羟基红花黄色素A对脑缺血再灌注损伤大鼠HSP70表达及相关性研究

发布时间：2018-05-05 17:04

本文选题：羟基红花黄色素A + 脑缺血再灌注　；参考：《云南中医学院》2017年硕士论文

【摘要】：目的:本研究通过线栓法阻塞右侧大脑中动脉构建大鼠缺血再灌注模型,在术前和术后分别尾静脉给予羟基红花黄色素A(HSYA)干预,通过神经功能缺损评分、脑梗死体积变化、免疫组化及蛋白质印迹法等方法,检测HSP70表达来探索HSYA对大脑中动脉缺血再灌注的预防和治疗作用、对脑缺血再灌注对抗凋亡蛋白HSP70表达的影响,并进一步探讨HSYA对缺血再灌注大鼠神经保护的相关分子机制,为HSYA在脑梗死的治疗上提供理论依据。方法:1.动物分组:将70只体重250-300g成年雄性SD大鼠随机分为7组(即正常组、假手术组、模型组、术前5mg/kg组、术前10mg/kg组、术后5mg/kg组、术后10mg/kg组,n=10。2.缺血再灌注模型建立:局灶性脑缺血再灌注模型采用尼龙丝线阻塞右侧大脑中动脉,通过将线栓有圆头的一端由颈总动脉插入到颈内动脉,进而到达大脑中动脉的起始端,阻塞大脑中动脉。阻塞2h后拔出栓线,再灌注24h。假手术组操作与之相同,但不阻塞大脑中动脉。3.给药:术前5mg/kg组、术前10mg/kg组组于术前30min分别按5mg/kg、10mg/kg的剂量给予HSYA尾静脉注射;术后5mg/kg组、术后10mg/kg组于术后分别按5mg/kg、10mg/kg的剂量给予HSYA尾静脉注射;假手术组与模型组给予生理盐水尾静脉注射。4.神经功能缺损评估:麻醉清醒后2h,采用五分法进行神经功能缺损评分。5.组织学评估:所有大鼠再灌注24h后处死迅速取脑,采用TTC染色,用Image J软件进行脑梗面积的测量,进而算出脑梗死比积;用免疫组化和蛋白质印迹法检测大鼠脑缺血再灌注后缺血半暗带区HSP70的表达情况。结果:1.神经功能评分示:HSYA(5mg/kg,10mg/kg)尾静脉注射治疗及预防用药均可减少大鼠脑缺血再灌注后神经功能缺损评分。与模型组相比,术前10mg/kg组、术后5mg/kg组、术后10mg/kg组神经功能缺损评分明显降低,差异具有统计学意义(P0.05)。术前5mg/kg组与模型组间的差异无统计学意义(P0.05)。2.脑梗死比积示:HSYA(5mg/kg,10mg/kg)的治疗和预防用药均可以明显减少大鼠脑缺血再灌注后脑梗死比积,差异具有统计学意义(P0.05)。术后5mg/kg组、术后10mg/kg组脑梗死比积较术前5mg/kg组明显减少,差异具有统计学意义(P0.05)。术后10mg/kg组治疗给药明显优于术后5mg/kg组预防给药,差异具有统计学意义(P0.05)。3.免疫组化示:HSP70主要表达于细胞质中,正常组阳性细胞表达量极少。假手术组较正常组表达稍多,但无明显统计学差异(P0.05);脑缺血再灌注会导致大鼠脑组织半暗带区阳性细胞大量增加,差异具有统计学意义(P0.01);给药各组与模型组相比,阳性细胞表达明显减少,且差异具有统计学意义(P0.01);与术前5mg/kg组相比,术后5mg/kg组及术后10mg/kg组阳性细胞表达明显减少,差异具有统计学意义(P0.05);与术前10mg/kg组相比,术后10mg/kg组阳性细胞表达明显减少,差异具有统计学意义(P0.01);与术后5mg/kg组相比,术后10mg/kg组阳性细胞表达明显减少,差异具有统计学意义(P0.01)。4.蛋白质印迹法示:与正常组相比,假手术组HSP70表达明显增加,差异具有统计学意义(P0.01);与假手术相比,模型组HSP70表达明显升高,差异具有统计学意义(P0.01);与术前5mg/kg组相比,术后5mg/kg组、术后10mg/kg组HSP70表达明显减少,且差异具有统计学意义(P0.01);与术前10mg/kg组相比,术后5mg/kg组、术后10mg/kg组HSP70表达明显增减少,且差异具有统计学意义(P0.01);与术后5mg/kg组相比,术后10mg/kg组HSP70表达明显增高,且差异具有统计学意义(P0.01)。结论1.HSYA的治疗及预防用药均可减少大鼠脑缺血再灌注后神经功能缺损评分,术前组10mg/kg、术后5mg/kg、10mg/kg剂量给药可以明显降低神经功能缺损评分,改善大鼠肢体偏瘫情况。2.HSYA的治疗和预防用药均可以明显减少大鼠脑缺血再灌注后脑梗死体积,在同等剂量下(5mg/kg、10mg/kg),术后治疗给药优于术前预防给药。3.免疫组化和免疫蛋白印记法示:HSP70主要表达于细胞质中,在正常组织中表达较少,手术损伤会导致HSP70表达稍增加。脑缺血再灌注后,大鼠脑组织半暗带区HSP70蛋白大量增加。然而,HSYA预防及治疗给药均可减少SD大鼠脑缺血再灌注后缺血半暗带区HSP70蛋白的表达。在同等剂量下(5mg/kg、10mg/kg),缺血再灌注后治疗给药降低HSP70表达的水平明显优于缺血前预防给药,且术后10mg/kg治疗组较术后5mg/kg组效果更佳。
[Abstract]:Objective: to block the right middle cerebral artery by blocking the right middle cerebral artery, the rat model of ischemia-reperfusion was constructed in this study. The tail vein was given hydroxysafflor A (HSYA) in the caudal vein before and after the operation, and the expression of HSP70 was detected by the methods of nerve function defect score, cerebral infarction volume, immunohistochemistry and Western blot, and HSYA was explored. The effect of the prevention and treatment of ischemia reperfusion in the middle cerebral artery, the effect of cerebral ischemia reperfusion against the expression of apoptotic protein HSP70, and further explore the molecular mechanism of HSYA on the neuroprotection of ischemia reperfusion rats, and provide a theoretical basis for the treatment of HSYA in cerebral infarction. Methods: 1. animals were divided into 70 weight male adult male S. D rats were randomly divided into 7 groups (normal group, sham operation group, model group, pre operation 5mg/kg group, group 10mg/kg, group 5mg/kg, 10mg/kg group after operation, n=10.2. ischemia reperfusion model. The focal cerebral ischemia reperfusion model was blocked by nylon thread to the right middle cerebral artery, by inserting one end of the thread with a round head of the neck to the common carotid artery. " The internal carotid artery, which then reached the beginning of the middle cerebral artery, blocked the middle cerebral artery and blocked the middle cerebral artery. After blocking 2h, the thrombus line was pulled out and the 24h. sham operation group operated the same, but did not block the.3. administration of the middle cerebral artery: before operation, group 5mg/kg, before operation, the 10mg/kg group was given 5mg/ kg, 10mg/kg dose to the tail vein of HSYA; 5mg/kg after the operation. Group 10mg/kg was given HSYA tail vein injection at the dose of 5mg/kg and 10mg/kg after operation respectively. The sham operation group and the model group were given the.4. nerve function defect of the tail vein of physiological saline: 2h after anesthesia sober, five points method was used to evaluate the neurological deficit score.5. histology evaluation: all rats were executed after 24h and executed quickly to get the brain. TTC staining was used to measure the area of cerebral infarction with Image J software, and then the cerebral infarction specific product was calculated. The expression of HSP70 in the ischemic penumbra region after cerebral ischemia and reperfusion was detected by immunohistochemistry and Western blotting. Results: the 1. nerve function score showed that the HSYA (5mg/kg, 10mg/kg) tail vein injection treatment and the preventive medication can reduce the large amount. Compared with the model group, the scores of nerve function defect in group 10mg/kg, group 5mg/kg and group 10mg/kg were significantly lower than those in the model group (P0.05). There was no statistically significant difference between the 5mg/kg group and the model group before operation (P0.05).2. infarction ratio: HSYA (5mg/kg, 10mg/kg). Treatment and prevention of drugs can significantly reduce the cerebral infarction ratio after cerebral ischemia and reperfusion in rats. The difference has statistical significance (P0.05). After operation, the cerebral infarction ratio in group 5mg/kg after operation is significantly lower than that of group 5mg/kg (P0.05). The treatment of 10mg/kg in group 10mg/kg is obviously better than that of group 5mg/kg after operation. The difference was statistically significant (P0.05).3. immunohistochemical staining: HSP70 was mainly expressed in the cytoplasm, and the expression of positive cells in the normal group was very few. The sham operation group was a little more expressed than the normal group, but there was no significant difference (P0.05). The cerebral ischemia reperfusion could lead to a large increase in the positive cells in the semi dark zone of the brain tissue of the rats, and the difference was statistically significant. Significance (P0.01). Compared with the model group, the expression of positive cells decreased significantly, and the difference was statistically significant (P0.01). Compared with the pre operation 5mg/kg group, the expression of positive cells in group 5mg/kg and 10mg/kg group decreased significantly (P0.05). The expression of positive cells in 10mg/kg group after operation was compared with that of group 10mg/kg before operation, and the expression of positive cells in 10mg/kg group after operation. The difference was statistically significant (P0.01). Compared with group 5mg/kg, the expression of positive cells in group 10mg/kg decreased significantly after operation, and the difference was statistically significant (P0.01).4. Western blot showed that the expression of HSP70 in the sham operation group increased significantly compared with the normal group, and the difference was statistically significant (P0.01); compared with the sham operation, the model group HS was compared. The expression of P70 was significantly higher, and the difference was statistically significant (P0.01). Compared with group 5mg/kg, HSP70 expression in group 10mg/kg after operation was significantly decreased and the difference was statistically significant (P0.01). Compared with group 10mg/kg, the expression of HSP70 expression in group 5mg/kg and 10mg/kg group after operation was significantly increased, and the difference was statistically significant (P0.01). The expression of HSP70 in group 10mg/kg after operation was significantly higher than that of group 5mg/kg, and the difference was statistically significant (P0.01). Conclusion the treatment and prevention of 1.HSYA can reduce the score of nerve function defect after cerebral ischemia-reperfusion in rats, 10mg/kg before operation, 5mg/kg and 10mg/kg dosage after operation can obviously reduce the score of nerve function defect. The treatment and prevention of hemiplegia in the good rat's limbs.2.HSYA can significantly reduce the volume of cerebral infarction after cerebral ischemia and reperfusion in rats, at the same dose (5mg/kg, 10mg/kg). The postoperative treatment is better than the pre operation prophylaxis.3. immunohistochemistry and immunoglobulin imprinting method: HSP70 is mainly expressed in the cytoplasm and expressed in normal tissues. Less, surgical injury may lead to a slight increase in the expression of HSP70. After cerebral ischemia and reperfusion, the HSP70 protein in the semi dark zone of the rat brain increases greatly. However, HSYA prevention and treatment can reduce the expression of HSP70 protein in the ischemic penumbra region of SD rats after cerebral ischemia reperfusion. At the same dose (5mg/kg, 10mg/kg), after ischemia-reperfusion treatment The level of HSP70 expression was significantly better than that before ischemia, and the 10mg/kg treatment group had better effect than the 5mg/kg group after operation.

【学位授予单位】：云南中医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.33

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