骨骼肌缺血再灌注损伤中血管功能障碍与丹参的干预效应

Vascular functional disturbance in skeletal muscle during ischemia/reperfusion injury and intervention of Danshen

目的:观察骨骼肌缺血再灌注损伤血清一氧化氮、诱导型一氧化氮合酶、内皮素1含量的变化及中药丹参的干预作用。方法:实验于2005-01/12在福建中医学院骨伤系实验室完成。实验分组:选用雄性SD大鼠84只,按随机数字表法分为丹参组、生理盐水组,每组42只,每组又分缺血再灌注10,20,30,40,50,60,90min7个时间点,每个时间点6只。实验方法:①制备大鼠左侧提睾肌缺血再灌注损伤模型。②丹参组于缺血2h时腹腔注射丹参注射液,生理盐水组给予相应剂量生理盐水;分别于缺血再灌注10,20,30,40,50,60,90min抽取腹主动脉血。实验评估:①采用硝酸还原酶法测定血清一氧化氮浓度。②采用比色法测定诱导型一氧化氮合酶活性。③采用放射免疫法测定内皮素1含量。结果:纳入大鼠84只,均进入结果分析。①一氧化氮、诱导型一氧化氮合酶的表达量:缺血再灌注10,20min两组一氧化氮、诱导型一氧化氮合酶的表达量无明显差异(P>0.05);缺血再灌注30,40,50,60,90min丹参组一氧化氮和诱导型一氧化氮合酶的表达量均高于生理盐水组[一氧化氮分别为(70.95±2.10),(68.21±2.23)μmol/L;(77.05±2.28),(72.20±1.56)μmol/L;(81.12±2.74),(74.60±1.90)μmol/L;(68.81±2.32),(62.03±2.80)μmol/L;(57.08±3.02),(46.77±3.01)μmol/L;诱导型一氧化氮合酶分别为(515.17±47.54),(459.78±37.27)μkat/L;(629.46±44.19),(499.37±29.46)μkat/L;(673.73±29.96),(584.77±58.48)μkat/L;(590.62±31.96),(507.78±31.82)μkat/L;(485.33±38.27),(378.64±38.04)μkat/L],差异有非常显著性意义(t=2.238,4.332,4.783,4.569,5.922;2.246,6.000,3.317,4.499,4.843,P<0.05,0.01)。缺血再灌注50min两组一氧化氮、诱导型一氧化氮合酶的表达量均达到最高峰,此后表达量开始下降。②内皮素1的表达量:缺血再灌注40min两组内皮素1的表达量均达到最高峰,此后表达量开始下降;缺血再灌注10min两组间内皮素1的表达量无明显差异(P>0.05),缺血再灌注20,30,40,50,60,90min丹参组内皮素1的表达量低于生理盐水组[分别为(145.77±26.54),(237.76±14.41)ng/L;(197.32±21.80),(258.50±40.20)ng/L;(124.44±6.00),(189.58±7.24)ng/L;(115.88±10.84),(165.93±10.43)ng/L;(103.96±3.84),(158.05±10.62)ng/L;(84.42±6.16),(113.69±10.41)ng/L],差异有非常显著性意义(t=7.462,3.278,16.959,8.149,11.731,5.926,P<0.01)。结论:骨骼肌缺血再灌注损伤导致血清中一氧化氮、诱导型一氧化氮合酶、内皮素1的表达量发生改变,丹参可能通过调整一氧化氮和内皮素1的平衡,改善缺血再灌注损伤造成的血管内皮功能障碍。

AIM： To evaluate the effect of Danshen on the content changes of serum nitrogen monoxidum （NO）, inducible nitricoxide synthase （iNOS）, endothelin-1 （ET-1） of skeletal muscle during ischemia/reperfusion （I/R）. METHODS： The experiment was conducted in the Department of Orthopaedics and Traumatology, Fujian College of Traditional Chinese Medicine from January to December 2005. Eighty-four male SD rats were randomly divided into Danshen group and saline group with 42 animals in each group. And each group was subdivided into I/R 10, 20, 30, 40, 50, 60, and 90 minutes groups with 6 rats in each group. ①Under anesthesia, the I/R models were established in the left cremaster muscles of rats. Danshen was intraperitoneally injected into Danshen group 2 hours after ischemia; the saline group was injected the same volume normal saline. ②The abdominal aorta blood was harvested after reperfusion 10, 20, 30, 40, 50, 60, and 90 minutes to detect the serum NO, iNOS and ET-1 by nitrate reductase, chromometry, and radioimmunity methods, respectively. RESULTS： All 84 rats were involved in the result analysis. ①There was no obvious difference in the expressions of NO, iNOS between Danshen group and normal saline group reperfusion 10, and 20 minutes （P 〉 0.05）; the expression of NO, and iNOS in reperfusion 30, 40, 50, 60, and 90 minutes Danshen groups were significantly higher than that of normal saline group [NO： （70.95±2.10）, （68.21±2.23） μmol/L; （77.05±2.28）, （72.20±1.56）μmol/L; （81.12±2.74）, （74.60±1.90）μmol/L, （68.81±2.32）, （62.03±2.80） μmol/L; （57.08±3.02）, （46.77±3.01） μmol/L; iNOS： （515.17±47.54）, （459.78± 37.27） μkat/L; （629.46±44.19）, （499.37±29.46） μkat/L; （673.73±29.96）, （584.77±58.48） μkat/L; （590.62±31.96）, （507.78±31.82）μkat/L; （485.33±38.27）, （378.64±38.04） μkat/L; t =2.238, 4.332, 4.783, 4.569, 5.922, 2.246, 6,000, 3.317, 4.499, 4.643, P 〈 0.05, 0.01]. The expression of NO, and iNOS reached the peak after 50 minutes reperfusion of both groups, then began to decrease. ②ET-1 expression of two groups reperfusion 40 minutes reached the peak value, and began to decrease afterward. There was no significantly difference between Danshen group and saline group after reperfusion for 10 minutes （P 〉 0.05）. But the expression of ET-1 in reperfusion 20, 30, 40, 50, 60, and 90 minutes Danshen groups ware significantly lower than that of normal saline group [（145.77±26.54）, （237.76±14.41） ng/L; （197.32±21.80）, （258.50±40.20） ng/L; （124.44±6.00）, （189.58±7.24） ng/L; （115.88±10.64）, （165.93±10.43） ng/L; （103.96±3.64）, （158.05±10.62） ng/L; （64.42±6.16）, （113.69±10.41） ng/L; t =7.462, 3.278, 16.959, 8.149, 11.731, 5.926, P〈 0.01]. CONCLUSION： I/R leads to the changes of NO, iNOS and ET-1. Danshen could improve the I/R-induced vascular endothelial function disturbance by balancing the expression of NO and ET-1.