硫化氢吸入对心搏骤停复苏大鼠脑蛋白激酶C同工酶的影响

Effect of inhalation of hydrogen sulfide on protein kinase C isoenzyme after cardiac arrest and resuscitation in rats

目的探讨吸入硫化氢对心搏骤停复苏大鼠脑蛋白激酶C(PKC)同工酶的影响。方法取清洁级健康SD雄性大鼠60只,采用数字表法,将其随机分为假手术组(10只)、对照组(25只)和硫化氢组(25只)。对照组和硫化氢组采用窒息性心搏骤停后心肺复苏模型,硫化氢组于自主循环恢复后立即吸入含有0.08‰硫化氢的30%氧气1 h。各组大鼠于假手术或自主循环恢复后24 h时,应用神经功能缺损评分(NDS)评价神经功能;于24 h监测PKC同工酶α、βⅠ、βⅡ、δ及ε的活性水平;于7 d计算存活率、计数海马CA1区存活神经细胞。结果 (1)假手术组大鼠全部存活,对照组和硫化氢组存活率分别为45.0%(9/20)和80.0%(16/20),与对照组相比,硫化氢组大鼠存活率明显增加,组间差异有统计学意义(χ2=8.087,P=0.018)。(2)自主循环恢复(ROSC)24 h,与假手术组相比,对照组和硫化氢组NDS评分均明显降低,组间中位数两两比较,差异均有统计学意义[46(43,52)、60(55,68)分比80(80,80),均P<0.05];与对照组比较,硫化氢组NDS评分升高,组间差异有统计学意义(P<0.05)。(3)ROSC后24 h,与假手术组比较,对照组PKC-α、PKC-βⅠ、PKC-βⅡ和PKC-δ活性水平明显升高,PKC-ε活性水平受到抑制,组间比较差异均有统计学意义[(1.63±0.14)比(1.00±0.02)、(2.00±0.08)比(1.00±0.04)、(1.51±0.10)比(1.00±0.01)、(2.02±0.12)比(1.00±0.02);均P<0.05];硫化氢组PKC-α、PKC-βⅠ、PKC-βⅡ、PKC-δ和PKC-ε活性水平的差异无统计学意义(均P>0.05);与对照组比较,硫化氢组PKC-α、PKC-βⅠ、PKC-βⅡ和PKC-δ活性水平[分别为(0.94±0.06)、(1.26±0.04)、(0.83±0.02)、(1.04±0.06)]明显下降,PKC-ε水平明显升高[(1.13±0.07)比(0.73±0.04)],组间差异均有统计学意义(均P<0.05)。(4)ROSC后7 d,假手术组、对照组和硫化氢组海马CA1区存活神经细胞计数分别为(53±3)、(23±2)和(34±1)个,与假手术组相比,对照组和硫化氢组存活神经细胞计数均明显降低(均P<0.01);与对照组比较,硫化氢组存活神经细胞计数明显升高(P<0.01)。结论心搏骤停/心肺复苏后,早期吸入硫化氢可抑制PKC-α、PKC-βⅠ、PKC-βⅡ及PKC-δ激活,促进PKC-ε激活,改善大鼠神经功能和促进神经细胞存活。

Objective To investigate the effect of inhalation of hydrogen sulfide on protein kinase C （PKC） isoenzyme after cardiac arrest and resuscitation in rats. Methods Sixty clean grade healthy SD male rats were selected. Using digital table method, they were randomly completely divided into 3 groups：a sham operation group （n = 10） ,a control group （n = 25 ） ,and a hydrogen sulfide group （n = 25 ）. A cardiopulmonary resuscitation model after asphyxia cardiac arrest was used in the control group and the hydrogen sulfide group. Immediately after the restoration of spontaneous circulation, the hydrogen sulfide group inhaled 0.08‰ hydrogen sulfide containing 30% oxygen for I h. The neurologic deficit score （NDS） was used to assess neurological function of each group at 24 h after sham operation or return of spontaneous circulation （ROSC）. The activation levels of PKC isozyme α, βI , β Ⅱ, δ and ε were detected at 24 h. The survival rate was calculated and the number of survival neurons of hippocampus in CA1 region was calculated at day 7. Results （1） Rats in sham operation group were all alive （5/5）, the survival rates of the control group and hydrogen sulfide group were 45.0% （9/20） and 80.0% （ 16/20）, respectively. Compared with the control group, the rat survival rate was significantly increased in the hydrogen sulfide group （X2 =8. 087,P =0. 018）. （2） Compared with the sham operation group,the NDS scores of the control group and the hydrogen sulfide group were decreased significantly. There was significant difference between the two groups （46[43,52] ,60155,683 vs. 80 [80,80] ,all P 〈0.05） ;compared with the control group,the NDS score of the hydrogen sulfide group was increased. There was significant difference between the two groups （P 〈 0.05）. （3） At 24 h after ROSC, compared with sham operation group, the activation levels of PKC-α,PKC-βⅠ,PKC-βⅡ,and PKC-δ were significantly increased in the control group,but the activation level of PKC-ε was inhibited. There were significant differences among the groups （ 1.63±0.14 vs. 1.00 ±0.02,2.00±0.08 vs. 1.00 ±0.04,1.51±0.10 vs. 1.00±0.01,2.02±0.12 vs. 1.00±0.02; all P 〈 0.05 ）. There were no significant differences in the activation levels of PKC-α,PKC-βⅠ,PKC-βⅡ,and PKC-δ of the hydrogen sulfide group （all P 〉 0.05 ）. Compared with the control group, the activation levels of PKC-α,PKC-βⅠ,PKC-βⅡ,and PKC-δ of the hydrogen sulfide group （0.94±0.06, 1.26±0.04,0.83±0.02, and 1.04±0. 06, respectively） were decreased significantly, and the PKC-ε level was increased significantly （ 1.13±0. 07 vs. 0. 73±0. 04） and there were significant differences among the groups （all P 〈0.05）. （4） At day 7 after ROSC,the counts of surviving neurons in hippocampal CA1 region of the sham operation group,control group and hydrogen sulfide group were 53±3,23±2, and 34±1 ,respectively. Compared with the sham operation group,the counts of survival neurons in hippocampal CA1 region of the control group and the hydrogen sulfide group were decreased significantly （ all P 〈 0. 01 ）. Compared with the control group, the count of survival neurons of the hydrogen sulfide group was increased significantly （ P 〈 0.01 ）. ConclusiOn After cardiac arrest/cardiopulmonary resuscitation, early inhalation of hydrogen sulfide may inhibit the activation of PKC-α,PKC-βⅠ,PKC-βⅡ,and PKC-δ, promote the activation of PKC-ε,improve the neuronal function and the survival of nerve cells in rats.