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
류화경복합천저온대돌촉내 N-갑기-D-천동안산수체급기반응원건결합단백신호통로적영향
Hydrogen sulfide and mild hypothermia can selectively activate synaptic NMDARs and trigger the CREB signaling pathway
  • 万方数据
    医学研究生学报 의학연구생학보
    JOURNAL OF MEDICAL POSTGRADUATE
    2014年 7期 686-689 ,共4页

    代海滨%胡益民%嵇晴%张利东%苗晓蕾%朱四海%李伟彦%段满林

    대해빈%호익민%혜청%장리동%묘효뢰%주사해%리위언%단만림

    硫化氢%低温%N-甲基-D-天冬氨酸受体%环磷酸腺苷反应元件结合蛋白%脑缺血%再灌注损伤 硫化氫%低溫%N-甲基-D-天鼕氨痠受體%環燐痠腺苷反應元件結閤蛋白%腦缺血%再灌註損傷
    류화경%저온%N-갑기-D-천동안산수체%배린산선감반응원건결합단백%뇌결혈%재관주손상
    Hydrogen sulfide%Hypothermia%N-methyl-D-aspartate receptor%Cyclic AMP response element binding protein%Brain ischemia%Reperfusion injury
    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。
    목적：연구표명류화경（ H2 S）능조절대뇌N-갑기-D-천동안산수체（ N-methyl-D-aspartate receptors ， NMDARs）적공능，단기재뇌복소중적작용잉수진일보천명。문중통과관찰H2 S복합천저온대대서전뇌결혈재관주후해마NMDARs적아단위NR2A、NR2B급기배린산선감반응원건결합단백（ phospho-cAMP response element binding protein ， p-CREB）신호통로적영향，지재탐토H2 S시부존재뇌복소작용급기발휘작용적잠재궤제。방법웅성SD대서수궤분위5조（ n＝20）：가수술조、모형조、천저온조、류경화납（ NaHS）조、천저온＋NaHS조。채용Pulsinelli-Brierley사혈관조새법건립대서전뇌결혈재관주손상모형，결혈15 min재관주즉각NaHS조화천저온＋NaHS조복강주사14μmol/kg NaHS，천저온조화천저온＋NaHS조행체표강온지항온32～33℃。6 h후단두취해마，분별채용분광광도계법측H2 S적함량，Western blot법측NR2A、NR2B이급p-CREB적표체，RT-PCR법측뇌원성신경영양인자（ brain derived neurotrophic factor ， BDNF） mRNA적수평，매조분별취4지우재관주72 h취뇌조직행HE염색관찰CA1구추체세포병리개변。결과①여가수술조해마조직H2S함량（15．2±2．0）nmol/g상비，각조균승고（P＜0．05）；여모형조적H2S함량（25．2±3．5）nmol/g상비，천저온조（26．5±3．5）nmol/g략승고（P＞0．05），이NaHS조（37．5±4．0）nmol/g화천저온＋NaHS조（38．7±4．4）nmol/g현저승고（P＜0．05）；②여가수술조상비，각조NR2A、NR2B적회도치균증고（P＜0．05），차모형조화천저온조NR2A/NR2B＜1，NaHS조화천저온＋NaHS조NR2A/NR2B＞1；③여모형조적p-CREB표체（0．55±0．06）상비，천저온조（0．99±0．15）、NaHS조（1．05±0．12）、천저온＋NaHS조（1．02±0．15）현저승고（P＜0．05）；여모형조적BDNF mRNA표체량（0．83±0．12）상비，천저온조（1．11±0．13）、NaHS조（1．27±0．16）、천저온＋NaHS조（1．35±0．16）야현저승고（P＜0．05）；④여모형조상비，천저온조、NaHS조、천저온＋NaHS조CA1구추체세포손상정도균명현감경，우이천저온＋NaHS조감경최명현。결론류화경복합천저온가능통과선택성작용우돌촉내적NMDARs，진이격활기하유촉존활CREB신호통로，발휘뇌복소적작용。
    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'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 .
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