2023, Vol. 44
2. 郑州大学药学院药物分析教研室, 郑州 450000;
3. 郑州大学附属郑州中心医院神经内科, 郑州 450001
2. Department of Pharmaceutical Analysis, School of Pharmacy, Zhengzhou University, Zhengzhou 450000, Henan, China;
3. Department of Neurology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450001, Henan, China
急性脑梗死（acute cerebral infarction，ACI）是临床常见的心、脑血管和神经系统疾病，发病率和病死率高，逐渐成为公共卫生面临的主要挑战之一[1]。ACI是脑血流短暂或永久减少的结果，仅涉及脑大动脉的范围[2]。目前，重组组织型纤溶酶原激活剂（recombinant tissue plasminogen activator，rt-PA）被认为是临床治疗ACI最有效的药物之一，但不良反应较多[3]。我国指南建议ACI患者发病3 h内应尽快静脉给予rt-PA溶栓治疗，发病6 h的ACI患者若不能使用rt-PA，可考虑静脉给予尿激酶[4]。替罗非班（tirofiban）是一种高选择性的糖蛋白Ⅱb/Ⅲa受体短效非肽抑制剂，可改善ACI患者的神经功能和体内炎症反应[5]，但其作用机制尚未明确。沉默信息调节因子2相关酶1（silent information regulator factor 2-related enzyme 1，SIRT1）是一种依赖于烟酰胺腺嘌呤二核苷酸的Ⅲ类组蛋白脱乙酰酶，在所有细胞类型中都有表达。它以广泛的转录因子为靶标，参与许多生理和病理过程如氧化应激、炎症和凋亡等[6-8]。研究表明，SIRT1通过调节血管内皮生长因子（vascular endothelial growth factor，VEGF）的表达参与脑缺血再灌注损伤过程[9]。本研究通过构建ACI大鼠模型，观察替罗非班能否通过影响SIRT1/VEGF信号通路对ACI大鼠起保护作用。
1 材料和方法 1.1 实验动物75只体重220~270 g的雄性SD大鼠购自珠海百试通生物科技有限公司[动物生产许可证号为SCXK（粤）2020-0051]。将SD大鼠（4只/笼）在（24±1）℃、光/暗循环（12 h/12 h）条件下饲养，予自由饮食、饮水。本研究通过郑州市第七人民医院动物伦理委员会审批，实验操作均遵循动物实验3R原则。
1.2 试剂与仪器替罗非班（纯度98%）购自上海源叶生物科技有限公司，SIRT1特异性抑制剂EX-527和ECL发光试剂盒购自上海碧云天生物技术有限公司，丙二醛（硫代巴比妥酸法）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）（比色法）、超氧化物歧化酶（superoxide dismutase，SOD）（微板法）检测试剂盒及氯化三苯基四氮唑（triphenyl tetrazolium chloride，TTC）染色、H-E染色试剂盒购自北京索莱宝科技有限公司，兔源SIRT1、VEGF、GAPDH一抗及HRP偶联的二抗、TUNEL检测试剂盒购自英国Abcam公司。BX61型光学显微镜购自日本Olympus公司，Gel Doc XR+凝胶成像系统购自美国BIO-RAD公司。
1.3 实验方法 1.3.1 分组及给药大鼠随机分为假手术组、模型组、替罗非班组、SIRT1抑制剂组、替罗非班+SIRT1抑制剂组，每组15只。除假手术组外，其他4组大鼠均采用大脑中动脉闭塞法（middle cerebral artery occlusion，MCAO）诱发ACI。替罗非班组大鼠术前3 d经尾静脉注射60 μg/kg替罗非班[10]；SIRT1抑制剂组大鼠术前3 d予5 mg/kg EX-527腹腔注射[11]；替罗非班+SIRT1抑制剂组大鼠术前3 d经尾静脉注射60 μg/kg替罗非班的同时予5 mg/kg EX-527腹腔注射；假手术组和模型组大鼠术前3 d分别通过尾静脉和腹腔注射等体积的生理盐水。
1.3.2 ACI模型的建立[12]使用戊巴比妥钠（40 mg/kg）麻醉大鼠后，仰卧位固定，使体核温度保持在37 ℃。将大鼠右侧颈部切开，充分露出右侧颈总动脉，分离并结扎颈外动脉及其分支。经颈动脉残端将3-0尼龙线插入颈内动脉，并延伸至大脑前动脉以阻断大脑中动脉。2 h后取出尼龙线使血流恢复，然后缝合皮肤、消毒，单笼饲养。假手术组除未阻断大脑中动脉外，其余手术操作与其他4组相同。各组大鼠于再灌注24 h后处死并进行取材和指标检测。
1.3.3 神经功能评分使用双盲法进行神经功能评分[13]。评分标准：0分，无神经损伤症状；1分，患侧前爪不能完全伸出；2分，行走时患侧前爪向内旋转；3分，行走时患侧前爪向内倾斜；4分，患侧足不能自主活动，失去意识；5分，患侧肢体完全不能动。得分＞3分说明ACI模型建立成功。
1.3.4 TTC染色检测脑梗死体积百分数神经功能评分完成后，每组取5只大鼠脑组织，切成2 mm厚的冠状切片，置于2% TTC溶液中在37 ℃孵育20 min，去除过量的TTC溶液，用4%多聚甲醛溶液固定。拍照并使用ImageJ 1.8.0软件分析脑梗死体积百分数。
1.3.5 血清丙二醛、GSH-Px、SOD的测定收集各组大鼠的外周血2 mL，分别按照试剂盒说明书操作，检测丙二醛、GSH-Px、SOD水平。
1.3.6 H-E染色检测脑组织海马区病理变化每组取5只大鼠脑组织，用4%多聚甲醛溶液固定过夜，经石蜡包埋后，用切片机切成4 μm厚的切片，经H-E染色后进行二甲苯透明、树胶封片。在光学显微镜下观察大鼠脑组织海马区病理变化并拍照。
1.3.7 TUNEL法检测神经元凋亡情况取脑组织海马区切片，用二甲苯脱蜡，乙醇梯度脱水至水化，按照TUNEL检测试剂盒说明书进行染色操作，然后在光学显微镜下随机选择5个视野拍照并进行细胞计数。正常细胞核呈蓝色，棕黄色为阳性细胞，按公式计算细胞凋亡率：细胞凋亡率（%）＝阳性细胞数/总细胞数×100%。
1.3.8 蛋白质印迹法检测SIRT1/VEGF通路相关蛋白表达每组取5只大鼠脑组织海马区，置于－80 ℃保存。取出后研磨成匀浆，利用预冷的RIPA裂解缓冲液提取总蛋白质，采用BCA法测定蛋白质浓度，10% SDS-PAGE分离蛋白质后转膜，然后用5%脱脂奶粉溶液孵育2 h以阻断非特异性结合位点。加入SIRT1一抗（稀释比例为1∶1 000）、VEGF一抗（稀释比例为1∶1 000）、GAPDH一抗（稀释比例为1∶3 000），在4 ℃孵育过夜。用TBST洗涤2次后，加入HRP偶联的二抗在室温下孵育2 h，通过ECL发光试剂盒检测蛋白质显色情况。以GAPDH为内参照，采用凝胶成像系统分析目的蛋白的相对表达量。
1.4 统计学处理应用SPSS 25.0软件进行统计学分析。计量资料以x±s表示，多组间比较采用单因素方差分析，多重比较采用SNK-q检验。检验水准（α）为0.05。
2 结果 2.1 替罗非班降低大鼠脑梗死体积百分数及神经功能评分TTC染色结果显示，假手术组大鼠脑组织呈均一红色，模型组、替罗非班组、SIRT1抑制剂组、替罗非班+SIRT1抑制剂组大鼠脑组织存在白色梗死区域（图 1A）。由图 1B可见，与假手术组比较，模型组大鼠脑梗死体积百分数升高（P＜0.05）；与模型组比较，替罗非班组、替罗非班+SIRT1抑制剂组大鼠脑梗死体积百分数降低，SIRT1抑制剂组大鼠脑梗死体积百分数升高（P均＜0.05）；与替罗非班组比较，替罗非班+SIRT1抑制剂组大鼠脑梗死体积百分数升高（P＜0.05）。由图 1C可见，与假手术组相比，模型组大鼠的神经功能评分升高（P＜0.05）；与模型组比较，替罗非班组、替罗非班+SIRT1抑制剂组大鼠的神经功能评分降低，SIRT1抑制剂组大鼠的神经功能评分升高（P均＜0.05）；与替罗非班组比较，替罗非班+SIRT1抑制剂组大鼠的神经功能评分升高（P＜0.05）。
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图 1 各组大鼠脑组织TTC染色、脑梗死体积百分数及神经功能评分 Fig 1 Brain tissue TTC staining, cerebral infarction volume percentage and neurological function score of rats in each group A: Representative TTC staining pictures of brain tissue; B: Comparison of cerebral infarction volume percentages in each group; C: Comparison of neurological function scores in each group. Sham group: The rats undergoing simulated surgery were injected with normal saline via tail vein 3 d before surgery; Model group: ACI model rats were injected intraperitoneally with normal saline 3 d before modeling; Tirofiban group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein 3 d before modeling; SIRT1 inhibitor group: ACI model rats were injected intraperitoneally with SITR1-specific inhibitor EX-527 (5 mg/kg) 3 d before modeling; Tirofiban+SITR1 inhibitor group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein and intraperitoneally administered with EX-527 (5 mg/kg) 3 d before modeling. *P < 0.05 vs sham group; △P < 0.05 vs model group; ▲P < 0.05 vs tirofiban group. n＝5 (B) or 15 (C), x±s. TTC: Triphenyl tetrazolium chloride; ACI: Acute cerebral infarction; SITR1: Silent information regulator factor 2-related enzyme 1. |
2.2 替罗非班抑制大鼠氧化应激
与假手术组相比，模型组大鼠血清丙二醛水平升高，GSH-Px、SOD水平降低（P均＜0.05）；与模型组相比，替罗非班组、替罗非班+SIRT1抑制剂组大鼠血清丙二醛水平降低，GSH-Px、SOD水平升高，而SIRT1抑制剂组大鼠血清丙二醛水平升高，GSH-Px、SOD水平降低（P均＜0.05）；与替罗非班组比较，替罗非班+SIRT1抑制剂组大鼠血清丙二醛水平升高，GSH-Px、SOD水平降低（P均＜0.05）。见图 2。
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图 2 各组大鼠血清MDA、GSH-Px、SOD水平比较 Fig 2 Comparison of MDA, GSH-Px and SOD levels in serum of rats in each group A: MDA level detected by thiobarbituric acid method; B: GSH-Px level detected by colorimetry; C: SOD level detected by microplate test. Sham group: The rats undergoing simulated surgery were injected normal saline via tail vein 3 d before surgery; Model group: ACI model rats were injected intraperitoneally normal saline 3 d before modeling; Tirofiban group: ACI model rats were injected tirofiban (60 μg/kg) via tail vein 3 d before modeling; SIRT1 inhibitor group: ACI model rats were injected intraperitoneally SIRT1-specific inhibitor EX-527 (5 mg/kg) 3 d before modeling; Tirofiban+SIRT1 inhibitor group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein and intraperitoneally administered with EX-527 (5 mg/kg) 3 d before modeling. *P < 0.05 vs sham group; △P < 0.05 vs model group; ▲P < 0.05 vs tirofiban group. n＝5, x±s. MDA: Malondialdehyde; GSH-Px: Glutathione peroxidase; SOD: Superoxide dismutase; ACI: Acute cerebral infarction; SIRT1: Silent information regulator factor 2-related enzyme 1. |
2.3 替罗非班减轻大鼠脑组织海马区病理损伤程度
H-E染色结果显示，假手术组大鼠脑组织海马区细胞结构完整，分布均匀，未见明显异常；模型组大鼠海马区大部分细胞排列紊乱，细胞间隙增大、细胞核破裂、凝缩变成固缩核，细胞坏死严重；替罗非班组大鼠海马区有少量细胞肿胀及坏死，病理损伤与模型组比较有所缓解；SIRT1抑制剂组大鼠海马区病理损伤程度较模型组明显加重；替罗非班+SIRT1抑制剂组大鼠脑组织海马区细胞排列紊乱，细胞间隙增大，大量细胞坏死，病变程度较替罗非班组严重，但较模型组有所缓解。见图 3。
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图 3 各组大鼠脑组织海马区苏木精-伊红染色 Fig 3 Hematoxylin-eosin staining of hippocampus of rats in each group Sham group: The rats undergoing simulated surgery were injected with normal saline via tail vein 3 d before surgery; Model group: ACI model rats were injected intraperitoneally with normal saline 3 d before modeling; Tirofiban group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein 3 d before modeling; SIRT1 inhibitor group: ACI model rats were injected intraperitoneally with SIRT1-specific inhibitor EX-527 (5 mg/kg) 3 d before modeling; Tirofiban+SIRT1 inhibitor group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein and intraperitoneally administered with EX-527 (5 mg/kg) 3 d before modeling. ACI: Acute cerebral infarction; SIRT1: Silent information regulator factor 2-related enzyme 1. |
2.4 替罗非班抑制大鼠脑组织海马区的神经元凋亡
TUNEL染色结果显示，与假手术组相比，模型组大鼠脑组织海马区神经元凋亡率升高（P＜0.05）；与模型组比较，替罗非班组、替罗非班+SIRT1抑制剂组大鼠脑组织海马区神经元凋亡率降低，SIRT1抑制剂组大鼠脑组织海马区神经元凋亡率升高（P均＜0.05）；与替罗非班组比较，替罗非班+SIRT1抑制剂组大鼠脑组织海马区神经元凋亡率升高（P＜0.05）。见图 4。
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图 4 TUNEL染色检测各组大鼠脑组织海马区神经元凋亡情况 Fig 4 Neuronal apoptosis in brain hippocampal regions in rats of each group detected by TUNEL staining A: Representative TUNEL staining pictures of brain hippocampal regions of rats in each group; B: Comparison of neuronal apoptosis rate in each group. Sham group: The rats undergoing simulated surgery were injected with normal saline via tail vein 3 d before surgery; Model group: ACI model rats were injected intraperitoneally with normal saline 3 d before modeling; Tirofiban group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein 3 d before modeling; SIRT1 inhibitor group: ACI model rats were injected intraperitoneally with SIRT1-specific inhibitor EX-527 (5 mg/kg) 3 d before modeling; Tirofiban+SIRT1 inhibitor group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein and intraperitoneally administered with EX-527 (5 mg/kg) 3 d before modeling. *P < 0.05 vs sham group; △P < 0.05 vs model group; ▲P < 0.05 vs tirofiban group. n＝5, x±s. TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; ACI: Acute cerebral infarction; SIRT1: Silent information regulator factor 2-related enzyme 1. |
2.5 替罗非班激活SIRT1/VEGF信号通路
蛋白质印迹法检测结果显示，与假手术组相比，模型组大鼠海马组织中SIRT1、VEGF蛋白表达水平均降低（P均＜0.05）；与模型组比较，替罗非班组、替罗非班+SIRT1抑制剂组大鼠海马组织中SIRT1、VEGF蛋白表达水平均升高，SIRT1抑制剂组大鼠海马组织中SIRT1、VEGF蛋白表达水平均降低（P均＜0.05）；与替罗非班组比较，替罗非班+SIRT1抑制剂组大鼠海马组织中SIRT1、VEGF蛋白表达水平均降低（P均＜0.05）。见图 5。
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图 5 蛋白质印迹法检测各组大鼠海马组织中SIRT1、VEGF蛋白表达 Fig 5 Expression of SIRT1 and VEGF proteins in hippocampal tissue of rats in each group detected by Western blotting Sham group: The rats undergoing simulated surgery were injected with normal saline via tail vein 3 d before surgery; Model group: ACI model rats were injected intraperitoneally with normal saline 3 d before modeling; Tirofiban group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein 3 d before modeling; SIRT1 inhibitor group: ACI model rats were injected intraperitoneally with SIRT1-specific inhibitor EX-527 (5 mg/kg) 3 d before modeling; Tirofiban+inhibitor group: ACI model rats were injected with tirofiban (60 μg/kg) via tail vein and intraperitoneally administered with EX-527 (5 mg/kg) 3 d before modeling. *P < 0.05 vs sham group; △P < 0.05 vs model group; ▲P < 0.05 vs tirofiban group. n＝5, x±s. SIRT1: Silent information regulator factor 2-related enzyme 1; VEGF: Vascular endothelial growth factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ACI: Acute cerebral infarction. |
3 讨论
大脑是高灌注、高耗氧量、高代谢和低能量储备的重要器官。发生ACI时，血管内血流中断导致缺氧缺血、梗死区缺氧、脑组织能量耗尽，最终导致脑组织坏死或软化。研究表明，MCAO可导致大鼠的行为、神经化学和组织学异常，该模型能够模拟人类ACI的许多特征[14-15]。本研究通过MCAO构建ACI模型，考察替罗非班对ACI的神经保护作用，结果证实替罗非班可抑制氧化应激损伤、神经元凋亡，调节SIRT1、VEGF蛋白表达。
脑缺血是导致神经功能损害的重要原因。神经功能缺损评分是评价脑损伤的常用指标[16]。本研究结果表明，闭塞大鼠大脑中动脉可导致神经功能缺损评分升高，而替罗非班可改善神经功能、减轻脑缺血的发展；ACI模型大鼠出现神经元受损，替罗非班可改善脑损伤、缩小梗死灶。以上结果提示替罗非班能抑制MCAO诱导的神经元损伤，对脑缺血有保护作用。
人体大约20%的氧气供给被脑组织利用。大量证据表明，脑氧化应激在ACI的发病中起着重要作用。SOD是抵御线粒体基质中超氧阴离子的主要防线[17]。GSH-Px是一种细胞内元件，在保护细胞免受氧化损伤方面起着至关重要的作用[18]。丙二醛是脂质过氧化的重要产物，可以间接反映细胞内的脂质过氧化水平[19]。缺血的大脑皮质抗氧化酶活性升高，脂质过氧化产物含量降低，表明脑抗氧化能力提高。本研究结果显示替罗非班提高了SOD和GSH-Px水平、降低了丙二醛含量，与既往研究[14]一致，表明替罗非班能够通过抑制氧化应激减轻脑损伤。
SIRT1参与氧化应激、自噬、神经保护和线粒体功能等多种生物学过程[20]。SIRT1在脑缺血再灌注大鼠脑组织中低表达，激活其表达能改善神经功能障碍[21]。VEGF由大脑中许多神经血管细胞产生和分泌，被认为是缺血后血管生成的关键因子[22]。缺血性脑卒中发生时SIRT1表达降低，下游VEGF表达减少，阻碍脑血管生成，从而使神经功能受损[9]。EX-527是SIRT1的特异性抑制剂[23]。本研究中EX-527抑制ACI大鼠SIRT1表达的同时降低了VEGF蛋白水平，增加了脑组织病理损伤、神经元凋亡，激活了大鼠体内的氧化应激反应；抑制SIRT1减弱了替罗非班对缺血再灌注损伤的保护作用，提示替罗非班通过激活SIRT1/VEGF信号通路对ACI大鼠的神经元损伤起到保护作用。
综上所述，替罗非班可能通过激活SIRT1/VEGF信号通路抑制ACI过程中的氧化应激及神经元损伤，发挥对脑的保护作用。
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