硫化氢复合浅低温对突触内N-甲基-D-天冬氨酸受体及其反应元件结合蛋白信号通路的影响

Hydrogen sulfide and mild hypothermia can selectively activate synaptic NMDARs and trigger the CREB signaling pathway

目的：研究表明硫化氢（ H2 S）能调节大脑N-甲基-D-天冬氨酸受体（ N-methyl-D-aspartate receptors ， NMDARs）的功能，但其在脑复苏中的作用仍需进一步阐明。文中通过观察H2 S复合浅低温对大鼠全脑缺血再灌注后海马NMDARs的亚单位NR2A、NR2B及其环磷酸腺苷反应元件结合蛋白（ phospho-cAMP response element binding protein ， p-CREB）信号通路的影响，旨在探讨H2 S是否存在脑复苏作用及其发挥作用的潜在机制。方法雄性SD大鼠随机分为5组（ n＝20）：假手术组、模型组、浅低温组、硫氢化钠（ NaHS）组、浅低温＋NaHS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NaHS组和浅低温＋NaHS组腹腔注射14μmol/kg NaHS，浅低温组和浅低温＋NaHS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H2 S的含量，Western blot法测NR2A、NR2B以及p-CREB的表达，RT-PCR法测脑源性神经营养因子（ brain derived neurotrophic factor ， BDNF） mRNA的水平，每组分别取4只于再灌注72 h取脑组织行HE染色观察CA1区锥体细胞病理改变。结果①与假手术组海马组织H2S含量（15．2±2．0）nmol/g相比，各组均升高（P＜0．05）；与模型组的H2S含量（25．2±3．5）nmol/g相比，浅低温组（26．5±3．5）nmol/g略升高（P＞0．05），而NaHS组（37．5±4．0）nmol/g和浅低温＋NaHS组（38．7±4．4）nmol/g显著升高（P＜0．05）；②与假手术组相比，各组NR2A、NR2B的灰度值均增高（P＜0．05），且模型组和浅低温组NR2A/NR2B＜1，NaHS组和浅低温＋NaHS组NR2A/NR2B＞1；③与模型组的p-CREB表达（0．55±0．06）相比，浅低温组（0．99±0．15）、NaHS组（1．05±0．12）、浅低温＋NaHS组（1．02±0．15）显著升高（P＜0．05）；与模型组的BDNF mRNA表达量（0．83±0．12）相比，浅低温组（1．11±0．13）、NaHS组（1．27±0．16）、浅低温＋NaHS组（1．35±0．16）也显著升高（P＜0．05）；④与模型组相比，浅低温组、NaHS组、浅低温＋NaHS组CA1区锥体细胞损伤程度均明显减轻，尤以浅低温＋NaHS组减轻最明显。结论硫化氢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CREB信号通路，发挥脑复苏的作用。

Objective Research has indicated that hydrogen sulfide（H2S） can regulate the function of N-methyl-D-aspartate re-ceptors（NMDARs） in the brain, but its effect on brain resuscitation requires further investigation.The study was to speculate the effect of H2 S on brain resuscitation as well as the underlying mechanism of neuroresuscitation by investigating the effects of hydrogen sulfide and hypo-thermia on the expression of NR2A, NR2B and phospho-cAMP response element binding protein （p-CREB） of NMDARs in the hippocampus after global cerebral ischemia following by reperfusion. Methods 100 male SD rats were randomly divided into five groups（n=20）：sham operation group, model group, mild hypothermia group, NaHS group, NaHS combined mild hypothermia group.Pulsinelli-Brierley four-ves-sel occlusion method was induced to build the injury rat model by reperfusion after global cerebral ischemia .After 15 minutes＆#39;ischemia, im-mediate injection of 14μmol/kg NaHS was performed intraperitoneally on NaHS group and NaHS combined mild hypothermia group , while skin cooling（rectal temperature=32-33℃） was done on mild hypothermia group and NaHS combined mild hypothermia group .6 hours late,r hip-pocampus were extracted from rat heads.Respectively, spectrophotometer was applied to measure the content of H2S, Western blot for the expres-sions of NR2 A,NR2 B and pC-REB, and RTP-CR for mRNA level of brain derived neurotrophic （BDNF）. HE staining was also performed on brain tissues 72hours after reperfusion on 4 rats from each group to evaluate the pathological changes of pyramidal neurons in CA1 region. R esul ts The content of H 2 S increased in each of the four groups after ischemia-reperfusion compared with sham operation group （ 15.2 ±2.0 nmol/g） （P〈0.05）.In comparison to model group （25.2 ±3.5 nmol/g）, NaHS group （37.5 ±4.0 nmol/g） and NaHS combined mild hypothermia group （38.7 ±4.4nmol/g ） resulted in significant high content of H2S（P〈0.05）, while mild hypothermia group（26.5 ±3.5nmol/g ） got a mild increase（P〉0.05）.The gray values of NR2A and NR2B in each group increased compared with sham operation group（P〈0.05）, re-sulting in NR2A/NR2B〈1 in model group and mild hypothermia group while NR2A/NR2B〉1 in NaHS group and NaHS combined mild hy-pothermia group.Compared with the expression of p-CREB（0.55 ±0.06） in model group, there were significant increases in mild hypother-mia group（0.99 ±0.15）, NaHS group（1.05 ±0.12）, NaHS combined mild hypothermia group（1.02 ±0.15）（P〈0.05）.Compared with the expression of BNDF mRNA（0.83 ±0.12） in model group, there were significant increases in mild hypothermia group （1.11 ±0.13）, NaHS group（1.27 ±0.16）, NaHS combined mild hypothermia group（1.35 ±0.16）（P〈0.05）.In comparison to model group, there were signifi-cant alleviation in the injury of pyramidal neurons in hippocampal CA1 region in mild hypothermia group, NaHS group, NaHS combined mild hypothermia group, with the best effect in NaHS combined mild hypothermia group . Conclusion Hydrogen sulfide combined mild hypo-thermia can selectively activate synaptic NMDA receptors and trigger the prosurvival CREB signaling pathway to exert brain resuscitation .