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

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目的:探讨Kuppel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 Kuppel-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 transcription-polymerase 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 factorα( 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-αlevels 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-αwere decreased. There were significant differences(all P<0. 05). There was a negative correlation between the KLF2 mRNA levels and the serum TNF-αlevels 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 mediated inflammatory reaction.
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