枸橼酸咖啡因对感染所致新生大鼠脑白质损伤的保护作用

Protective effect of caffeine citrate on neonatal rat brain white matter damage induced by infection

目的探讨枸橼酸咖啡因对出生后感染所致2日龄新生大鼠脑白质损伤的保护作用。方法将48只2日龄新生SD大鼠采用随机数字表法随机分为对照组（A组）、脂多糖（LPS）感染组（B组）、枸橼酸咖啡因干预组（C组），每组16只。B、C2组新生大鼠于2日龄时连续5d给予0．6mg／kgLPS腹腔注射，A组连续5d给予等体积的9g／L盐水腹腔注射；4日龄时，C组连续7d另给予10mg／kg枸橼酸咖啡因腹腔注射，同时A组和B组连续7d另给予等体积的9g／L盐水腹腔注射。至12日龄时，3组均采用随机数字表法随机抽取8只处死，免疫组织化学法检测皮质下白质髓鞘碱性蛋白（MBP）表达情况；各组剩余8只大鼠于冰面上迅速取出左右海马组织并保存，左侧海马组织采用Westernblot法检测MBP及Caspase-3的表达，右侧海马组织采用实时荧光定量PCR（qPCR）技术检测MBP及Caspase-3的表达。结果A组、B组、C组皮质下MBP阳性表达的积分光密度值（IOD）分别为132．64±1．94、102．45±2．12、114．25±2.04，组间比较差异有统计学意义（F=22．912，P〈0．05）。3组海马区MBP mRNA的相对表达水平分别为0．79±0．01、0．39±0．03、0．55±0．02，组间比较差异有统计学意义（F=18．584，P〈0．05）；3组海马区MBP蛋白的相对表达水平分别为0．64±0．03、0．31±0．03、0．51±0．05，组间比较差异有统计学意义（F=25．780，P〈0．05）。3组Caspase-3mRNA的相对表达量分别为0．34±0．02、0．74±0．03、0．57±0．04，组间比较差异有统计学意义（F=6．105，P〈0．05）；3组Caspase-3蛋白的相对表达量分别为0．11±0．03、0．36±0．02、0．23±O．03，组间比较差异有统计学意义（F=40．541，P〈0．05）。结论枸橼酸咖啡因对感染所致2日龄新生大鼠脑白质损伤可能具有保护作用，其机制之一可能与抑制炎性反应、减少细胞凋亡有关。

Objective To investigate the protective effect of caffeine citrate on white matter damage in 2 - day - old neonatal rats induced by postnatal infection. Methods Forty - eight 2 - day - old SD rats were randomly di- vided into the control group （group A,n = 16） ,lipopolysaccharide（LPS） infection group （group B,n = 16）,and caffeine citrate intervention group （group C, n = 16 ） according to the random table method. The newborn rats of group B and C were continuously injected LPS 0.6 mg/kg intraperitoneally for 5 days from 2 days old ,and the newborn rats of group A were continuously injected by an equal volume of 9 g/L saline intraperitoneally. Group C was continuously injected by caffeine citrate 10 mg/kg intraperitoneally for 7 days from 4 clays old ; equal volume of 9 g/L saline was injected into group A and B for 7 days continuously. At 12 days old ,8 rats of each group were sacrificed randomly to evaluate the ex- pression of myelin basic protein（MBP） subcortical white matter by immunohistochemical method. Both sides of hippo- campus of the rest 8 rats of each group were taken out in ice surface rapidly. The left hippocampus was used to detect the expression of MBP and Caspase - 3 by Western blot method, and the right hippocampus was used to evaluate the MBP and Caspase -3 protein level by quantitative real -time PCR（qPCR） method. Results The mean integral opti- cal density （IOD） of subcortical MBP positive expression in group A, group B and group C were 132.64 ± 1.94, 102.43 ±2. 12,114.25 ± 2.04, and the difference was statistically significant among 3 groups （ F = 22. 912, P 〈 0. 05 ）. The relative expression levels of MBP mRNA of 3 groups in hippocampus were 0.79 ± 0.01,0.39 ± 0.03, 0. 55 ± 0. 02, and the difference was statistically significant among 3 groups （ F = 18. 584, P 〈 0. 05 ）. The relative ex- pression levels of MBP protein of 3 groups in hippocampus were 0.64 ± 0.03,0.31 ± 0.03,0.51 ± 0.05, and the differ- ence was statistically significant among 3 groups （ F = 25. 780, P 〈 0.05 ）. The relative expression levels of Caspase - 3 mRNA of 3 groups in hippocampus were 0. 34 ± 0.02,0.74 ± 0.03,0.57 ± 0.04, and the difference was statistically significant among 3 groups （F = 6. 105 ,P 〈 0.05 ）. The relative expression of Caspase -3 protein of 3 groups in hippo- campus were 0.11 ± 0.03,0.36 ± 0.02,0.23 ± 0.03, and the difference was statistically significant among 3 groups （F = 40.541 ,P 〈 0.05 ）. Conclusions Caffeine citrate has showed protective effect on white matter damage in neo- natal rats of 2 days old induced by postnatal infection. The mechanism may be related to inhibition of inflammatory response and reducing apoptosis.