κ阿片受体激动剂(U50488H)对缺血性脑卒中大鼠神经元自噬、迟发性神经元损伤及NOTCH表达水平的影响

Effects of κ opioid receptor agonist(U50488H)on autophagy,delayed nerve injury and NOTCH expression in ischemic stroke rats

目的探讨κ阿片受体激动剂(U50488H)对缺血性脑卒中大鼠神经元自噬、迟发性神经元损伤及NOTCH表达水平的影响。方法选取40只清洁级(Specific pathogen free,SPF)级Sprague Dawley(SD)雄性大鼠,随机分为正常(N)组、模型(M)组、丁苯酞(B)组、U50488H(U)组,每组各10只,对M,B,U组采用线栓法建立缺血性脑卒中模型,N组不建立该模型,建模成功后对B组给予灌胃4.5 mg/kg的丁苯酞,对U组给予脑内注射1.5 mg/kg的U50488H,N,M组同期给予灌胃同体积生理盐水,生物机能实验系统检测大鼠脑血流动力学,Zea longa评分及神经症状评分(Neurosymptom score,NSS)评分检测大鼠迟发性神经损伤,HE染色法检测脑组织病理形态,免疫印迹法检测脑组织中自噬相关蛋白表达水平,免疫组化法检测NOTCH表达水平。结果与N组比较,M组心率(Heart rate,HR)、收缩压(Systolic blood pressure,SBP)、舒张压(Diastolic blood pressure,DBP)均显著升高(P<0.05);与M组比较,B,U组大鼠HR,SBP,DBP均显著降低(P<0.05),且U组比B组降低显著(P<0.05)。与N组比较,M组Zea longa评分、NSS评分均显著升高(P<0.05);与M组比较,B,U组大鼠Zea longa评分、NSS评分均显著降低(P<0.05),且U组比B组降低显著(P<0.05)。N组大鼠脑组织及皮质结构完整且致密均匀,神经细胞核形态正常完整,核仁清晰,神经细胞排列整齐,未见细胞周围间隙水肿;M组脑组织结构遭到破坏,细胞排列紊乱且着色变浅,核仁及结构消失,出现蹄网状结构,并可见大量空泡;与M组比较,B,U组病理状态明显,细胞体积明显缩小,细胞排列少且散乱。与N组比较,M组脑组织中LC3,Beclin1蛋白表达水平显著升高(P<0.05);与M组比较,B,U组脑组织中LC3,Beclin1蛋白表达水平显著降低(P<0.05),且U组比B组降低显著(P<0.05)。与N组比较,M组脑组织NOTCH蛋白表达水平显著升高(P<0.05);与M组比较,B,U组脑组织NOTCH蛋白表达水平显著降低(P<0.05),且U组比B组降低显著(P<0.05)。结论κ阿片受体激动剂可显著降低缺血性脑卒中大鼠神经元自噬及NOTCH表达水平,并有效改善迟发性神经元损伤。

Objective To investigate the effects of κ opioid receptor agonist(U50488 H)on autophagy,delayed nerve injury,and NOTCH expression in ischemic stroke rats.Methods Forty male Sprague Dawley(SD)rats of specific pathogen free(SPF)were randomly divided into normal(N)group,model(M)group,butylphthalide(B)group,and U50488 H(U)group with10 animals in each group.The ischemic stroke model was established for the M,B,and U groups by suture method.After the modeling was successful,the B group was given 4.5 mg/kg butylbenzene Phthaloin,the U group was injected with U50488 H into the brain at 1.5 mg/kg,and the N and M groups were given the same volume of normal saline by gavage at the same time.Neurosymptom score(NSS)was used to detect delayed nerve injury in rats.HE staining was used to detect the pathological morphology of brain tissue.Western blotting was used to detect the expression level of autophagy-related proteins in brain tissue.Immunohistochemistry was used to detect the expression level of NOTCH.Results Compared with the N group,the heart rate(HR),systolic blood pressure(SBP),diastolic blood pressure(DBP),Zea longa score and NSS score of the M group were significantly increased(P<0.05).Compared with the M group,the Zea longa score and the NSS score of the B and U groups were significantly decreased(P<0.05).HR,SBP,DBP,Zea longa score and the NSS score of U group were significantly decreased(P<0.05),and U group was significantly lower than B group(P<0.05).The brain tissue and cortex of the rats were intact,dense and uniform;the nerve cell nucleus was normal and complete;the nucleoli were clear;the nerve cells were arranged neatly,and there was no edema in the space around the cells in the N group.The brain structure was destroyed;the cell arrangement was disordered and colored in the M group.Compared with the M group,the pathological state of the B and U groups was obvious,the cell volume was reduced,and the cell arrangement was less scattered.Compared with the N group,the levels of LC3,Beclin1 and NOTCH in the brain tissue of the M group were significantly increased(P<0.05).Compared with the M group,the levels of LC3,Beclin1 and NOTCH in the brain tissue of the B and U groups were significantly decreased(P<0.05),and group U was significantly lower than group B(P<0.05).Conclusion κ opioid receptor agonists can significantly reduce neuronal autophagy and NOTCH expression in rats with ischemic stroke,and effectively improve delayed neuronal damage.