JJ40821)。

This work was supported by the Natural Science Foundation of Hunan Province, China (2021JJ40821).

利益冲突声明

作者声称无任何利益冲突。

作者贡献

朱雨薇、郑海平 实验操作，数据分析，论文写作；陈春丽 论文修改，实验指导。所有作者阅读并同意最终的文本。

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202302172.pdf

参考文献

  • 1. Etminan N, Chang HS, Hackenberg K, et al. Worldwide incidence of aneurysmal subarachnoid hemorrhage according to region, time period, blood pressure, and smoking prevalence in the population: A systematic review and Meta-analysis[J]. JAMA Neurol, 2019, 76(5): 588-597. 10.1001/jamaneurol.2019.0006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Dodd WS, Noda I, Martinez M, et al. NLRP3 inhibition attenuates early brain injury and delayed cerebral vasospasm after subarachnoid hemorrhage[J]. J Neuroinflammation, 2021, 18(1): 163. 10.1186/s12974-021-02207-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Qu W, Cheng Y, Peng W, et al. Targeting iNOS alleviates early brain injury after experimental subarachnoid hemorrhage via promoting ferroptosis of M1 microglia and reducing neuroinflammation[J]. Mol Neurobiol, 2022, 59(5): 3124-3139. 10.1007/s12035-022-02788-5. [DOI] [PubMed] [Google Scholar]
  • 4. Rass V, Helbok R. Early brain injury after poor-grade subarachnoid hemorrhage[J]. Curr Neurol Neurosci Rep, 2019, 19(10): 78. 10.1007/s11910-019-0990-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Li X, Liu W, Li R, et al. TSG-6 attenuates oxidative stress-induced early brain injury in subarachnoid hemorrhage partly by the HO-1 and Nox2 pathways[J]. J Stroke Cerebrovasc Dis, 2020, 29(12): 104986. 10.1016/j.jstrokecerebrovasdis.2020.104986. [DOI] [PubMed] [Google Scholar]
  • 6. Zhu Q, Enkhjargal B, Huang L, et al. Aggf1 attenuates neuroinflammation and BBB disruption via PI3K/Akt/NF-κB pathway after subarachnoid hemorrhage in rats[J]. J Neuroinflammation, 2018, 15(1): 178. 10.1186/s12974-018-1211-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Chen J, Chen G, Li J, et al. Melatonin attenuates inflammatory response-induced brain edema in early brain injury following a subarachnoid hemorrhage: a possible role for the regulation of pro-inflammatory cytokines[J]. J Pineal Res, 2014, 57(3): 340-347. 10.1111/jpi.12173. [DOI] [PubMed] [Google Scholar]
  • 8. Onoue H, Tsutsui M, Smith L, et al. Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage[J]. Stroke, 1998, 29(9): 1959-1966. 10.1161/01.str.29.9.1959. [DOI] [PubMed] [Google Scholar]
  • 9. Stoodley M, Weihl CC, Zhang ZD, et al. Effect of adenovirus-mediated nitric oxide synthase gene transfer on vasospasm after experimental subarachnoid hemorrhage[J]. Neurosurgery, 2000, 46(5): 1193-1203. 10.1097/00006123-200005000-00034. [DOI] [PubMed] [Google Scholar]
  • 10. Minucci S, Pelicci PG. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[J]. Nat Rev Cancer, 2006, 6(1): 38-51. 10.1038/nrc1779. [DOI] [PubMed] [Google Scholar]
  • 11. de Ruijter AJ, van Gennip AH, Caron HN, et al. Histone deacetylases (HDACs): characterization of the classical HDAC family[J]. Biochem J, 2003, 370(Pt3): 737-749. 10.1042/bj20021321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Valenzuela-Fernández A, Cabrero JR, Serrador JM, et al. HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions[J]. Trends Cell Biol, 2008, 18(6): 291-297. 10.1016/j.tcb.2008.04.003. [DOI] [PubMed] [Google Scholar]
  • 13. Seidel C, Schnekenburger M, Dicato M, et al. Histone deacetylase 6 in health and disease[J]. Epigenomics, 2015, 7(1): 103-118. 10.2217/epi.14.69. [DOI] [PubMed] [Google Scholar]
  • 14. Rivieccio MA, Brochier C, Willis DE, et al. HDAC6 is a target for protection and regeneration following injury in the nervous system[J]. Proc Natl Acad Sci USA, 2009, 106(46): 19599-19604. 10.1073/pnas.0907935106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Zhang L, Liu C, Wu J, et al. Tubastatin A/ACY-1215 improves cognition in Alzheimer’s disease transgenic mice[J]. J Alzheimers Dis, 2014, 41(4): 1193-1205. 10.3233/jad-140066. [DOI] [PubMed] [Google Scholar]
  • 16. Wang Z, Leng Y, Wang J, et al. Tubastatin A, an HDAC6 inhibitor, alleviates stroke-induced brain infarction and functional deficits: potential roles of α-tubulin acetylation and FGF-21 up-regulation[J]. Sci Rep, 2016, 6: 19626. 10.1038/srep19626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Garcia JH, Wagner S, Liu KF, et al. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation[J]. Stroke, 1995, 26(4): 627-635. 10.1161/01.str.26.4.627. [DOI] [PubMed] [Google Scholar]
  • 18. Wang Y, Liu Y, Li Y, et al. Protective effects of astaxanthin on subarachnoid hemorrhage-induced early brain injury: Reduction of cerebral vasospasm and improvement of neuron survival and mitochondrial function[J]. Acta Histochem, 2019, 121(1): 56-63. 10.1016/j.acthis.2018.10.014. [DOI] [PubMed] [Google Scholar]
  • 19. Chuang DM, Leng Y, Marinova Z, et al. Multiple roles of HDAC inhibition in neurodegenerative conditions[J]. Trends Neurosci, 2009, 32(11): 591-601. 10.1016/j.tins.2009.06.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Wang ZF, Fessler EB, Chuang DM. Beneficial effects of mood stabilizers lithium, valproate and lamotrigine in experimental stroke models[J]. Acta Pharmacol Sin, 2011, 32(12): 1433-1445. 10.1038/aps.2011.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Kazantsev AG, Thompson LM. Therapeutic application of histone deacetylase inhibitors for central nervous system disorders[J]. Nat Rev Drug Discov, 2008, 7(10): 854-868. https:// doi.10.1038/nrd2681. [DOI] [PubMed] [Google Scholar]
  • 22. Butler KV, Kalin J, Brochier C, et al. Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A[J]. J Am Chem Soc, 2010, 132(31): 10842-10846. 10.1021/ja102758v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. d’Ydewalle C, Krishnan J, Chiheb DM, et al. HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease[J]. Nat Med, 2011, 17(8): 968-974. 10.1038/nm.2396. [DOI] [PubMed] [Google Scholar]
  • 24. Vishwakarma S, Iyer LR, Muley M, et al. Tubastatin, a selective histone deacetylase 6 inhibitor shows anti-inflammatory and anti-rheumatic effects[J]. Int Immunopharmacol, 2013, 16(1): 72-78. 10.1016/j.intimp.2013.03.016. [DOI] [PubMed] [Google Scholar]
  • 25. Zhang Y, Liu CM, Cao XC, et al. Involvement of transcription factor XBP1s in the resistance of HDAC6 inhibitor tubastatin A to superoxidation via acetylation-mediated proteasomal degradation[J]. Biochem Biophys Res Commun, 2014, 450(1): 433-439. 10.1016/j.bbrc.2014.05.134. [DOI] [PubMed] [Google Scholar]
  • 26. Chen YT, Zang XF, Pan J, et al. Expression patterns of histone deacetylases in experimental stroke and potential targets for neuroprotection[J]. Clin Exp Pharmacol Physiol, 2012, 39(9): 751-758. 10.1111/j.1440-1681.2012.05729.x. [DOI] [PubMed] [Google Scholar]
  • 27. Yuan L, Wang Z, Liu L, et al. Inhibiting histone deacetylase 6 partly protects cultured rat cortical neurons from oxygen‑glucose deprivation‑induced necroptosis[J]. Mol Med Rep, 2015, 12(2): 2661-2667. 10.3892/mmr.2015.3779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Zhang J, Tan J, Hu Z, et al. HDAC6 Inhibition protects against ogdr-induced Golgi fragmentation and apoptosis[J]. Oxid Med Cell Longev, 2019, 2019: 6507537. 10.1155/2019/6507537.eCollection2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Bruhn PJ, Nikolian VC, Halaweish I, et al. Tubastatin A prevents hemorrhage-induced endothelial barrier dysfunction[J]. J Trauma Acute Care Surg, 2018, 84(2): 386-392. 10.1097/ta.0000000000001753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Yu J, Ma Z, Shetty S, et al. Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema[J]. Am J Physiol Lung Cell Mol Physiol, 2016, 311(1): L39-47. 10.1152/ajplung.00051.2016. [DOI] [PubMed] [Google Scholar]
  • 31. Sabri M, Ai J, Knight B, et al. Uncoupling of endothelial nitric oxide synthase after experimental subarachnoid hemorrhage[J]. J Cereb Blood Flow Metab, 2011, 31(1): 190-199. 10.1038/jcbfm.2010.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Lin CL, Shih HC, Dumont AS, et al. The effect of 17beta-estradiol in attenuating experimental subarachnoid hemorrhage-induced cerebral vasospasm[J]. J Neurosurg, 2006, 104(2): 298-304. 10.3171/jns.2006.104.2.298. [DOI] [PubMed] [Google Scholar]
  • 33. Hino A, Tokuyama Y, Weir B, et al. Changes in endothelial nitric oxide synthase mRNA during vasospasm after subarachnoid hemorrhage in monkeys[J]. Neurosurgery, 1996, 39(3): 562-567. 10.1097/00006123-199609000-00026. [DOI] [PubMed] [Google Scholar]
  • 34. Pluta RM. Dysfunction of nitric oxide synthases as a cause and therapeutic target in delayed cerebral vasospasm after SAH[J]. Acta Neurochir Suppl, 2008, 104: 139-147. 10.1007/978-3-211-75718-5_28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. McGirt MJ, Lynch JR, Parra A, et al. Simvastatin increases endothelial nitric oxide synthase and ameliorates cerebral vasospasm resulting from subarachnoid hemorrhage[J]. Stroke, 2002, 33(12): 2950-2956. 10.1161/01.str.0000038986.68044.39. [DOI] [PubMed] [Google Scholar]
  • 36. Khurana VG, Smith LA, Baker TA, et al. Protective vasomotor effects of in vivo recombinant endothelial nitric oxide synthase gene expression in a canine model of cerebral vasospasm[J]. Stroke, 2002, 33(3): 782-789. 10.1161/hs0302.103735. [DOI] [PubMed] [Google Scholar]
  • 37. Güleç İ, Şengelen A, Karagöz-Güzey F, et al. The calcimimetic R-568 attenuates subarachnoid hemorrhage-induced vasospasm through PI3K/Akt/eNOS signaling pathway in the rat model[J]. Brain Res, 2021, 1765: 147508. 10.1016/j.brainres.2021.147508. [DOI] [PubMed] [Google Scholar]
  • 38. 冯九庚, 封荣华, 段剑, 等. 诱导型一氧化氮合成酶在OxyHb诱导的血管痉挛细胞模型中的表达[J]. 中华实验外科杂志, 2008, 25(6): 807. [Google Scholar]; FENG Jiugeng, FENG Ronghua, DUAN Jian, et al. Expression of inducible nitric oxide synthase in OxyHb-induced vasospasm model in vitro[J]. Chinese Journal of Experimental Surgery, 2008, 25(6): 807. [Google Scholar]
  • 39. Zheng B, Zheng T, Wang L, et al. Aminoguanidine inhibition of iNOS activity ameliorates cerebral vasospasm after subarachnoid hemorrhage in rabbits via restoration of dysfunctional endothelial cells[J]. J Neurol Sci, 2010, 295(1/2): 97-103. 10.1016/j.jns.2010.04.012. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Central South University Medical Sciences are provided here courtesy of Central South University

RESOURCES