抑制高迁移率族蛋白B1减少大鼠脊髓损伤后微血管内皮细胞的葡萄糖转运体1表达

Inhibiting high mobility group box-1 decreased the expression of glucose transporter 1 in microvascular endothelial cells in rats after spinal cord injury

目的探讨高迁移率族蛋白B1(HMGB1)对大鼠脊髓损伤(SCI)后微血管内皮细胞(EC)葡萄糖转运体1(GLUT1)表达的作用及机制。方法改良Allen’s法制作Sprague-Dawley大鼠SCI模型,观察SCI 1 d后抑制HMGB1作用脊髓GLUT1表达。培养大鼠脑脊髓EC,建立氧糖剥夺/复氧(OGD/R)模型,观察减少HMGB1对OGD 6 h/R 12 h处理EC的GLUT1表达的作用。使用Toll样受体4(TLR4)、TRIF及核转录因子-κB(NF-κB)的激动剂或抑制剂,激动或抑制通路蛋白作用,观察TLR4/TRIF/NF-κB通路在体内外HMGB1调节GLUT1表达中的作用。统计学方法采用单因素方差分析,两两比较采用Tukey’s检验。结果SCI 1 d后大鼠脊髓GLUT1表达增高(SCI 1 d组高于sham组,1.655±0.083比1.000±0,q=24.580,P<0.01)。丙酮酸乙酯(实验组低于SCI组,1.356±0.096比2.107±0.115,q=14.730,P<0.01)或甘草甜素(实验组低于SCI组,1.311±0.093比2.107±0.115,q=15.620,P<0.01)抑制HMGB1作用减少SCI 1 d大鼠脊髓GLUT1表达。SC 1 d大鼠激动TLR4增加GLUT1表达(实验组高于SCI组,1.880±0.120比1.655±0.076,q=5.032,P<0.05),抑制TRIF(实验组低于SCI组1.427±0.118比1.655±0.076,q=5.109,P<0.05)或抑制NF-κB(实验组低于SCI组1.301±0.076比1.655±0.076,q=7.948,P<0.01)都可减少GLUT1表达。体外培养的脑脊髓EC,加入含HMGB1的星形胶质细胞条件培养基(ACM)并进行OGD 6 h/R 12 h处理后增加GLUT1表达(OGD 6 h/R 12 h组高于Normal组,2.122±0.075比1.000±0,q=38.720,P<0.01),减少ACM内HMGB1含量降低GLUT1表达(实验组低于OGD 6 h/R 12 h组,1.316±0.131比1.950±0.166,q=8.737,P<0.01)。抑制TLR4(CLI-095:实验组低于OGD 6 h/R 12 h组,1.753±0.103比2.230±0.103,q=13.515,P<0.01;C34:实验组低于OGD 6 h/R 12 h组,1.306±0.038比2.230±0.103,q=16.440,P<0.05),抑制TRIF(实验组低于OGD 6 h/R 12 h组,1.383±0.027比2.230±0.103,q=23.750,P<0.05)或抑制NF-κB(实验组低于OGD 6 h/R 12 h组,1.149±0.064比2.230±0.103,q=17.510,P<0.05)都可减少加入ACM并进行OGD 6 h/R 12 h处理的EC的GLUT1表达。结论抑制HMGB1作用减少SCI后EC的GLUT1表达,HMGB1通过TLR4/TRIF/NF-κB信号通路发挥上述调节作用。

Objective To study the roles of high mobility group box-1(HMGB1)in the expression of glucose transporter 1(GLUT1)in microvascular endothelial cells(ECs)in rats after spinal cord injury(SCI)and the underlying mechanisms.Methods The SCI model in Sprague-Dawley rats was established using a modified Allen’s method,and the effect of HMGB1 inhibition on GLUT1 expression in spinal cord was observed at 1st day after SCI.ECs in central nervous system of rats were incubated under oxygen-glucose deprivation/reoxygenation(OGD/R)procedure,and the effect of decreasing HMGB1 on GLUT1 expression was observed after OGD 6 h/R 12 h.The toll-like receptor 4(TLR4)agonist,TLR4 inhibitor,TRIF inhibitor,or nuclear factor-kappa B(NF-κB)inhibitor was used to study the roles of TLR4/TRIF/NF-κB signaling pathway during the regulation of HMGB1 on GLUT1 in vivo and in vitro.Intergroup data were compared using one-way analysis of variance followed by Tukey’s test.Results Firstly,the expression of GLUT1 in spinal cord was increased at 1st day after SCI in rats as compared with that in Sham group(1.655±0.083 vs.1.000±0,q=24.580,P<0.01).Inhibiting HMGB1 using ethyl pyruvate(1.356±0.096 vs.2.107±0.115,q=14.730,P<0.01)or glycyrrhizin(1.311±0.093 vs.2.107±0.115,q=15.620,P<0.01)reduced GLUT1 expression in spinal cord.Furthermore,activating TLR4 increased GLUT1 expression(1.880±0.120 vs.1.655±0.076,q=5.032,P<0.05),and both inhibiting TRIF(1.427±0.118 vs.1.655±0.076,q=5.109,P<0.05)and NF-κB(1.301±0.076 vs.1.655±0.076,q=7.948,P<0.01)reduced GLUT1 expression in spinal cord at 1st day after SCI in rats.Secondly,compared with normally cultured ECs,the expression of GLUT1 was increased in ECs exposed to astrocyte conditioned media(ACM),which contained HMGB1,after OGD 6 h/R 12 h(2.122±0.075 vs.1.000±0,q=38.720,P<0.01).After the HMGB1 decreased in ACM,the expression of GLUT1 was reduced in ECs after OGD 6 h/R 12 h(1.316±0.131 vs.1.950±0.166,q=8.737,P<0.01).In cultured ECs exposed to ACM,inhibiting TLR4 reduced GLUT1 expression after OGD 6 h/R 12 h(CLI-095:1.753±0.103 vs.2.230±0.103,q=13.515,P<0.01;C34:1.306±0.038 vs.2.230±0.103,q=16.440,P<0.05;TRIF:1.383±0.027 vs.2.230±0.103,q=23.750,P<0.05;NF-κB:1.149±0.064 vs.2.230±0.103,q=17.510,P<0.05).Conclusion Inhibiting HMGB1 resulted in a reduction in GLUT1 expression in both spinal cord of SCI rats and cultured ECs after OGD/R.These effects occur through HMGB1/TLR4/TRIF/NF-κB signaling pathway.