水蛭联合淮山药对创伤性脑损伤大鼠的干预作用

Intervention effect of Leech combined with Dioscorea Opposita Thunb on rats with traumatic brain injury

目的探讨水蛭联合淮山药对创伤性脑损伤(TBI)大鼠的干预作用。方法选择36只健康SD大鼠,按随机数字表法分为正常对照组、假手术组、模型组、水蛭淮山药组,每组9只。采用Feeney自由落体法复制TBI大鼠模型。制模前和制模后4 d,水蛭淮山药组给予水蛭加淮山药中药液10 mL/kg灌胃,每日2次;其余3组给予等量生理盐水。制模后24 h,采用头颅磁共振成像(MRI)观察各组大鼠脑组织的变化。制模后4 d,处死大鼠后取血,用全自动血凝仪检测各组血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)水平;光镜下观察脑组织病理学改变;采用酶联免疫吸附试验(ELISA)检测各组脑组织内皮素(ET)、一氧化氮(NO)和血浆D-二聚体含量;采用免疫组化法检测各组脑组织组织型纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制剂-1(PAI-1)、磷酸化p65(p-p65)的阳性细胞表达水平;采用流式细胞术检测各组脑组织CD61、CD62表达水平。结果MRI和光镜下均可见模型组脑组织水肿和梗死程度均较正常对照组更明显;水蛭淮山药组脑组织水肿和梗死程度均较模型组明显减轻。与正常对照组比较,模型组ET、D-二聚体含量和t-PA、PAI-1、p-p65阳性细胞比例均显著升高〔ET(ng/L):307.6±20.7比102.6±5.5,D-二聚体(μg/L):3193.5±622.0比882.3±231.7,t-PA:(71.4±1.1)%比(40.7±3.2)%,PAI-1:(76.9±4.7)%比(30.4±4.4)%,p-p65:(115.8±2.8)%比(57.9±2.1)%,均P<0.05〕,PT、APTT均明显延长〔PT(s):19.2±1.4比9.4±0.7,APTT(s):27.5±1.5比12.5±0.6,均P<0.05〕,NO含量明显降低(μmol/g:5.0±0.3比7.9±0.3,P<0.05)。与模型组比较,水蛭淮山药组PT、APTT明显缩短〔PT(s):17.6±1.5比19.2±1.4,APTT(s):25.3±1.8比27.5±1.5,均P<0.05〕,t-PA、p-p65阳性细胞比例均显著下降〔t-PA:(57.9±4.2)%比(71.4±1.1)%,p-p65:(79.0±0.1)%比(115.8±2.8)%〕。模型组和水蛭淮山药组ET、NO、D-二聚体含量和PAI-1阳性细胞比例比较差异均无统计学意义(均P>0.05)。流式细胞术分析可见:模型组大鼠脑组织中CD61和CD62的表达量均较假手术组上调;水蛭淮山药组大鼠脑组织中CD61和CD62的表达量均较模型组上调。结论水蛭联合淮山药可通过缩短PT和APTT,减少t-PA、p-p65阳性细胞比例以及增加CD61、CD62,调节TBI大鼠的凝血状态和纤溶过程,改善凝血功能,进而降低进展性出血性脑损伤发生的风险,起到对TBI的保护作用。

Objective To investigate the intervention effects of Leech combined with Dioscorea Opposita Thunb on rats with traumatic brain injury(TBI).Methods Thirty-six healthy Sprague-Dawley(SD)rats were selected and divided into four groups:normal control group,sham operation group,model group,Leech combined with Dioscorea Opposita Thunb traditional Chinese medicine(TCM)group(combined TCM group)according to the random number table method,with 9 rats in each group.The TBI rat model was replicated by Feeney body free-fall method.Before and 4 days after model establishment,the rats in combined TCM group were given 10 mL/kg of combined TCM solution by gavage,twice a day;the other three groups were given the same amount of normal saline.The brain tissue changes in each group were observed by cranial magnetic resonance imaging(MRI)at 24 hours after modeling.Four days after modeling,the rats were killed and their blood was collected.The plasma prothrombin time(PT),activated partial thromboplastin time(APTT)in each group were detected by automatic blood coagulation apparatus;under light microscope,the brain histopathological changes were observed;the brain tissue contents of endothelin(ET),nitric oxide(NO)and D-dimer levels were determined by enzyme-linked immunosorbent assay(ELISA);immunohistochemistry method was used to detect the positive cell expression levels of brain tissue plasminogen activator(t-PA),plasminogen activator inhibitor-1(PAI-1)and phosphated p65(p-p65)in various groups;the brain tissue expression levels of CD61 and CD62 were detected by flow cytometry.Results Both MRI and light microscopic examinations showed that the degree of brain edema and infarction in the model group were more obvious than those in the normal control group;the degree of brain edema and infarction in combined TCM group were significantly reduced compared with those in model group.Compared with the normal control group,the contents of ET,D-dimer and the proportions of t-PA,PAI-1 and p-p65 positive cells were significantly increased in the model group[ET(ng/L):307.6±20.7 vs.102.6±5.5,D-dimer(μg/L):3193.5±622.0 vs.882.3±231.7,t-PA:(71.4±1.1)%vs.(40.7±3.2)%,PAI-1:(76.9±4.7)%vs.(30.4±4.4)%,p-p65:(115.8±2.8)%vs.(57.9±2.1)%,all P<0.05],PT and APTT were significantly prolonged[PT(s):19.2±1.4 vs.9.4±0.7,APTT(s):27.5±1.5 vs.12.5±0.6,both P<0.05],the content of NO was decreased significantly(μmol/g:5.0±0.3 vs.7.9±0.3,P<0.05).Compared with the model group,PT and APTT were significantly shortened in the combined TCM group[PT(s):17.6±1.5 vs.19.2±1.4,APTT(s):25.3±1.8 vs.27.5±1.5,both P<0.05],the proportions of t-PA and p-p65 positive cells were decreased significantly[t-PA:(57.9±4.2)%vs.(71.4±1.1)%,p-p65:(79.0±0.1)%vs.(115.8±2.8)%].There were no significant differences in ET,NO,D-dimer contents and PAI-1 positive cell ratio between the model group and the combined TCM group(all P>0.05).Flow cytometry analysis showed that the expression levels of CD61 and CD62 in the brain tissues of the model group were significantly up-regulated compared with those in the sham operation group,and the expressions of CD61 and CD62 in the brain tissues of the combined TCM group were significantly up-regulated compared with those of the model group.Conclusion By shortening PT and APTT,reducing the proportions of t-PA and p-p65 positive cells and increasing CD61 and CD62,Leech combined with Dioscorea Opposita Thunb can regulate the coagulation state and fibrinolysis process of TBI rats,improve the coagulation function,and then decrease the risk of progressive hemorrhagic brain injury and play a protective role on TBI.