目的 观察大鼠创伤性脑损伤（traumatic brain injury，TBI）后脑微血管内皮细胞间紧密连接蛋白Claudin-5表达的时序性规律，探讨大麻二酚（cannabidiol，CBD）的干预作用。 方法 采用改良Feeney自由落体撞击法建立SD大鼠TBI模型，随机分为对照组（Sham组）、模型组（TBI+vehicle组）和干预组（TBI+CBD组），每组分8 h、1 d、2 d、3 d、5 d和7 d 6个亚组（n=3）。运用RT-PCR、免疫组织化学染色和免疫荧光双标染色实验，观察CBD干预对TBI后Claudin-5阳性表达与星形胶质细胞（astrocyte，AST）激活的影响。 结果 PCR结果表明：TBI后不同时间段Claudin-5的mRNA表达下降，CBD干预后，其表达有明显回升。免疫组化及免疫荧光结果表明，Sham组Claudin-5蛋白表达于脑微血管内皮细胞连接处，呈点状、线条状，AST胞体较小、突起少呈细长状；TBI后Claudin-5阳性细胞线条断裂，其表达显著减弱且呈下降趋势，以2 d组表达最低（P<0.05），此外，AST被激活，胞体变大、突起增多肿胀；CBD干预后Claudin-5阳性表达显著升高，AST激活状态明显减弱，胞体有所回缩，足板肿胀减轻。胶质纤维酸性蛋白阳性表达的AST和Claudin-5蛋白不存在共表达，但是AST伸出的足板与微血管内皮细胞直接相贴附。 结论 TBI后AST激活并参与Claudin-5蛋白表达的调节；CBD可能通过调控AST的激活状态调节Claudin-5阳性表达，进而改善血脑屏障的通透性。
Abstract
Objective To observe the expression of Claudin-5 in brain microvascular endothelial cells after traumatic brain injury (TBI), and to investigate the intervention effect of cannabidiol (CBD). Methods TBI model of SD rats was established by modified Feeney free-fall impingement method. After modeling, the rats were randomly divided into a sham group (sham group), a TBI + Vehicle group and a TBI+CBD group (intervention group), and each group was divided into 6 subgroups (n=3) at 8 h, 1 d, 2 d, 3 d, 5 d and 7 d. RT-PCR, Immunohistochemistry staining and immunofluorescence double-standard staining were used to observe the effect of the CBD intervention on positive Claudin-5 expression of and the astrocytes (AST) activation after TBI. Results The mRNA expression of Claudin-5 decreased at different time periods after TBI, while the mRNA expression of Claudin-5 increased significantly after CBD intervention. Claudin-5 protein expression was located at the junction of brain microvascular endothelial cells, and the AST cell body was small, with few protrusions and elongated in the sham group. After TBI, Claudin-5 positive cell lines were broken, and the expression of Claudin-5 was significantly decreased and showed a downward trend, and the expression of them was the lowest in the 2 d group (P<0.05). In addition, ATS were activated, and the cell bodies became larger, the protrusions became increased and swollen. After CBD intervention, the positive expression of Claudin-5 was significantly increased, the AST activation state was significantly weakened, the cell body was increased, and the foot plate swelling was reduced. It was also observed that there was no co-expression between GFAP positive AST and Claudin-5 protein, but the foot plate extended by AST was directly attached to microvascular endothelial cells. Conclusions AST is activated and involved in the regulation of Claudin-5 protein expression after TBI. CBD may indirectly regulate the positive expression of Claudin-5 by regulating the activation state of AST, thus improving the permeability of blood brain barrier.
关键词
TBI； /
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紧密连接蛋白Claudin-5； /
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CBD； /
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大鼠
Key words
TBI; /
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Tight junction protein Claudin-5; /
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CBD; /
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Rat
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参考文献
[1] Yu P, Li Yan Y, Zhong GL, et al. Claudin-5 affects endothelial autophagy in response to early hypoxia[J]. Front Physiol, 2021, 12: 737474. DOI: 10.3389/fphys.2021.737474.
[2] 蒋鸿雁. 大麻二酚(CBD)改善脑损伤血脑屏障渗透性的研究[D]. 昆明: 昆明医科大学, 2021. DOI: 10.27202/d.cnki.gkmyc.2021.000740.
[3] Iffland K, Grotenhermen F. An update on safety and side effects of cannabidiol: a review of clinical data and relevant animal studies[J]. Cannabis Cannabinoid Res, 2017, 2(1): 139-154. DOI: 10.1089/can.2016.0034.
[4] 韩宗博, 戚克进, 王少峰, 等. 大麻二酚的生物学功效与制备研究[J]. 植物学研究, 2019, 8(3): 190-195. DOI: 10.12677/BR.2019.83025.
[5] McPartland JM, Duncan M, Di Marzo V, et al. Are cannabidiol and Delta(9)-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review[J]. Br J Pharmacol, 2015, 172(3): 737-753. DOI: 10.1111/bph.12944.
[6] Huestis MA, Solimini R, Pacifici R, et al. Cannabidiol adverse effects and toxicity[J]. Curr Neuropharmacol, 2019, 17(10): 974-989. DOI: 10.2174/1570159X17666190603171901.
[7] 米杨. 大麻二酚在大鼠蛛网膜下腔出血早期脑损伤中促进自噬反应的研究[D]. 贵州: 贵州医科大学, 2021. DOI: 10.27045/d.cnki.ggyyc.2021.000035.
[8] McDonald BZ, Gee CC, Kievit FM. The nanotheranostic researcher's guide for use of animal models of traumatic brain injury[J]. J Nanotheranostics. 2021, 2(4): 224-268. DOI: 10.3390/jnt2040014.
[9] 曹艳. 创伤性脑损伤中大麻二酚下调星形胶质细胞活化的机制研究[D]. 昆明医科学, 2022. DOI: 10.27202/d.cnki.gkmyc.2022.000975.
[10] 衣昕, 毛伟峰, 秦建兵, 等. 大鼠创伤性脑损伤后星形胶质细胞的变化[J]. 中国临床解剖学杂志, 2006, 24(5): 536-540. DOI: 10.3969/j.issn.1001-165X.2006.05.016.
[11] 张玉鹏, 李芳, 陈竹, 等. PJ34对脑缺血再灌注大鼠应用rt-PA后血脑屏障完整性的影响[J]. 中国临床解剖学杂志, 2019, 37(3): 287-291, 298. DOI: 10.13418/j.issn.1001-165x.2019.03.010.
[12] 野战鹰. 丁苯酚对血管性认知功能障碍大鼠血脑屏障的保护及对紧密连接蛋白调控机制研究[D]. 石家庄: 河北医科大学, 2019.
[13] 许静红, 齐铁伟, 郭少雷, 等. 急性创伤性脑损伤大鼠血脑屏障通透性变化与Claudin-5的相关性研究[J]. 中华神经医学杂志, 2012, 11(9): 954-957. DOI: 10.3760/cma.j.issn.1671-8925.2012.09.022.
[14] Roschel H, Gualano B, Ostojic SM, Rawson ES. Creatine supplementation and brain health[J]. Nutrients, 2021, 13(2): 586. DOI: 10.3390/nu13020586.
[15] Neuhaus W, Wirth M, Plattner VE, et al. Expression of Claudin-1, Claudin-3 and Claudin-5 in human blood-brain barrier mimicking cell line ECV304 is inducible by glioma-conditioned media[J]. Neurosci Lett, 2008, 446(2-3): 59-64. DOI: 10.1016/j.neulet.2008.09.025.
[16] Gao Y, Li D, Lin J, et al. Cerebral small vessel disease: pathological mechanisms and potential therapeutic targets[J]. Front Aging Neurosci, 2022, 14: 961661. DOI: 10.3389/fnagi.2022.961661.
[17] Hongyan Jiang, Hengxi Li, Yan Cao, et al. Effects of cannabinoid (CBD) on blood brain barrier permeability after brain injury in rats[J]. Brain Res, 2021, 1768: 147586. DOI: 10.1016/j.brainres.
[18] Belardo C, Iannotta Ms Boccella S, Rubino RC, et al. Oral cannabidiol prevents allodynia and neurological dysfunctions in a mouse model of mild traumatic brain injury[J]. Front Pharmacol, 2019, 10: 352. DOI: 10.3389/fphar.2019.00352.
[19] 黄腾飞, 闫超. 大麻二酚在胶质瘤中的抗肿瘤作用及其机制研究[J]. 中国药理学与毒理学杂志, 2021, 35(10): 725-726. DOI: 10.3867/j.issn.1000-3002.2021.10.009.
基金
国家自然科学基金项目（82060241）；云南省科技厅-昆明医科大学应用基础研究联合专项重点项目（202101AY070001-002）
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