肌肽对血管性认知障碍大鼠氧化应激及NF-κB信号途径的影响

doi:10.13481/j.1671-587x.20200220

摘要/Abstract

摘要： 目的：探讨肌肽对大鼠血管性认知功能障碍（VCI）的保护作用，并阐明其作用机制。方法：50只SD大鼠随机分为假手术组、模型组（双侧颈总动脉夹闭10 min→再灌注10 min→夹闭10 min）和不同剂量肌肽组（双侧颈总动脉夹闭10 min→再灌注10 min→夹闭10 min+100、300和900 mg·kg^-1·d^-1肌肽），每组10只。肌肽于造模前21d至造模后12d灌胃给药，每日1次。于术后第7天开始进行水迷宫实验测定各组大鼠学习记忆能力；术后第12天取大鼠海马，高效液相色谱（HPLC）法测定各组大鼠海马组织中肌肽和还原性谷胱甘肽（GSH）水平。免疫组织化学法观察各组大鼠海马CA1区中胶质纤维酸性蛋白（GFAP）表达情况，Western blotting法检测各组大鼠海马组织中磷酸化核因子κB p65（p-NF-κB p65）蛋白表达水平，ELISA法检测各组大鼠海马组织中白细胞介素1β（IL-1β）和肿瘤坏死因子α（TNF-α）水平。结果：与假手术组比较，模型组大鼠海马CA1区锥体细胞部分缺失，逃避潜伏期明显延长（P<0.01），在平台象限停留时间明显缩短（P<0.01）；大鼠海马组织中肌肽和GSH水平降低（P<0.01），GFAP阳性细胞数增加，p-NF-κB p65蛋白表达水平升高（P<0.01），IL-1β和TNF-α水平升高（P<0.01）。与模型组比较，不同剂量肌肽组大鼠海马CA1区锥体细胞排列整齐，逃避潜伏期明显缩短（P<0.01），平台象限停留时间明显增加（P<0.01）；大鼠海马组织中肌肽和GSH水平升高（P<0.05或P<0.01），GFAP阳性细胞数减少，p-NF-κB p65蛋白表达水平降低（P<0.01），IL-1β和TNF-α水平降低（P<0.01）。结论：肌肽对大鼠VCI具有保护作用，其作用机制可能与提高大鼠抗氧化能力、抑制NF-κB途径激活、进而抑制星形胶质细胞的异常激活并减少炎症有关。

关键词: 肌肽, 血管性认知障碍, 核因子κB, 谷胱甘肽, 星形胶质细胞

Abstract: Objective: To investigate the protective effect of carnosine on the vascular cognitive impairment(VCI) of the rats, and to elucidate its mechanism. Methods: Fifty SD rats were randomly divided into sham operation group, model group (bilateral common carotid artery occlusion for 10 min→reperfusion for 10 min→occlusion for 10 min) and different doses of carnosine group (bilateral common carotid artery occlusion for 10 min→reperfusion for 10 min→occlusion for 10 minutes + 100, 300,and 900 mg·kg^-1·d^-1 carnosine);there were 10 rats in each group. The carnosine was administered by gavage once a day from 21 d before modeling to 12 d after modeling. Morris water maze test was carried out on the 7th day after operation to measure the learning and memory ability of the rats. The hippocampus tissue of the rats was obtained on the 12th day; the levels of carnosine and reduced glutathione(GSH) in hippocampus tissue of the rats in various groups were detected by high performance liquid chromatography (HPLC),the expression of glial fibrillary acidic protein(GFAP) in CA1 area of hippocampus of the rats in various groups was detected by immunohistochemistry method.The expression levels of phosphate nuclear factor-kappa B p65(p-NF-κB p65) protein in hippocampus tissue of the rats in various groups were determined by Western blotting method. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in hippocampus tissue of the rats in various groups were detected by ELISA method. Results: Compared with sham operation group, the pyramidal cells in the CA1 area of hippocampus of the rats in model group were partially absent, the escape latency was significantly prolonged (P<0.01), and the time staying in the platform quadrant was significantly shortened (P<0.01);the levels of carnosine and GSH in hippocampus tissue were decreased (P<0.01), the number of GFAP positive cells was increased, the expression level of p-NF-κB p65 protein was increased (P<0.01),and the levels of IL-1β and TNF-α were increased (P<0.01).Compared with model group, the pyramidal cells in CA1 area of hippocampus of the rats in different doses of carnosine groups were arranged in order, the escape latency was significantly shortened (P<0.01), and the time staying in the platform quadrant was significantly increased (P<0.01);the levels of carnosine and GSH in hippocampus tissue of the rats were increased (P<0.05 or P<0.01), the number of GFAP positive cells was decreased, the expression levels of p-NF-κB p65 protein were decreased(P<0.01),and the levels of IL-1β and TNF-α were decreased (P<0.01). Conclusion: Carnosine has the protective effect in the rats with VCI,and its mechanism may be related to the improvement of antioxidant capacity,inhibition of NF-κB pathway activation, thus inhibiting the abnormal activation of astrocytes and reducing inflammation.

Key words: carnosine, vascular cognitive impairment, nuclear factor kappa B, glutathione, astrocyte

中图分类号:

R963

杨文强, 何鑫, 白雪, 于露, 李宗泽, 张家悦, 杨菁. 肌肽对血管性认知障碍大鼠氧化应激及NF-κB信号途径的影响[J]. 吉林大学学报(医学版), 2020, 46(02): 329-334.

YANG Wenqiang, HE Xin, BAI Xue, YU Lu, LI Zongze, ZHANG Jiayue, YANG Jing. Effects of carnosine on oxidative stress and NF-κB signaling pathway in rats with vascular cognitive impairment[J]. Journal of Jilin University(Medicine Edition), 2020, 46(02): 329-334.

参考文献

[1] KALARIA R N. Neuropathological diagnosis of vascular cognitive impairment and vascular dementia with implications for Alzheimer's disease[J].ActaNeuropathol,2016,131(5):659-685.
[2] RAZ L, KNOEFEL J, BHASKAR K. The neuropathology and cerebrovascular mechanisms of dementia[J]. J Cereb Blood Flow Metab,2016,36(1):172-186.
[3] 方超,李晴,鲁美丽,等. 肌肽对大鼠脑片缺氧缺糖/再灌损伤的保护作用[J].中国生化药物杂志,2015,35(9):41-47.
[4] 白剑,迟戈,张俊,等.肌肽对低氧所致大鼠血管内皮细胞损伤的保护作用[J].中国应用生理学杂志,2010,26(1):30-32.
[5] XIE RX,LI DW,LIU XC,et al. Carnosine attenuates brain oxidative stress and apoptosis after intracerebral hemorrhage in rats[J]. Neurochem Res, 2017,42(2):541-551.
[6] 王蕊,杨秦飞,唐一鹏,等.大鼠拟血管性痴呆模型的建立及中药9602防治作用初探[J].北京中医药大学学报,2000,23(5):30-32.
[7] MORRIS R. Developments of a water-maze procedure for studying spatial learning in the rat[J]. J Neurosci Methods,1984,11(1):47-60.
[8] 杨菁,吴国强,白剑,等.邻苯二甲醛柱前衍生化高效液相色谱法测定组织中肌肽含量[J].中国生化药物杂志,2009,30(4):258-260.
[9] 魏斌,李鹏,崔胜云. HPLC法测定莲子中还原型谷胱甘肽(GSH)和总巯基(-SH)含量[J].食品研究与开发,2015,36(8):68-72.
[10] HORT J,VALIS M,KUC A,et al. Vascular cognitive impairment:Information from animal models on the pathogenic mechanisms of cognitive deficits[J]. Int J Mol Sci,2019,20(10):E2405.
[11] HOSSEINI AA,MENG D,SIMPSON RJ,et al. Mesiotemporal atrophy and hippocampal diffusivity distinguish amnestic from non-amnestic vascular cognitive impairment[J].Eur J Neurol,2017,24(7):902-911.
[12] PENG T M,JIANG Y Z,FARHAN M,et al. Anti-inflammatory effects of traditional Chinese medicines on preclinical in vivo models of brain ischemia-reperfusion-injury:Prospects for neuroprotective drug discovery and therapy[J]. Front Pharmacol,2019,10:204.
[13] YANG CM,LIN CC,HSIEH HL. High-glucose-derived oxidative stress-dependent heme oxygenase-1 expression from astrocytes contributes to the neuronal apoptosis[J]. MolNeurobiol,2017,54(1):470-483.
[14] ROY CHOUDHURY G,RYOU M G,POTEET E,et al. Involvement of p38 MAPK in reactive astrogliosis induced by ischemic stroke[J]. Brain Res,2014,1551:45-58.
[15] BAO Y,QIN L Y,KIM E,et al. CD36 is involved in astrocyte activation and astroglial scar formation[J].J Cereb Blood Flow Metab,2012,32(8):1567-1577.
[16] ABEYSINGHE H C,PHILLIPS E L,CHIN-CHENG H,et al. Modulating astrocyte transition after stroke to promote brain rescue and functional recovery:emerging targets include rho kinase[J].Int J MolSci,2016,17(3):288.
[17] LIU Z,CHOPP M. Astrocytes,therapeutic targets for neuroprotection and neurorestoration in ischemic stroke[J]. ProgNeurobiol,2016,144:103-120.
[18] JAYARAJ R L,AZIMULLAH S,BEIRAM R,et al. Neuroinflammation:friend and foe for ischemic stroke[J]. J Neuroinflammation,2019,16(1):142.
[19] KEMPURAJ D,THANGAVEL R,NATTERU P A,et al. Neuroinflammation induces neurodegeneration[J]. J Neurol Neurosurg Spine,2016,1(1):1003.
[20] THOMPSON W L,VAN ELDIK L J. Inflammatory cytokines stimulate the chemokines CCL2/MCP-1 and CCL7/MCP-7 through NF-κB and MAPK dependent pathways in rat astrocytes[J]. Brain Res,2009,1287:47-57.
[21] DVORIANTCHIKOVA G,BARAKAT D,BRAMBILLA R, et al.Inactivation of astroglial NF-kappaB promotes survival of retinal neurons following ischemic injury[J].Eur J Neurosci,2009,30(2):175-185.