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目的:探讨丹红注射液对原代培养的海马神经元细胞因缺氧缺糖所致损伤的保护作用及其机制。方法:在体外培养新生SD乳鼠原代海马神经元细胞,并以神经元特异性烯醇化酶(NSE)免疫组化染色法对其进行鉴定。运用MTT法确定丹红注射液安全剂量,将培养的细胞随机分成6组:正常对照组、模型组、尼莫地平(200μg/m L)阳性对照组及丹红注射液低(40μL/m L)、中(80μL/m L)、高(120μL/m L)浓度组。加药并建立乳鼠海马神经元缺氧缺糖模型。观察丹红注射液对糖氧剥夺损伤乳鼠海马神经元细胞丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及细胞上清液乳酸脱氢酶(LDH)水平,并采用流式细胞仪检测各组细胞早期凋亡率,利用试剂盒检测各组细胞Caspase-3活性,利用Hoechst33342染色观察各组细胞凋亡情况。结果:与模型组比较,丹红注射液能显著减少海马细胞上清液内LDH漏出量,增强糖氧剥夺损伤海马神经元细胞内SOD与GSH-Px活性,降低MDA含量,有效减轻缺氧缺糖损伤导致的海马神经元早期细胞凋亡,抑制细胞内Caspase-3活性。结论:丹红注射液能有效抑制缺氧缺糖所致原代海马神经元凋亡,增强机体抗氧化能力,对脑缺血具有保护作用。
Objective: To investigate the protective effect and its mechanism of Danhong injection on injury induced by hypoxia and hypoglycemia in primary cultured hippocampal neurons. Methods: Primary cultured hippocampal neurons of neonatal SD rats were cultured in vitro and identified by neuron specific enolase (NSE) immunohistochemical staining. The safe dose of Danhong injection was determined by MTT method. The cultured cells were randomly divided into 6 groups: normal control group, model group, nimodipine (200μg / m L) positive control group and Danhong injection low (40μL / ) Medium (80 μL / m L) and high (120 μL / m L) concentrations. Dosing and establishment of hypoxia - glucose deprivation model in neonatal rat hippocampal neurons. To observe the effect of Danhong injection on malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and cell supernatant Lactate dehydrogenase (LDH) were measured. Caspase-3 activity in each group was detected by flow cytometry. The apoptosis of each group was observed by Hoechst33342 staining. Results: Compared with the model group, Danhong injection can significantly reduce the leakage of LDH in the hippocampal supernatant, enhance the activity of SOD and GSH-Px in the hippocampal neurons induced by glucose and oxygen deprivation, reduce the content of MDA, Early injury of hippocampal neurons caused by glucose-induced apoptosis and inhibition of intracellular Caspase-3 activity. CONCLUSION: Danhong injection can effectively inhibit the apoptosis of primary hippocampal neurons induced by oxygen-glucose-deprivation and enhance the body’s antioxidant capacity, which has a protective effect on cerebral ischemia.