环孢素和褪黑素联合作用对急性脑缺血再灌注损伤的保护作用。

Synergic effect of combined cyclosporin and melatonin protects the brain against acute ischemic reperfusion injury.

机构信息

Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.

Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University, College of Medicine, Kaohsiung, Taiwan.

出版信息

Biomed Pharmacother. 2021 Apr;136:111266. doi: 10.1016/j.biopha.2021.111266. Epub 2021 Jan 19.

DOI:10.1016/j.biopha.2021.111266

PMID:33465677

Abstract

BACKGROUND

This study tested whether combined cyclosporin-A (CsA) and melatonin (Mel) was superior to either one on protecting the brain against ischemia (occluded left-middle-cerebral-artery for 90-min)-reperfusion (for 14 days) injury.

METHODS AND RESULTS

Neuro-2a cells (N2a) were categorized into groups 1 (N2a), 2 (N2a-IR), 3 (N2a-IR-Mel), 4 (N2a-IR-CsA) and 5 (N2a-IR-CsA-Mel). in vitro results showed the protein expressions of cytosolic-cytochrome-C/mitochondrial-Bax/cleaved-capase-3/NOX-1/NOX-2 and flow-cytometric results of ROS (DCFDA/Mito-SOX) were highest in group 2, lowest in group 1, significantly lower in group 5 than in groups 3/4, but they showed no difference in groups 3/4 (all p < 0.001). Male-adult-SD rats (50) were equally categorized into groups 1 (sham-operated-control), 2 (IR), 3 (IR-CsA/20.0 mg/kg at 0.5/24/48 h intraperitoneally after IR), 4 (IR-Mel/50.0 mg/kg intraperitoneally at 30 min and 30 mg/kg at 6/24/48 h after IR) and 5 (IR-CsA-Mel). The brain-infarct-area (BIA) (at day-3 by TTC-stain) was lowest in group 1, highest in group 2, significantly lower in group 5 than groups 3/4, but it showed no difference between groups 3/4 whereas the brain-infarct-volume (at day 14 by MRI) was similar as BIA except for significantly lower in group 4 than in group 3 (all p < 0.0001). By day 14, microscopic finding showed the numbers of glial+/GFAP+/AQP + cells expressed an identical trend whereas the number of NeuN + cells exhibited an opposite pattern of BIA among the groups (all p < 0.0001). The protein expressions of oxidative-stress (NOX-1/NOX-2/p22phox/oxidized-protein), inflammatory (TNF-α/p-NF-κB/MMP-9), apoptotic (mitochondrial-Bax/caspase-3/PARP) and mitochondrial-damaged (Cyclophilin-D/DRP1/cytosolic-cytochrome-C) biomarkers displayed an identical pattern of BIA among the five groups (all p < 0.0001).

CONCLUSION

Combined CsA-Mel was superior to either CsA or Mel on protecting the brain against IR injury.

摘要

背景

本研究旨在检验环孢素 A（CsA）和褪黑素（Mel）联合应用是否优于单一药物，以保护大脑免受缺血再灌注（缺血左中大脑动脉闭塞 90 分钟，再灌注 14 天）损伤。

方法和结果

将神经 2a 细胞（N2a）分为以下 5 组：1 组（N2a）、2 组（N2a-IR）、3 组（N2a-IR-Mel）、4 组（N2a-IR-CsA）和 5 组（N2a-IR-CsA-Mel）。体外实验结果显示，第 2 组细胞胞浆细胞色素 C/线粒体 Bax/裂解 caspase-3/NOX-1/NOX-2 蛋白表达和 ROS 流式细胞术结果（DCFDA/Mito-SOX）最高，第 1 组最低，第 5 组明显低于第 3/4 组，但第 3/4 组之间无差异（均 p<0.001）。50 只雄性成年 SD 大鼠（50）等分为以下 5 组：1 组（假手术对照）、2 组（IR）、3 组（IR 后 0.5、24、48 小时腹腔内注射 20.0 mg/kg CsA）、4 组（IR 后 30 分钟腹腔内注射 50.0 mg/kg Mel，6、24、48 小时腹腔内注射 30 mg/kg Mel）和 5 组（IR 后 0.5、24、48 小时腹腔内注射 20.0 mg/kg CsA 和 Mel）。第 3 组脑梗死面积（TTC 染色，第 3 天）最低，第 2 组最高，第 5 组明显低于第 3/4 组，但与第 3/4 组无差异，而第 4 组脑梗死体积（MRI，第 14 天）与脑梗死面积相似，第 4 组明显低于第 3 组（均 p<0.0001）。第 14 天，显微镜观察结果显示，各组胶质细胞+/GFAP+/AQP + 细胞表达的数量呈相同趋势，而 NeuN + 细胞数量呈与脑梗死面积相反的趋势（均 p<0.0001）。氧化应激（NOX-1/NOX-2/p22phox/氧化蛋白）、炎症（TNF-α/p-NF-κB/MMP-9）、凋亡（线粒体 Bax/caspase-3/PARP）和线粒体损伤（环孢菌素 D/DRP1/胞浆细胞色素 C）生物标志物的蛋白表达在 5 组中呈相同的脑梗死面积模式（均 p<0.0001）。