乳果糖对大鼠脊髓缺血再灌注损伤的影响

Effect of lactulose on spinal cord ischemia-reperfusion injury in rats

目的 评价乳果糖对大鼠脊髓缺血再灌注损伤的影响.方法 选择胃管置入成功的健康雄性清洁级SD大鼠144只,3月龄,体重300～ 350 g,采用随机数字表法,将其分为4组（n=36）：假手术组（S组）、脊髓缺血再灌注组（I/R组）、乳果糖组（L组）和乳果糖＋抗生素组（LA组）.采用阻断胸主动脉联合体循环低血压的方法诱导脊髓缺血9 min,然后恢复灌注,建立脊髓缺血再灌注损伤模型.L组于再灌注即刻胃内灌注乳果糖0.5 g/kg.LA组于术前1～3d胃内灌注甲硝唑和庆大霉素,每日单次剂量分别为30和40 mg/kg,3次/d,余处理同L组.于缺血前、再灌注10、30、60、90、120、150和180 min时采用氢气探针测定脑脊液氢气浓度.于再灌注12、24和48 h时,采用Westernblot法测定脊髓核因子E2相关因子-2（Nrf2）表达水平,黄嘌呤氧化酶法测定超氧化物歧化酶（SOD）活性,钼酸铵法测定过氧化氢酶（CAT）活性,ELISA法测定脊髓8-羟基脱氧鸟苷酸（8-OHdG）、3-硝基酪氨酸（3-NT）和丙二醛（MDA）的含量.于再灌注48 h时行后肢神经行为学评分,然后处死大鼠,取L3-5脊髓组织,测定神经元存活率和凋亡率.结果 与S组比较,I/R组再灌注各时点脑脊液氢气浓度差异无统计学意义,再灌注12、24和48 h时脊髓Nrf2、CAT和SOD水平差异无统计学意义（P＞0.05）,8-OHdG、3-NT、MDA含量增加,后肢神经行为学评分和神经元存活率降低,神经元凋亡率升高,L组再灌注30～ 180 min时脑脊液氢气浓度升高,再灌注12、24和48 h时脊髓Nrf2、CAT和SOD水平增加（P＜0.05）,8-OHdG、3-NT、MDA含量、后肢神经行为学评分、神经元存活率和凋亡率差异无统计学意义（P＞0.05）.与I/R组比较,L组再灌注30～ 180 min时脑脊液氢气浓度升高,脊髓Nrf2、CAT和SOD水平增加,8-OHdG、3-NT和MDA含量降低,后肢神经行为学评分和神经元存活率升高,神经元凋亡率降低（P＜0.05）,LA组上述指标差异无统计学意义（P＞0.05）.结论 乳果糖可减轻大鼠脊髓缺血再灌注损伤.

Objective To evaluate the effect of lactulose on spinal cord ischemia-reperfusion （I/R） injury in rats.Methods One hundred forty-four male Sprague-Dawley rats, aged 3 months, weighing 300-350 g, in which gastric tube was successfully inserted, were randomly divided into 4 groups （n=36 each） using a random number table： sham operation group （group S）, spinal cord I/R group （group I/R）, lactulose group （group L）, and lactulose ＋ antibiotics group （group LA）.Spinal cord ischemia was induced by occlusion of the thoracic aorta combined with controlled hypotension for 9 min, followed by reperfusion.Lactulose 0.5 g/kg was administered intragastrically immediately after onset of reperfusion in group L.Metronidazole 30 mg/kg and gentamicin 40 mg/kg were administered intragastrically three times a day during 1-3 days before operation in group LA, and the other procedures were similar to those previously described in group L.Hydrogen concentration in cerebrospinal fluid was detected before ischemia and at 10, 30, 60, 90, 120, 150 and 180 min after reperfusion.At 12, 24 and 48 h of reperfusion, 8 rats in each group were sacrificed, and the L3-5 segment of the spinal cord was isolated for determination of nuclear factor E2-related factor 2 （Nrf2） expression （by Western blot）, superoxide dismutase （SOD） activity （by using xanthine oxidase method）, catalase （CAT） activity （ammonium molybdate method）, and 8-hydroxy-2＆#39;-deoxyguanosine （8-OHdG）, 3-nitrotyrosine （3-NT） and malondialdehyde （MDA） contents （by ELISA）.Neurological function was assessed and scored at 48 h of reperfusion.Six animals in each group were then sacrificed after assessment of neurological function, and the L3-5 segment of the spinal cord was removed for detection of apoptotic neurons.The cell survival rate and apoptotic rate were calculated.Results Compared with group S, no significant change was found in the hydrogen concentration in the cerebrospinal fluid at each time point, and in the level of Nrf2, CAT and SOD at 12, 24 and 48 h of reperfusion, the contents of 8-OHdG, 3-NT and MDA were significantly increased, the neurological scores and cell survival rate were decreased, and the apoptotic rate was increased in group I/R, and the hydrogen concentration in the cerebrospinal fluid at 30-180 min of reperfusion was increased, the level of Nrf2, CAT and SOD was increased at 12, 24 and 48 h of reperfusion, and no significant change was found in the contents of 8-OHdG, 3-NT and MDA, neurological scores, cell survival rate, and apoptotic rate in group L.Compared with group I/R, the hydrogen concentration in the cerebrospinal fluid at 30-180 min of reperfusion was increased, the level of Nrf2, CAT and SOD was increased, the contents of 8-OHdG, 3-NT and MDA were decreased, the neurological scores and cell survival rate were increased, and the apoptotic rate was decreased in group L, and no significant change was found in the parameters mentioned above in group LA.Conclusion Lactulose can reduce spinal cord I/R injury in rats.