p38 MAPK信号通路在异氟醚诱导新生大鼠海马神经细胞凋亡的作用

Effect of p38 MAPK pathway on isoflurane-induced neuronal apoptosis in hippocampus of neonatal rats

目的探讨异氟醚麻醉对新生大鼠海马组织的p38丝裂原活化蛋白激酶通路(mitogen-activated protein kinase,MAPK)激活的影响,以及p38 MAPK信号通路对异氟醚诱导海马神经细胞凋亡的影响。方法 48只出生后7 d的新生大鼠随机分为DMSO对照组(Air+DMSO组)、p38 MAPK抑制剂SB203580对照组(Air+SB20组)、异氟醚+DMSO组(Iso+DMSO组)和异氟醚+SB203580组(Iso+SB20组)。对照组吸入空气,异氟醚组吸入体积分数为0.011异氟醚4 h。麻醉前30 min,分别侧脑室注射SB203580(20 nmol)或者体积分数为0.1 DMSO 5μl。麻醉结束后6 h,部分幼鼠灌注取脑,TUNEL荧光染色检测幼鼠脑海马CA1区神经细胞凋亡(n=6);部分幼鼠取新鲜脑海马,Western blot法检测磷酸化p38(phospho-p38,p-p38)、p38、cleaved caspase-3、磷酸化NF-κB(phospho-NF-κB,p-NF-κB)、Bcl-2、Bax等蛋白表达的变化(n=6)。结果 Iso+DMSO组海马CA1区TUNEL阳性细胞数比Air+DMSO组增加了4.8倍(P<0.01),与Iso+DMSO组相比,Iso+SB20组海马CA1区TUNEL阳性细胞数降低了约3/5(P<0.01);Iso+DMSO组海马cleaved caspase-3蛋白表达较Air+DMSO组表达增加(P=0.003),Iso+SB20组减少了异氟醚引起的海马cleaved caspase-3增加(P=0.007);与Air+DMSO组比较,异氟醚增加了p-p38及其下游p-NF-κB的表达,增加了Bax的表达,降低了Bcl-2的表达;而p38抑制剂SB203580则减少了异氟醚引起的pp38、p-NF-κB和Bax的增加,增加了Bcl-2的表达。结论异氟醚通过激活p38 MAPK信号通路诱导发育期大鼠海马神经细胞凋亡。

Aim To investigate the effect of isoflurane on the phosphorylation of p38 mitogen-activated protein kinase （MAPK）in the hippocampus of neonatal rats, and the effect of p38 MAPK pathway on isoflurane-in-duced neuronal apoptosis.Methods Forty-eight neo-natal rats on postnatal day 7 were assigned randomly into four groups：DMSO group （group Air ＋DMSO）, p38 MAPK inhibitor SB203580 group （group Air ＋SB20 ）,isoflurane ＋DMSO group （group Iso ＋DM-SO）,and isoflurane ＋SB203580 group （group Iso ＋SB20 ）.Rats were exposed to air or isoflurane （volume fraction of 0.01 1 ）for 4h.The p38 inhibitor SB203580 （20 nmol）or DMSO （volume fraction of 0.1 ）5μl was intraventricularly administered 30 min before the expo-sure.The brains of some rats in each group were per-fused and embedded by paraffin 6h after the exposure. Neuronal apoptosis in the hippocampal CA1 area was detected by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling （TUNEL）（n =6）. The hippocampal tissues of the other rats in each group were dissected 6h after the exposure,and the protein expressions of phospho-p38 （p-p38 ）,p38,cleaved caspase-3,phospho-NF-κB （p-NF-κB ）,Bcl-2 and nbsp;Bax were detected by Westem blot （n =6）.Results The number of TUNEL positive cells in the hippocam-pal CA1 region in group Iso ＋DMSO increased by 4.8 fold compared with that in group Air ＋DMSO （P ＆lt;0.01 ）,while the number of TUNEL positive cells in group Iso ＋SB20 decreased by 3 /5 compared with that in group Iso ＋DMSO （P ＆lt;0.01 ）.The protein expres-sion of cleaved caspase-3 in group Iso ＋DMSO signifi-cantly increasd （P =0.003）compared to that in group Air ＋DMSO,which was significantly decreasd in group Iso ＋SB20 （P =0.007 ）.In addition,isoflurane also increased the protein expression of p-p38,p-NF-κB and Bax,decreased the level of Bcl-2,and reduced the ratio of Bcl-2 /Bax compared with control animals （P〈0.01 ,P =0.004,P〈0.01 ,P〈0.01 ,P〈0.01 ,respectively）.Howerver,SB203580 partly at-tenuated the isoflurane-induced protein change above. Conclusion Isoflurane induces neuroapoptosis in neo-natal rat hippocampus by the activation of p38 MAPK pathway.