丹红注射液对大鼠皮质神经元体外模拟缺血再灌注损伤的保护作用

目的 探讨体外模拟缺血再灌注后大鼠皮质神经元内质网应激诱导凋亡的机制及丹红注射液对其的干预作用.方法 体外培养大鼠皮质神经元,神经元特异性烯醇酶(NSE)鉴定神经元纯度.建立体外培养大鼠皮质神经元模拟缺血再灌注模型,随机分为对照组(始终正常培养)、缺血再灌注组(体外模拟缺血再灌注)、治疗组(缺血再灌注加丹红注射液干预),应用免疫组织化学、免疫荧光染色方法鉴定神经元纯度;流式细胞术Annexin V、PI双标检测神经元凋亡率及活性半胱天冬酶(Caspase)-3、Caspase-8、Caspase-9蛋白表达;Western-blot免疫印迹分析法检测活性Caspase-12、葡萄糖调节蛋白78(GRP78)、Bcl-2及细胞色素C蛋白表达.结果 大鼠皮质神经元可纯化体外培养.皮质神经元体外模拟缺血6 h再灌注24、 48 h细胞凋亡率分别为(17.95±1.03)%、(22.62±0.98)%,丹红治疗组(8 mL/L)分别为(9.74±0.56)%、 (3.93±0.23)%,两组比较差异有统计学意义(χ2=7.164、10.650,P<0.05).体外模拟缺血再灌注诱导神经元GRP78表达上调,活性Caspase-12表达增加,但与治疗组比较差异无显著性(t=0.591、1.772,P>0.05).神经元缺血再灌注后细胞色素C表达增加,Bcl-2表达减少,活性Caspase-3、Caspase-8、Caspase-9表达增加,丹红注射液治疗后细胞Bcl-2表达增加,细胞色素C释放减少,活性Caspase-3、 Caspase-8、Caspase-9含量降低,两组比较差异有显著性(t=8.374～25.235,P<0.05、0.01).结论 内质网应激参与神经元体外模拟缺血再灌注后细胞凋亡,丹红注射液能抑制模拟缺血再灌注后细胞凋亡.

PROTECTIVE EFFECT OF DANHONG INJECTION ON RAT CORTICAL NEURONAL INJURY CAUSED BY SIMULATED ISCHEMIA/REPERFUSION IN VITRO

Objective To study the protection effect of danhong injection on rat cortical neurons during apoptotic induction of simulated ischemia/reperfusion in vitro. Methods Rat cortical neurons were cultured in vitro and identified by NSE-immunohistological staining and immunofluorescence staining.Neuronal ischemia/reperfusion models in vitro were established as followings: cultures were rinsed twice with PBS,then subjected to ischemia for 6 h in Earle's without glucose by placing culture wells into an anaerobic ch...