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目的探讨Küppel样转录因子2(KLF2)在大鼠局灶性脑缺血-再灌注(I/R)损伤后的表达及核因子κB(NF-κB)抑制剂的干预作用。方法取健康雄性SD大鼠60只,按照随机数字表法分为假手术组、I/R组、NF-κB抑制剂组,每组20只,采用大脑中动脉线栓法制作大鼠局灶性脑I/R模型,并给予NF-κB抑制剂——吡咯烷二硫代氨基甲酸盐(PDTC)进行干预,观察时间点为I/R后6、12、24、48 h。采用逆转录PCR及Western Blot测定缺血脑组织KLF2 mRNA及其蛋白的表达,采用ELISA法测定血清中肿瘤坏死因子α(TNF-α)水平并进行各组间的比较。结果与假手术组比较,I/R组6、12、24、48 h缺血脑组织中KLF2 mRNA及蛋白表达水平均降低(KLF2 mRNA相对表达量分别为:0.46±0.03比0.82±0.04,0.30±0.04比0.78±0.05,0.18±0.04比0.76±0.02,0.26±0.02比0.81±0.04;KLF2蛋白相对表达量分别为:0.46±0.04比0.80±0.02,0.30±0.02比0.79±0.02,0.15±0.02比0.77±0.01,0.24±0.01比0.79±0.02),I/R后24 h达最低值,而血清TNF-α水平升高,差异均有统计学意义(均P<0.05);给予NF-κB抑制剂PDTC后,I/R后6、12、24、48 h KLF2 mRNA及蛋白表达水平较I/R组出现不同程度的上调,KLF2 mRNA相对表达量分别为0.61±0.04、0.44±0.03、0.34±0.02、0.43±0.04,KLF2蛋白水平的相对表达量分别为0.60±0.02、0.43±0.02、0.33±0.01、0.44±0.03,而TNF-α含量降低,差异均有统计学意义(均P<0.05)。I/R组及PDTC组各时间点脑组织中KLF2 mRNA水平与血清中TNF-α水平呈负相关(r=-0.728,P<0.05)。结论脑I/R后脑组织中KLF2 mRNA表达水平降低,且与血清TNF-α水平存在负相关,其可能通过NF-κB通路介导炎性反应参与脑I/R病理过程。
Objective To investigate the expression of Küppel-like transcription factor 2 (KLF2) after focal cerebral ischemia-reperfusion (I / R) injury in rats and the effect of NF-κB inhibitor. Methods Sixty healthy male Sprague-Dawley rats were randomly divided into sham-operated group, I / R group and NF-κB inhibitor group, with 20 rats in each group. The focal middle cerebral artery occlusion Brain I / R model, and given NF-κB inhibitor - pyrrolidine dithiocarbamate (PDTC) for intervention, observation time point after I / R 6,12,24,48 h. Reverse transcription PCR and Western Blot were used to detect the expression of KLF2 mRNA and protein in ischemic brain tissue. The level of TNF-α in serum was determined by ELISA and the comparison among groups was made. Results Compared with the sham operation group, the expression of KLF2 mRNA and protein in the ischemic brain tissue of I / R group was decreased at 6, 12, 24 and 48 h (the relative expression of KLF2 mRNA was 0.46 ± 0.03 vs 0.82 ± 0.04 and 0.30, respectively ± 0.04 to 0.78 ± 0.05, 0.18 ± 0.04 to 0.76 ± 0.02, 0.26 ± 0.02 to 0.81 ± 0.04; the relative expression levels of KLF2 protein were: 0.46 ± 0.04 vs. 0.80 ± 0.02, 0.30 ± 0.02, 0.79 ± 0.02, 0.15 ± 0.02 (0.77 ± 0.01,0.24 ± 0.01 vs 0.79 ± 0.02). After 24 h of I / R, the serum TNF-α level reached the lowest value, and the difference was statistically significant (all P <0.05) After PDTC, the mRNA and protein expression of KLF2 at 6, 12, 24 and 48 h after I / R were significantly up-regulated compared with I / R group. The relative expression of KLF2 mRNA was 0.61 ± 0.04, 0.44 ± 0.03 and 0.34 ± 0.02,0.43 ± 0.04 and KLF2 protein levels were 0.60 ± 0.02,0.43 ± 0.02,0.33 ± 0.01,0.44 ± 0.03 respectively, while the levels of TNF-α were decreased (all P <0.05 ). The levels of KLF2 mRNA in brain tissue of I / R group and PDTC group were negatively correlated with the level of TNF-α in serum (r = -0.728, P <0.05). Conclusions The expression of KLF2 mRNA in brain I / R brain tissue is decreased and negatively correlated with the level of serum TNF-α. It may be involved in the pathological process of brain I / R through inflammatory reaction mediated by NF-κB pathway.