氧化苦参碱对缺氧缺血性脑损伤新生大鼠脑组织Casepase-3、Bcl-2及Bax表达的影响

The effect of oxymatrine on the hypoxic ischemic Brain Damage of Neonatal rats and the expression of Caspase-3,Bax and Bcl-2

目的观察氧化苦参碱(OMT)对缺氧缺血性脑损伤(HIBD)新生大鼠的保护作用及对Caspase-3、Bax、Bcl-2蛋白表达的影响。方法清洁级新生7 d龄SD大鼠72只,随机分为假手术组、模型组、OMT 120 mg/kg治疗组。后2组采用经典Rice法制作HIBD动物模型,假手术组未给予缺氧处理及左侧颈总动脉结扎。模型组和假手术组在造模完成后,即刻开始腹腔注射无菌注射用水,剂量为10 m L/kg,每12 h 1次。OMT治疗组在造模完成后,即刻开始腹腔注射OMT 120 mg/kg,每12 h 1次,共注射4次。末次注射后12 h断头取材,用HE染色方法观察脑组织病理变化,采用免疫荧光及Western blot检测损伤侧脑组织内凋亡相关基因Caspase-3、Bax、Bcl-2蛋白表达情况。结果 HE染色结果表明假手术组大鼠皮层及海马CA3区锥体神经元结构完整、排列紧凑;与假手术组比较,模型组神经元边界不清、排列紊乱、组织疏松、基质不完整,OMT治疗组可以改善HIBD新生大鼠脑组织神经元的异常形态变化。免疫荧光显示假手术组脑组织中Caspase-3、Bax荧光细胞数目少、荧光强度低,Bcl-2阳性细胞较多、荧光明显;模型组Caspase-3、Bax阳性细胞数增加、荧光增强,Bcl-2阳性细胞数减少、荧光减弱,OMT治疗组显著逆转上述改变。Western blot显示模型组脑组织中Caspase-3、Bax蛋白表达增强(P<0.05),Bcl-2蛋白表达减少(P<0.05);与模型组比较,OMT治疗组Caspase-3、Bax蛋白表达减少(P<0.05),Bcl-2蛋白表达增加(P<0.05)。结论 OMT可能是通过上调Bcl-2表达,抑制Caspase-3、Bax表达,减轻缺氧缺血引起的神经元凋亡,从而对HIBD新生大鼠发挥保护作用。

Objective To observe the protective effect of Oxymatrine on the hypoxic ischemic brain damage of neonatal rats and the expression of Caspase -3,Bax and Bcl - 2. Methods A total of 72 7 - day - old health SD rats were randomly divided into sham group,model group and oxymatrine （120mg/kg） group. Classic Rice method was used to establish HIBD model in the latter 2 goups. After ischemic, oxymatrine at dose of 120mg/kg as given to rats by intraperitoneal injection immediately,twice a day for two successive days,sham and model groups were given equal physiological saline 4 times. Eight rats in each group were sacrificed at 3rd day 12h after model establishmentlast injec- tion. Histomorphology changes were performed by hematoxylin - eosin （HE） staining. Expression of apeptosis - related proteins Caspase - 3,Bax and Bcl -2 were analyzed by western- blot analysis and immunofluorescence. Result In sham group,neurons in cortex and hippocampus CA3 region remained intact and the distribution of neurons possessed regularity. In model group, neurons arrangement was disordered with loosened cytoplasms ,interstitial edema. Immunofluorescence analysis revealed the expressions of Caspase -3 and Bax were significantly greater in ipsilat- eral region of model group but were reduced by OMT post -treatment. On the other hand,the expression of Bc1-2 was decreased in region of model group,which were restored by OMT post -treatment. Western blots results indicated that The level of Caspase -3 ,Bax expression in brain were increased （P 〈0.05） and the expression of Bc1-2 was decreased（P 〈0.05） in model group. Compared with model group,in oxymatrine group,a remarkable reduction in the extension of injury was observed in neurons, The level of Caspase- 3 ,Bax expression in brain were de- creased （P 〈 0. 05） and the expression of Bcl - 2 was increased （P 〈 0. 05）. Conclusion Oxymatrine maybe reduce the apoptosis of neuron after severe brain injury through improving the expression of Bcl -2 protein and inhibiting expression of Bax and thus provides neuroprotection against HIBD in neonatal rats.