陇中消肿止痛合剂对脊髓损伤后大鼠脊髓中水通道蛋白4表达的影响

Effect of Longzhong Xiaozhu Zhitong Mixture on Aquaporin 4 Expression in Spinal Cord of Rats after Spinal Cord Injury

目的 探索陇中消肿止痛合剂对脊髓损伤后大鼠脊髓中水通道蛋白4(AQP-4)的表达情况及其作用机制。方法 选取成年雄性清洁级SD大鼠175只,按照Allen’s法建立脊髓损伤大鼠模型。造模成功后将其随机分为7组,分别为对照组、假损伤组、模型组、七叶皂苷钠组及陇中消肿止痛合剂高、中、低剂量组(简称陇中高、中、低剂量组),每组各25只。采取灌胃给药的方式,根据巴索、比蒂和布雷斯纳汉运动评级(BBB)法评分标准,每隔3天观察评分并记录,总计观察15天。在上述7组随机分别选取SD大鼠15只,提取损伤脊髓样本,分别采用逆转录-聚合酶链反应(RT-PCR)、蛋白质免疫印迹(Western Blot)及免疫组织化学染色检测AQP-4蛋白及基因表达,并进行统计分析。结果模型组大鼠BBB评分各时间段均低于对照组和假损伤组(P<0.05);第3、6、9、12、15天于七叶皂苷钠组及陇中高、中、低剂量组BBB评分均高于模型组(P<0.05);第6、9、12、15天陇中高剂量组BBB评分高于七叶皂苷钠组及陇中低剂量组(P<0.05,P<0.01);且第15天陇中低剂量组BBB评分低于七叶皂苷钠组(P<0.05);陇中高剂量组第9、12、15天BBB评分高于陇中中剂量组(P<0.01);模型组AQP-4蛋白及mRNA表达高于对照组及假损伤组(P<0.05);与模型组比较,七叶皂苷钠组及陇中高、中剂量组AQP-4蛋白及mRNA表达明显降低(P<0.05);与七叶皂苷钠组比较,陇中高、中、低剂量组AQP-4蛋白及mRNA表达降低(P<0.05);与陇中高剂量组比较,陇中中、低剂量组AQP-4蛋白及mRNA表达降低(P<0.05)。结论 陇中消肿止痛合剂通过抑制AQP-4在脊髓组织中的表达,从而减轻脊髓水肿,促进神经功能恢复。

Objective To study the expression of aquaporin 4(AQP-4) in spinal cord of rats after spinal cord injury with Longzhong Xiaozhong Zhitong Mixture(LXZM) and its mechanism. Methods Totally 175 adult male SD rats of clean grade were selected to establish spinal cord injury model according to Allen’s method.After successful modeling, the rats were randomly divided into 7 groups: control group, sham-injury group, model group, sodium aescinate saponin group, high, medium, and low dose LXZM groups, 25 rats in each group. The drugs were administered by gastrogavage. According to Basso, Beatty, and Bresnahan Exercise Rating(BBB) criteria, the scores were observed and recorded every 3 days, with a total of 15 days. Fifteen SD rats were randomly selected from the above seven groups to extract injured spinal cord samples. AQP-4 protein and gene expressions were detected by RT-PCR, Western Blot, and immunohistochemical staining, and statistical analyses were conducted. Results BBB score of model group was lower than that of control group and sham-injury group(P<0.05). On the 3rd, 6th, 9th, 12th, and 15th day, BBB scores in sodium aescinate saponin group and each dose LXZM groups were higher than those in model group(P<0.05). On the 6th,9th,12th, and 15th day, BBB score in high dose LXZM group was higher than that of sodium aescinate saponin group and low dose LXZM group(P<0.05, P<0.01). On the 15th day BBB score of low dose LXZM group was lower than that of sodium aescinate saponin group(P<0.05). BBB score of high dose LXZM group was higher than that of middle dose LXZM group on the 9th,12th, and 15th day(P<0.01). The expressions of AQP-4 protein and mRNA in model group was higher than that in control group and sham-injury group(P<0.05). Compared with the model group, the expressions of AQP-4 protein and mRNA in sodium aescinate saponin group and high and middle dose LXZM groups decreased significantly(P<0.05). Compared with sodium aescinate saponin group, the expressions of AQP-4 protein and mRNA in high, medium, and low dose LXZM groups decreased(P<0.05). Compared with high dose LXZM group, the expressions of AQP-4 protein and mRNA in medium and low dose LXZM groups decreased(P<0.05). Conclusion LXZM attenuated spinal edema and promoted the recovery of nerve function by inhibiting the expression of AQP-4 in spinal cord tissue.