硫化氢复合浅低温可选择性激活突触内NMDARs及其下游PI3K/Akt信号通路

目的观察硫化氢（H₂S）复合浅低温对大鼠全脑缺血-再灌注后海马N-甲基-D-天冬氨酸受体（NMDARs）的亚单位NR2A、NR2B、磷酸化蛋白激酶B（p-Akt）及磷酸化糖原合成酶激酶3β（p-GSK 3β）的影响,旨在探讨其发挥脑复苏作用的潜在机制。方法雄性SD大鼠100只,随机均分为五组:假手术组（Ⅰ组）、模型组（Ⅱ组）、浅低温组（Ⅲ组）、硫氢化钠（NaHS）组（Ⅳ组）、浅低温+NaHS组（Ⅴ组）。采用Pulsinelli-Brierley四血管阻塞法建立大鼠全脑缺血再灌注损伤模型,缺血15 min再灌注即刻Ⅳ组和Ⅴ组腹腔注射14μmol/kg NaHS,Ⅲ组和Ⅴ组行体表降温至肛温32～33℃。6 h后断头取海马,分别采用分光光度计法测H₂S的含量,western blot法测NR2A、NR2B、p-Akt及p-GSK 3β的表达,每组分别取4只于再灌注24 h取脑行Tunel染色观察CA1区锥体细胞凋亡情况。结果与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ、Ⅴ组海马组织H₂S含量均升高（P<0.05）;与Ⅱ组相比,Ⅳ和Ⅴ组H₂S含量显著升高（P<0.05）;与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ、Ⅴ组NR2A、NR2B的表达、凋亡指数（AI）均增高（P<0.05）,且Ⅱ和Ⅲ组NR2A/NR2B<1,Ⅰ、Ⅳ和Ⅴ组NR2A/NR2B>1;与Ⅰ组相比,Ⅱ、Ⅲ、Ⅳ、Ⅴ组海马p-Akt及p-GSK 3β的表达均上调（P<0.05）;与Ⅱ组相比,Ⅲ、Ⅳ、Ⅴ组海马p-Akt及p-GSK 3β的表达均上调,AI降低（P<0.05）;与Ⅲ和Ⅳ组相比,Ⅴ组海马p-GSK 3β的表达上调（P<0.05）。与Ⅱ组相比,Ⅲ、Ⅳ、Ⅴ组CA1区锥体细胞凋亡程度均明显减轻,尤以Ⅴ组效果最明显。结论 H₂S复合浅低温可能通过选择性作用于突触内的NMDARs,进而激活其下游促存活磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt）信号通路,抑制锥体细胞凋亡,从而发挥脑复苏的作用。

Hydrogen sulfide and mild hypothermia can selectively activating synaptic NMDARs and triggering the PI3K/Akt signaling pathway

Objective To investigate the effects of hydrogen sulfide and mild hypothermia on the expression of NR2A,NR2B,phospho-Akt and phospho-GSK 3βin the rat hippocampus following global cerebral ischemia-reperfusion.so as to speculate the underlying possible mechanisms of neuroresuscitation.Methods One hundred male SD rats were randomly divided into five groups with 20 each:sham operation group（Ⅰ）,model group（Ⅱ）.hypothermia group（Ⅲ）,NaHS group（Ⅳ）, NaHS combined hypothermia group（Ⅴ）.Transient（15 min） global cerebral ischemia was induced by the four-vessel occlusion method described by Pulsinelli.Immediate general mild hypothermia after reperfusion was performed and lasted for 6 hours in groupⅢandⅤ.GroupⅣandⅤwere injected the NaHS 14μM/kg intraperitoneally at the same time.We used spectrophotometer to measure the content of H₂S in hippocampus.The expression of NR2A,NR2B,ρ-Akt andρ-GSK 3βwere detected by western blotting.TUNEL staining was also performed to evaluate the apoptotic index of hippocampal neurons.Results The content of H₂ S was increased in all of the four groups after ischemia-reperfusion.Compare to groupⅡ,groupⅣandⅤ,which administrate NaHS,resulted in significant high content of H₂S（P<0.05）.There was also a mild increase in groupⅢ.The level of NR2B was greater than NR2A in groupⅡandⅢ,which was opposite in groupⅣandⅤ.The expression ofρ-Akt andρ-GSK 3βwere both up-regulated in groupⅢtoⅤ（P<0.05）.Giving NaHS or mild hypothermia could alleviant the damage in CA1 region of hippocampus,with the best effect in groupⅤ,which administer NaHS combined hypothermia.Conclusion Hydrogen sulfide combined mild hypothermia could induce neuroresuscitation by selectively activating synaptic NMDA receptors, which was predominately NR2A-containing,and triggering the prosurvival PI3K/Akt signaling pathway.