&nbsp;沉默排斥性导向分子A对脑缺血再灌注损伤大鼠血脑屏障的保护作用

李敏 伏红霞 张艳梅 钟义良 秦新月*

doi:10.16098/j.issn.0529-1356.2019.03.001

PDF(4483 KB)

解剖学报 ›› 2019, Vol. 50 ›› Issue (3) : 269-274. DOI: 10.16098/j.issn.0529-1356.2019.03.001

神经生物学

沉默排斥性导向分子A对脑缺血再灌注损伤大鼠血脑屏障的保护作用

李敏¹ 伏红霞¹ 张艳梅¹ 钟义良² 秦新月^2*

作者信息 +

Silencing repulsive guidance molecule A showing protective effects on blood brain barrier after the injury of cerebral ischemia and reperfusion in rats

LI Min¹ FU Hong-xia¹ ZHANG Yan-mei¹ ZHONG Yi-liang² QIN Xin-yue ^2*

Author information +

文章历史 +

摘要

目的观察沉默排斥性导向分子A（RGMa）对脑缺血再灌注损伤大鼠血脑屏障及紧密连接蛋白的影响。方法雄性成年 SD 大鼠立体定位侧脑室注射腺病毒后建立大脑中动脉闭塞(MCAO) /再灌注（I/R）模型。将成年雄性SD大鼠40只，分为空白对照组（sh-con）和RGMa干扰组（sh-RGMa），分别在注射后1 d和3d观察腺病毒对RGMa的影响。将其余120只SD大鼠随机分为假手术组（sham）、缺血再灌注组（I/R）、空病毒组（I/R+sh-con）及病毒组（I/R+sh-RGMa）；I/R 72 h 后采用神经功能缺损评分评估各组大鼠神经功能恢复情况; 采用2，3，5-氯化三苯基四氮唑(TTC)染色测量脑梗死体积；采用静脉注射伊文思蓝观察血脑屏障通透性的改变；应用Western blotting和免疫组织化学染色检测RGMa的变化；应用Western blotting检测血脑屏障完整性相关紧密连接蛋白5（claudin-5）、基质金属蛋白酶9（MMP-9）和闭锁小带蛋白1（ZO-1）的表达。结果缺血再灌注后 72 h，I/R组较 sham 组神经功能评分降低，沉默RGMa能改善血脑屏障通透性，减少脑梗死体积；可下调MMP-9及上调claudin-5和ZO-1的表达。结论沉默RGMa对脑缺血再灌注损伤大鼠的血脑屏障有保护作用。

Abstract

Objective To investigate whether silencing repulsive guidance molecule A (RGMa) shows protective effects on blood brain barrier (BBB) and tight junction protein after the injury of cerebral ischemia and reperfusion (I/R) in rats. Methods Middle cerebral artery occlusion (MCAO) /reperfusion was employed to establish the models in the male adult rats. Fourty male Sprague-Dawley rats were divided into blank control group (sh-con) and RGMa interference group (sh-RGMa). The effects of adenovirus on RGMa were observed at day 1 and day 3 after injection. The remaining 120 SD rats were randomly divided into sham group, I/R group, I/R+sh-con group and I/R+sh-RGMa group. After reperfusion for 72 hours, the neurological recovery was evaluated in rats by neurological deficit score，the infarcted volume was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the permeability of BBB was performed through evans blue by tail vein injection. The expression of RGMa was detected by Western blotting and immunohistochemistry assay. The expression of claudin-5, matrix metalloprotein 9 (MMP-9）and zonula occludin 1（ZO-1）were detected by Western blotting. Results Silencing RGMa could improve the permeability of BBB, the infarcted volume, down-regulate the expression of MMP-9, and up-regulate the expression of claudin-5 and ZO-1. Conclusion Silencing RGMa shows protective effects on BBB after I/R injury in rats.

导出引用

李敏伏红霞张艳梅钟义良秦新月. 沉默排斥性导向分子A对脑缺血再灌注损伤大鼠血脑屏障的保护作用[J]. 解剖学报. 2019, 50(3): 269-274 https://doi.org/10.16098/j.issn.0529-1356.2019.03.001

LI Min FU Hong-xia ZHANG Yan-mei ZHONG Yi-liang QIN Xin-yue. Silencing repulsive guidance molecule A showing protective effects on blood brain barrier after the injury of cerebral ischemia and reperfusion in rats[J]. Acta Anatomica Sinica. 2019, 50(3): 269-274 https://doi.org/10.16098/j.issn.0529-1356.2019.03.001

参考文献

［1］ Yellon DM, Hausenloy DJ. Myocardial reperfusion injury［J］. N Engl J Med, 2007,357(11):1121-1135.

［2］ Shi H, Liu KJ. Cerebral tissue oxygenation and oxidative brain injury during ischemia and reperfusion［J］. Front Biosci, 2007，12:1318-1328.

［3］ Gu Y, Dee CM, Shen J. Interaction of free radicals, matrix metalloproteinases and caveolin-1 impacts blood-brain barrier permeability［J］. Front Biosci (Schol Ed), 2011，3:1216-1231.

［4］ Rubin LL, Staddon JM. The cell biology of the blood-brain barrier［J］. Annu Rev Neurosci, 1999，22:11-28.

［5］ Corradini E, Babitt JL, Lin HY. The RGM/DRAGON family of BMP co-receptors［J］. Cytokine Growth Factor Rev, 2009,20(5-6):389-398.

［6］ Satoh J, Tabunoki H, Ishida T, et al. Accumulation of a repulsive axonal guidance molecule RGMa in amyloid plaques: a possible hallmark of regenerative failure in Alzheimer’s disease brains［J］. Neuropathol Appl Neurobiol, 2013,20(2):109-120.

［7］ Schwab JM, Conrad S, Monnier PP, et al. Spinal cord injury-induced lesional expression of the repulsive guidance molecule (RGM)［J］. Eur J Neurosci, 2005,21(6):1569-1576.

［8］ Chen L, Gao B, Fang M, et al. Lentiviral vector-induced overexpression of RGMa in the hippocampus suppresses seizures and mossy fiber sprouting［J］. Mol Neurobiol, 2017,54(2):1379-1391.

［9］ Schnichels S, Heiduschka P, Julien S. RGMa and neogenin protein expression are influenced by lens injury following optic nerve crush in the rat retina［J］. Graefes Arch Clin Exp Ophthalmol， 2012, 250(1):39-50.

［10］ Wang T, Wu X, Yin C, et al. CRMP-2 is involved in axon growth inhibition induced by RGMa in vitro and in vivo［J］. Neurobiology, 2013, 47(3):903-913.

［11］ Mothe AJ, Tassew NG, Shabanzadeh AP, et al.RGMa inhibition with human monoclonal antibodies promotes regeneration, plasticity and repair, and attenuates neuropathic pain after spinal cord injury［J］. Sci Rep， 2017, 7(1):10529.

［12］ Zhang R, Wu Y, Xie F, et al. RGMa mediates reactive astrogliosis and glial scar formation through TGFbeta1/Smad2/3 signaling after stroke［J］. Cell Death Differ， 2018，25(8):1503-1516.

［13］ Feng J，Wang T，Li Q，et al．ＲNA interference against repulsive guidance molecule a improves axon sprout and neural function recovery of rats after MCAO/reperfusion［J］． Exp Neurol，2012，238 (2): 235-242．

［14］ Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats［J］. Stroke, 1989,20(1):84-91.

［15］ Schallert T, Fleming SM, Leasure JL, et al. CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury［J］. Neuro Pharmacol, 2000, 39(5): 777-787.

［16］ Wang T, Wu X, Yin C, et al. CRMP-2 is involved in axon growth inhibition induced by RGMa in vitro and in vivo［J］. Mol Neurobiol, 2013,47(3):903-913.

［17］ Wong CH, Crack PJ. Modulation of neuro-inflammation and vascular response by oxidative stress following cerebral ischemia-reperfusion injury［J］. Curr Med Chem, 2008,15(1):1-14.

［18］ Zhang DD, Zou MJ, Zhang YT, et al. A novel IL-1RA-PEP fusion protein with enhanced brain penetration ameliorates cerebral ischemia-reperfusion injury by inhibition of oxidative stress and neuroinflammation［J］. Exp Neurol, 2017，297:1-13.

［19］ Zhang H, Park JH, Maharjan S, et al. Sac-1004, a vascular leakage blocker, reduces cerebral ischemia-reperfusion injury by suppressing blood-brain barrier disruption and inflammation［J］. J Neuroinflammation, 2017,14(1):122.

［20］ Yang GY, Betz AL. Reperfusion-induced injury to the blood-brain barrier after middle cerebral artery occlusion in rats［J］. Stroke, 1994,25(8):1658-1665.

［21］ Wu Y, Wang YP, Guo P, et al. A lipoxin A4 analog ameliorates blood-brain barrier dysfunction and reduces MMP-9 expression in a rat model of focal cerebral ischemia-reperfusion injury［J］. J Mol Neurosci, 2012,46(3):483-491.

［22］ Monnier PP, Sierra A, Macchi P, et al. RGM is a repulsive guidance molecule for retinal axons［J］. Nature, 2002,419(6905):392-395.

［23］ O’Leary C, Cole SJ, Langford M, et al. RGMa regulates cortical interneuron migration and differentiation［J］. PLoS One, 2013,8(11):e81711.

［24］ Schwab JM, Monnier PP, Schluesener HJ, et al. Central nervous system injury-induced repulsive guidance molecule expression in the adult human brain［J］. Arch Neurol, 2005,62(10):1561-1568.

［25］ Schwab JM, Monnier PD, Schluesener HJ, et al. Central nervous system injaryinduced repulsive guidance molecule expression in the adult human brain［J］. Arch Neurol, 62(10):1561-1568.

［26］ Jiang F, Yin H, Qin X. Fastigial nucleus electrostimulation reduces the expression of repulsive guidance molecule, improves axonal growth following focal cerebral ischemia［J］. Neurochem Res, 2012,37(9):1906-1914.

［27］ Kong Y, Rogers MR, Qin X. Effective neuroprotection by ischemic postconditioning is associated with a decreased expression of RGMa and inflammation mediatiors in ischemic rats ［J］. Neurochem Res, 2013,38(4):815-825.

［28］ Conrad S, Genth H, Hofmann F, et al. Neogenin-RGMa signaling at the growth cone is bone morphogenetic protein-independent and involves RhoA, ROCK, and PKC［J］. J Biol Chem, 2007,282(22):16423-16433.