局灶性脑缺血-再灌注大鼠脑中Küppel样转录因子2表达及核因子κB抑制剂的作用

Expression of cerebral Küppel-like factor 2 in focal cerebral ischemia-reperfusion injury in rats and intervention effect of nuclear factor kappa B inhibitor

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 factor 2（ KLF2） after focal cerebral ischemia-reperfusion（ I / R） injury in rats and the intervention effect of nuclear factor kappa B（ NF-κB） inhibitor. Methods Sixty healthy male SD rats were randomly divided into a sham operation group,an I / R group,and a NF-κB inhibitor group（ n = 20 in each group）. A focal cerebral I / R model was induced by the intraluminal suture method,and NF-κB inhibitor（ pyrrolidinedithio carbamate,PDTC） was given to intervene. The observation time points were 6,12,24,and 48 hours after I / R. Reverse transcriptionpolymerase chain reaction（ PCR） and Western blot were used to measure KLF2 mRNA and protein expression in ischemic brain tissue. Enzyme-linked immunosorbent assay（ ELISA） was used to detect the levels of serum tumor necrosis factor α（ TNF-α）,and they were compared among groups. Results Compared with the sham operation group,the expression levels of KLF2 mRNA and protein in I / R group in the ischemic brain tissue at each time point were averagely decreased（ the relative expression levels of KLF2 mRNA： 0. 46 ±0. 03 vs. 0. 82 ± 0. 04,0. 30 ± 0. 04 vs. 0. 78 ± 0. 05,0. 18 ± 0. 04 vs. 0. 76 ± 0. 02,0. 26 ± 0. 02 vs. 0. 81 ±0. 04,respectively; the relative expression levels of KLF2 protein： 0. 46 ± 0. 04 vs. 0. 80 ± 0. 02,0. 30 ± 0. 02 vs. 0. 79 ± 0. 02,0. 15 ± 0. 02 vs. 0. 77 ± 0. 01,0. 24 ± 0. 01 vs. 0. 79 ± 0. 02,respectively）. They reached the lowest values at 24 hours after I / R,while the serum TNF-α levels were increased. There were significant differences（ all P〈 0. 05）. After giving NF-κB inhibitor PDTC,the expression levels of KLF2 mRNA and protein at 6,12,24,and 48 hours after I / R were upregulated differently compared with the I / R group. The relative expression levels of KLF2 mRNA were 0. 61 ± 0. 04,0. 44 ± 0. 03,0. 34 ± 0. 02,and 0. 43 ± 0. 04,respectively. Those of KLF2 protein were 0. 60 ± 0. 02,0. 43 ± 0. 02,0. 33 ± 0. 01,and 0. 44 ± 0. 03,respectively,while the levels of TNF-α were decreased. There were significant differences（ all P 〈0. 05）.There was a negative correlation between the KLF2 mRNA levels and the serum TNF-α levels at each time point in the I / R group and the PDTC group（ r =- 0. 728,P 〈0. 05）. Conclusions The expression levels of KLF2 mRNA in brain tissue are decreased after I / R,and it is negatively correlated with the serum TNF-α levels. It may be involved in the pathological process of I / R by NF-κB pathway m ediated inflam m atory reaction.