鼠脑缺血再灌注时葛根素对核因子_(κ)B表达的影响(英文)

Effect of puerarin on the expression of nuclear factor kappa B in rats with cerebral ischemia reperfusion

背景:近年研究证明,炎症反应是脑缺血再灌注损伤的主要机制之一,核因子κB作为一种转录因子在脑缺血再灌注炎症反应中起着调控多种炎性细胞因子表达的关键作用。先期实验表明,葛根素具有抗氧自由基和神经细胞凋亡的作用,如果还具有抗炎作用,则可进一步阐述葛根素的脑保护作用机制。目的:探讨葛根素在全脑缺血再灌注损伤中对核因子κB的影响。设计:随机平行对照设计。单位:卫生部北京医院急诊科、华中科技大学同济医学院附属同济医院急诊科、华中科技大学同济医学院的病理学教研室、实验动物中心和公共卫生学院卫生统计学教研室。材料:实验于2003-04/12在同济医学院病理学教研室进行。将健康清洁级的Wistar大鼠75只随机分为假手术组,脑缺血再灌注组和葛根素组3组各25只,每组按再灌注的时间又分为2,6,12,24和48h共5个观察时间点,每个时间点5只大鼠。方法:①假手术组:不电凝双侧椎动脉,分离双侧颈总动脉不夹闭,不给药。②脑缺血再灌注组:电凝双侧椎动脉后用无创动脉夹夹闭双侧颈总动脉10min后松开,行再灌注,在再灌注开始时腹腔注射1mL的生理盐水,以后每隔6h注射1次。③葛根素组:处理程序同脑缺血再灌注组,只是将生理盐水改为葛根素100mg/kg。主要观察指标:在大鼠全脑缺血10min后再灌注2,6,12,24和48h时,用免疫组织化学法检测海马CA1区核因子κB和抑制蛋白κB的活性;用原位杂交法检测肿瘤坏死因子αmRNA的表达,用苏木精-伊红染色检测存活神经元数目。结果:经补充后75只大鼠进入结果分析。①核因子κB的活性:脑缺血再灌注组再灌注2h即明显升高,6h达到高峰,48h仍高于假手术组(P均<0.01);葛根素组在各个时间点均低于脑缺血再灌注组(P<0.01)。②肿瘤坏死因子αmRNA的表达:脑缺血再灌注组再灌注2h即明显升高,12h达到高峰,48h仍高于假手术组(P均<0.01);葛根素组在再灌注6～48h均低于脑缺血再灌注组(P<0.01)。③抑制蛋白κB的活性:脑缺血再灌注组再灌注2h有明显下降,6h降至最低点,以后逐渐升至2h水平,葛根素组在各个时间点均高于脑缺血再灌注组(P<0.01或0.05)。④存活神经元数目:脑缺血再灌注组随再灌注时间的延长而逐渐减少(P均<0.01)。在各对应时间点,葛根素组均多于脑缺血再灌注组(P<0.05或0.01)。结论:全脑缺血再灌注时,葛根素可通过抑制抑制蛋白κB的降解及核因子κB的活性,下调肿瘤坏死因子αmRNA的表达,抑制炎症反应而起到脑保护作用。

BACKGROUND： The studies in recent years proved that the inflammatory reaction is of the main reasons in the damage of cerebral ischemia reperfusion. The nuclear factor KB （NF-KB）, as a kind of transcription factor, plays an important role in regulating the expressions of various inflammatory cell factors in the inflammatory reaction of cerebral isehemia reperfusion. The previous experiments show that puerarin functions to resist the oxidated free radicals and the apoptosis of nerve cells. In case it has the functions of anti-inflammation, its brain protection can be explained further. OBJECTIVE： To study the effect of puerarin on NF-κB for the rats with the damage of ischemia reperfusion. DESIGN： A random parallel controlled study. SETTING： The Emergency Department of Beijing Hospital, Emergency Department of Tongji Hospital, Pathology Department and Experimental Animal Center of Tongji Medical College, and Health Statistics Department of Public Health College of Huazhong University of Science and Technology. MATERIALS： The experiment was started on April 12, 2003 in the Pathology Department of Tongji Medical College. The 75 healthy and clean Wistar rats were randomized into 3 groups with 25 in each, Sham operation group, cerebral ischemia reperfusion group, and puerarin group. Each group was reperfused at 2, 6, 12, 24, and 48 hours after ischemia and 5 rats were used at each time point.METHODS： ① Sham operation group： Without electric coagulation of bilateral vertebral arteries, without blockage of bilateral common carotid arteries, without medicinal administration. ② Cerebral ischemia reperfusion group： Ten minutes after the blockage of bilateral common carotid arteries with non-invasive artery clamp, the reperfusion was given. At the beginning of reperfusion, the abdominal injection of normal saline 1 mL was applied and later every 6 hours the injection was repeated once. ③ Puerarin group： The procedure was the same as for the reperfusion group, only with normal saline changed to puerarin 100 mg/kg. MAIN OUTCOME MEASURES： At the time points of 2, 6, 12, 24, and 48 hours after reperfusion, the activity of NF-KB and inhibitory protein κB （IP-κB） in the hippocampus CA1 region was examined with immunohistochemical method; the expression of tumor necrosis factoror （TNF-α） mRNA was measured with in situ hybridization method; and the number of surviving neurons was detected with hematoxylin-eosin （HE） staining. RESULTS： After supplement, 75 rats entered the result analysis. ① Activity of NF-κB： In the ischemia reperfusion group, it was obviously increased at 2 hours after reperfusion, to the highest at 6 hours, and still higher than that of the sham operation group, （P〈0.01）. In the puerarin group, it was lower at each time point than that of the ischemia reperfusion group （P〈0.01）. ② Expression of TNF-α mRNA： In the ischemia reperfusion group, it was obviously increased at 2 hours after reperfusion, to the highest at 12 hours, and still higher than that of the sham operation group at 48 hours （P 〈 0.01）. In the puerarin group, it was lower than that of the ischemia reperfusion group at 6-48 hours （P〈0.01）. ③ Activity of IP-κB： In the ischemia reperfusion group, it was obviously decreased at 2 hours after reperfusion, to the lowest at 6 hours, and then gradually increased to the level of 12 hours. In the puerarin group, it was higher than that of the ischemia reperfusion group at each time point （P 〈 0.01 or 0.05）. ④ Number Of surviving neurons： In the ischemia reperfusion group, it was decreased gradually with the time prolonging after reperfusion （P 〈 0.01）. In the puerarin group, at each time point, it was higher than that of the ischemia reperfusion group （P 〈 0.05 or 0.01）. CONCLUSION： In the cerebral ischemia reperfusion, puerarin can protect the brain through decreasing the degradation of IP-κB, the activity of NF-κB, the expression of TNF-α mRNA, and the inflammatory reaction.