硫化氢联合浅低温对大鼠脑缺血再灌注损伤的影响

Effect of hydrogen sulfide combined with mild hypothermia on cerebral ischemia-reperfusion in rats

目的 评价硫化氢（H2S）联合浅低温对大鼠脑缺血再灌注损伤的影响.方法 雄性SD大鼠80只,体重250～300 g,月龄3月,随机分为5组（n=16）：假手术组（S组）、脑缺血再灌注组（IR组）、浅低温组（M组）、硫氢化钠组（NaHS组）和硫氢化钠＋浅低温组（NM组）.IR组、M组、NaHS组、和NM组采用四血管阻塞法建立大鼠脑缺血再灌注模型,缺血15 min后恢复灌注.NaHS组和NM组于再灌注即刻腹腔注射NaHS 14μmol/kg,其余组注射等容量生理盐水.同时M组和NM组于15 min内将直肠温降至32～33 ℃,并维持6 h;其余组采用白炽灯维持直肠温36～37 ℃.再灌注6 h时,各组处死12只大鼠,取海马组织,测定H2S的含量,采用Western b1ot法测定磷酸化cAMP反应原件结合蛋白（p-CREB）的表达,采用RT-PCR法测定脑源性神经营养因子（BDNF）mRNA的表达.于再灌注72 h时,各组处死4只大鼠,取海马组织,观察CA1区病理学结果.结果 M组、NaHS组和NM组病理学损伤较IR组减轻,其中NM组减轻最明显.与S组比较,IR组、M组、NaHS组和NM组海马H2S含量升高（P＜0.05）;与IR组和M组比较,NaHS组和NM组海马H2S含量升高（P＜0.05）.与S组比较,IR组、M组、NaHS组和NM组海马p-CREB和BDNF mRNA的表达上调（P＜0.05）;与IR组比较,M组、NaHS组和NM组海马p-CREB和BDNF mRNA的表达上调（P＜0.05）;与NaHS组和M组比较,NM组海马p-CREB表达差异无统计学意义（P＞0.05）,BDNF mRNA表达上调（P＜0.05）.NahS组和M组各指标比较差异无统计学意义（P＞0.05）.结论 H2S联合浅低温可减轻大鼠脑缺血再灌注损伤,其机制与上调海马p-CREB和BDNF mRNA的表达有关.

Objective To evaluate the effect of hydrogen sulfide combined with mild hypothermia on cerebral ischemia-reperfusion （I/R） injury in rats. Methods Eighty male SD rats, aged 3 months, weighing 250-300 g, were randomly divided into 5 groups （ n = 16 each）： sham operation group （group S）, cerebral I/R group,mild hypothermia group （group M）, sodium hydrosulfide group （group NaHS） and NaHS ＋ mild hypothermia group （group NM）. In group I/R, M, NaHS and NM, cerebral I/R was induced by occlusion of 4 vessels （cauterization of bilateral vertebral arteries and 15 min occlusion of bilateral common carotid arteries） followed by reperfusion. In group NaHS and NM, intraperitoneal NaHS 14 μmol/kg was injected immediately after reperfusion, while the equal volume of normal saline was injected in the other three groups. At the same time, the rectal temperature was reduced to 32-33 ℃ within 15 min, lasting for 6 h, in group M and NM, while it was maintained at 36-37 ℃by physical method in other groups. Twelve rats of each group were sacrificed after 6 h of reperfusion, and then the hippocampus was removed for determination of the content of H2 S by using spectrophotometer and the expression of p-CREB and BDNF mRNA by using Western blot and RT-PCR respectively. Four rats in each group were sacririced after 72 h of reperfusion and then the hippocampus was removed for microscopic examination. Results The cerebral I/R injury was attenuated in group M, NaHS and NM compared with group I/R, with the slightest injury in group NM. The H2S content was significantly higher in group I/R, M, NaHS and NM than in group S, and in group NaHS and NM than in group I/R and M. The expression of p-CREB and BNDF mRNA was significantly higher in group I/R, M, NaHS and NM than in group S, and in group M, NaHS and NM than in group I/R. The BDNF mRNA expression was significantly higher in group NM than in group M and NaHS. There was no significant difference in the H2S content and the expression of p-CREB and BNDF mRNA between group NaHS and M.Conclusion Hydrogen sulfide combined with mild hypothermia can attenuate cerebral I/R injury by up-regulating the expression of p-CREB and BDNF mRNA in hippocampus in rats.