联合水蛭注射液后处理对脑缺血损伤大鼠GSK-3β、eNOS蛋白表达的变化

The effect of combined leech injection postconditioning and ischemic postconditioning on the expression of GSK-3β and eNos against ischemia-reperfusion injury in rats

目的观察联合应用水蛭注射液后处理对脑缺血损伤大鼠GSK-3β、eNOS蛋白表达的影响,以探讨其对脑缺血再灌注损伤的神经保护机制。方法 60只成年雄性SD大鼠,随机分为假手术组(S)、缺血-再灌注组(I/R)、缺血后处理组(P)、小剂量水蛭后处理组(S-H)、大剂量水蛭后处理组(L-H)、缺血后处理+大剂量水蛭组(P-H)等6组。采用线栓法制备大鼠局灶性脑缺血-再灌注损伤动物模型及缺血后处理模型,24 h后比较各组神经功能缺损评分,应用Western blot技术,观察GSK-3β、eNOS蛋白在大鼠缺血半暗带表达的变化。结果神经行为学评分:I/R组评分显著增高(P<0.01);与I/R组比较,其余各组评分均显著减少(P<0.01);其中P-H组评分最低(P<0.01)。GSK-3β/eNOS蛋白表达:与S组比较,I/R组GSK-3β含量上升,eNOS含量下降(P<0.01);与I/R组比较,其余各组GSK-3β表达显著下降、eNOS表达显著增高(P<0.01);P-H组此种表达趋势变化最明显(P<0.01)。结论水蛭注射液和缺血后处理可减轻神经功能缺损,下调GSK-3β、上调eNOS蛋白的表达,减轻脑缺血损害,两者联合具有协同神经保护作用,这种保护作用与水蛭的剂量可能有一定的关系。

Objective To observe the effect of combined leech injection postconditioning and ischemic postconditioning on the protein expression of GSK -3β and eNOS, and explore their protection mechanism against cerebral ischemia -reperfusion injury. Methods 60 adult male SD rats were randomly divided into six groups：control group （S） , ischemia- reperfusion （I/R） , ische- mic postconditioning group （P）, small dose of leech postconditioning （S -H） , high dose of leeeh postconditioning group （L -H） and ischemic postconditioning ＋ high - dose leech （ P - H）. Using the suture method to make the rat models of focal cerebral is-chemia-reperfusion injury and ischemic postconditioning, then compared neural function defect score of every group after 24 hours. Using western blot technique to detect the protein expression change of GSK -3β and eNOS in penumbra. Results ①Neurologie deficit score ：There is a higher score in I/R group than S group（ P〈0.01 ） ; The score in other groups were significantly reduced than that in L/R group （ P〈0.01 ） ; Score is the lowest in P - H group （ P 〈0.01 ）. ②Protein expression of GSK - 3 β and eNOS： Compared with S group,the protein content of GSK -3β increased in I/R group, but the content of eNOS decreased （P〈0.01 ） ; Compared with I/R group, the protein expression of GSK -3β decreased significantly, and eNOS increased considerably （P〈0.01 ） in the rest of the group,such expression trend change was most obviously in P- H group（P 〈0.01 ）. Conclusion - Leech injection and ischemic postconditioning can alleviate cerebral ischemia damage through reducing neurological deficit, down -regulating GSK - 3β, or up - regulating the eNOS protein expression. And there is a synergetic protective effect of combination of the two intervention methods. This protective effect may have a certain relationship with the dose of leeches.